Registration Dossier

Diss Factsheets

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 November 2020 - ..June 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study presented herein is a guideline study without restrictions performed under GLP conditions.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 November 2020 - ...June 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study presented herein is a guideline study without restrictions performed under GLP conditions.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016-07-29
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 426 (Developmental Neurotoxicity Study)
Version / remarks:
2007-10-16
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
not applicable
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL

- Purity, including information on contaminants, isomers, etc.: 93.8% for T0421.
- Test substance No.: 20/0051-1 for T0421.
- Batch identification: T0421.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at room temperature. The stability under the storage condition over the exposure period is guaranteed by the sponsor, and the sponsor holds this responsibility. Expiry date of the test substance: Aug 2020 for T0421.

INFORMATION ON NANOMATERIALS
- Chemical Composition:
- Density:
- Particle size & distribution:
- Specific surface area:
- Isoelectric point:
- Dissolution (rate):

Test substance preparation:
- Generation procedure: For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage; mixed with conditioned dilution air and passed into the inhalation system.

OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added: homogenous, white solid.
Species:
rat
Strain:
Wistar
Remarks:
Wistar rats, Crl:WI(Han) Rats were selected since this rodent species is recommended in the respective test guidelines. Wistar rats were selected since there is extensive experience available in the laboratory with this strain of rats.
Details on species / strain selection:
not specified
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
- Females nulliparous and non-pregnant: yes
- Age at study initiation: about 7 weeks (female), about 8 weeks (male)
- Weight at study initiation: The weight variation of the animals used did not exceed +/- 20 percent of the mean weight of each sex.
- Fasting period before study: The animals did not have access to food or water during exposure.
- Housing:
From delivery until mating and male animals after mating: Typ 2000P: ca. 2065 cm2 (polysulfone cages) / up to 5 animals
During mating: type III polycarbonate cages, 1 male/1 female per cage
During rearing: up to PND 22: type III polycarbonate cages, 1 dam with her litter
After weaning the females from study day 90 after exposure onward until sacrifice: Typ 2000P: ca. 2065 cm2 (polysulfone cages) / up to 5 animals. Remaining females with litters will be
maintained in type III cages until weaning.
For Motor Activity Measurement: Typ III polycarbonate cages (floor area about 800 cm²) / 1 animal
During Exposure: Wire cages, type DK III / up to 2 animals Females from PND 4 until study day 94 (and females without litter from the same time period onwards): perforated polycarbonate cages type II. From study day 95 onward wire cages, type DK III
- Diet (ad libitum): mouse and rat maintenance diet, GLP, 12 mm pellets, Granovit AG, Kaiseraugst, Switzerland before and after exposure. Food was withdrawn during exposure.
- Water (ad libitum): tap water
- Acclimation period: 11 days

DETAILS OF FOOD AND WATER QUALITY: The food used in the study was assayed for chemical as well as for microbiological contaminants. In view of the aim and duration of the study, the contaminants occurring in commercial food should not influence the results. The drinking water is regularly assayed for chemical contaminants both by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring of BASF SE as well as for bacteria by a contract laboratory. The Drinking Water Regulation will serve as the guideline for maximum tolerable contaminants. In view of the aim and duration of the study, there are no special requirements exceeding the specification of drinking water.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 45 - 65%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: beginning of experiment To: end of experiment
Route of administration:
inhalation: aerosol
Type of inhalation exposure (if applicable):
whole body
Remarks:
whole-body exposure for the reasons explained see IUCLID section 13.2 'Human health requirements Final Decision: protocol deviations and rationale'
Mass median aerodynamic diameter (MMAD):
>= 0.6 - <= 2.53 µm
Remarks on MMAD:
MMAD / GSD: MMAD = 0.60-2.53 μm (geometric standard deviation = 3.08-2.23)
Vehicle:
unchanged (no vehicle)
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Generation of the inhalation atmospheres via a solid particle generators (brush-generator; BASF SE, Ludwigshafen, Germany) & Aerosol mixing tube (stainless steel; BASF SE, Ludwigshafen, Germany). Whole body exposure systems were used. The animals were kept singly in wire cages located in a glass steel inhalation chamber, volume of 1.1 m³ (BASF SE).
- Method of holding animals in test chamber: Whole body exposure systems. The animals were kept singly in wire cages located in a glass steel inhalation chamber, volume of 1.1 m³ (BASF SE). The chambers were located in exhaust hoods in an air conditioned room.
- Source and rate of air: Conditioned air from the central air conditioning system, compressed and exhaust air. Compressed air was produced by an oil-free compressor (HT 6, Josef Mehrer GmbH & Co KG, Germany). For this purpose, air is filtered by an inlet air strainer and introduced into the compressor. After passing through an second ultra filter (SMF 5/3, 108 mm, Donalson), the compressed air (15 bar) is stored in a storage of 1500 or 5000 L. The compressed air is conducted to the laboratories via pipes, where the pressure is reduced to 5 - 6 bar. In the laboratory, the compressed air can be taken as required.
- Method of conditioning air: Conditioned air from the central air conditioning system provides cold air of about 15°C. This cold air passes through an activated charcoal filter, is adjusted to room temperature of 20 to 24°C and passes through a second particle filter (H13 (HEPA) Camfil Farr, Germany). The so generated conditioned air was used to generate inhalation atmospheres.
- System of generating particulates/aerosols: The particles/aerosol was generated via a solid particle generator (brush-generator; BASF SE, Ludwigshafen, Germany) and an aerosol mixing tube (stainless steel; BASF SE, Ludwigshafen, Germany), according to the following method: For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage; mixed with conditioned dilution air and passed into the inhalation system.
- Temperature, humidity, pressure in air chamber: Daily mean relative humidities in the inhalation systems ranged between 41.6 and 60.8 %. Daily mean temperatures in the inhalation systems ranged between 21.4 and 23.7°C. They are within the range suggested by the respective testing guidelines.
- Air flow rate: The air flows were constantly maintained in the desired range.
- Air change rate: An air change of about 24 to 25 times per hour can be calculated by dividing the supply air flow through the volume of each inhalation system.
- Method of particle size determination: The particle size analysis was carried out with a cascade impactor.Equipment for particle size analysis: Stack sampler Marple 298 (New Star Environmental, Inc., Roswell, Georgia 30075, USA) ; Vacuum compressed air pump (Millipore Corporation, Billerica, MA 01821, USA) ; Limiting orifice 3 L/min (Millipore Corporation, Billerica, MA 01821, USA) ; Sampling probe internal diameter 7 mm ; Balance Sartorius MSA 6.6S-000-DF (Sartorius AG, Göttingen, Germany). The calculation of the particle size distribution was carried out in the Laboratory for Inhalation Toxicology of the Experimental Toxicology and Ecology of BASF SE on the basis of mathematical methods for evaluating particle measurements (OECD guidance document No. 39). Particle Size distribution of the test atmosphere were determined also with the Aerodynamic Particle Spectrometer APS 3321 (TSI, USA). MMAD and GSD is obtained directly by the piece of equipment used APS 3321. Frequency: On two days during the exposure period, with 3 repeats on each day. To determine the particle size distribution in the submicrometer range, each test atmosphere was measured with the Scanning Mobility Particle Sizer (SMPS; Grimm Aerosol Technik GmbH & Co KG, Ainring, Germany). The SMPS system comprises an Electrostatic Classifier (Model Vienna U-DMA) which separates the particles into known size fractions, and a Condensation Particle Counter (CPC) which measures particle count concentrations. The DMA was equipped with Am-241 neutralizer. The instrument measures particles in the size range from 0.011 to 1.083 µm. Using a conductive sample hose, the SMPS sampled at 0.3 liters per minute (LPM) with a sheath flow of 3 LPM. At this setting the single-stage, inertial impactor incorporated into the inlet of the SMPS to remove larger particles had a 50% cut size of 1.082 µm according to the software calculation. The sampling duration was about 7 minutes. As a rule 10 repeats were measured for each exposure concentration.
- Treatment of exhaust air: Exhaust air was filtered and conducted into the exhaust air of the building.

TEST ATMOSPHERE
- Brief description of analytical method used: The concentrations of the inhalation atmospheres were determined by gravimetrical measurements of filter samples in all test groups. Control group was not sampled. This analytical method was judged to be valid because the test substances did not possess an appreciable vapor pressure.
- Samples taken from breathing zone: yes
Details on mating procedure:
Mating of the F0 generation parental animals
After 44 days premating period, the male and female parental animals were mated overnight in a 1:1 ratio until there was evidence of copulation or the maximum period of 14 days has elapsed. Throughout the mating period, each female was mated with a predetermined male.

Normally, the female was placed into the cage of her male partner about 16:00 h and separated from the male between 06:30 and 09:00 h, the following morning. Deviations from the specified times are possible on Saturdays, Sundays and public holidays and were documented in the raw data.

A vaginal smear was prepared for each pair after each mating and examined for sperm. If sperm was detected, mating of the pair was discontinued. The day on which sperm were detected, was referred to as gestation day (GD) 0 and the following day as GD 1.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
The nominal concentration could be/was calculated from the study means of the test-substance flow and the supply air flows used during exposure to generate the respective concentrations. The concentrations of the inhalation atmospheres were determined by gravimetrical measurements of filter samples in all test groups. Control group was not sampled. This analytical method was judged to be valid because the test substances did not possess an appreciable vapor pressure.
Duration of treatment / exposure:
The animals were exposed for 44 days before mating. The mating period were
maximal 2 weeks. After the mating period, the exposure of all male F0 animals were continued
until they are exposed for total minimal 90 days. After the mating period, the female F0 animals
were exposed further until gestation day 19. To allow them deliver and rearing their pups (F1
generation), they were not exposed from gestation day 20 to postnatal day (PND) 3. From
PND 4 through to PND 21, the dams were exposed with their pups in exposure cages
containing beddings. The first parental female animals were in gestation stage already after
the first few mating days, therefore, the post-weaning period were adjusted in such a way, that
a total of minimum 90 exposure will be achieved for females.
Frequency of treatment:
7 consecutive days per week, 6 hours per day
from PND4 through PND21
Details on study schedule:
not specified
Dose / conc.:
0 mg/m³ air
Remarks:
Test Group 0 (>Parental animals F0) - air control
Dose / conc.:
0.52 mg/m³ air (analytical)
Remarks:
SD: 0.16 mg/m3; target concentration: 0.5 mg/m³: Test Group 1 (>Parental animals F0)
Dose / conc.:
2.01 mg/m³ air (analytical)
Remarks:
SD: 0.20 mg/m3; target concentration: 2.0 mg/m³: Test Group 2 (>Parental animals F0)
Dose / conc.:
10.07 mg/m³ air (analytical)
Remarks:
SD: 0.96 mg/m3, target concentration: 10 mg/m³: Test Group 3 (>Parental animals F0)
No. of animals per sex per dose:
16/sex/dose group (parental animals)
5/sex at the high dose (recovery animals)
3 males at the high dose (for particle detection)

Subset Number of pups selected Day of examination Examination
I 10/sex/group PND 22 Measurement of thyroid hormones
II 10/sex/group PND 22 Perfusion fixation, brain weights
and neuropathology
III 5/sex/group PND 22 Histopathological examination and
organ burden
IV 10/sex/group PND 13 and 21 Open field observation
PND 13, 17 and 21 Motor activity
V 3 males/group PND 22 Perfusion fixation and electron
(highest dose) microscopic for particle detection
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of the 14-day range finding study (BASF study no 36I0050/20I005 - Ma- Hock 2021), upon approval of the sponsor, nominal aerosol concentrations of 0.5, 2.0 and 10.0 mg/m³ were used for the test substance in the low, mid and high dose groups, respectively.
- Rationale for animal assignment:
Prior to the pre-exposure period, the animals were distributed according to weight among the
individual test groups, separated by sex. The weight variation of the animals used did not
exceed ± 20 percent of the mean weight of each sex. The list of randomization instructions
was compiled with a computer.
For each neurofunctional test and motor activity measurement, separate randomization lists
were created. The list of randomization instructions were compiled with a computer (Laboratory
data processing, Experimental Toxicology and Ecology, BASF SE).
- Fasting period before blood sampling for clinical biochemistry: not specified
- Post-exposure recovery period in satellite groups: 45 days recovery period
Positive control:
none
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The clinical observation was performed on each animal at least three times (before, during and after exposure) on exposure days and once a day during pre-exposure and post exposure observation days. On non-exposure days a cage-side examination will be conducted at least once daily for any signs of morbidity, pertinent behavioral changes and/or signs of overall toxicity.

MORTALITY: The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) on working days and once a day (in the morning) on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS: YES
- Time schedule: All parental animals and recovery group animals were subjected to detailed clinical observations (DCO) outside their cages once before the beginning of the administration period and once during the first two weeks of the exposure, once monthly thereafter. DCO was performed in the morning before exposure. For observation, the animals were removed from their cages and placed in a standard arena (50 x 37.5 cm with a lateral border of 25 cm) for at least 20 seconds/animal.

BODY WEIGHT: Yes
- Time schedule for examinations:
The body weight of the animals was determined at the start of the pre-exposure, at the start of
the exposure period and then, as a rule, once a week as well as prior to gross necropsy. The
body weight of the recovery animals were determined at the start of the recovery period, and
once a week during the recovery period.
The following exceptions were notable for the female parental animals:
• During the mating period, the females were weighed on the day of positive evidence of
sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day after parturition (PND1) and on PND 4, 7, 14, and 21.
• In the females without positive evidence of sperm, body weight was determined once a week during mating and gestation periods and in the females without litter during lactation period.

As a rule, the animals were weighed at the same time of the day (in the morning).

Body weight change was calculated as the difference between body weight on the respective exposure day and body weight and the weight of previous weighing. Group means were derived from the individual differences.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was determined weekly and calculated as mean food consumption in grams
per animal and day.
Generally, food consumption was determined once a week for the male and female animals
and post mating period (males), with the following exceptions:
• Food consumption was not determined during the mating period (male and female
parental animals).
• Food consumption of the females with evidence of sperm was determined for GD 0-7, 7-
14 and 14-20.
• Food consumption of the females which gave birth to a litter was determined for PND 1-
4, 4-7, 7-13.
During recovery period, food consumption was determined in the animals of test groups 20 –
28 of the recovery animals. It was determined at the start of the recovery period and once a
week during the recovery period.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OPHTHALMOSCOPIC EXAMINATION: YES
Before the beginning of exposure, the eyes of all parental animals were examined with an ophthalmoscope (HEINE OPTOTECHNIK, Herrsching, Germany) after administration of a mydriatic agent (Mydrum, Dr. Gerhard Mann chem.-pharm. Fabrik GmbH and Bausch & Lomb GmbH, Germany). At the end of the exposure period, only animals selected for examinations according to OECD 413, 10 males and 10 females per group, were subjected to ophthalmological examination. In the first step, only control (test group 0) and high concentration groups (test groups 3, 6, 7 and 8) were examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in the morning
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes
- How many animals: 10 M + 10 F per dose group
-Parameters checked: leukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets, differential blood count, reticulocytes, preparation of blood smears, prothrombin time (PT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in the morning
- Animals fasted: Yes
- How many animals: 10 M + 10 F per dose group
- Parameters checked: alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-glutamyl transpeptidase (GGT), sodium (Na), potassium (K), chloride (CL), Inorganic phosphate (INP), calcium (Ca), urea (UREA), creatinine (CREA), glucose (GLUC), total biluribin (TBIL), total protein (TP), albumin (ALB), globulin (GLB), triglycerides (TRIG), cholesterol (CHOL)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the 90days exposure period
- Dose groups that were examined: 10 M + 10 F per dose group
- Battery of functions tested: sensory activity / grip strength / motor activity / reflexes


IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: Not specified
- Dose groups that were examined: 10 M + 10 F per dose group and recovery groups (highest dose: 5M + 5F)
- Number of animals: 10 M + 10 F per dose group and recovery groups (highest dose: 5M + 5F)
- Parameters checked: Cytological parameters: total cell count, cell differential analysis of cytospin preparations; protein; Enzymes: lactate dehydrogenase, alkaline phosphatase, N-acetyl-beta-D-Glucosaminidase (NAG BAL), gamma−Glutamyltransferase

ORGAN (lung, liver, heart, brain, olfactory bulb) BURDEN: Yes
- Time schedule for analysis: at the end of the exposure period and after the recovery period (45days post exposure)
- Dose groups that were examined: all
- Number of animals: 3 /sex / group
- Parameters checked: Zn content

OTHER: - Electron microscope analysis of particulate matter in organs and tissues: 3 male animals of the highest dose group



Oestrous cyclicity (parental animals):
In all parental females in the premating phase, estrous cycle length and normality were
evaluated by preparing vaginal smears during a minimum of 2 weeks prior to mating and
throughout cohabitation until there is evidence of sperm in the vaginal smear.
Additionally, on the day of scheduled sacrifice, the estrous status was also determined in all
parental female rats.
Sperm parameters (parental animals):
not specified
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum:
On PND 4, the individual litters were standardized in such a way that, whenever possible, each litter contains 5 male and 5 female pups (always the first 5 surviving pups/sex in each litter were taken for further rearing). If individual litters did not have 5 pups/sex, the litters were processed in such a way that the most evenly distributed 10 pups per litter were present for further rearing (e.g., 6 male and 4 female pups). Standardization of litters was not performed in litters with 10 pups or less.

PARAMETERS EXAMINED
The following parameters were examined in offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups, open field observations, motor activity

GROSS EXAMINATION OF DEAD PUPS: yes

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: yes
Necropsy:
On postnatal day 22, 10 animals per sex and group were weighed, subjected to deep
anesthesia (i.p. pentobarbital) and sacrificed by perfusion fixation.
SOERENSEN phosphate buffer was used as the rinsing solution, and neutrally buffered, 4% formaldehyde solution was used as a fixative.
The perfusion fixed animals were necropsied with regard to the question of neuropathology, and the visible organs were assessed by gross pathology as accurately as is possible after a perfusion fixation. The cranial vault was opened and the skin was removed from the head. In this state, the perfused animals were stored in neutrally buffered, 4% formaldehyde solution for at least 48 hours.
Organ weights:
The following weights were determined (the brain was weighed after its removal but before
further preparation):
1. Final body weight
2. Brain (including olfactory bulb)
The final body weights were recorded to calculate the relative organ weights.
Length and width of the brain:
The length and maximum width of the brain were measured in all animals. The length of the brain was measured on a line extending from the rostral end of the frontal lobe to the caudal medulla oblongata of the cerebellum, width: pituitary region.
Organ/Tissue fixation:
The following organs/tissue specimens were carefully removed and processed histotechnically in accordance with the data given in the respective sections of this report:
1. All gross lesions
2. Brain with olfactory bulb
3. Eyes with retina and optic nerve
4. Nose (nasal cavity level III) with olfactory epithelium
5. Pituitary gland
6. Trigeminal ganglia
The animals and the tissue or organ material remaining after trimming was stored in neutrally buffered, 4% formaldehyde solution.
Neurohistopathology:
Morphometry of the brains (PND 22) :
Morphometry was performed in all animals of test groups 0 (control), 3, 6, 7 and 8.
Thickness measurements of major brain layers (neocortex: frontal and parietal cortices, caudate nucleus/putamen, hippocampus, corpus callosum, cerebellum) were performed. Measurements were carried out bilaterally in the left and right halves of the brain except for the corpus callosum and the cerebellum.

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no
Postmortem examinations (parental animals):
SACRIFICE / GROSS NECROPSY / ORGAN WEIGHTS
At the time of sacrifice, adult animals were examined macroscopically for any abnormalities or pathological changes.
The following weights were determined in all animals sacrificed on schedule:
1.Anesthetized animals (final body weight)
2. Adrenal glands (fixed)
3. Brain
4. Epididymides
5. Heart
6. Kidneys
7. Liver
8. Lung
9. Ovaries
10. Prostate (ventral and dorsolateral part together, fixed)
11. Seminal vesicles with coagulating glands (fixed)
12. Spleen
13. Testes
14. Thymus (fixed)
15. Thyroid glands (with parathyroid glands) (fixed)
16. Uterus with cervix
All paired organs were weighed together (left and right).

HISTOPATHOLOGY
Organs and tissues of F0 animals histologically processed:
1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Epididymides
12. Esophagus
13. Eyes with optic nerve
14. Extraorbital lacrimal gland
15. Femur with knee joint
16. Harderian glands
17. Heart
18. Ileum
19. Jejunum
20. Kidneys
21. Larynx (3 levels)
22. Liver
23. Lungs
24. Lymph nodes (tracheobronchial and mediastinal)
25. Lymph nodes (mesenteric)
26. Mammary gland (female)
27. Nasal cavity (4 levels)
28. Olfactory bulb
29. Ovaries
30. Oviducts
31. Pancreas
32. Pharynx
33. Parathyroid glands
34. Peyer’s patches
35. Pituitary gland
36. Prostate
37. Rectum
38. Salivary glands
(mandibular and sublingual glands)
39. Sciatic nerve
40. Seminal vesicles
41. Skeletal muscle
42. Skin
43. Spinal cord
(cervical, thoracic and lumbar cord)
44. Spleen
45. Sternum with marrow
46. Stomach
(forestomach and glandular stomach)
47. Teeth
48. Testes
49. Thymus
50. Thyroid glands
51. Trachea
52. Urinary bladder
53. Uterus
54. Vagina



Postmortem examinations (offspring):
GROSS NECROPSY/ORGAN WEIGHTS:
Five pups per sex and group were sacrificed under pentobarbitone anesthesia by
exsanguination from the abdominal aorta and vena cava. They were necropsied and assessed
by gross pathology.
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (final body weight)
2. Brain
3. Epididymides
4. Heart
5. Kidneys
6. Liver
7. Lungs
8. Ovaries
9. Spleen
10. Testes
11. Thymus (fixed)
12. Uterus with cervix


HISTOPATHOLOGY:
Organs and tissues of PND 22 pups that were histologically processed and
examined by light microscopy.
1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Cecum
6. Cervix
7. Coagulating glands
8. Colon
9. Duodenum
10. Epididymides
11. Esophagus
12. Eyes with optic nerve
13. Extraorbital lacrimal gland
14. Femur with knee joint
15. Harderian glands
16. Heart
17. Ileum
18. Jejunum
19. Kidneys
20. Larynx (level II)
21. Liver
22. Lungs
23. Lung associated lymph nodes
24. Lymph nodes (mesenteric)
25. Mammary gland (female)
26. Nasal cavity (3 levels)
27. Olfactory bulb
28. Ovaries
29. Oviducts
30. Pancreas
31. Pharynx
32. Parathyroid glands
33. Pituitary gland
34. Prostate
35. Rectum
36. Salivary glands
(mandibular and sublingual glands)
37. Sciatic nerve
38. Seminal vesicles
39. Skeletal muscle
40. Skin
41. Spinal cord
(cervical, thoracic and lumbar cord)
42. Spleen
43. Sternum with marrow
44. Stomach
(forestomach and glandular stomach)
45. Teeth
46. Testes
47. Thymus
48. Thyroid glands
49. Trachea
50. Urinary bladder
51. Uterus
52. Vagina
Statistics:
Statistical evaluation for test groups low, mid, high in comparison with air control group
- Food consumption (parental animals), body weight and body weight change (parental animals
and pups (for the pup weights, the litter means were used)), gestation days, anogenital distance,
anogenital index
--> DUNNETT test (two-sided)
- Male and female mating indices, male and female fertility indices, gestation index, females mated, females delivering, females with liveborn pups, females with stillborn pups, females with all stillborn pups
--> FISHER'S EXACT test (one-sided)
-Mating days until day 0 pc, %postimplantation loss, pups stillborn, %perinatal loss, nipple development
--> WILCOXON test (one-sided+) with BONFERRONI-HOLM
-Implantation sites, pups delivered, pups liveborn, live pups day x, viability Index, lactation index
--> WILCOXON test (one sided-) with BONFERRONI-HOLM
-% live male day x, %live female day x
--> WILCOXON test (two-sided)
- Rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity
--> KRUSKAL-WALLIS and WILCOXON test (two-sided)
-Number of cycles and Cycle Length
--> KRUSKAL-WALLIS test (two-sided) and WILCOXON test (two-sided)
-Blood parameters
--> For parameters with bidirectional changes: WILCOXON-test (two-sided)
For parameters with unidirectional changes: WILCOXON-test (one-sided)
-Broncho-alveolar lavage fluid (BALF)
--> Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians
-Weight parameters in pathology (adult animals and PND 22 pups)
-->Non-parametric one-way analysis using KRUSKAL-WALLIS H test (two-sided).

Statistical evaluation of neuropathological parameters of PND 22 pups (subset II)
-Weight parameters (brain)
-->KRUSKAL-WALLIS test (two-sided)
-Brain width and length
--> WILCOXON test (two-sided) with Bonferroni-Holm-
-Brain morphometry: linear measurements of selected brain regions
--> WILCOXON test (two-sided)
Reproductive indices:
MALES:

Male mating index (%) = (number of males with confirmed mating*/number of males placed with females) x 100

* defined by a female with vaginal sperm or with implants in utero

Male fertility index (%) = (number of males proving their fertility* /number of males placed with females) x 100

* defined by a female with implants in utero

FEMALES:

Female mating index (%) = (number of females mated*/number of females placed with males) x 100

* defined as the number of females with vaginal sperm or with implants in utero


Female fertility index (%) = (number of females pregnant*/number of females mated**)x 100

* defined as the number of females with implants in utero
** defined as the number of females with vaginal sperm or with implants in utero

Gestation index (%) = (number of females with live pups on the day of birth/number of females pregnant*) x 100

* defined as the number of females with implants in utero

Live birth index (%) = (number of liveborn pups at birth/total number of pups born) x 100


Postimplantation loss (%) =[(number of implantations – number of pups delivered)/number of implantations] x 100


Offspring viability indices:
Viability index (%) = (number of live pups on day 4* after birth/ number of live pups on the day of birth) x 100

* before standardization of litters (i.e. before culling)


Lactation index (%) = (number of live pups on day 21 after birth/number of live pups on day 4* after birth) x 100

* after standardization of litters (i.e. after culling)

Clinical signs:
no effects observed
Description (incidence and severity):
-During the pre-exposure period and the exposure period the animals showed no clinical signs and findings different from normal.
-Exposure period, control group animals (test groups 0, 10, 20):
There were no clinical signs and findings different from normal.
-Exposure period, test item 2 (test groups 4, 5, 6, 16 and 26):
One male animal (No.: 82) of test group 5 showed protruding eyeball during exposure period
on study days 26 – 31. No clinical signs of toxicity were noted in any other animals of these
groups. The findings in the eye was considered incidental due to missing concentration response
relationship.
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
No deaths were recorded throughout the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In animals exposed to low (0.5 mg/m³) and mid concentration (2 mg/m³) of test item 2 (Zinc oxide T0421), there were no statistically significant deviation from the concurrent control group was observed in body weight.

The following statistically significant body weight changes were determined in male animals of group 4 and 5:
- Test group 4: day 60 -> 67: 1.3g (p< 0.05), whereas the control group was 7.8g
- Test group 4: day 67 -> 74: 10.7g (p< 0.05), whereas the control group was 6.6g
- Test group 5: day 74 -> 81: 10.2g (p< 0.01), whereas the control group was 5.7g
- Test group 5: day 81 -> 88: 8.2g (p< 0.05), whereas the control group was 5.4g
These values were mostly higher than the control value and were of transient nature. They did not influence the mean body weight. Thus, they were considered incidental.


The following statistically significant body weight changes were determined in male animals:
- Test group 6: day 18: 329.8g (p< 0.05), whereas the control group was 345.9g
- Test group 6: day 25: 340.2g (p< 0.01), whereas the control group was 363.4g
- Test group 6: day 32: 351.3g (p< 0.01), whereas the control group was 379.4g
- Test group 6: day 39: 365.7g (p< 0.01), whereas the control group was 390.9g
- Test group 6: day 46: 373.3g (p< 0.05), whereas the control group was 394.9g

The following statistically significant body weight changes were determined in male animals:

- Test group 6: day 0 -> 4: 6.9 g (p< 0.05), whereas the control group was 10.4g
- Test group 6: day 11 -> 18: 17.2g (p< 0.05), whereas the control group was 22.0g
- Test group 6: day 18 -> 25: 10.4g (p< 0.01), whereas the control group was 17.5g
- Test group 6: day 25 -> 32: 11.1g (p< 0.05), whereas the control group was 16.1g
- Test group 6: day 81 -> 88: 8.1g (p< 0.05), whereas the control group was 5.4g

The mean body weights of test group 6 were lower than the concurrent control group animals. On study days 18, 25, 32 and 39 they co-incidence with statistically significantly lowered mean body weight change, they are likely attributed to the exposure to high concentration of test item 2 (10 mg/m³, Zinc oxide T0421). The lower body weight change from study day 0 to study day 4 could be considered initial response to the exposure. The changes were very minor, and the mean body weight at the end of the exposure period of this group was not statistically lower than the control. Thus, they were considered not biologically relevant and not adverse.

The following statistically significant changes of body weight were determined in female
animals:
- Test group 6: day 32: 210.9g (p< 0.05), whereas the control group was 221.0g
- Test group 6: day 93: 234.4g (p< 0.01), whereas the control group was 251.8g
The following statistically significant body weight changes were determined in female
animals:
- Test group 6: day 11-> 18: 9.1 (p< 0.05), whereas the control group was 13.8g
- Test group 6: day 102 -> 109: -1.4 (p< 0.05), whereas the control group was 14.3g
- Test group 6: day 116 -> 123: 3.7 (p< 0.05), whereas the control group was -3.2g

The significant changes of body weight and body weight changes in female animals exposed to high concentration of test item 2 (10 mg/m³, Zinc oxide T0421) were all of transient nature. As there were already transient effects observed in male animals of these group, these changes in females may be also attributed to the test substance. As the mean body weight at the end of the exposure period of this group was not statistically lower than the control, these effects in body weights and body weight changes were considered not biologically relevant and not adverse.

The following significant changes were observed in recovery group male animals exposed to high concentration of test item 2 (10 mg/m³, Zinc oxide T0421):
- Test group 26: day 18: 316.6g (p< 0.01), whereas the control group was 344.0g
- Test group 26: day 25: 327.2g (p< 0.01), whereas the control group was 360.9g
- Test group 26: day 32: 338.7g (p< 0.01), whereas the control group was 375.0g
- Test group 26: day 39: 353.3g (p< 0.01), whereas the control group was 390.9g
- Test group 26: day 46: 365.0g (p< 0.01), whereas the control group was 399.8g
- Test group 26: day 53: 373.3g (p< 0.01), whereas the control group was 411.9g
- Test group 26: day 60: 378.8g (p< 0.01), whereas the control group was 414.4g
- Test group 26: day 67: 386.4g (p< 0.01), whereas the control group was 425.4g
- Test group 26: day 74: 394.6g (p< 0.01), whereas the control group was 429.7g
- Test group 26: day 81: 400.7g (p< 0.01), whereas the control group was 439.5g
- Test group 26: day 88: 404.5g (p< 0.01), whereas the control group was 446.1g
- Test group 26: day 92: 409.9g (p< 0.01), whereas the control group was 449.2g
- Test group 26: day 102: 415.4g (p< 0.01), whereas the control group was 450.4g
- Test group 26: day 109: 425.0g (p< 0.01), whereas the control group was 457.6g
Test group 26: day 116: 431.1g (p< 0.01), whereas the control group was 459.8g
- Test group 26: day 123: 437.3g (p< 0.01), whereas the control group was 465.5g
- Test group 26: day 130: 441.0g (p< 0.01), whereas the control group was 470.0g
- Test group 26: day 137: 447.8g (p< 0.05), whereas the control group was 472.3g
- Test group 26: day 144: 450.7g (p< 0.05), whereas the control group was 475.3g
- Test group 26: day 146: 452.6g (p< 0.05), whereas the control group was 475.9g

The following significant deviations from the control were observed in male recovery group animals exposed to high concentration of test item 2:
- Test group 26: day 0 -> 4: 5.5 (p< 0.01), whereas the control group was 12.6g
- Test group 26: day 11 -> 18: 10.7 (p< 0.01), whereas the control group was 21.7g
- Test group 26: day 18 -> 25: 10.6 (p< 0.01), whereas the control group was 16.9g
- Test group 26: day 67 -> 74: 8.2 (p< 0.05), whereas the control group was 4.4g
- Test group 26: day 130 -> 137: 6.8 (p< 0.01), whereas the control group was 2.3g
The mean body weights of the recovery group animals were statistically lower than the concurrent control group throughout the whole exposure and post-exposure period. The mean body weight changes were only significantly decreased during the initial period of the exposure period. This showed that the body weight development of the male animals of this groups was impaired at the initial time of the exposure. During the course of continuous exposure, as well as the recovery period, the body weight did not increase in such an extent that could compensate the initially reduced body weight gain. The retarded body weight development was also observed in main group animals exposed at the same concentration in the same chamber. Thus, the effect was considered treatment-related. As the final mean body weight was only about 5 % lower than the control, the body weight effect was considered not biologically relevant and not adverse.

Further, the following significant body weight changes were observed in male animals of test group 16 (10 mg/m³, Zinc oxide T0421)
- Test group 16: day 0 -> 4: 4.4g (p< 0.05), whereas the control group was 11.6g
- Test group 16: day 18 -> 25: 9.8g (p< 0.05), whereas the control group was 20.6g
- Test group 16: day 67 -> 7 4: -1.1g (p< 0.05), whereas the control group was 8.5g
- Test group 16: day 74 -> 81: 10.0g (p< 0.01), whereas the control group was 0.9g
As discussed above, the findings were considered treatment-related, but of no biological relevance and not adverse.

Body weight of F0 females during gestation/lactation of F1 litters:
Significantly lower body weights were noted for animals exposed to test item T0421 at a concentration of 10 mg/m³ during gestation and lactation. The maximum difference to the control was 6.7%. Body weight gain of these females was also lower than control throughout gestation and lactation, but the difference was not large enough to become statistically significant, except on GD 7 – 14.
--> at 10mg/m3: decreased body weights/body weight gain during gestation and lactation of parental females as treatment-related adverse effects
Body weight/body weight gain of test groups 4 and 5 (0.5 and 2 mg/m³) was comparable to the concurrent control.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant changes of mean food consumption were determined in
male animals:
• Test group 6: day 0 - 4: +22.6 g (p≤ 0.05), whereas the control group was +24.4 g
• Test group 6: day 18 - 25: +22.5 g (p≤ 0.05), whereas the control group was +25.2 g
The lowered food consumption in test group 6 coincidenced with lower mean body weights and mean body weight change of these groups in the same time range. They may be related to the daily inhalation exposure to the test and reference substance. They were of transient nature, thus, they were considered not of biological relevance.

Food consumption of F0 animals during gestation of F1 litters:
Food consumption of females exposed to 10 mg/m3 (test group 6) was consistently and significantly below concurrent control throughout gestation (about 9.5%), the difference became, in addition, statistically significant during GD 7 – 14 (about 14% below control). This effect was still present but somewhat lessened through lactation, showing no significant differences to control anymore. In contrast, during lactation episodes of significantly decreased food consumption were noted for test group 5 (PND 1 – 7 and PND 1 – 13). However, as the effects in test group 5 were rather mild and not concentration-related they may well be incidental.
--> at 10mg/m3: decreased food consumption during gestation and lactation as treatment related adverse effects
Food consumption of test group 4 (0.5 mg/m3) was comparable to the concurrent control.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
The ophthalmologic examinations did not show any impairment of the refracting media.
Spontaneous findings such as remainders of the pupillary membrane or corneal stippling were
observed in several animals of all test groups and the control group without any concentration response relationship.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the administration period, in males of test group 6 (10 mg/m3 Zinc oxide T0421) total white blood cell (WBC) counts as well as absolute neutrophil and lymphocyte counts were slightly but significantly increased. These changes were regarded as treatment-related and adverse.

The following significant changes were regarded as incidental and not treatment related, because the values were within historical control ranges: decreased relative eosinophil cell counts in males of test groups 4 and 6 (0.5 and 10 mg/m3 Zinc oxide T0421)

The following significant changes were regarded as incidental and not treatment related, because the alteration was not dose dependent: increased hematocrit value in males of test group 5 (2 mg/m3 Zinc oxide T0421); increased absolute monocyte counts in females of test group 4 (0.5 mg/m3 Zinc oxide T0421).

After the recovery period, in males of test group 26 (10 mg/m3 Zinc oxide T0421) absolute large unstained cell (LUC) counts were significantly increased. This was the only change of the differential blood cell counts among these individuals. Therefore, it was regarded as if at all treatment related as non-adverse.

Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Significantly increased potassium values in males of test group 6 (10 mg/m3 Zinc oxide T0421). This was regarded if at all treatment related as non-adverse (ECETOC Technical Report No. 85, 2002).
The following significant changes were regarded as incidental and not treatment related because the values were within historical control ranges: increased inorganic phosphate levels in males of test group 6 and 8 (10 mg/m3 Zinc oxide T0421
; decreased albumin values in females of test group 6 (10 mg/m3 Zinc oxide T0421);
Significantly increased alkaline phosphatase activities in females of test groups 5 and 6 (2 and 10 mg/m3 Zinc oxide T0421) were regarded as incidental and not treatment related, because the alteration was not dose dependent.
After the 8-week recovery period, in males of test group 26 (10 mg/m3 Zinc oxide T0421) total bilirubin values were significantly increased.
Endocrine findings:
no effects observed
Description (incidence and severity):
After the administration period, in parental males and in male and female pups at PND22 of all
test groups, no treatment-related alterations of T4 and TSH levels were observed.
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional observational battery:
Quantitative parameters: no substance-related findings were observed.
Home cage observations: no substance-related findings were observed.
Open field observations: no substance-related findings were observed.
Sensorimotor tests/reflexes: no substance-related findings were observed

Overall motor activity (summation of all intervals):
Test item 2 (Zinc oxide T0421) (Test groups 4, 5 and 6):
there were no statistically significant deviations from the control group 0.

Single intervals:
Comparing the single intervals with the control group, the following statistically significant
deviations were seen:
• Decrease of activity in the male animals of test group 6 (10 mg/m³, test item 2) at interval
9 on day 87 (p ≤ 0.01).
-->No other abnormalities were detected.
These changes were considered incidental because they were of transient nature and the
overall motor activity was not changed in the respective group.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings were observed in in the larynx, lungs, nasal cavity and the tracheobronchial lymph nodes. These are further described in the details on results section

The following treatment-related, adverse effects were observed:
Main group (F0)
Test item 2 (Zinc oxide T0421)
Test group 6 (10 mg/m³)
Minimal to severe numbers of foamy macrophages in the lungs in all male and all female
animals
• Minimal to severe cellular debris in the lungs in all male and all female animals
• Minimal to slight infiltration of neutrophils of alveoli of the lungs in all male and all female
animals
• Minimal to slight hyperplasia of type II pneumocytes in 8 males and 9 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes
(exemplarily) in 8 males and 3 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes
(exemplarily) in 4 males and 2 females
• Minimal to slight degeneration/regeneration of the olfactory epithelium (nasal cavity,
level IV, exemplarily) in 6 males and 10 females

Test group 26 (Recovery group R1, 10 mg/m³)
• No treatment-related adverse findings

Test group 5 (2 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium (nasal cavity, level IV,
exemplarily) in 1 male and 1 female
Test group 4 (0.5 mg/m³)
No treatment-related adverse findings
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
BAL
Main group (F0)
The following treatment-related, adverse effects were observed:

Test item 2 (Zinc oxide T0421)
Test group 6 (10 mg/m³)

• Increased total cell counts as well as absolute and relative lymphocyte, neutrophil cell
and monocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute eosinophil cell counts in males
• Increased total protein levels lactate dehydrogenase (LDH) and alkaline phosphatase
(ALP) activities in BAL of both sexes
• Increased β-Glutamyl-transferase (GGT) activity in BAL of males

Test group 26 (Recovery group R1, 10 mg/m³)
• No treatment-related adverse findings in lavage
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Estrous cycle data, generated during the last 3 weeks prior to mating to produce the F1 litter, revealed regular cycles in the females of all test groups. The mean estrous cycle duration was comparable: 3.9 / 3.9 / 4.0, 4.0, 3.9, 3.9, 4.0 and 4.0 days in test groups 1 – 6 as well as 7 and 8.
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
Description (incidence and severity):
MALE REPRODUCTION DATA:
For all F0 parental males of all test groups, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100% in all test groups.

Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter. However, two males (No. 105, 112) of test group 6 (test item T0421, 10 mg/m3) did not generate F1 pups.

Thus, the male fertility index was 87.5% in test group 6 and 100% in all other groups. This reflects the normal range of biological variation inherent in the strain of rats used for this study.

FEMALE REPRODUCTION DATA AND DELIVERY DATA:
The female mating index was 100% in all test groups. The mean duration until copulation was detected (GD 0) varied between 1.9 and 2.8 days without any relation to test item and concentration.

All female rats delivered pups or had implants in utero with the following exception:
• Test group 6 (test item T0421)
female No. 305 (mated with male No. 105) did not become pregnant
female No. 312 (mated with male No. 112) did not become pregnant

The female fertility index was 87.5% in test group 6 and 100% in all other groups. This reflects the normal range of biological variation inherent in the strain of rats used for this study.

The gestation index was 100% in in all test groups. The mean duration of gestation was comparable in all test groups (i.e. between 21.9 and 22.4 days).

Litter and delivery parameters:
Implantation was not affected by the treatment since the mean number of implantation sites was comparable between the test substance-treated groups and the control, taking normal biological variation into account (13.2 / 12.4 / 12.3 and 12.5 implants/dam in test groups 0 and 4 - 6, respectively). All values are well within the historical control range (HCD: 11.1 - 13.9).

Post-implantation loss was 2.8 / 5.7 / 5.4 and 5.6 mean% in test groups 0 and 4 - 6, respectively. All values are well within the historical control range (HCD: 2.4 - 17.7).

Consequently, the mean number of F1 pups delivered per dam remained unaffected in all exposure groups (12.9 / 11.7 / 11.6 and 11.8 pups/dam in test groups 0 and 4 - 6, respectively). While the control was above, numbers of F1 pups delivered per dam in all exposure groups are well within the historical control range (HCD: 10.3 - 12.7).

The rate of liveborn pups was not affected by the test substance, as indicated by live birth indices of 100% / 97.9% / 98.4% and 100% in test groups 0 and 4 - 6. Moreover, the rate of stillborn pups was not significantly different between the groups and within the historical control range (0 – 5.5%).

CLINICAL SIGNS AND MORTALITY:
Mortality:
No deaths were recorded throughout the study.
Clinical observations:

-During the pre-exposure period and the exposure period the animals showed no clinical signs and findings different from normal.
-Exposure period, control group animals (test groups 0, 10, 20):
There were no clinical signs and findings different from normal.
-Exposure period, test item 2 (test groups 4, 5, 6, 16 and 26):
One male animal (No.: 82) of test group 5 showed protruding eyeball during exposure period on study days 26 – 31. No clinical signs of toxicity were noted in any other animals of these groups. The findings in the eye was considered incidental due to missing concentration response relationship.

BODY WEIGHT AND WEIGHT GAIN
In animals exposed to low (0.5 mg/m³) and mid concentration (2 mg/m³) of test item 2 (Zinc oxide T0421), there were no statistically significant deviation from the concurrent control group was observed in body weight.

The following statistically significant body weight changes were determined in male animals of group 4 and 5:
- Test group 4: day 60 -> 67: 1.3g (p< 0.05), whereas the control group was 7.8g
- Test group 4: day 67 -> 74: 10.7g (p< 0.05), whereas the control group was 6.6g
- Test group 5: day 74 -> 81: 10.2g (p< 0.01), whereas the control group was 5.7g
- Test group 5: day 81 -> 88: 8.2g (p< 0.05), whereas the control group was 5.4g
These values were mostly higher than the control value and were of transient nature. They did not influence the mean body weight. Thus, they were considered incidental.


The following statistically significant body weight changes were determined in male animals:
- Test group 6: day 18: 329.8g (p< 0.05), whereas the control group was 345.9g
- Test group 6: day 25: 340.2g (p< 0.01), whereas the control group was 363.4g
- Test group 6: day 32: 351.3g (p< 0.01), whereas the control group was 379.4g
- Test group 6: day 39: 365.7g (p< 0.01), whereas the control group was 390.9g
- Test group 6: day 46: 373.3g (p< 0.05), whereas the control group was 394.9g

The following statistically significant body weight changes were determined in male animals:

- Test group 6: day 0 -> 4: 6.9 g (p< 0.05), whereas the control group was 10.4g
- Test group 6: day 11 -> 18: 17.2g (p< 0.05), whereas the control group was 22.0g
- Test group 6: day 18 -> 25: 10.4g (p< 0.01), whereas the control group was 17.5g
- Test group 6: day 25 -> 32: 11.1g (p< 0.05), whereas the control group was 16.1g
- Test group 6: day 81 -> 88: 8.1g (p< 0.05), whereas the control group was 5.4g

The mean body weights of test group 6 were lower than the concurrent control group animals. On study days 18, 25, 32 and 39 they co-incidence with statistically significantly lowered mean body weight change, they are likely attributed to the exposure to high concentration of test item 2 (10 mg/m³, Zinc oxide T0421). The lower body weight change from study day 0 to study day 4 could be considered initial response to the exposure. The changes were very minor, and the mean body weight at the end of the exposure period of this group was not statistically lower than the control. Thus, they were considered not biologically relevant and not adverse.

The following statistically significant changes of body weight were determined in female
animals:
- Test group 6: day 32: 210.9g (p< 0.05), whereas the control group was 221.0g
- Test group 6: day 93: 234.4g (p< 0.01), whereas the control group was 251.8g
The following statistically significant body weight changes were determined in female
animals:
- Test group 6: day 11-> 18: 9.1 (p< 0.05), whereas the control group was 13.8g
- Test group 6: day 102 -> 109: -1.4 (p< 0.05), whereas the control group was 14.3g
- Test group 6: day 116 -> 123: 3.7 (p< 0.05), whereas the control group was -3.2g

The significant changes of body weight and body weight changes in female animals exposed to high concentration of test item 2 (10 mg/m³, Zinc oxide T0421) were all of transient nature. As there were already transient effects observed in male animals of these group, these changes in females may be also attributed to the test substance. As the mean body weight at the end of the exposure period of this group was not statistically lower than the control, these effects in body weights and body weight changes were considered not biologically relevant and not adverse.

The following significant changes were observed in recovery group male animals exposed to high concentration of test item 2 (10 mg/m³, Zinc oxide T0421):
- Test group 26: day 18: 316.6g (p< 0.01), whereas the control group was 344.0g
- Test group 26: day 25: 327.2g (p< 0.01), whereas the control group was 360.9g
- Test group 26: day 32: 338.7g (p< 0.01), whereas the control group was 375.0g
- Test group 26: day 39: 353.3g (p< 0.01), whereas the control group was 390.9g
- Test group 26: day 46: 365.0g (p< 0.01), whereas the control group was 399.8g
- Test group 26: day 53: 373.3g (p< 0.01), whereas the control group was 411.9g
- Test group 26: day 60: 378.8g (p< 0.01), whereas the control group was 414.4g
- Test group 26: day 67: 386.4g (p< 0.01), whereas the control group was 425.4g
- Test group 26: day 74: 394.6g (p< 0.01), whereas the control group was 429.7g
- Test group 26: day 81: 400.7g (p< 0.01), whereas the control group was 439.5g
- Test group 26: day 88: 404.5g (p< 0.01), whereas the control group was 446.1g
- Test group 26: day 92: 409.9g (p< 0.01), whereas the control group was 449.2g
- Test group 26: day 102: 415.4g (p< 0.01), whereas the control group was 450.4g
- Test group 26: day 109: 425.0g (p< 0.01), whereas the control group was 457.6g
Test group 26: day 116: 431.1g (p< 0.01), whereas the control group was 459.8g
- Test group 26: day 123: 437.3g (p< 0.01), whereas the control group was 465.5g
- Test group 26: day 130: 441.0g (p< 0.01), whereas the control group was 470.0g
- Test group 26: day 137: 447.8g (p< 0.05), whereas the control group was 472.3g
- Test group 26: day 144: 450.7g (p< 0.05), whereas the control group was 475.3g
- Test group 26: day 146: 452.6g (p< 0.05), whereas the control group was 475.9g

The following significant deviations from the control were observed in male recovery group animals exposed to high concentration of test item 2:
- Test group 26: day 0 -> 4: 5.5 (p< 0.01), whereas the control group was 12.6g
- Test group 26: day 11 -> 18: 10.7 (p< 0.01), whereas the control group was 21.7g
- Test group 26: day 18 -> 25: 10.6 (p< 0.01), whereas the control group was 16.9g
- Test group 26: day 67 -> 74: 8.2 (p< 0.05), whereas the control group was 4.4g
- Test group 26: day 130 -> 137: 6.8 (p< 0.01), whereas the control group was 2.3g
The mean body weights of the recovery group animals were statistically lower than the concurrent control group throughout the whole exposure and post-exposure period. The mean body weight changes were only significantly decreased during the initial period of the exposure period. This showed that the body weight development of the male animals of this groups was impaired at the initial time of the exposure. During the course of continuous exposure, as well as the recovery period, the body weight did not increase in such an extent that could compensate the initially reduced body weight gain. The retarded body weight development was also observed in main group animals exposed at the same concentration in the same chamber. Thus, the effect was considered treatment-related. As the final mean body weight was only about 5 % lower than the control, the body weight effect was considered not biologically relevant and not adverse.

Further, the following significant body weight changes were observed in male animals of test group 16 (10 mg/m³, Zinc oxide T0421)
- Test group 16: day 0 -> 4: 4.4g (p< 0.05), whereas the control group was 11.6g
- Test group 16: day 18 -> 25: 9.8g (p< 0.05), whereas the control group was 20.6g
- Test group 16: day 67 -> 7 4: -1.1g (p< 0.05), whereas the control group was 8.5g
- Test group 16: day 74 -> 81: 10.0g (p< 0.01), whereas the control group was 0.9g
As discussed above, the findings were considered treatment-related, but of no biological relevance and not adverse.

Body weight of F0 females during gestation/lactation of F1 litters:
Significantly lower body weights were noted for animals exposed to test item T0421 at a concentration of 10 mg/m³ during gestation and lactation. The maximum difference to the control was 6.7%. Body weight gain of these females was also lower than control throughout gestation and lactation, but the difference was not large enough to become statistically significant, except on GD 7 – 14.
--> at 10mg/m3: decreased body weights/body weight gain during gestation and lactation of parental females as treatment-related adverse effects
Body weight/body weight gain of test groups 4 and 5 (0.5 and 2 mg/m³) was comparable to the concurrent control.

FOOD CONSUMPTION
The following statistically significant changes of mean food consumption were determined in
male animals:
• Test group 6: day 0 - 4: +22.6 g (p≤ 0.05), whereas the control group was +24.4 g
• Test group 6: day 18 - 25: +22.5 g (p≤ 0.05), whereas the control group was +25.2 g
The lowered food consumption in test group 6 coincidenced with lower mean body weights and mean body weight change of these groups in the same time range. They may be related to the daily inhalation exposure to the test and reference substance. They were of transient nature, thus, they were considered not of biological relevance.

Food consumption of F0 animals during gestation of F1 litters:
Food consumption of females exposed to 10 mg/m3 (test group 6) was consistently and significantly below concurrent control throughout gestation (about 9.5%), the difference became, in addition, statistically significant during GD 7 – 14 (about 14% below control). This effect was still present but somewhat lessened through lactation, showing no significant differences to control anymore. In contrast, during lactation episodes of significantly decreased food consumption were noted for test group 5 (PND 1 – 7 and PND 1 – 13). However, as the effects in test group 5 were rather mild and not concentration-related they may well be incidental.
--> at 10mg/m3: decreased food consumption during gestation and lactation as treatment related adverse effects
Food consumption of test group 4 (0.5 mg/m3) was comparable to the concurrent control.


HAEMATOLOGICAL FINDINGS:
At the end of the administration period, in males of test group 6 (10 mg/m3 Zinc oxide T0421) total white blood cell (WBC) counts as well as absolute neutrophil and lymphocyte counts were slightly but significantly increased. These changes were regarded as treatment-related and adverse.

The following significant changes were regarded as incidental and not treatment related, because the values were within historical control ranges: decreased relative eosinophil cell counts in males of test groups 4 and 6 (0.5 and 10 mg/m3 Zinc oxide T0421)

The following significant changes were regarded as incidental and not treatment related, because the alteration was not dose dependent: increased hematocrit value in males of test group 5 (2 mg/m3 Zinc oxide T0421); increased absolute monocyte counts in females of test group 4 (0.5 mg/m3 Zinc oxide T0421).

After the recovery period, in males of test group 26 (10 mg/m3 Zinc oxide T0421) absolute large unstained cell (LUC) counts were significantly increased. This was the only change of the differential blood cell counts among these individuals. Therefore, it was regarded as if at all treatment related as non-adverse.


CLINICAL CHEMISTRY:
Significantly increased potassium values in males of test group 6 (10 mg/m3 Zinc oxide T0421). This was regarded if at all treatment related as non-adverse (ECETOC Technical Report No. 85, 2002).
The following significant changes were regarded as incidental and not treatment related because the values were within historical control ranges: increased inorganic phosphate levels in males of test group 6 and 8 (10 mg/m3 Zinc oxide T0421
; decreased albumin values in females of test group 6 (10 mg/m3 Zinc oxide T0421);
Significantly increased alkaline phosphatase activities in females of test groups 5 and 6 (2 and 10 mg/m3 Zinc oxide T0421) were regarded as incidental and not treatment related, because the alteration was not dose dependent.
After the 8-week recovery period, in males of test group 26 (10 mg/m3 Zinc oxide T0421) total bilirubin values were significantly increased.


NEUROBEHAVIOUR:
Functional observational battery:
Quantitative parameters: no substance-related findings were observed.
Home cage observations: no substance-related findings were observed.
Open field observations: no substance-related findings were observed.
Sensorimotor tests/reflexes: no substance-related findings were observed

Overall motor activity (summation of all intervals):
Test item 2 (Zinc oxide T0421) (Test groups 4, 5 and 6):
there were no statistically significant deviations from the control group 0.

Single intervals:
Comparing the single intervals with the control group, the following statistically significant
deviations were seen:
• Decrease of activity in the male animals of test group 6 (10 mg/m³, test item 2) at interval
9 on day 87 (p ≤ 0.01).
-->No other abnormalities were detected.
These changes were considered incidental because they were of transient nature and the
overall motor activity was not changed in the respective group.

ORGAN WEIGHTS
When compared with control group 0 (=100%), Test item 2 (Zinc oxide T0421):
Test group 6 (10 mg/m³): Increase of absolute/relative lung weights in males (136%/143%) and females (131%/137%)
--> These effects were observed as treatment-related, adverse effects


GROSS PATHOLOGY

Test item 2 (Zinc oxide T0421):
Test group 6 (10 mg/m³):
• Macroscopically observed white foci in the lungs of 6 males and 8 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial,
highest number is given) in 10 males and 5 females
--> These effects were observed as treatment-related, adverse effects
Test group 26 (Recovery group R1, 10 mg/m³)
No treatment-related adverse findings

HISTOPATHOLOGICAL FINDINGS: NON-NEOPLASTIC:

Larynx (level I):
In the larynx, the most severe findings were observed in level I, therefore only findings in level I of the larynx are given

Parental animals:
Minimal epithelial alteration was observed in several test groups treated with test item 1 or test item 2 as well as in control animals. This finding is characterized by an increase of cell layers and replacement of respiratory epithelium by squamous epithelial cells, which may exhibit slight nuclear polymorphism and cellular atypia. The site most susceptible for this lesion, is the base of the epiglottis as it was observed in the present study. This finding was regarded to be treatment-related (inhalation).

Recovery animals: One female of test group 26 (test item 2, 10 mg/m³) revealed also a minimal epithelial alteration at the base of the epiglottis. These findings were considered to be treatment-related.


Lungs:

Parental animals:
Mainly, high dose group males and females (test group 3 and 6 [test item 1 and 2, 2 mg/m³]) were affected. Within alveoli, mainly in the bronchio-alveolar transition region, a multifocal accumulation of alveolar macrophages with vacuolar (foamy) cytoplasm was seen. The alveolar macrophages often revealed nuclei of increased size and occasionally multiple nuclei.
Intermingled with the foamy macrophages, cellular debris of presumable fragmented
macrophages and neutrophils were observed. In the region of these cellular accumulations, proliferation (hyperplasia) of type II pneumocytes was observed.
Males of test group 2 and 4 (test item 1 and 2, 2 mg/m³) revealed also an accumulation of foamy macrophages, only.

Recovery animals:
The same findings as described for the main group animals were observed in the recovery animals. These findings were regarded to be treatment-related.

Lymph nodes (mediastinal):
Parental animals:
The mediastinal and tracheobronchial lymph nodes revealed comparable findings.
In general, the high dose group males and females (test group 3 and 6 [test item 1 and 2, 2 mg/m³]) were more severely affected. A lympho-reticular cell hyperplasia was observed, which can be explained by an activation of the draining lymph nodes of the lungs. Furthermore, aggregates of macrophages were seen within the lymph nodes. These findings were considered as treatment-related.
Single animals of test group 1, 2 (test item 1, 0.5 and 2 mg/m³), and test group 5 (test item 2, 2 mg/m³) revealed similar findings.

Recovery animals:
The same findings as described for the main group animals were observed in the recovery animals. These findings were regarded to be treatment-related.

Nasal cavity:

Parental animals:
The nasal cavity was investigated in four levels. The most severely affected levels were level III and IV
In general, the high dose group males and females (test group 3 and 6 [test item 1 and 2, 2 mg/m³]) were affected. The finding was characterized by loss of olfactory epithelial cells and occasionally regeneration. Mainly the dorsal meatus and areas on the nasal septum were affected. This finding was regarded to be treatment-related.
One female of test group 1 (test item 1, 0.5 mg/m³) and three males and one female of test group 5 (test item 2, 2 mg/m³) showed minimal to slight degeneration of the olfactory epithelium. As this finding normally does not occur as a background lesion, it was assumed to have been most likely caused by the test substances.

Recovery animals:
Findings occurred either individually or were biologically equally distributed over
control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Trachea
In the trachea, two male animals of test group 8 (reference item 2, 22 mg/m³) revealed a flattening of the respiratory epithelium at the carina. This finding was considered to be treatment-related.
All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.



BRONCHOALVEOLAR LAVAGE FLUID (BALF):
Cytology:
Parental animals:
After the administration period, in BAL of males and females of test group 6 (10 mg/m3 Zinc oxide T0421) total cell counts as well as absolute and relative lymphocyte, neutrophil cell and monocyte counts were significantly increased. Additionally, absolute eosinophil cell counts were increased (not significantly) in BAL of males whereas relative macrophage counts were significantly decreased in BAL of males and females of test group 6. These alterations were regarded as treatment related and adverse.
In BAL of males in test group 5 (2 mg/m3 Zinc oxide T0421) absolute and relative neutrophil cell and monocyte counts as well as relative lymphocyte counts were already significantly increased. In BAL of both sexes of test group 5 relative macrophage counts were significantly decreased, and in females of this test group relative neutrophil cell counts were already significantly increased. However, in both sexes total cell counts in BAL were not significantly altered, and the differential cell counts were only marginally changed (below 10fold).
Therefore, these alterations in BAL cytology were regarded as treatment related but nonadverse.
Recovery animals:
After the 8-week administration period, in BAL of females of test group 26 (10 mg/m3 Zinc oxide T0421) absolute epithelial cells were marginally but significantly increased. This isolated change was regarded as incidental and not treatment related.

Proteins/enzymes:
Parental animals:
After the administration period, in BAL of males and females of test group 6 (10 mg/m3 Zinc oxide T0421) total protein levels as well as lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) activity were moderately, significantly increased whereas γ-Glutamyltransferase (GGT) activity in males of this test group was marginally but also significantly increased. These alterations were regarded as treatment related and adverse.
Additionally, in BAL of both sexes of test group 6 (10 mg/m3 Zinc oxide T0421) β-
-N-Acetyl glucosaminidase (NAG) activity as well as in females of this test group GGT activity were significantly increased. In BAL of males of test group 5 (2 mg/m3 Zinc oxide T0421) LDH, ALP and GGT activities and in females ALP and GGT activities were significantly increased.
However, the changes were marginally (below 2fold). Therefore, these alterations were regarded as treatment related but non-adverse.
Recovery animals:
After the 8-week recovery period, in males of test group 26 (10 mg/m3 Zinc oxide T0421) total protein levels in BAL were still significantly increased. However, this was the only altered parameter in BAL among these individuals and the increase was only marginal (below 2fold).
Therefore, this isolated change was regarded as incidental and not treatment related.

OTHER FINDINGS
- Electron microscopy:
Dose descriptor:
NOAEC
Remarks:
local toxicity
Effect level:
0.52 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: histological findings in the nasal cavity of one male and one female rat. at the target mid concentration of 2 mg/m³
Dose descriptor:
NOAEC
Remarks:
systemic toxicity
Effect level:
10.07 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology
clinical biochemistry
histopathology: non-neoplastic
Remarks on result:
other: No systemic toxicity was observed in hematology, clinical chemistry ; increased neutrophils and lymphocytes in blood at the target conc of 10 mg/m3
Dose descriptor:
NOEC
Remarks:
fertility and reproductive performance
Effect level:
10.07 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance
Critical effects observed:
yes
Lowest effective dose / conc.:
2.01 other: mg/m3 air (analytical)
System:
respiratory system: lower respiratory tract
Organ:
lungs
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Clinical signs:
no effects observed
Description (incidence and severity):
No test or reference item related adverse clinical signs were observed in any of the F1 generation pups of the different test groups. Individual findings in few pups, like dehydrated appearance or gasping, were noted in several groups including control. They were most likely related to the technical procedure of inhalation exposure rather than any test or reference item.
Dermal irritation (if dermal study):
not specified
Mortality / viability:
no mortality observed
Description (incidence and severity):
The viability index indicating pup survival during early lactation (PND 0 - 4) varied between 99.5% / 99.0% / 100% / 100% / 95.7% / 97.9% / 100% / 99.6% and 99.5% in test groups 0 - 8 without showing significant differences between the groups. All values were within the historical control range (94 – 100%).

The lactation index indicating pup survival on PND 4 - 21 was 98.8% in test group 5 and 100% in all remaining test groups. All values were within the historical control range (95.7 – 100%).
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weights of all male and female pups in all test and reference item-treated groups 1 – 8 were comparable to the concurrent control values throughout the entire study.

The statistically significantly higher body weights in male pups and in both sexes combined in test group 4 on PND 4 were considered to be spontaneous in nature.

Calculation of body weight change resulted in a number of statistical changes in various groups, sometimes higher, sometimes lower than the concurrent control:

Increased:
- test group 3 males and both sexes combined on PND 1
- test group 4 males, females and both sexes combined on PND 1
- test group 7 males, females and both sexes combined on PND 1

Decreased:
- test group 5 females and both sexes combined on PND 4
- test group 7 males, females and both sexes combined on PND 13
- test group 8 males, females and both sexes combined on PND 4
- test group 8 males, females and both sexes combined on PND 13

None of these apparent changes is considered to be associated with the respective test or reference items.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not specified
Sexual maturation:
not specified
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
The anogenital distance and anogenital index of all treated male and female pups was comparable to the concurrent control values.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
The apparent number and percentage of male pups having areolae was not influenced by the test item when examined on PND 13. Likewise, no test item-related effect was detected in any of the test groups during the re-examination on PND 20.
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings were observed in in the lungs and nasal cavity.

The following treatment-related, adverse effects were observed:
PUPS (F1)
Test item 2 (Zinc oxide T0421)
Test group 6 (10 mg/m³)
• Minimal cellular debris in the lungs in 1 male and 4 female animals
• Minimal infiltration of neutrophils of alveoli of the lungs in 1 male and 1 female animals
• Minimal to moderate degeneration/regeneration of the olfactory epithelium (nasal
cavity, level III) in 4 male and 2 female animals
Test group 5 (2 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium (nasal cavity, level III) in
2 female animals
Test group 4 (0.5 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium (nasal cavity, level III) in
1 male animal
Other effects:
no effects observed
Description (incidence and severity):
OPEN FIELD OBSERVATIONS (OFO):
None of the animals in all test groups showed abnormalities attributable to the exposure to the test substance.

MOTOR ACTIVITY MEASUREMENT (MA):
Motor activity of male and female F1 offspring was not influenced by the test item at all concentration levels and at any of the testing dates PND 13, 17 and 21. Overall activity levels and habituation to the test environment corresponded to the age of these animals at the specific testing date, if usual biological variation inherent in rats used for this type of experiment was considered.

Across the test groups, there were a number of statistically significant changes in either the number of beam interrupts or in the number of rearings, in single intervals on various testing dates, sometimes higher, sometimes lower than the concurrent control:

Test item T0421
Increased:
- test group 4 males, rearings, interval 1, PND 21

Decreased:
- test group 6 males, beam interrupts, interval 9, PND 21
- test group 5 males, rearings, interval 5, PND 17
- test group 4 females, rearings, interval 6, PND 17
- test group 5 females, rearings, interval 6 and 11, PND 17

None of these apparent changes is considered to be associated with the respective test or reference items.


Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Regarding neuropathology, no treatment-related findings were seen in pups of PND 22: Neuropathology, brain weight determination, necropsy, gross measurements of the brain,
neuropathology examination by light microscopy and morphometry did not reveal any
neuropathological, treatment-related findings.
--> There was no developmental neurotoxicity in all examined pups.
Developmental immunotoxicity:
not examined
Dose descriptor:
NOAEC
Remarks:
local toxicity
Generation:
F1
Effect level:
0.52 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: minimal cellular debris, neutrophilic infiltration in the lungs and minimal to moderate degeneration/regeneration of the olfactory epithelium in nasal cavity in a few male and female animals at 10 mg/m³ At 2mg/m3: changes in nasal cavity still observed
Dose descriptor:
NOAEC
Remarks:
systemic toxicity
Generation:
F1
Effect level:
10.07 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology
clinical biochemistry
histopathology: non-neoplastic
Remarks on result:
other: No systemic toxicity was observed in hematology, clinical chemistry and histopathology
Critical effects observed:
yes
Lowest effective dose / conc.:
2.01 other: mg/m3 air (analytical)
System:
respiratory system: lower respiratory tract
Organ:
lungs
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Reproductive effects observed:
no
Conclusions:
Overall assessment for adult animals:

With regards to systemic toxicity, none of the test or reference substances caused any systemic toxicity that were not triggered by the local toxicity.

Comparing the local effects of the two nano Zinc oxide materials, the overall finding in the lungs, mediastinal lymph nodes, in the nasal cavity were comparable at the tested concentrations, as well as the changes of lavage parameters. The small differences are considered biological variations. There were no considerable differences between the effects caused by zinc oxide nanoparticles and those caused by micron-size zinc oxide particle.

For reference substance 2 (zinc sulfate monohydrate), lower incidence and severity was found in the lungs than in the other zinc oxide treated groups, but higher incidence and severity in nasal cavity and larynx. This difference is considered being related to the different deposition pattern, caused by the different aerodynamic diameter. The aerodynamic diameter of zinc sulfate monohydrate was larger than the different types of zinc oxide. The mean MMAD of zinc sulfate monohydrate was with 2.3 µm considerably higher than those measured at the high concentrations of the test items 1 (1.19 µm) and 2 (0.97 µm). The deposited dose at the upper respiratory tract was higher, while those deposited in the lung was lower.

After the recovery period, all parameters in lavage fluid returned to the control level in all animals, irrespective of the exposed test and reference substance. With regards of histological findings in the respiratory tract, all changes reduced greatly in incidence and severity. Only single animals showed still some mild effects.

Overall assessment for PND 22 animals:
With regards to effects observed in PND 22 pups that were exposed whole-body to zinc oxide nanomaterials from PND 4 to PND 21, the findings were limited to lungs and nasal cavities. The effects observed in the parental animals showed much higher incidence and severity than in the pups exposed at the same concentration. This could be explained by the duration of the exposure, because pups were only exposed for 17 days, while the adult animals were exposed for 90-days.

Comparing the toxicity of the two nano Zinc oxide materials in pups of PND 22, the overall finding in the lungs were comparable at the high concentration of 10 mg/m³ for test items 1 and 2. However, lesions were also observed in nasal cavity in pups exposed to test item 2. While lesions in nasal cavity were still observed at 0.5 mg/m³ test item 2, there were no effect observed in animals exposed to 2 mg/m³ and 0.5 mg/m³ test item 1.

Comparing the toxicity caused by the two nano zinc oxide materials with those caused by the microscale zinc oxide material, or by the soluble zinc sulfate monohydrate, the changes in lungs were comparable in incidence and severity in exposed pups on PND 22. However, no nasal cavity lesions were observed in animals exposed to test item 1, while similar lesions were seen in those exposed to test item 2 and the reference items.

None of the substances cause any systemic toxicity, nor were there any developmental neurotoxicity in exposed pups.
Executive summary:

This study was a 90-Day Study (OECD test guideline (TG) 413) combined with the Reproduction/ Developmental Toxicity Screening Test (OECD TG 421) in rat with neurotoxicity and developmental (neuro)toxicity evaluation, including detailed clinical observations addressing potential neurobehavioral effects, histological and morphological evaluations of the brains of the pups on post-natal day 22.


To compare the toxicity of uncoated and coated nano Zinc oxide, these two materials (Zinc oxide T0420 was uncoated, Zinc oxide T0421 was coated) were tested at each three concentrations. In addition, micronsize Zinc oxide T0242 and a soluble salt zinc sulfate monohydrate was tested as reference items. 


Groups of male and female Wistar rats were whole-body exposed to the aerosols of ZnO nano materials, Zinc oxide T0420 and Zinc oxide T0421, for 6 hours daily, at least 90 days. Zinc oxide T0420 was uncoated, Zinc oxide T0421 was coated.


The target concentrations for Zinc oxide T0420 and T0421 were 0.5, 2 and 10 mg/m³ referring to the non-volatile fraction. For the reference item 1 microscale Zinc oxide T0242, 10 mg/m³ was tested. For the reference item 2, Zinc sulfate monohydrate a target concentration of 22 mg/m³ was tested because this is equimolar to zinc ion of the ZnO materials. Concurrent control groups were exposed to humidified air (control group 0, 10 and 20).


All animals were exposed to the respective concentrations of test substance for 6 hours a day according to the time schedule (exception: no exposure on the day of FOB/MA and parental females from GD20 – PND 3)). Control animals were exposed to conditioned air. Male and female rats aged about 6 or 7 weeks when supplied, were used as F0 generation parental animals. The animals were exposed for 43 days before mating. The mating period were maximal 2 weeks. After the mating period, the exposure of all male F0 animals were continued until they are exposed for total minimal 90 days. After the mating period, the female F0 animals were exposed further until gestation day 19. To allow them to deliver and rearing their pups (F1 generation), they were not exposed from gestation day 20 to postnatal day (PND) 3. From PND 4 through to PND 21, the dams were exposed with their pups in exposure cages containing beddings. During the exposure food was withdrawn. Water was provided in form of hydrogel pads from PND 14 to 16 onward. The first parental female animals were in gestation stage already after the first few mating days, therefore, the post-weaning period were adjusted in such a way, that a total of minimum 90 exposure will be achieved for females.


Daily clinical observations, body weights, food consumption, ophthalmology, detailed clinical observation and FOB/MA were recorded. Moreover, male and female fertility were determined. Additional assessments including hematology and clinical chemistry in blood, bronchoalveolar lavage, and histopathology according to the referenced guidelines were carried out at the termination of exposure period. In addition, recovery groups of male and female animals were included; after an exposure period of about 90 days, these animals were kept for an additional period of ca. 60 days without exposure (control group 20, and test groups 23, 26, 27 and 28, respectively).


To assess the reproductive/developmental toxicity of the test substances (incl. reference substances), estrus cycles, male and female reproduction, delivery data were collected. In the pups, open field observations were performed on PND 13 and 21, motor activity measurements were performed on PND 13, 17 and 21. On PND 22, thyroid hormones, brain weights, neuropathology, general histopathology were examined in separate subsets of animals.


The following treatment-related, adverse effects were observed:
Main group (F0)
Test item 1 (Zinc oxide T0420)
Test group 3 (10 mg/m³)


• Decreased food consumption during gestation and lactation of parental females
• Increased total cell counts as well as absolute and relative lymphocyte, neutrophil cell and monocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute eosinophil cell counts in males in BAL
• Increased total protein levels lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and γ-Glutamyl-transferase (GGT) activities in BAL of both sexes
• Increased β-N-Acetyl glucosaminidase (NAG) activity in BAL of males
• Increase of absolute/relative lung weights in males (140%/150%) and females (128%/130%)
• Macroscopically observed white foci in the lungs of 5 males and 8 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial, highest number is given) in 6 males and 9 females
• Slight to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to severe cellular debris in the lungs in all male and all female animals
• Minimal to moderate infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 9 males and all females
• Minimal to slight degeneration/regeneration of the olfactory epithelium 



Test group 23 (Recovery group R1, 10 mg/m³)
• Macroscopically observed white foci in the lungs of 1 male and 2 females
• Macroscopically enlarged draining lymph nodes (mediastinal) in 1 female
• Minimal to moderate numbers of foamy macrophages in the lungs in 3 male and 4 female animals
• Minimal cellular debris in the lungs in 1 male and 2 female animals
• Minimal infiltration of neutrophils of alveoli of the lungs in 1 male and 2 female animals
• Minimal hyperplasia of type II pneumocytes in 3 females


Test group 2 (2 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium in one male animal


Test group 1 (0.5 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium in 1 female



Conclusion for adult animals exposed to test item 1 (Zinc oxide T0420):
Inhalation exposure to Zinc oxide T0420 caused changes in lung, lung-draining lymph nodes and nasal cavity at the high concentration of 10 mg/m³. These findings were almost, though not completely resolved during the post-exposure observation period. At 2 mg/m³, minimal degeneration/regeneration in the nasal cavity was noted in one male animal, and at 0.5 mg/m³ in one female animal. Due to findings in nasal cavity, the NOAEC for local toxicity at the respiratory tract was 0.5 mg/m³ for male rats. a No Observed Adverse Effect Concentration (NOAEC) for local toxicity for females could not be unequivocally determined.


No systemic toxicity was observed in hematology, clinical chemistry and histopathology the NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity was 10 mg/m³ for Zinc oxide T0420.



Test item 2 (Zinc oxide T0421)
Test group 6 (10 mg/m³)
• Decreased food consumption during gestation and lactation of parental females
• Decreased body weights/body weight gain during gestation and lactation of parental females
• Increased total white blood cell (WBC) as well as absolute neutrophil and lymphocyte counts in blood of males
• Slightly increased total cell counts as well as absolute and relative lymphocyte, neutrophil cell and monocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute eosinophil cell counts in males in BAL
• Increased total protein levels lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) activities in BAL of both sexes
• Increased  γ-Glutamyl-transferase (GGT) activity in BAL of males
• Increase of absolute/relative lung weights in males (136%/143%) and females (131%/137%)
• Macroscopically observed white foci in the lungs of 6 males and 8 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial, highest number is given) in 10 males and 5 females
• Minimal to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to severe cellular debris in the lungs in all male and all female animals
• Minimal to slight infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 8 males and 9 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes (exemplarily) in 8 males and 3 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes (exemplarily) in 4 males and 2 females
• Minimal to slight degeneration/regeneration of the olfactory epithelium 

Test group 26 (Recovery group R1, 10 mg/m³)
• No treatment-related adverse findings in lavage and histopathology

Test group 5 (2 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium 

Test group 4 (0.5 mg/m³)
No treatment-related adverse findings


Conclusion for adult animals exposed to test item 2 (Zinc oxide T0421):
Inhalation exposure to Zinc oxide T0421 caused changes several lavage parameters, as well as histological changes in lung, lung-draining lymph nodes and nasal cavity at the highest tested concentration of 10 mg/m³. All these effects were completely resolved after the postexposure observation period. In blood, increased neutrophils and lymphocyte was notice at the concentration of 10 mg/m³, which is considered secondary to the inflammation in the lung.
At the mid concentration of 2 mg/m³, histological findings were still observed in the nasal cavity of three male and two female rats. Thus, the No Observed Adverse Effect Concentration (NOAEC) for local toxicity was 0.5 mg/m³ under the current study conditions. 
Besides the increased neutrophils and lymphocytes in blood, no other changes were observed in hematology, clinical chemistry. No histopathological changes were observed in any organs and tissues that are not part of the respiratory tract. The NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity, that were not attributed to the local effect, was 10 mg/m³ for Zinc oxide T0421.



Reference item 1 (Zinc oxide T0242)
Test group 7 (10 mg/m³)
• Retarded body weight development in male animals
• Increased total cell counts as well as absolute and relative neutrophil cell and monocyte counts in BAL of both sexes
• Increased absolute lymphocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute macrophage and eosinophil cell counts in BAL of males
• Increased total protein levels lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and γ-Glutamyl-transferase (GGT) activities in BAL of both sexes
• Increased β-N-Acetyl glucosaminidase (NAG) activity in BAL of males
• Increase of absolute/relative lung weights in males (130%/141%) and females (137%/141%)
• Macroscopically observed white foci in the lungs of 5 males and 8 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial, highest number is given) in 7 males and 10 females
• Minimal to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to severe cellular debris in the lungs in all male and all female animals
• Minimal to moderate infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 6 males and 8 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes (exemplarily) in 4 males and 6 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes (exemplarily) in 6 males and 8 females
• Minimal degeneration/regeneration of the olfactory epithelium 


Test group 27 (Recovery group R1, 10 mg/m³)
• Macroscopically observed white foci in the lungs of 3 males and 3 females
• Minimal to slight numbers of foamy macrophages in the lungs in 2 male and 4 female animals
• Minimal cellular debris in the lungs in 1 male animal
• Minimal infiltration of neutrophils of lung alveoli in 1 male animal
• Minimal hyperplasia of type II pneumocytes in 4 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes in 2 males and 3 females


Conclusion for adult animals exposed to reference item 1 (Zinc oxide T0242):
Inhalation exposure to Zinc oxide T0242 caused changes in lung, lung-draining lymph nodes and nasal cavity at the highest tested concentration of 10 mg/m³. These findings were greatly, though not completely, resolved during the post-exposure observation period.
No systemic toxicity was observed in hematology, clinical chemistry and histopathology the NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity was 10 mg/m³ for Zinc oxide T0242.


Reference item 2 (Zinc sulfate monohydrate)
Test group 8 (22 mg/m³)
• During exposure period, salivation and respiration sounds were detected in several male and female animals.
• Retarded body weight development in all male and female animals. 
• Decreased food consumption during gestation and lactation of parental female animals
• Recreased body weights/body weight gain during gestation and lactation of parental female animals
• Increased total cell counts as well as absolute and relative lymphocyte, neutrophil cell and monocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute macrophage and eosinophil cell counts in BAL of males
• Increased total protein levels lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and γ-Glutamyl-transferase (GGT) activities in BAL of both sexes
• Increase of absolute/relative lung weights in males (125%/138%) and females (114%/119%)
• Macroscopically observed white foci in the lungs of 3 males and 7 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial, highest number is given) in 5 males and 8 females
• Erosion/ulcer of the laryngeal epithelium at the base of the epiglottis in 1 female
• Minimal to slight squamous metaplasia of the laryngeal epithelium at the base of the epiglottis in all males and all females
• Minimal to slight inflammatory cell infiltrates of the laryngeal epithelium in 1 male and 9 females
• Minimal to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to moderate cellular debris in the lungs in all male and all female animals
• Minimal to slight infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 6 males and 8 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes (exemplarily) in 5 males and 5 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes (exemplarily) in 8 males and 8 females
• Minimal to moderate degeneration/regeneration of the olfactory epithelium in all males and all females

Test group 28 (Recovery group R1: 22 mg/m³)
• Macroscopically observed white foci in the lungs of 2 males and 2 females
• Macroscopically enlarged draining lymph nodes (mediastinal) in 1 male and 1 female
• Minimal squamous metaplasia of the laryngeal epithelium at the base of the epiglottis in 1 male and 1 female animal
• Minimal to slight inflammatory cell infiltrates in the laryngeal epithelium in 4 males and 3 females
• Minimal to slight numbers of foamy macrophages in the lungs in 2 male and 4 female animals
• Minimal hyperplasia of type II pneumocytes in 3 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes in 1 male and 1 female animal
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes in 4 males and 4 females
• Minimal degeneration/regeneration of the olfactory epithelium in 1 male and 1 female 


Conclusion for adult animals exposed to reference item 2 (Zinc sulfate monohydrate):
Inhalation exposure to Zinc sulfate monohydrate caused changes in lung, lung-draining lymph nodes, larynx and nasal cavity at the highest tested concentration of 22 mg/m³. These findings were partly resolved during the post-exposure observation period.
No systemic toxicity was observed in hematology, clinical chemistry and histopathology the NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity was 22 mg/m³ for Zinc sulfate monohydrate.


Overall assessment for adult animals:


With regards to systemic toxicity, none of the test or reference substances caused any systemic toxicity that were not triggered by the local toxicity.


Comparing the local effects of the two nano Zinc oxide materials, the overall finding in the lungs, mediastinal lymph nodes, in the nasal cavity were comparable at the tested concentrations, as well as the changes of lavage parameters. The small differences are considered biological variations. There were no considerable differences between the effects caused by zinc oxide nanoparticles and those caused by micron-size zinc oxide particle.


For reference substance 2 (zinc sulfate monohydrate), lower incidence and severity was found in the lungs than in the other zinc oxide treated groups, but higher incidence and severity in nasal cavity and larynx. This difference is considered being related to the different deposition pattern, caused by the different aerodynamic diameter. The aerodynamic diameter of zinc sulfate monohydrate was larger than the different types of zinc oxide. The mean MMAD of zinc sulfate monohydrate was with 2.3 µm considerably higher than those measured at the high concentrations of the test items 1 (1.19 µm) and 2 (0.97 µm). The deposited dose at the upper respiratory tract was higher, while those deposited in the lung was lower.


After the recovery period, all parameters in lavage fluid returned to the control level in all animals, irrespective of the exposed test and reference substance. With regards of histological findings in the respiratory tract, all changes reduced greatly in incidence and severity. Only single animals showed still some mild effects.


PUPS on PND 22 
Test item 1 (Zinc oxide T0420)
Test group 3 (10 mg/m³)
Minimal cellular debris in the lungs in 2 male and 4 female animals
• Minimal infiltration of neutrophils of alveoli of the lungs in 1 male and 2 female animals
Test group 2 (2 mg/m³) and test group 1 (0.5 mg/m³)
No treatment-related adverse findings observed
Conclusion for pups exposed to test item 1 (Zinc oxide T0420) sacrificed on PND 22:
Inhalation exposure to Zinc oxide T0420 caused minimal cellular debris and neutrophilic infiltration in a few male and female animals at 10 mg/m³. These findings were considered treatment-related and adverse. The NOAEC for local toxicity was 2 mg/m³. 


There were no test-item related adverse findings in clinical examination/OFO/MA and gross necropsy up to the highest tested concentration of 10 mg/m³. No systemic toxicity was observed in hematology, clinical chemistry, no changes of thyroid hormones. There were no histopathological changes in organs and tissues other than those of the respiratory tract.



Test item 2 (Zinc oxide T0421)
Test group 6 (10 mg/m³)
• Minimal cellular debris in the lungs in 1 male and 4 female animals
• Minimal infiltration of neutrophils of alveoli of the lungs in 1 male and 1 female animals
• Minimal to moderate degeneration/regeneration of the olfactory epithelium (nasal cavity, level III) in 4 male and 2 female animals
Test group 5 (2 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium (nasal cavity, level III) in 2 female animals
Test group 4 (0.5 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium (nasal cavity, level III) in 1 male animal 


Conclusion for pups exposed to test item 2 (Zinc oxide T0421) sacrificed on PND 22:
Inhalation exposure to Zinc oxide T0421 caused minimal cellular debris, neutrophilic infiltration in the lungs and minimal to moderate degeneration/regeneration of the olfactory epithelium in nasal cavity in a few male and female animals at 10 mg/m³. The changes in nasal cavity was still observed at the mid concentration of 2 mg/m³. This findings were considered treatmentrelated and adverse. The NOAEC for local toxicity was 0.5 mg/m³.


There were no test-item related adverse findings in clinical examination/OFO/MA and gross necropsy up to the highest tested concentration of 10 mg/m³. No systemic toxicity was observed in hematology, clinical chemistry, no changes in thyroid hormones. There were no histopathological changes in organs and tissues other than those of the respiratory tract.



Reference item 1 (Zinc oxide T0242)
Test group 7 (10 mg/m³)
• Minimal cellular debris in the lungs in 5 male and 5 female animals
• Minimal infiltration of neutrophils of lung alveoli in 2 male and 3 female animals
• Minimal degeneration/regeneration of the olfactory epithelium (nasal cavity, level III) in 1 male and 1 female


Conclusion for pups exposed to reference item 1 (Zinc oxide T0242) sacrificed on PND 22:
Inhalation exposure to Zinc oxide T0242 caused minimal cellular debris, neutrophilic infiltration in the lungs and minimal degeneration/regeneration of the olfactory epithelium in nasal cavity in a one male and one female animals at 10 mg/m³.


There were no test-item related adverse findings in clinical examination/OFO/MA and gross necropsy up to the highest tested concentration of 10 mg/m³. No systemic toxicity was observed in hematology, clinical chemistry, no changes in thyroid hormones. There were no histopathological changes in organs and tissues other than those of the respiratory tract.


Reference item 2 (Zinc sulfate monohydrate)
Test group 8 (22 mg/m³)
• Minimal cellular debris in the lungs in 2 male and 1 female animals
• Minimal infiltration of neutrophils of lung alveoli in 2 male and 1 female animals
• Minimal degeneration/regeneration of the olfactory epithelium (nasal cavity, level III) in 1 male and 1 female


Conclusion for pups exposed to reference item 2 (zinc sulfate monohydrate) sacrificed on PND 22:
Inhalation exposure to zinc sulfate monhydrate caused minimal cellular debris, neutrophilic infiltration in the lungs and minimal degeneration/regeneration of the olfactory epithelium in nasal cavity in a few male and female animals at 22 mg/m³.


There were no test-item related adverse findings in clinical examination/OFO/MA and gross necropsy up to the highest tested concentration of 10 mg/m³. No systemic toxicity was observed in hematology, clinical chemistry, no changes in thyroid hormones. There were no histopathological changes in organs and tissues other than those of the respiratory tract.


Overall assessment for PND 22 animals:
With regards to effects observed in PND 22 pups that were exposed whole-body to zinc oxide nanomaterials from PND 4 to PND 21, the findings were limited to lungs and nasal cavities. The effects were observed also in the parental animals with much higher incidence and severity than in the pups exposed at the same concentration. This could be explained by the duration of the exposure, because pups were only exposed for 17 days, while the adult animals were exposed for 90-days.


Comparing the toxicity of the two nano Zinc oxide materials in pups of PND 22, the overall finding in the lungs were comparable at the high concentration of 10 mg/m³ for test items 1 and 2. However, lesions were also observed in nasal cavity in pups exposed to test item 2. While lesions in nasal cavity were still observed at 0.5 mg/m³ test item 2, there were no effect observed in animals exposed to 2 mg/m³ and 0.5 mg/m³ test item 1.


Comparing the toxicity caused by the two nano zinc oxide materials with those caused by the microscale zinc oxide material, or by the soluble zinc sulfate monohydrate, the changes in lungs were comparable in incidence and severity in exposed pups on PND 22. However, no nasal cavity lesions were observed in animals exposed to test item 1, while similar lesions were seen in those exposed to test item 2 and the reference items.


None of the substances cause any systemic toxicity, nor were there any developmental neurotoxicity in exposed pups.

Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
24 November 2020 - ...June 2022
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study presented herein is a guideline study with a major deficiency under GLP conditions. Only one concentration level was tested.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016-07-29
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 426 (Developmental Neurotoxicity Study)
Version / remarks:
2007-10-16
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
not applicable
Specific details on test material used for the study:
micro sized Zinc Oxide
Purity: 99.8%
Name of reference substance 1: Zinc oxide T0242
Reference substance No.: 20/0201-1
Batch identification: 56589
Appearance - physical state / color: Solid / white
Storage conditions: Room temperature
BET: 4.48 m2/g
Species:
rat
Strain:
Wistar
Remarks:
Wistar rats, Crl:WI(Han) Rats were selected since this rodent species is recommended in the respective test guidelines. Wistar rats were selected since there is extensive experience available in the laboratory with this strain of rats.
Details on species / strain selection:
not specified
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
- Females nulliparous and non-pregnant: yes
- Age at study initiation: about 7 weeks (female), about 8 weeks (male)
- Weight at study initiation: The weight variation of the animals used did not exceed +/- 20 percent of the mean weight of each sex.
- Fasting period before study: The animals did not have access to food or water during exposure.
- Housing:
From delivery until mating and male animals after mating: Typ 2000P: ca. 2065 cm2 (polysulfone cages) / up to 5 animals
During mating: type III polycarbonate cages, 1 male/1 female per cage
During rearing: up to PND 22: type III polycarbonate cages, 1 dam with her litter
After weaning the females from study day 90 after exposure onward until sacrifice: Typ 2000P: ca. 2065 cm2 (polysulfone cages) / up to 5 animals. Remaining females with litters will be
maintained in type III cages until weaning.
For Motor Activity Measurement: Typ III polycarbonate cages (floor area about 800 cm²) / 1 animal
During Exposure: Wire cages, type DK III / up to 2 animals Females from PND 4 until study day 94 (and females without litter from the same time period onwards): perforated polycarbonate cages type II. From study day 95 onward wire cages, type DK III
- Diet (ad libitum): mouse and rat maintenance diet, GLP, 12 mm pellets, Granovit AG, Kaiseraugst, Switzerland before and after exposure. Food was withdrawn during exposure.
- Water (ad libitum): tap water
- Acclimation period: 11 days

DETAILS OF FOOD AND WATER QUALITY: The food used in the study was assayed for chemical as well as for microbiological contaminants. In view of the aim and duration of the study, the contaminants occurring in commercial food should not influence the results. The drinking water is regularly assayed for chemical contaminants both by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring of BASF SE as well as for bacteria by a contract laboratory. The Drinking Water Regulation will serve as the guideline for maximum tolerable contaminants. In view of the aim and duration of the study, there are no special requirements exceeding the specification of drinking water.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 45 - 65%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: beginning of experiment To: end of experiment
Route of administration:
inhalation: aerosol
Type of inhalation exposure (if applicable):
whole body
Remarks:
whole-body exposure for the reasons explained see IUCLID section 13.2 'Human health requirements Final Decision: protocol deviations and rationale'
Mass median aerodynamic diameter (MMAD):
>= 0.67 - <= 1.48 µm
Remarks on MMAD:
MMAD / GSD: MMAD = 0.67- 1.48 μm (geometric standard deviation = 3.71-2.40)
Vehicle:
unchanged (no vehicle)
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Generation of the inhalation atmospheres via a solid particle generators (brush-generator; BASF SE, Ludwigshafen, Germany) & Aerosol mixing tube (stainless steel; BASF SE, Ludwigshafen, Germany). Whole body exposure systems were used. The animals were kept singly in wire cages located in a glass steel inhalation chamber, volume of 1.1 m³ (BASF SE).
- Method of holding animals in test chamber: Whole body exposure systems. The animals were kept singly in wire cages located in a glass steel inhalation chamber, volume of 1.1 m³ (BASF SE). The chambers were located in exhaust hoods in an air conditioned room.
- Source and rate of air: Conditioned air from the central air conditioning system, compressed and exhaust air. Compressed air was produced by an oil-free compressor (HT 6, Josef Mehrer GmbH & Co KG, Germany). For this purpose, air is filtered by an inlet air strainer and introduced into the compressor. After passing through an second ultra filter (SMF 5/3, 108 mm, Donalson), the compressed air (15 bar) is stored in a storage of 1500 or 5000 L. The compressed air is conducted to the laboratories via pipes, where the pressure is reduced to 5 - 6 bar. In the laboratory, the compressed air can be taken as required.
- Method of conditioning air: Conditioned air from the central air conditioning system provides cold air of about 15°C. This cold air passes through an activated charcoal filter, is adjusted to room temperature of 20 to 24°C and passes through a second particle filter (H13 (HEPA) Camfil Farr, Germany). The so generated conditioned air was used to generate inhalation atmospheres.
- System of generating particulates/aerosols: The particles/aerosol was generated via a solid particle generator (brush-generator; BASF SE, Ludwigshafen, Germany) and an aerosol mixing tube (stainless steel; BASF SE, Ludwigshafen, Germany), according to the following method: For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage; mixed with conditioned dilution air and passed into the inhalation system.
- Temperature, humidity, pressure in air chamber: Daily mean relative humidities in the inhalation systems ranged between 41.6 and 60.8 %. Daily mean temperatures in the inhalation systems ranged between 21.4 and 23.7°C. They are within the range suggested by the respective testing guidelines.
- Air flow rate: The air flows were constantly maintained in the desired range.
- Air change rate: An air change of about 24 to 25 times per hour can be calculated by dividing the supply air flow through the volume of each inhalation system.
- Method of particle size determination: The particle size analysis was carried out with a cascade impactor.Equipment for particle size analysis: Stack sampler Marple 298 (New Star Environmental, Inc., Roswell, Georgia 30075, USA) ; Vacuum compressed air pump (Millipore Corporation, Billerica, MA 01821, USA) ; Limiting orifice 3 L/min (Millipore Corporation, Billerica, MA 01821, USA) ; Sampling probe internal diameter 7 mm ; Balance Sartorius MSA 6.6S-000-DF (Sartorius AG, Göttingen, Germany). The calculation of the particle size distribution was carried out in the Laboratory for Inhalation Toxicology of the Experimental Toxicology and Ecology of BASF SE on the basis of mathematical methods for evaluating particle measurements (OECD guidance document No. 39). Particle Size distribution of the test atmosphere were determined also with the Aerodynamic Particle Spectrometer APS 3321 (TSI, USA). MMAD and GSD is obtained directly by the piece of equipment used APS 3321. Frequency: On two days during the exposure period, with 3 repeats on each day. To determine the particle size distribution in the submicrometer range, each test atmosphere was measured with the Scanning Mobility Particle Sizer (SMPS; Grimm Aerosol Technik GmbH & Co KG, Ainring, Germany). The SMPS system comprises an Electrostatic Classifier (Model Vienna U-DMA) which separates the particles into known size fractions, and a Condensation Particle Counter (CPC) which measures particle count concentrations. The DMA was equipped with Am-241 neutralizer. The instrument measures particles in the size range from 0.011 to 1.083 µm. Using a conductive sample hose, the SMPS sampled at 0.3 liters per minute (LPM) with a sheath flow of 3 LPM. At this setting the single-stage, inertial impactor incorporated into the inlet of the SMPS to remove larger particles had a 50% cut size of 1.082 µm according to the software calculation. The sampling duration was about 7 minutes. As a rule 10 repeats were measured for each exposure concentration.
- Treatment of exhaust air: Exhaust air was filtered and conducted into the exhaust air of the building.

TEST ATMOSPHERE
- Brief description of analytical method used: The concentrations of the inhalation atmospheres were determined by gravimetrical measurements of filter samples in all test groups. Control group was not sampled. This analytical method was judged to be valid because the test substances did not possess an appreciable vapor pressure.
- Samples taken from breathing zone: yes
Details on mating procedure:
Mating of the F0 generation parental animals
After 44 days premating period, the male and female parental animals were mated overnight in a 1:1 ratio until there was evidence of copulation or the maximum period of 14 days has elapsed. Throughout the mating period, each female was mated with a predetermined male.

Normally, the female was placed into the cage of her male partner about 16:00 h and separated from the male between 06:30 and 09:00 h, the following morning. Deviations from the specified times are possible on Saturdays, Sundays and public holidays and were documented in the raw data.

A vaginal smear was prepared for each pair after each mating and examined for sperm. If sperm was detected, mating of the pair was discontinued. The day on which sperm were detected, was referred to as gestation day (GD) 0 and the following day as GD 1.

Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
The nominal concentration could be/was calculated from the study means of the test-substance flow and the supply air flows used during exposure to generate the respective concentrations. The concentrations of the inhalation atmospheres were determined by gravimetrical measurements of filter samples in all test groups. Control group was not sampled. This analytical method was judged to be valid because the test substances did not possess an appreciable vapor pressure.
Duration of treatment / exposure:
The animals were exposed for 44 days before mating. The mating period were
maximal 2 weeks. After the mating period, the exposure of all male F0 animals were continued
until they are exposed for total minimal 90 days. After the mating period, the female F0 animals
were exposed further until gestation day 19. To allow them deliver and rearing their pups (F1
generation), they were not exposed from gestation day 20 to postnatal day (PND) 3. From
PND 4 through to PND 21, the dams were exposed with their pups in exposure cages
containing beddings. The first parental female animals were in gestation stage already after
the first few mating days, therefore, the post-weaning period were adjusted in such a way, that
a total of minimum 90 exposure will be achieved for females.
Frequency of treatment:
7 consecutive days per week, 6 hours per day
from PND4 through PND21
Details on study schedule:
not specified
Dose / conc.:
0 mg/m³ air
Remarks:
Test Group 0 (>Parental animals F0) - air control
Dose / conc.:
9.68 mg/m³ air (analytical)
Remarks:
SD: 1.47 mg/m3, target concentration: 10 mg/m³: Test Group 7 (>Parental animals F0); Test Group 17 (male animals for particle detection); Test Group 27 (Recovery animals)
No. of animals per sex per dose:
16/sex/dose group (parental animals)
5/sex at the high dose (recovery animals)
3 males at the high dose (for particle detection)

Subset Number of pups selected Day of examination Examination
I 10/sex/group PND 22 Measurement of thyroid hormones
II 10/sex/group PND 22 Perfusion fixation, brain weights
and neuropathology
III 5/sex/group PND 22 Histopathological examination and
organ burden
IV 10/sex/group PND 13 and 21 Open field observation
PND 13, 17 and 21 Motor activity
V 3 males/group PND 22 Perfusion fixation and electron
(highest dose) microscopic for particle detection
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of the 14-day range finding study (BASF study no 36I0050/20I005 - Ma- Hock 2021), upon approval of the sponsor, nominal aerosol concentrations of 0.5, 2.0 and 10.0 mg/m³ were used for the test substance in the low, mid and high dose groups, respectively.
- Rationale for animal assignment:
Prior to the pre-exposure period, the animals were distributed according to weight among the
individual test groups, separated by sex. The weight variation of the animals used did not
exceed ± 20 percent of the mean weight of each sex. The list of randomization instructions
was compiled with a computer.
For each neurofunctional test and motor activity measurement, separate randomization lists
were created. The list of randomization instructions were compiled with a computer (Laboratory
data processing, Experimental Toxicology and Ecology, BASF SE).
- Fasting period before blood sampling for clinical biochemistry: not specified
- Post-exposure recovery period in satellite groups: 45 days recovery period
Positive control:
none
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The clinical observation was performed on each animal at least three times (before, during and after exposure) on exposure days and once a day during pre-exposure and post exposure observation days. On non-exposure days a cage-side examination will be conducted at least once daily for any signs of morbidity, pertinent behavioral changes and/or signs of overall toxicity.

MORTALITY: The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) on working days and once a day (in the morning) on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS: YES
- Time schedule: All parental animals and recovery group animals were subjected to detailed clinical observations (DCO) outside their cages once before the beginning of the administration period and once during the first two weeks of the exposure, once monthly thereafter. DCO was performed in the morning before exposure. For observation, the animals were removed from their cages and placed in a standard arena (50 x 37.5 cm with a lateral border of 25 cm) for at least 20 seconds/animal.

BODY WEIGHT: Yes
- Time schedule for examinations:
The body weight of the animals was determined at the start of the pre-exposure, at the start of
the exposure period and then, as a rule, once a week as well as prior to gross necropsy. The
body weight of the recovery animals were determined at the start of the recovery period, and
once a week during the recovery period.
The following exceptions were notable for the female parental animals:
• During the mating period, the females were weighed on the day of positive evidence of
sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day after parturition (PND1) and on PND 4, 7, 14, and 21.
• In the females without positive evidence of sperm, body weight was determined once a week during mating and gestation periods and in the females without litter during lactation period.

As a rule, the animals were weighed at the same time of the day (in the morning).

Body weight change was calculated as the difference between body weight on the respective exposure day and body weight and the weight of previous weighing. Group means were derived from the individual differences.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was determined weekly and calculated as mean food consumption in grams
per animal and day.
Generally, food consumption was determined once a week for the male and female animals
and post mating period (males), with the following exceptions:
• Food consumption was not determined during the mating period (male and female
parental animals).
• Food consumption of the females with evidence of sperm was determined for GD 0-7, 7-
14 and 14-20.
• Food consumption of the females which gave birth to a litter was determined for PND 1-
4, 4-7, 7-13.
During recovery period, food consumption was determined in the animals of test groups 20 –
28 of the recovery animals. It was determined at the start of the recovery period and once a
week during the recovery period.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OPHTHALMOSCOPIC EXAMINATION: YES
Before the beginning of exposure, the eyes of all parental animals were examined with an ophthalmoscope (HEINE OPTOTECHNIK, Herrsching, Germany) after administration of a mydriatic agent (Mydrum, Dr. Gerhard Mann chem.-pharm. Fabrik GmbH and Bausch & Lomb GmbH, Germany). At the end of the exposure period, only animals selected for examinations according to OECD 413, 10 males and 10 females per group, were subjected to ophthalmological examination. In the first step, only control (test group 0) and high concentration groups (test groups 3, 6, 7 and 8) were examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in the morning
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes
- How many animals: 10 M + 10 F per dose group
-Parameters checked: leukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets, differential blood count, reticulocytes, preparation of blood smears, prothrombin time (PT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in the morning
- Animals fasted: Yes
- How many animals: 10 M + 10 F per dose group
- Parameters checked: alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-glutamyl transpeptidase (GGT), sodium (Na), potassium (K), chloride (CL), Inorganic phosphate (INP), calcium (Ca), urea (UREA), creatinine (CREA), glucose (GLUC), total biluribin (TBIL), total protein (TP), albumin (ALB), globulin (GLB), triglycerides (TRIG), cholesterol (CHOL)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the 90days exposure period
- Dose groups that were examined: 10 M + 10 F per dose group
- Battery of functions tested: sensory activity / grip strength / motor activity / reflexes


IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: Not specified
- Dose groups that were examined: 10 M + 10 F per dose group and recovery groups (highest dose: 5M + 5F)
- Number of animals: 10 M + 10 F per dose group and recovery groups (highest dose: 5M + 5F)
- Parameters checked: Cytological parameters: total cell count, cell differential analysis of cytospin preparations; protein; Enzymes: lactate dehydrogenase, alkaline phosphatase, N-acetyl-beta-D-Glucosaminidase (NAG BAL), gamma−Glutamyltransferase

ORGAN (lung, liver, heart, brain, olfactory bulb) BURDEN: Yes
- Time schedule for analysis: at the end of the exposure period and after the recovery period (45days post exposure)
- Dose groups that were examined: all
- Number of animals: 3 /sex / group
- Parameters checked: Zn content

OTHER: - Electron microscope analysis of particulate matter in organs and tissues: 3 male animals of the highest dose group



Oestrous cyclicity (parental animals):
In all parental females in the premating phase, estrous cycle length and normality were
evaluated by preparing vaginal smears during a minimum of 2 weeks prior to mating and
throughout cohabitation until there is evidence of sperm in the vaginal smear.
Additionally, on the day of scheduled sacrifice, the estrous status was also determined in all
parental female rats.
Sperm parameters (parental animals):
not specified
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum:
On PND 4, the individual litters were standardized in such a way that, whenever possible, each litter contains 5 male and 5 female pups (always the first 5 surviving pups/sex in each litter were taken for further rearing). If individual litters did not have 5 pups/sex, the litters were processed in such a way that the most evenly distributed 10 pups per litter were present for further rearing (e.g., 6 male and 4 female pups). Standardization of litters was not performed in litters with 10 pups or less.

PARAMETERS EXAMINED
The following parameters were examined in offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups, open field observations, motor activity

GROSS EXAMINATION OF DEAD PUPS: yes

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: yes
Necropsy:
On postnatal day 22, 10 animals per sex and group were weighed, subjected to deep
anesthesia (i.p. pentobarbital) and sacrificed by perfusion fixation.
SOERENSEN phosphate buffer was used as the rinsing solution, and neutrally buffered, 4% formaldehyde solution was used as a fixative.
The perfusion fixed animals were necropsied with regard to the question of neuropathology, and the visible organs were assessed by gross pathology as accurately as is possible after a perfusion fixation. The cranial vault was opened and the skin was removed from the head. In this state, the perfused animals were stored in neutrally buffered, 4% formaldehyde solution for at least 48 hours.
Organ weights:
The following weights were determined (the brain was weighed after its removal but before
further preparation):
1. Final body weight
2. Brain (including olfactory bulb)
The final body weights were recorded to calculate the relative organ weights.
Length and width of the brain:
The length and maximum width of the brain were measured in all animals. The length of the brain was measured on a line extending from the rostral end of the frontal lobe to the caudal medulla oblongata of the cerebellum, width: pituitary region.
Organ/Tissue fixation:
The following organs/tissue specimens were carefully removed and processed histotechnically in accordance with the data given in the respective sections of this report:
1. All gross lesions
2. Brain with olfactory bulb
3. Eyes with retina and optic nerve
4. Nose (nasal cavity level III) with olfactory epithelium
5. Pituitary gland
6. Trigeminal ganglia
The animals and the tissue or organ material remaining after trimming was stored in neutrally buffered, 4% formaldehyde solution.
Neurohistopathology:
Morphometry of the brains (PND 22) :
Morphometry was performed in all animals of test groups 0 (control), 3, 6, 7 and 8.
Thickness measurements of major brain layers (neocortex: frontal and parietal cortices, caudate nucleus/putamen, hippocampus, corpus callosum, cerebellum) were performed. Measurements were carried out bilaterally in the left and right halves of the brain except for the corpus callosum and the cerebellum.

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no
Postmortem examinations (parental animals):
SACRIFICE / GROSS NECROPSY / ORGAN WEIGHTS
At the time of sacrifice, adult animals were examined macroscopically for any abnormalities or pathological changes.
The following weights were determined in all animals sacrificed on schedule:
1.Anesthetized animals (final body weight)
2. Adrenal glands (fixed)
3. Brain
4. Epididymides
5. Heart
6. Kidneys
7. Liver
8. Lung
9. Ovaries
10. Prostate (ventral and dorsolateral part together, fixed)
11. Seminal vesicles with coagulating glands (fixed)
12. Spleen
13. Testes
14. Thymus (fixed)
15. Thyroid glands (with parathyroid glands) (fixed)
16. Uterus with cervix
All paired organs were weighed together (left and right).

HISTOPATHOLOGY
Organs and tissues of F0 animals histologically processed:
1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Epididymides
12. Esophagus
13. Eyes with optic nerve
14. Extraorbital lacrimal gland
15. Femur with knee joint
16. Harderian glands
17. Heart
18. Ileum
19. Jejunum
20. Kidneys
21. Larynx (3 levels)
22. Liver
23. Lungs
24. Lymph nodes (tracheobronchial and mediastinal)
25. Lymph nodes (mesenteric)
26. Mammary gland (female)
27. Nasal cavity (4 levels)
28. Olfactory bulb
29. Ovaries
30. Oviducts
31. Pancreas
32. Pharynx
33. Parathyroid glands
34. Peyer’s patches
35. Pituitary gland
36. Prostate
37. Rectum
38. Salivary glands
(mandibular and sublingual glands)
39. Sciatic nerve
40. Seminal vesicles
41. Skeletal muscle
42. Skin
43. Spinal cord
(cervical, thoracic and lumbar cord)
44. Spleen
45. Sternum with marrow
46. Stomach
(forestomach and glandular stomach)
47. Teeth
48. Testes
49. Thymus
50. Thyroid glands
51. Trachea
52. Urinary bladder
53. Uterus
54. Vagina



Postmortem examinations (offspring):
GROSS NECROPSY/ORGAN WEIGHTS:
Five pups per sex and group were sacrificed under pentobarbitone anesthesia by
exsanguination from the abdominal aorta and vena cava. They were necropsied and assessed
by gross pathology.
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (final body weight)
2. Brain
3. Epididymides
4. Heart
5. Kidneys
6. Liver
7. Lungs
8. Ovaries
9. Spleen
10. Testes
11. Thymus (fixed)
12. Uterus with cervix


HISTOPATHOLOGY:
Organs and tissues of PND 22 pups that were histologically processed and
examined by light microscopy.
1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Cecum
6. Cervix
7. Coagulating glands
8. Colon
9. Duodenum
10. Epididymides
11. Esophagus
12. Eyes with optic nerve
13. Extraorbital lacrimal gland
14. Femur with knee joint
15. Harderian glands
16. Heart
17. Ileum
18. Jejunum
19. Kidneys
20. Larynx (level II)
21. Liver
22. Lungs
23. Lung associated lymph nodes
24. Lymph nodes (mesenteric)
25. Mammary gland (female)
26. Nasal cavity (3 levels)
27. Olfactory bulb
28. Ovaries
29. Oviducts
30. Pancreas
31. Pharynx
32. Parathyroid glands
33. Pituitary gland
34. Prostate
35. Rectum
36. Salivary glands
(mandibular and sublingual glands)
37. Sciatic nerve
38. Seminal vesicles
39. Skeletal muscle
40. Skin
41. Spinal cord
(cervical, thoracic and lumbar cord)
42. Spleen
43. Sternum with marrow
44. Stomach
(forestomach and glandular stomach)
45. Teeth
46. Testes
47. Thymus
48. Thyroid glands
49. Trachea
50. Urinary bladder
51. Uterus
52. Vagina
Statistics:
Statistical evaluation for main groups 0 (air control) versus 7 (micro ZnO) as well as 0 versus 8 (Zn sulphate), and recovery groups 20 (air control) versus 23 (T0420), 20 versus 26 (T0421), 20 versus 27 and 20 versus 28
- Food consumption (parental animals), body weight and body weight change (parental animals
and pups (for the pup weights, the litter means were used)), gestation days, anogenital distance,
anogenital index
--> Student's t-test (two-sided)
- Male and female mating indices, male and female fertility indices, gestation index, females mated, females delivering, females with liveborn pups, females with stillborn pups, females with all stillborn pups
--> FISHER'S EXACT test (one-sided)
-Mating days until day 0 pc, %postimplantation loss, pups stillborn, %perinatal loss, nipple development
--> WILCOXON test (one-sided+)
-Implantation sites, pups delivered, pups liveborn, live pups day x, viability Index, lactation index
--> WILCOXON test (one-sided-)
- Rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity
--> KRUSKAL-WALLIS and WILCOXON test (two-sided)
-Number of cycles and Cycle Length
--> KRUSKAL-WALLIS test (two-sided) and WILCOXON test (two-sided)
-Blood parameters
--> For parameters with bidirectional changes: WILCOXON-test (two-sided) for the hypothesis of equal medians
-Broncho-alveolar lavage fluid (BALF)
--> Pairwise comparison of each dose group with the control group using the WILCOXON-test (onesided) for the hypothesis of equal medians
-Weight of the anesthetized animals and absolute and relative organ weights (adult animals and PND 22 pups)
--> WILCOXON test (two-sided)

Statistical evaluation of neuropathological parameters of PND 22 pups (subset II)
-Weight parameters (brain)
-->KRUSKAL-WALLIS test (two-sided)
-Brain width and length
--> WILCOXON test (two-sided) with Bonferroni-Holm-
-Brain morphometry: linear measurements of selected brain regions
-->-> WILCOXON test (two-sided)
Reproductive indices:
MALES:

Male mating index (%) = (number of males with confirmed mating*/number of males placed with females) x 100

* defined by a female with vaginal sperm or with implants in utero

Male fertility index (%) = (number of males proving their fertility* /number of males placed with females) x 100

* defined by a female with implants in utero

FEMALES:

Female mating index (%) = (number of females mated*/number of females placed with males) x 100

* defined as the number of females with vaginal sperm or with implants in utero


Female fertility index (%) = (number of females pregnant*/number of females mated**)x 100

* defined as the number of females with implants in utero
** defined as the number of females with vaginal sperm or with implants in utero

Gestation index (%) = (number of females with live pups on the day of birth/number of females pregnant*) x 100

* defined as the number of females with implants in utero

Live birth index (%) = (number of liveborn pups at birth/total number of pups born) x 100


Postimplantation loss (%) =[(number of implantations – number of pups delivered)/number of implantations] x 100


Offspring viability indices:
Viability index (%) = (number of live pups on day 4* after birth/ number of live pups on the day of birth) x 100

* before standardization of litters (i.e. before culling)


Lactation index (%) = (number of live pups on day 21 after birth/number of live pups on day 4* after birth) x 100

* after standardization of litters (i.e. after culling)

Clinical signs:
no effects observed
Description (incidence and severity):
During the pre-exposure period and the exposure period the animals showed no clinical signs and findings different from normal.
-Exposure period, control group animals (test groups 0, 10, 20): There were no clinical signs and findings different from normal.
-Exposure period, reference item 1 (test groups 7, 17 and 27): One male animal of test group 7 (No. 126) showed a mass that was palpable through skin on study days 66 - 149. No clinical signs of toxicity were noted in any other animals of these groups.
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
No deaths were recorded throughout the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant changes of body weight were determined in male
animals:
- Test group 7: day 25: 350.5g (p< 0.05), whereas the control group was 363.4g
- Test group 7: day 32: 362.2g (p< 0.05), whereas the control group was 379.4g
- Test group 7: day 39: 373.5g (p< 0.05), whereas the control group was 390.9g
- Test group 7: day 46: 377.4g (p< 0.05), whereas the control group was 394.9g
- Test group 7: day 60: 398.5g (p< 0.05), whereas the control group was 416.7g
- Test group 7: day 67: 405.2g (p< 0.05), whereas the control group was 424.6g
- Test group 7: day 94: 423.8g (p< 0.01), whereas the control group was 453.5g
- Test group 27: day 32: 361.7g (p< 0.05), whereas the control group was 375.0g
- Test group 27: day 39: 375.9g (p< 0.05), whereas the control group was 390.9g

The following statistically significant body weight changes were determined in male animals:
- Test group 7: day 25-> 32: 11.7 (p< 0.01), whereas the control group was 16.1g
- Test group 27: day 18-> 25: 12.7 (p< 0.01), whereas the control group was 16.9g
- Test group 27: day 74-> 81: 5.4 (p< 0.01), whereas the control group was 9.8g

-> Retarded body weight development in male animals as treatment-related, adverse effects

Body weight of F0 females during gestation/lactation of F1 litters:
no adverse effects on body weights/body weight gain at a concentration of 10 mg/m³ during gestation and lactation.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
The ophthalmologic examinations did not show any impairment of the refracting media.
Spontaneous findings such as remainders of the pupillary membrane or corneal stippling were
observed in several animals of all test groups and the control group without any concentration response relationship.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The following significant changes were regarded as incidental and not treatment related, because the values were within historical control ranges:
decreased relative basophil counts in males of test group 7 (10 mg/m3 Zinc oxide T0242); decreased absolute and relative monocyte counts in females of test group 7 (10 mg/m3 Zinc oxide T0242); increased red blood cell (RBC) counts in females of test group 7 (10 mg/m3 Zinc oxide T0242); decreased mean corpuscular volume (MCV) and mean corpuscular hemoglobin content (MCH) in females of test group 7 (10 mg/m3 Zinc oxide T0242);
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The following significant changes were regarded as incidental and not treatment related
because the values were within historical control ranges: increased total bilirubin and sodium values in females of test group 7 (10 mg/m3 Zinc oxide T0242)(males, inorganic phosphate 1.48-1.85 mmol/L; females, albumin 35.27-40.13 g/L; total bilirubin 1.25-2.20 μmol/L; sodium 140.7-143.0 mmol/L).
Endocrine findings:
no effects observed
Description (incidence and severity):
After the administration period, in parental males and in male and female pups at PND22 of all
test groups, no treatment-related alterations of T4 and TSH levels were observed.
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional observational battery:
Quantitative parameters: no substance-related findings were observed.
Home cage observations: no substance-related findings were observed.
Open field observations: no substance-related findings were observed.
Sensorimotor tests/reflexes: no substance-related findings were observed.

Overall motor activity (summation of all intervals):
Reference item 1 (Test group 7):
there were no statistically significant deviations from the control group 0.

Single intervals:
Comparing the single intervals with the control group, the following statistically significant
deviations were seen:
Decrease of activity in the female animals of test group 7 (10 mg/m³, reference item 1)
at interval 5 on day 87 (p ≤ 0.05).
-->No other abnormalities were detected.
These changes were considered incidental because they were of transient nature and the
overall motor activity was not changed in the respective group.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings were observed in in the larynx, lungs, nasal cavity and the tracheobronchial lymph nodes. These are further described in the details on results section

The following treatment-related, adverse effects were observed:
Main group (F0)
Reference item 1 (Zinc oxide T0242)
Test group 7 (10 mg/m³):
• Minimal to severe numbers of foamy macrophages in the lungs in all male and all female
animals
• Minimal to severe cellular debris in the lungs in all male and all female animals
• Minimal to moderate infiltration of neutrophils of alveoli of the lungs in all male and all
female animals
• Minimal to slight hyperplasia of type II pneumocytes in 6 males and 8 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes
(exemplarily) in 4 males and 6 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes
(exemplarily) in 6 males and 8 females
• Minimal degeneration/regeneration of the olfactory epithelium (nasal cavity, level IV,
exemplarily) in 4 females

Test group 27 (Recovery group R1, 10 mg/m³)
• Minimal cellular debris in the lungs in 1 male animal
• Minimal infiltration of neutrophils of lung alveoli in 1 male animal
• Minimal hyperplasia of type II pneumocytes in 4 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes in
2 males and 3 females
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
BAL
Main group (F0)
The following treatment-related, adverse effects were observed:

Reference item 1 (Zinc oxide T0242)
Test group 7 (10 mg/m³)
• Retarded body weight development in male animals
• Increased total cell counts as well as absolute and relative neutrophil cell and monocyte
counts in BAL of both sexes
• Increased absolute lymphocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute macrophage and eosinophil cell counts in BAL of males
• Increased total protein levels lactate dehydrogenase (LDH), alkaline phosphatase
(ALP) and γ-Glutamyl-transferase (GGT) activities in BAL of both sexes
• Increased β--N-Acetyl glucosaminidase (NAG) activity in BAL of males

Test group 27 (Recovery group R1, 10 mg/m³)
• No treatment-related adverse findings in lavage
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Estrous cycle data, generated during the last 3 weeks prior to mating to produce the F1 litter, revealed regular cycles in the females of all test groups. The mean estrous cycle duration was comparable: 3.9 / 3.9 / 4.0, 4.0, 3.9, 3.9, 4.0 and 4.0 days in test groups 1 – 6 as well as 7 and 8.
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
Description (incidence and severity):
MALE REPRODUCTION DATA:
For all F0 parental males of all test groups, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100% in all test groups.

Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter. However, two males (No. 105, 112) of test group 6 (test item T0421, 10 mg/m3) did not generate F1 pups.

Thus, the male fertility index was 87.5% in test group 6 and 100% in all other groups. This reflects the normal range of biological variation inherent in the strain of rats used for this study.

FEMALE REPRODUCTION DATA AND DELIVERY DATA:
The female mating index was 100% in all test groups. The mean duration until copulation was detected (GD 0) varied between 1.9 and 2.8 days without any relation to test item and concentration.

All female rats delivered pups or had implants in utero with the following exception:
• Test group 6 (test item T0421)
female No. 305 (mated with male No. 105) did not become pregnant
female No. 312 (mated with male No. 112) did not become pregnant

The female fertility index was 87.5% in test group 6 and 100% in all other groups. This reflects the normal range of biological variation inherent in the strain of rats used for this study.

The gestation index was 100% in in all test groups. The mean duration of gestation was comparable in all test groups (i.e. between 21.9 and 22.4 days).

Litter and delivery parameters:
Implantation was not affected by the treatment since the mean number of implantation sites was comparable between the test substance-treated group and the control (13.2 and 12.5 implants/dam in test groups 0 and 7). All values are well within the historical control range (HCD: 11.1 - 13.9).

Post-implantation loss was 2.8 and 7.7* mean% in test groups 0 and 7, respectively. The apparently changed value in test group 7 is well within the historical control range and thus considered spontaneous in nature and not treatment related (HCD: 2.4 - 17.7).

Also, the mean number of F1 pups delivered per dam remained unaffected in the exposure group (12.9 and 11.6 pups/dam in test groups 0 and 7, respectively). While the control was above, numbers of F1 pups delivered per dam in the exposure group are well within the historical control range (HCD: 10.3 - 12.7).

The rate of liveborn pups was not affected by the test substance, as indicated by live birth indices of 100% in both control and exposure group.
CLINICAL SIGNS AND MORTALITY:
Mortality:
No deaths were recorded throughout the study.
Clinical observations:

During the pre-exposure period and the exposure period the animals showed no clinical signs and findings different from normal.
-Exposure period, control group animals (test groups 0, 10, 20): There were no clinical signs and findings different from normal.
-Exposure period, reference item 1 (test groups 7, 17 and 27): One male animal of test group 7 (No. 126) showed a mass that was palpable through skin on study days 66 - 149. No clinical signs of toxicity were noted in any other animals of these groups.


BODY WEIGHT AND WEIGHT GAIN
The following statistically significant changes of body weight were determined in male
animals:
- Test group 7: day 25: 350.5g (p< 0.05), whereas the control group was 363.4g
- Test group 7: day 32: 362.2g (p< 0.05), whereas the control group was 379.4g
- Test group 7: day 39: 373.5g (p< 0.05), whereas the control group was 390.9g
- Test group 7: day 46: 377.4g (p< 0.05), whereas the control group was 394.9g
- Test group 7: day 60: 398.5g (p< 0.05), whereas the control group was 416.7g
- Test group 7: day 67: 405.2g (p< 0.05), whereas the control group was 424.6g
- Test group 7: day 94: 423.8g (p< 0.01), whereas the control group was 453.5g
- Test group 27: day 32: 361.7g (p< 0.05), whereas the control group was 375.0g
- Test group 27: day 39: 375.9g (p< 0.05), whereas the control group was 390.9g

The following statistically significant body weight changes were determined in male animals:
- Test group 7: day 25-> 32: 11.7 (p< 0.01), whereas the control group was 16.1g
- Test group 27: day 18-> 25: 12.7 (p< 0.01), whereas the control group was 16.9g
- Test group 27: day 74-> 81: 5.4 (p< 0.01), whereas the control group was 9.8g

-> Retarded body weight development in male animals as treatment-related, adverse effects

Body weight of F0 females during gestation/lactation of F1 litters:
no adverse effects on body weights/body weight gain at a concentration of 10 mg/m³ during gestation and lactation.

FOOD CONSUMPTION
No effect observed

HAEMATOLOGICAL FINDINGS:
The following significant changes were regarded as incidental and not treatment related, because the values were within historical control ranges:
decreased relative basophil counts in males of test group 7 (10 mg/m3 Zinc oxide T0242); decreased absolute and relative monocyte counts in females of test group 7 (10 mg/m3 Zinc oxide T0242); increased red blood cell (RBC) counts in females of test group 7 (10 mg/m3 Zinc oxide T0242); decreased mean corpuscular volume (MCV) and mean corpuscular hemoglobin content (MCH) in females of test group 7 (10 mg/m3 Zinc oxide T0242);

CLINICAL CHEMISTRY:
The following significant changes were regarded as incidental and not treatment related
because the values were within historical control ranges: increased total bilirubin and sodium values in females of test group 7 (10 mg/m3 Zinc oxide T0242)(males, inorganic phosphate 1.48-1.85 mmol/L; females, albumin 35.27-40.13 g/L; total bilirubin 1.25-2.20 μmol/L; sodium 140.7-143.0 mmol/L).

NEUROBEHAVIOUR:
Functional observational battery:
Quantitative parameters: no substance-related findings were observed.
Home cage observations: no substance-related findings were observed.
Open field observations: no substance-related findings were observed.
Sensorimotor tests/reflexes: no substance-related findings were observed.

Overall motor activity (summation of all intervals):
Reference item 1 (Test group 7):
there were no statistically significant deviations from the control group 0.

Single intervals:
Comparing the single intervals with the control group, the following statistically significant
deviations were seen:
Decrease of activity in the female animals of test group 7 (10 mg/m³, reference item 1)
at interval 5 on day 87 (p ≤ 0.05).
-->No other abnormalities were detected.
These changes were considered incidental because they were of transient nature and the
overall motor activity was not changed in the respective group.

ORGAN WEIGHTS
When compared with control group 0 (=100%),
Reference item 1- Test group 7 (10mg/m3) (Zinc oxide T0242) : Increase of absolute/relative lung weights in males (130%/141%) and females (137%/141%)
--> These effects were observed as treatment-related, adverse effects

GROSS PATHOLOGY

Reference item 1 (Zinc oxide T0242)
Test group 7 (10 mg/m³):
•Macroscopically observed white foci in the lungs of 5 males and 8 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial,
highest number is given) in 7 males and 10 females
--> These effects were observed as treatment-related, adverse effects
Test group 27 (Recovery group R1, 10 mg/m³)
• Macroscopically observed white foci in the lungs of 3 males and 3 females
• Minimal to slight numbers of foamy macrophages in the lungs in 2 male and 4 female Animals

HISTOPATHOLOGICAL FINDINGS: NON-NEOPLASTIC:

Larynx (level I):
In the larynx, the most severe findings were observed in level I, therefore only findings in level I of the larynx are given

Parental animals:
No findings

Recovery animals:
No findings


Lungs:

Parental animals:
Mainly, high dose group males and females (test group 3 and 6 [test item 1 and 2, 2 mg/m³]) were affected. Within alveoli, mainly in the bronchio-alveolar transition region, a multifocal accumulation of alveolar macrophages with vacuolar (foamy) cytoplasm was seen. The alveolar macrophages often revealed nuclei of increased size and occasionally multiple nuclei.
Intermingled with the foamy macrophages, cellular debris of presumable fragmented
macrophages and neutrophils were observed. In the region of these cellular accumulations, proliferation (hyperplasia) of type II pneumocytes was observed.
Males of test group 2 and 4 (test item 1 and 2, 2 mg/m³) revealed also an accumulation of foamy macrophages, only.

In males and females of the two reference items, similar findings were observed as described for test group 3 and 6 (test item 1 and 2, 2 mg/m³). Only the severity was slightly higher when compared with the other test items, especially in test group 7 animals.

Recovery animals:
The same findings as described for the main group animals were observed in the recovery animals. These findings were regarded to be treatment-related.

Lymph nodes (mediastinal):
Parental animals:
The mediastinal and tracheobronchial lymph nodes revealed comparable findings.
In general, the high dose group males and females (test group 3 and 6 [test item 1 and 2, 2 mg/m³]) were more severely affected. A lympho-reticular cell hyperplasia was observed, which can be explained by an activation of the draining lymph nodes of the lungs. Furthermore, aggregates of macrophages were seen within the lymph nodes. These findings were considered as treatment-related.
Single animals of test group 1, 2 (test item 1, 0.5 and 2 mg/m³), and test group 5 (test item 2, 2 mg/m³) revealed similar findings.

The males and females exposed to the reference items showed similar findings as the animals exposed to the test substances.

Recovery animals:
The same findings as described for the main group animals were observed in the recovery animals. These findings were regarded to be treatment-related.

Nasal cavity:

Parental animals:
The nasal cavity was investigated in four levels. The most severely affected levels were level III and IV
Females of test group 7 (reference item 1, 10 mg/m³) and males and females of test group 8 (reference item 2, 22 mg/m³) revealed the same findings in the nasal cavity.

Recovery animals:
Findings occurred either individually or were biologically equally distributed over
control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Trachea
In the trachea, two male animals of test group 8 (reference item 2, 22 mg/m³) revealed a flattening of the respiratory epithelium at the carina. This finding was considered to be treatment-related.
All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.



BRONCHOALVEOLAR LAVAGE FLUID (BALF):
Cytology:
Parental animals:
After the administration period, in BAL of males and females of test group 7 (10 mg/m3 Zinc oxide T0422) total cell counts as well as absolute and relative neutrophil cell and monocyte counts and absolute lymphocyte counts were significantly increased whereas relative macrophage counts were significantly decreased. Additionally, in males of this test group absolute macrophage and eosinophil counts (not significantly) were increased. These alterations were regarded as treatment related and adverse.

Recovery animals:
After the 8-week recovery period, no changes were observed in BAL cytology of males and females of test group 27 (10 mg/m3 Zinc oxide T0422).

Proteins/enzymes:

Parental animals:
After the administration period, in BAL of males and females of test group 7 (10 mg/m3 Zinc oxide T0422) total protein levels as well as lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) activity were moderately, significantly increased whereas β -N-Acetyl glucosaminidase (NAG) activity in males and γ-Glutamyl-transferase (GGT) activity in both sexes were marginally but also significantly increased. These alterations were regarded as treatment related and adverse.
Additionally, in females of test group 7 (10 mg/m3 Zinc oxide T0422) NAG activity was also significantly increased, but the change was below 2fold and therefore it was regarded as maybe treatment related but non-adverse.


Recovery animals:
After the 8-week recovery period, in males of test group 27 (10 mg/m3 Zinc oxide T0422) total protein levels and NAG activity were marginally but significantly increased. However, the small increases (below 2fold) of both parameters and no changed BAL cytology counts among these individuals indicated that the total protein and NAG changes were rather incidental than treatment related.

OTHER FINDINGS
- Electron microscopy:
Dose descriptor:
LOAEC
Remarks:
local toxicity
Effect level:
9.68 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: changes in lung, lung-draining lymph nodes and nasal cavity at the highest tested target concentration of 10 mg/m³
Dose descriptor:
NOAEC
Remarks:
systemic toxicity
Effect level:
9.68 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology
clinical biochemistry
histopathology: non-neoplastic
Remarks on result:
other: No systemic toxicity was observed in hematology, clinical chemistry ; increased neutrophils and lymphocytes in blood at the target conc of 10 mg/m3
Dose descriptor:
NOEC
Effect level:
9.68 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance
Critical effects observed:
yes
Lowest effective dose / conc.:
9.68 other: mg/m3 air (analytical)
System:
respiratory system: lower respiratory tract
Organ:
lungs
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Clinical signs:
no effects observed
Description (incidence and severity):
No test or reference item related adverse clinical signs were observed in any of the F1 generation pups of the different test groups. Individual findings in few pups, like dehydrated appearance or gasping, were noted in several groups including control. They were most likely related to the technical procedure of inhalation exposure rather than any test or reference item.
Dermal irritation (if dermal study):
not specified
Mortality / viability:
no mortality observed
Description (incidence and severity):
The viability index indicating pup survival during early lactation (PND 0 - 4) varied between 99.5% / 99.0% / 100% / 100% / 95.7% / 97.9% / 100% / 99.6% and 99.5% in test groups 0 - 8 without showing significant differences between the groups. All values were within the historical control range (94 – 100%).

The lactation index indicating pup survival on PND 4 - 21 was 98.8% in test group 5 and 100% in all remaining test groups. All values were within the historical control range (95.7 – 100%).
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weights of all male and female pups in all test and reference item-treated groups 1 – 8 were comparable to the concurrent control values throughout the entire study.

The statistically significantly higher body weights in male pups and in both sexes combined in test group 4 on PND 4 were considered to be spontaneous in nature.

Calculation of body weight change resulted in a number of statistical changes in various groups, sometimes higher, sometimes lower than the concurrent control:

Increased:
- test group 3 males and both sexes combined on PND 1
- test group 4 males, females and both sexes combined on PND 1
- test group 7 males, females and both sexes combined on PND 1

Decreased:
- test group 5 females and both sexes combined on PND 4
- test group 7 males, females and both sexes combined on PND 13
- test group 8 males, females and both sexes combined on PND 4
- test group 8 males, females and both sexes combined on PND 13

None of these apparent changes is considered to be associated with the respective test or reference items.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not specified
Sexual maturation:
not specified
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
The anogenital distance and anogenital index of all treated male and female pups was comparable to the concurrent control values.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
The apparent number and percentage of male pups having areolae was not influenced by the test item when examined on PND 13. Likewise, no test item-related effect was detected in any of the test groups during the re-examination on PND 20.
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings were observed in in the lungs and nasal cavity.

The following treatment-related, adverse effects were observed:
PUPS (F1)
Reference item 1 (Zinc oxide T0242)
Test group 7 (10 mg/m³)
• Minimal cellular debris in the lungs in 5 male and 5 female animals
• Minimal infiltration of neutrophils of lung alveoli in 2 male and 3 female animals
• Minimal degeneration/regeneration of the olfactory epithelium (nasal cavity, level III) in
1 male and 1 female
Other effects:
no effects observed
Description (incidence and severity):
OPEN FIELD OBSERVATIONS (OFO):
None of the animals in all test groups showed abnormalities attributable to the exposure to the test substance.

MOTOR ACTIVITY MEASUREMENT (MA):
Motor activity of male and female F1 offspring was not influenced by the test item at all concentration levels and at any of the testing dates PND 13, 17 and 21. Overall activity levels and habituation to the test environment corresponded to the age of these animals at the specific testing date, if usual biological variation inherent in rats used for this type of experiment was considered.

Across the test groups, there were a number of statistically significant changes in either the number of beam interrupts or in the number of rearings, in single intervals on various testing dates, sometimes higher, sometimes lower than the concurrent control:

Test item T0242
Increased:
- test group 7 females, rearings, interval 1, PND 21
- test group 7 females, beam interrupts, interval 8, PND 21


Decreased:
- test group 7 males, beam interrupts, interval 9 and 10, PND 13
- test group 7 males, beam interrupts, interval 4, 5, 7 and 1-12, PND 17
- test group 7 males, rearings, interval 9 and 10, PND 13
- test group 7 males, rearings, interval 2, 5 and 1-12, PND 17
- test group 7 females, beam interrupts, interval 5, 9 and 1-12, PND 13
- test group 7 females, beam interrupts, interval 3, 5 and 6, PND 17
- test group 7 females, beam interrupts, interval 4, PND 21
- test group 7 females, rearings, interval 3, 4, 5, 6 and 1-12, PND 17

None of these apparent changes is considered to be associated with the respective test or reference items.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Regarding neuropathology, no treatment-related findings were seen in pups of PND 22: Neuropathology, brain weight determination, necropsy, gross measurements of the brain,
neuropathology examination by light microscopy and morphometry did not reveal any
neuropathological, treatment-related findings.
--> There was no developmental neurotoxicity in all examined pups.
Developmental immunotoxicity:
not examined
Dose descriptor:
LOAEC
Remarks:
local toxicity
Generation:
F1
Effect level:
9.68 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: minimal cellular debris, neutrophilic infiltration in the lungs and minimal degeneration/regeneration of the olfactory epithelium in nasal cavity in 1 male and 1 female animal at 10 mg/m³
Dose descriptor:
NOAEC
Remarks:
systemic toxicity
Generation:
F1
Effect level:
9.68 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology
clinical biochemistry
histopathology: non-neoplastic
Remarks on result:
other: No systemic toxicity was observed in hematology, clinical chemistry and histopathology
Critical effects observed:
yes
Lowest effective dose / conc.:
9.68 other: mg/m3 air (analytical)
System:
respiratory system: lower respiratory tract
Organ:
lungs
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Reproductive effects observed:
no
Conclusions:
Overall assessment for adult animals:

With regards to systemic toxicity, none of the test or reference substances caused any systemic toxicity that were not triggered by the local toxicity.

Comparing the local effects of the two nano Zinc oxide materials, the overall finding in the lungs, mediastinal lymph nodes, in the nasal cavity were comparable at the tested concentrations, as well as the changes of lavage parameters. The small differences are considered biological variations. There were no considerable differences between the effects caused by zinc oxide nanoparticles and those caused by micron-size zinc oxide particle.

For reference substance 2 (zinc sulfate monohydrate), lower incidence and severity was found in the lungs than in the other zinc oxide treated groups, but higher incidence and severity in nasal cavity and larynx. This difference is considered being related to the different deposition pattern, caused by the different aerodynamic diameter. The aerodynamic diameter of zinc sulfate monohydrate was larger than the different types of zinc oxide. The mean MMAD of zinc sulfate monohydrate was with 2.3 µm considerably higher than those measured at the high concentrations of the test items 1 (1.19 µm) and 2 (0.97 µm). The deposited dose at the upper respiratory tract was higher, while those deposited in the lung was lower.

After the recovery period, all parameters in lavage fluid returned to the control level in all animals, irrespective of the exposed test and reference substance. With regards of histological findings in the respiratory tract, all changes reduced greatly in incidence and severity. Only single animals showed still some mild effects.

Overall assessment for PND 22 animals:
With regards to effects observed in PND 22 pups that were exposed whole-body to zinc oxide nanomaterials from PND 4 to PND 21, the findings were limited to lungs and nasal cavities. The effects observed in the parental animals showed much higher incidence and severity than in the pups exposed at the same concentration. This could be explained by the duration of the exposure, because pups were only exposed for 17 days, while the adult animals were exposed for 90-days.

Comparing the toxicity of the two nano Zinc oxide materials in pups of PND 22, the overall finding in the lungs were comparable at the high concentration of 10 mg/m³ for test items 1 and 2. However, lesions were also observed in nasal cavity in pups exposed to test item 2. While lesions in nasal cavity were still observed at 0.5 mg/m³ test item 2, there were no effect observed in animals exposed to 2 mg/m³ and 0.5 mg/m³ test item 1.

Comparing the toxicity caused by the two nano zinc oxide materials with those caused by the microscale zinc oxide material, or by the soluble zinc sulfate monohydrate, the changes in lungs were comparable in incidence and severity in exposed pups on PND 22. However, no nasal cavity lesions were observed in animals exposed to test item 1, while similar lesions were seen in those exposed to test item 2 and the reference items.

None of the substances cause any systemic toxicity, nor were there any developmental neurotoxicity in exposed pups.
Executive summary:

This study was a 90-Day Study (OECD test guideline (TG) 413) combined with the Reproduction/ Developmental Toxicity Screening Test (OECD TG 421) in rat with neurotoxicity and developmental (neuro)toxicity evaluation, including detailed clinical observations addressing potential neurobehavioral effects, histological and morphological evaluations of the brains of the pups on post-natal day 22.


To compare the toxicity of uncoated and coated nano Zinc oxide, these two materials (Zinc oxide T0420 was uncoated, Zinc oxide T0421 was coated) were tested at each three concentrations. In addition, micronsize Zinc oxide T0242 and a soluble salt zinc sulfate monohydrate was tested as reference items. 


Groups of male and female Wistar rats were whole-body exposed to the aerosols of ZnO nano materials, Zinc oxide T0420 and Zinc oxide T0421, for 6 hours daily, at least 90 days. Zinc oxide T0420 was uncoated, Zinc oxide T0421 was coated.


The target concentrations for Zinc oxide T0420 and T0421 were 0.5, 2 and 10 mg/m³ referring to the non-volatile fraction. For the reference item 1 microscale Zinc oxide T0242, 10 mg/m³ was tested. For the reference item 2, Zinc sulfate monohydrate a target concentration of 22 mg/m³ was tested because this is equimolar to zinc ion of the ZnO materials. Concurrent control groups were exposed to humidified air (control group 0, 10 and 20).


All animals were exposed to the respective concentrations of test substance for 6 hours a day according to the time schedule (exception: no exposure on the day of FOB/MA and parental females from GD20 – PND 3)). Control animals were exposed to conditioned air. Male and female rats aged about 6 or 7 weeks when supplied, were used as F0 generation parental animals. The animals were exposed for 43 days before mating. The mating period were maximal 2 weeks. After the mating period, the exposure of all male F0 animals were continued until they are exposed for total minimal 90 days. After the mating period, the female F0 animals were exposed further until gestation day 19. To allow them to deliver and rearing their pups (F1 generation), they were not exposed from gestation day 20 to postnatal day (PND) 3. From PND 4 through to PND 21, the dams were exposed with their pups in exposure cages containing beddings. During the exposure food was withdrawn. Water was provided in form of hydrogel pads from PND 14 to 16 onward. The first parental female animals were in gestation stage already after the first few mating days, therefore, the post-weaning period were adjusted in such a way, that a total of minimum 90 exposure will be achieved for females.


Daily clinical observations, body weights, food consumption, ophthalmology, detailed clinical observation and FOB/MA were recorded. Moreover, male and female fertility were determined. Additional assessments including hematology and clinical chemistry in blood, bronchoalveolar lavage, and histopathology according to the referenced guidelines were carried out at the termination of exposure period. In addition, recovery groups of male and female animals were included; after an exposure period of about 90 days, these animals were kept for an additional period of ca. 60 days without exposure (control group 20, and test groups 23, 26, 27 and 28, respectively).


To assess the reproductive/developmental toxicity of the test substances (incl. reference substances), estrus cycles, male and female reproduction, delivery data were collected. In the pups, open field observations were performed on PND 13 and 21, motor activity measurements were performed on PND 13, 17 and 21. On PND 22, thyroid hormones, brain weights, neuropathology, general histopathology were examined in separate subsets of animals.


The following treatment-related, adverse effects were observed:
Main group (F0)
Test item 1 (Zinc oxide T0420)
Test group 3 (10 mg/m³)


• Decreased food consumption during gestation and lactation of parental females
• Increased total cell counts as well as absolute and relative lymphocyte, neutrophil cell and monocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute eosinophil cell counts in males in BAL
• Increased total protein levels lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and γ-Glutamyl-transferase (GGT) activities in BAL of both sexes
• Increased β-N-Acetyl glucosaminidase (NAG) activity in BAL of males
• Increase of absolute/relative lung weights in males (140%/150%) and females (128%/130%)
• Macroscopically observed white foci in the lungs of 5 males and 8 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial, highest number is given) in 6 males and 9 females
• Slight to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to severe cellular debris in the lungs in all male and all female animals
• Minimal to moderate infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 9 males and all females
• Minimal to slight degeneration/regeneration of the olfactory epithelium 



Test group 23 (Recovery group R1, 10 mg/m³)
• Macroscopically observed white foci in the lungs of 1 male and 2 females
• Macroscopically enlarged draining lymph nodes (mediastinal) in 1 female
• Minimal to moderate numbers of foamy macrophages in the lungs in 3 male and 4 female animals
• Minimal cellular debris in the lungs in 1 male and 2 female animals
• Minimal infiltration of neutrophils of alveoli of the lungs in 1 male and 2 female animals
• Minimal hyperplasia of type II pneumocytes in 3 females


Test group 2 (2 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium in one male animal


Test group 1 (0.5 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium in 1 female



Conclusion for adult animals exposed to test item 1 (Zinc oxide T0420):
Inhalation exposure to Zinc oxide T0420 caused changes in lung, lung-draining lymph nodes and nasal cavity at the high concentration of 10 mg/m³. These findings were almost, though not completely resolved during the post-exposure observation period. At 2 mg/m³, minimal degeneration/regeneration in the nasal cavity was noted in one male animal, and at 0.5 mg/m³ in one female animal. Due to findings in nasal cavity, the NOAEC for local toxicity at the respiratory tract was 0.5 mg/m³ for male rats. a No Observed Adverse Effect Concentration (NOAEC) for local toxicity for females could not be unequivocally determined.


No systemic toxicity was observed in hematology, clinical chemistry and histopathology the NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity was 10 mg/m³ for Zinc oxide T0420.



Test item 2 (Zinc oxide T0421)
Test group 6 (10 mg/m³)
• Decreased food consumption during gestation and lactation of parental females
• Decreased body weights/body weight gain during gestation and lactation of parental females
• Increased total white blood cell (WBC) as well as absolute neutrophil and lymphocyte counts in blood of males
• Slightly increased total cell counts as well as absolute and relative lymphocyte, neutrophil cell and monocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute eosinophil cell counts in males in BAL
• Increased total protein levels lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) activities in BAL of both sexes
• Increased  γ-Glutamyl-transferase (GGT) activity in BAL of males
• Increase of absolute/relative lung weights in males (136%/143%) and females (131%/137%)
• Macroscopically observed white foci in the lungs of 6 males and 8 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial, highest number is given) in 10 males and 5 females
• Minimal to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to severe cellular debris in the lungs in all male and all female animals
• Minimal to slight infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 8 males and 9 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes (exemplarily) in 8 males and 3 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes (exemplarily) in 4 males and 2 females
• Minimal to slight degeneration/regeneration of the olfactory epithelium 

Test group 26 (Recovery group R1, 10 mg/m³)
• No treatment-related adverse findings in lavage and histopathology

Test group 5 (2 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium 

Test group 4 (0.5 mg/m³)
No treatment-related adverse findings


Conclusion for adult animals exposed to test item 2 (Zinc oxide T0421):
Inhalation exposure to Zinc oxide T0421 caused changes several lavage parameters, as well as histological changes in lung, lung-draining lymph nodes and nasal cavity at the highest tested concentration of 10 mg/m³. All these effects were completely resolved after the postexposure observation period. In blood, increased neutrophils and lymphocyte was notice at the concentration of 10 mg/m³, which is considered secondary to the inflammation in the lung.
At the mid concentration of 2 mg/m³, histological findings were still observed in the nasal cavity of three male and two female rats. Thus, the No Observed Adverse Effect Concentration (NOAEC) for local toxicity was 0.5 mg/m³ under the current study conditions. 
Besides the increased neutrophils and lymphocytes in blood, no other changes were observed in hematology, clinical chemistry. No histopathological changes were observed in any organs and tissues that are not part of the respiratory tract. The NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity, that were not attributed to the local effect, was 10 mg/m³ for Zinc oxide T0421.



Reference item 1 (Zinc oxide T0242)
Test group 7 (10 mg/m³)
• Retarded body weight development in male animals
• Increased total cell counts as well as absolute and relative neutrophil cell and monocyte counts in BAL of both sexes
• Increased absolute lymphocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute macrophage and eosinophil cell counts in BAL of males
• Increased total protein levels lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and γ-Glutamyl-transferase (GGT) activities in BAL of both sexes
• Increased β-N-Acetyl glucosaminidase (NAG) activity in BAL of males
• Increase of absolute/relative lung weights in males (130%/141%) and females (137%/141%)
• Macroscopically observed white foci in the lungs of 5 males and 8 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial, highest number is given) in 7 males and 10 females
• Minimal to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to severe cellular debris in the lungs in all male and all female animals
• Minimal to moderate infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 6 males and 8 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes (exemplarily) in 4 males and 6 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes (exemplarily) in 6 males and 8 females
• Minimal degeneration/regeneration of the olfactory epithelium 


Test group 27 (Recovery group R1, 10 mg/m³)
• Macroscopically observed white foci in the lungs of 3 males and 3 females
• Minimal to slight numbers of foamy macrophages in the lungs in 2 male and 4 female animals
• Minimal cellular debris in the lungs in 1 male animal
• Minimal infiltration of neutrophils of lung alveoli in 1 male animal
• Minimal hyperplasia of type II pneumocytes in 4 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes in 2 males and 3 females


Conclusion for adult animals exposed to reference item 1 (Zinc oxide T0242):
Inhalation exposure to Zinc oxide T0242 caused changes in lung, lung-draining lymph nodes and nasal cavity at the highest tested concentration of 10 mg/m³. These findings were greatly, though not completely, resolved during the post-exposure observation period.
No systemic toxicity was observed in hematology, clinical chemistry and histopathology the NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity was 10 mg/m³ for Zinc oxide T0242.


Reference item 2 (Zinc sulfate monohydrate)
Test group 8 (22 mg/m³)
• During exposure period, salivation and respiration sounds were detected in several male and female animals.
• Retarded body weight development in all male and female animals. 
• Decreased food consumption during gestation and lactation of parental female animals
• Recreased body weights/body weight gain during gestation and lactation of parental female animals
• Increased total cell counts as well as absolute and relative lymphocyte, neutrophil cell and monocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute macrophage and eosinophil cell counts in BAL of males
• Increased total protein levels lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and γ-Glutamyl-transferase (GGT) activities in BAL of both sexes
• Increase of absolute/relative lung weights in males (125%/138%) and females (114%/119%)
• Macroscopically observed white foci in the lungs of 3 males and 7 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial, highest number is given) in 5 males and 8 females
• Erosion/ulcer of the laryngeal epithelium at the base of the epiglottis in 1 female
• Minimal to slight squamous metaplasia of the laryngeal epithelium at the base of the epiglottis in all males and all females
• Minimal to slight inflammatory cell infiltrates of the laryngeal epithelium in 1 male and 9 females
• Minimal to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to moderate cellular debris in the lungs in all male and all female animals
• Minimal to slight infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 6 males and 8 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes (exemplarily) in 5 males and 5 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes (exemplarily) in 8 males and 8 females
• Minimal to moderate degeneration/regeneration of the olfactory epithelium in all males and all females

Test group 28 (Recovery group R1: 22 mg/m³)
• Macroscopically observed white foci in the lungs of 2 males and 2 females
• Macroscopically enlarged draining lymph nodes (mediastinal) in 1 male and 1 female
• Minimal squamous metaplasia of the laryngeal epithelium at the base of the epiglottis in 1 male and 1 female animal
• Minimal to slight inflammatory cell infiltrates in the laryngeal epithelium in 4 males and 3 females
• Minimal to slight numbers of foamy macrophages in the lungs in 2 male and 4 female animals
• Minimal hyperplasia of type II pneumocytes in 3 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes in 1 male and 1 female animal
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes in 4 males and 4 females
• Minimal degeneration/regeneration of the olfactory epithelium in 1 male and 1 female 


Conclusion for adult animals exposed to reference item 2 (Zinc sulfate monohydrate):
Inhalation exposure to Zinc sulfate monohydrate caused changes in lung, lung-draining lymph nodes, larynx and nasal cavity at the highest tested concentration of 22 mg/m³. These findings were partly resolved during the post-exposure observation period.
No systemic toxicity was observed in hematology, clinical chemistry and histopathology the NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity was 22 mg/m³ for Zinc sulfate monohydrate.


Overall assessment for adult animals:


With regards to systemic toxicity, none of the test or reference substances caused any systemic toxicity that were not triggered by the local toxicity.


Comparing the local effects of the two nano Zinc oxide materials, the overall finding in the lungs, mediastinal lymph nodes, in the nasal cavity were comparable at the tested concentrations, as well as the changes of lavage parameters. The small differences are considered biological variations. There were no considerable differences between the effects caused by zinc oxide nanoparticles and those caused by micron-size zinc oxide particle.


For reference substance 2 (zinc sulfate monohydrate), lower incidence and severity was found in the lungs than in the other zinc oxide treated groups, but higher incidence and severity in nasal cavity and larynx. This difference is considered being related to the different deposition pattern, caused by the different aerodynamic diameter. The aerodynamic diameter of zinc sulfate monohydrate was larger than the different types of zinc oxide. The mean MMAD of zinc sulfate monohydrate was with 2.3 µm considerably higher than those measured at the high concentrations of the test items 1 (1.19 µm) and 2 (0.97 µm). The deposited dose at the upper respiratory tract was higher, while those deposited in the lung was lower.


After the recovery period, all parameters in lavage fluid returned to the control level in all animals, irrespective of the exposed test and reference substance. With regards of histological findings in the respiratory tract, all changes reduced greatly in incidence and severity. Only single animals showed still some mild effects.


PUPS on PND 22 
Test item 1 (Zinc oxide T0420)
Test group 3 (10 mg/m³)
Minimal cellular debris in the lungs in 2 male and 4 female animals
• Minimal infiltration of neutrophils of alveoli of the lungs in 1 male and 2 female animals
Test group 2 (2 mg/m³) and test group 1 (0.5 mg/m³)
No treatment-related adverse findings observed
Conclusion for pups exposed to test item 1 (Zinc oxide T0420) sacrificed on PND 22:
Inhalation exposure to Zinc oxide T0420 caused minimal cellular debris and neutrophilic infiltration in a few male and female animals at 10 mg/m³. These findings were considered treatment-related and adverse. The NOAEC for local toxicity was 2 mg/m³. 


There were no test-item related adverse findings in clinical examination/OFO/MA and gross necropsy up to the highest tested concentration of 10 mg/m³. No systemic toxicity was observed in hematology, clinical chemistry, no changes of thyroid hormones. There were no histopathological changes in organs and tissues other than those of the respiratory tract.



Test item 2 (Zinc oxide T0421)
Test group 6 (10 mg/m³)
• Minimal cellular debris in the lungs in 1 male and 4 female animals
• Minimal infiltration of neutrophils of alveoli of the lungs in 1 male and 1 female animals
• Minimal to moderate degeneration/regeneration of the olfactory epithelium (nasal cavity, level III) in 4 male and 2 female animals
Test group 5 (2 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium (nasal cavity, level III) in 2 female animals
Test group 4 (0.5 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium (nasal cavity, level III) in 1 male animal 


Conclusion for pups exposed to test item 2 (Zinc oxide T0421) sacrificed on PND 22:
Inhalation exposure to Zinc oxide T0421 caused minimal cellular debris, neutrophilic infiltration in the lungs and minimal to moderate degeneration/regeneration of the olfactory epithelium in nasal cavity in a few male and female animals at 10 mg/m³. The changes in nasal cavity was still observed at the mid concentration of 2 mg/m³. This findings were considered treatmentrelated and adverse. The NOAEC for local toxicity was 0.5 mg/m³.


There were no test-item related adverse findings in clinical examination/OFO/MA and gross necropsy up to the highest tested concentration of 10 mg/m³. No systemic toxicity was observed in hematology, clinical chemistry, no changes in thyroid hormones. There were no histopathological changes in organs and tissues other than those of the respiratory tract.



Reference item 1 (Zinc oxide T0242)
Test group 7 (10 mg/m³)
• Minimal cellular debris in the lungs in 5 male and 5 female animals
• Minimal infiltration of neutrophils of lung alveoli in 2 male and 3 female animals
• Minimal degeneration/regeneration of the olfactory epithelium (nasal cavity, level III) in 1 male and 1 female


Conclusion for pups exposed to reference item 1 (Zinc oxide T0242) sacrificed on PND 22:
Inhalation exposure to Zinc oxide T0242 caused minimal cellular debris, neutrophilic infiltration in the lungs and minimal degeneration/regeneration of the olfactory epithelium in nasal cavity in a one male and one female animals at 10 mg/m³.


There were no test-item related adverse findings in clinical examination/OFO/MA and gross necropsy up to the highest tested concentration of 10 mg/m³. No systemic toxicity was observed in hematology, clinical chemistry, no changes in thyroid hormones. There were no histopathological changes in organs and tissues other than those of the respiratory tract.


Reference item 2 (Zinc sulfate monohydrate)
Test group 8 (22 mg/m³)
• Minimal cellular debris in the lungs in 2 male and 1 female animals
• Minimal infiltration of neutrophils of lung alveoli in 2 male and 1 female animals
• Minimal degeneration/regeneration of the olfactory epithelium (nasal cavity, level III) in 1 male and 1 female


Conclusion for pups exposed to reference item 2 (zinc sulfate monohydrate) sacrificed on PND 22:
Inhalation exposure to zinc sulfate monhydrate caused minimal cellular debris, neutrophilic infiltration in the lungs and minimal degeneration/regeneration of the olfactory epithelium in nasal cavity in a few male and female animals at 22 mg/m³.


There were no test-item related adverse findings in clinical examination/OFO/MA and gross necropsy up to the highest tested concentration of 10 mg/m³. No systemic toxicity was observed in hematology, clinical chemistry, no changes in thyroid hormones. There were no histopathological changes in organs and tissues other than those of the respiratory tract.


Overall assessment for PND 22 animals:
With regards to effects observed in PND 22 pups that were exposed whole-body to zinc oxide nanomaterials from PND 4 to PND 21, the findings were limited to lungs and nasal cavities. The effects were observed also in the parental animals with much higher incidence and severity than in the pups exposed at the same concentration. This could be explained by the duration of the exposure, because pups were only exposed for 17 days, while the adult animals were exposed for 90-days.


Comparing the toxicity of the two nano Zinc oxide materials in pups of PND 22, the overall finding in the lungs were comparable at the high concentration of 10 mg/m³ for test items 1 and 2. However, lesions were also observed in nasal cavity in pups exposed to test item 2. While lesions in nasal cavity were still observed at 0.5 mg/m³ test item 2, there were no effect observed in animals exposed to 2 mg/m³ and 0.5 mg/m³ test item 1.


Comparing the toxicity caused by the two nano zinc oxide materials with those caused by the microscale zinc oxide material, or by the soluble zinc sulfate monohydrate, the changes in lungs were comparable in incidence and severity in exposed pups on PND 22. However, no nasal cavity lesions were observed in animals exposed to test item 1, while similar lesions were seen in those exposed to test item 2 and the reference items.


None of the substances cause any systemic toxicity, nor were there any developmental neurotoxicity in exposed pups.

Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
24 November 2020 - ...June 2022
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study presented herein is a guideline study with a major deficiency under GLP conditions. Only one concentration level was tested.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016-07-29
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 426 (Developmental Neurotoxicity Study)
Version / remarks:
2007-10-16
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
not applicable
Specific details on test material used for the study:
Name of substance: Zinc sulfate monohydrate
Reference substance No.: 20/0420-1
Batch identification: 201126
Content: Zinc: 36.2 weight-%
Storage stability: Dec 2021
The stability of the test substance under storage conditions over the test period was guaranteed by the manufacturer, and the manufacturer holds this responsibility
Storage conditions: Room temperature
Appearance - physical state / color solid / white
By Products Chlorides: 0.32 weight-%
Species:
rat
Strain:
Wistar
Remarks:
Wistar rats, Crl:WI(Han) Rats were selected since this rodent species is recommended in the respective test guidelines. Wistar rats were selected since there is extensive experience available in the laboratory with this strain of rats.
Details on species / strain selection:
not specified
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
- Females nulliparous and non-pregnant: yes
- Age at study initiation: about 7 weeks (female), about 8 weeks (male)
- Weight at study initiation: The weight variation of the animals used did not exceed +/- 20 percent of the mean weight of each sex.
- Fasting period before study: The animals did not have access to food or water during exposure.
- Housing:
From delivery until mating and male animals after mating: Typ 2000P: ca. 2065 cm2 (polysulfone cages) / up to 5 animals
During mating: type III polycarbonate cages, 1 male/1 female per cage
During rearing: up to PND 22: type III polycarbonate cages, 1 dam with her litter
After weaning the females from study day 90 after exposure onward until sacrifice: Typ 2000P: ca. 2065 cm2 (polysulfone cages) / up to 5 animals. Remaining females with litters will be
maintained in type III cages until weaning.
For Motor Activity Measurement: Typ III polycarbonate cages (floor area about 800 cm²) / 1 animal
During Exposure: Wire cages, type DK III / up to 2 animals Females from PND 4 until study day 94 (and females without litter from the same time period onwards): perforated polycarbonate cages type II. From study day 95 onward wire cages, type DK III
- Diet (ad libitum): mouse and rat maintenance diet, GLP, 12 mm pellets, Granovit AG, Kaiseraugst, Switzerland before and after exposure. Food was withdrawn during exposure.
- Water (ad libitum): tap water
- Acclimation period: 11 days

DETAILS OF FOOD AND WATER QUALITY: The food used in the study was assayed for chemical as well as for microbiological contaminants. In view of the aim and duration of the study, the contaminants occurring in commercial food should not influence the results. The drinking water is regularly assayed for chemical contaminants both by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring of BASF SE as well as for bacteria by a contract laboratory. The Drinking Water Regulation will serve as the guideline for maximum tolerable contaminants. In view of the aim and duration of the study, there are no special requirements exceeding the specification of drinking water.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 45 - 65%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: beginning of experiment To: end of experiment
Route of administration:
inhalation: aerosol
Type of inhalation exposure (if applicable):
whole body
Remarks:
whole-body exposure for the reasons explained see IUCLID section 13.2 'Human health requirements Final Decision: protocol deviations and rationale'
Mass median aerodynamic diameter (MMAD):
>= 1.86 - <= 2.74 µm
Remarks on MMAD:
MMAD / GSD: MMAD = 1.86- 2.74 μm (geometric standard deviation = 1.99-2.01)
Vehicle:
unchanged (no vehicle)
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Generation of the inhalation atmospheres via a solid particle generators (brush-generator; BASF SE, Ludwigshafen, Germany) & Aerosol mixing tube (stainless steel; BASF SE, Ludwigshafen, Germany). Whole body exposure systems were used. The animals were kept singly in wire cages located in a glass steel inhalation chamber, volume of 1.1 m³ (BASF SE).
- Method of holding animals in test chamber: Whole body exposure systems. The animals were kept singly in wire cages located in a glass steel inhalation chamber, volume of 1.1 m³ (BASF SE). The chambers were located in exhaust hoods in an air conditioned room.
- Source and rate of air: Conditioned air from the central air conditioning system, compressed and exhaust air. Compressed air was produced by an oil-free compressor (HT 6, Josef Mehrer GmbH & Co KG, Germany). For this purpose, air is filtered by an inlet air strainer and introduced into the compressor. After passing through an second ultra filter (SMF 5/3, 108 mm, Donalson), the compressed air (15 bar) is stored in a storage of 1500 or 5000 L. The compressed air is conducted to the laboratories via pipes, where the pressure is reduced to 5 - 6 bar. In the laboratory, the compressed air can be taken as required.
- Method of conditioning air: Conditioned air from the central air conditioning system provides cold air of about 15°C. This cold air passes through an activated charcoal filter, is adjusted to room temperature of 20 to 24°C and passes through a second particle filter (H13 (HEPA) Camfil Farr, Germany). The so generated conditioned air was used to generate inhalation atmospheres.
- System of generating particulates/aerosols: The particles/aerosol was generated via a solid particle generator (brush-generator; BASF SE, Ludwigshafen, Germany) and an aerosol mixing tube (stainless steel; BASF SE, Ludwigshafen, Germany), according to the following method: For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage; mixed with conditioned dilution air and passed into the inhalation system.
- Temperature, humidity, pressure in air chamber: Daily mean relative humidities in the inhalation systems ranged between 41.6 and 60.8 %. Daily mean temperatures in the inhalation systems ranged between 21.4 and 23.7°C. They are within the range suggested by the respective testing guidelines.
- Air flow rate: The air flows were constantly maintained in the desired range.
- Air change rate: An air change of about 24 to 25 times per hour can be calculated by dividing the supply air flow through the volume of each inhalation system.
- Method of particle size determination: The particle size analysis was carried out with a cascade impactor.Equipment for particle size analysis: Stack sampler Marple 298 (New Star Environmental, Inc., Roswell, Georgia 30075, USA) ; Vacuum compressed air pump (Millipore Corporation, Billerica, MA 01821, USA) ; Limiting orifice 3 L/min (Millipore Corporation, Billerica, MA 01821, USA) ; Sampling probe internal diameter 7 mm ; Balance Sartorius MSA 6.6S-000-DF (Sartorius AG, Göttingen, Germany). The calculation of the particle size distribution was carried out in the Laboratory for Inhalation Toxicology of the Experimental Toxicology and Ecology of BASF SE on the basis of mathematical methods for evaluating particle measurements (OECD guidance document No. 39). Particle Size distribution of the test atmosphere were determined also with the Aerodynamic Particle Spectrometer APS 3321 (TSI, USA). MMAD and GSD is obtained directly by the piece of equipment used APS 3321. Frequency: On two days during the exposure period, with 3 repeats on each day. To determine the particle size distribution in the submicrometer range, each test atmosphere was measured with the Scanning Mobility Particle Sizer (SMPS; Grimm Aerosol Technik GmbH & Co KG, Ainring, Germany). The SMPS system comprises an Electrostatic Classifier (Model Vienna U-DMA) which separates the particles into known size fractions, and a Condensation Particle Counter (CPC) which measures particle count concentrations. The DMA was equipped with Am-241 neutralizer. The instrument measures particles in the size range from 0.011 to 1.083 µm. Using a conductive sample hose, the SMPS sampled at 0.3 liters per minute (LPM) with a sheath flow of 3 LPM. At this setting the single-stage, inertial impactor incorporated into the inlet of the SMPS to remove larger particles had a 50% cut size of 1.082 µm according to the software calculation. The sampling duration was about 7 minutes. As a rule 10 repeats were measured for each exposure concentration.
- Treatment of exhaust air: Exhaust air was filtered and conducted into the exhaust air of the building.

TEST ATMOSPHERE
- Brief description of analytical method used: The concentrations of the inhalation atmospheres were determined by gravimetrical measurements of filter samples in all test groups. Control group was not sampled. This analytical method was judged to be valid because the test substances did not possess an appreciable vapor pressure.
- Samples taken from breathing zone: yes
Details on mating procedure:
Mating of the F0 generation parental animals
After 44 days premating period, the male and female parental animals were mated overnight in a 1:1 ratio until there was evidence of copulation or the maximum period of 14 days has elapsed. Throughout the mating period, each female was mated with a predetermined male.

Normally, the female was placed into the cage of her male partner about 16:00 h and separated from the male between 06:30 and 09:00 h, the following morning. Deviations from the specified times are possible on Saturdays, Sundays and public holidays and were documented in the raw data.

A vaginal smear was prepared for each pair after each mating and examined for sperm. If sperm was detected, mating of the pair was discontinued. The day on which sperm were detected, was referred to as gestation day (GD) 0 and the following day as GD 1.

Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
The nominal concentration could be/was calculated from the study means of the test-substance flow and the supply air flows used during exposure to generate the respective concentrations. The concentrations of the inhalation atmospheres were determined by gravimetrical measurements of filter samples in all test groups. Control group was not sampled. This analytical method was judged to be valid because the test substances did not possess an appreciable vapor pressure.
Duration of treatment / exposure:
The animals were exposed for 44 days before mating. The mating period were
maximal 2 weeks. After the mating period, the exposure of all male F0 animals were continued
until they are exposed for total minimal 90 days. After the mating period, the female F0 animals
were exposed further until gestation day 19. To allow them deliver and rearing their pups (F1
generation), they were not exposed from gestation day 20 to postnatal day (PND) 3. From
PND 4 through to PND 21, the dams were exposed with their pups in exposure cages
containing beddings. The first parental female animals were in gestation stage already after
the first few mating days, therefore, the post-weaning period were adjusted in such a way, that
a total of minimum 90 exposure will be achieved for females.
Frequency of treatment:
7 consecutive days per week, 6 hours per day
from PND4 through PND21
Details on study schedule:
not specified
Dose / conc.:
0 mg/m³ air
Remarks:
Test Group 0 (>Parental animals F0) - air control
Dose / conc.:
21.92 mg/m³ air (analytical)
Remarks:
SD: 1.30 mg/m3, target concentration: 22 mg/m³: Test Group 8 (>Parental animals F0); Test Group 18 (male animals for particle detection); Test Group 28 (Recovery animals)
No. of animals per sex per dose:
16/sex/dose group (parental animals)
5/sex at the high dose (recovery animals)
3 males at the high dose (for particle detection)

Subset Number of pups selected Day of examination Examination
I 10/sex/group PND 22 Measurement of thyroid hormones
II 10/sex/group PND 22 Perfusion fixation, brain weights
and neuropathology
III 5/sex/group PND 22 Histopathological examination and
organ burden
IV 10/sex/group PND 13 and 21 Open field observation
PND 13, 17 and 21 Motor activity
V 3 males/group PND 22 Perfusion fixation and electron
(highest dose) microscopic for particle detection
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of the 14-day range finding study (BASF study no 36I0050/20I005 - Ma- Hock 2021), upon approval of the sponsor, nominal aerosol concentrations of 0.5, 2.0 and 10.0 mg/m³ were used for the test substance in the low, mid and high dose groups, respectively.
- Rationale for animal assignment:
Prior to the pre-exposure period, the animals were distributed according to weight among the
individual test groups, separated by sex. The weight variation of the animals used did not
exceed ± 20 percent of the mean weight of each sex. The list of randomization instructions
was compiled with a computer.
For each neurofunctional test and motor activity measurement, separate randomization lists
were created. The list of randomization instructions were compiled with a computer (Laboratory
data processing, Experimental Toxicology and Ecology, BASF SE).
- Fasting period before blood sampling for clinical biochemistry: not specified
- Post-exposure recovery period in satellite groups: 45 days recovery period
Positive control:
none
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The clinical observation was performed on each animal at least three times (before, during and after exposure) on exposure days and once a day during pre-exposure and post exposure observation days. On non-exposure days a cage-side examination will be conducted at least once daily for any signs of morbidity, pertinent behavioral changes and/or signs of overall toxicity.

MORTALITY: The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) on working days and once a day (in the morning) on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS: YES
- Time schedule: All parental animals and recovery group animals were subjected to detailed clinical observations (DCO) outside their cages once before the beginning of the administration period and once during the first two weeks of the exposure, once monthly thereafter. DCO was performed in the morning before exposure. For observation, the animals were removed from their cages and placed in a standard arena (50 x 37.5 cm with a lateral border of 25 cm) for at least 20 seconds/animal.

BODY WEIGHT: Yes
- Time schedule for examinations:
The body weight of the animals was determined at the start of the pre-exposure, at the start of
the exposure period and then, as a rule, once a week as well as prior to gross necropsy. The
body weight of the recovery animals were determined at the start of the recovery period, and
once a week during the recovery period.
The following exceptions were notable for the female parental animals:
• During the mating period, the females were weighed on the day of positive evidence of
sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day after parturition (PND1) and on PND 4, 7, 14, and 21.
• In the females without positive evidence of sperm, body weight was determined once a week during mating and gestation periods and in the females without litter during lactation period.

As a rule, the animals were weighed at the same time of the day (in the morning).

Body weight change was calculated as the difference between body weight on the respective exposure day and body weight and the weight of previous weighing. Group means were derived from the individual differences.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was determined weekly and calculated as mean food consumption in grams
per animal and day.
Generally, food consumption was determined once a week for the male and female animals
and post mating period (males), with the following exceptions:
• Food consumption was not determined during the mating period (male and female
parental animals).
• Food consumption of the females with evidence of sperm was determined for GD 0-7, 7-
14 and 14-20.
• Food consumption of the females which gave birth to a litter was determined for PND 1-
4, 4-7, 7-13.
During recovery period, food consumption was determined in the animals of test groups 20 –
28 of the recovery animals. It was determined at the start of the recovery period and once a
week during the recovery period.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OPHTHALMOSCOPIC EXAMINATION: YES
Before the beginning of exposure, the eyes of all parental animals were examined with an ophthalmoscope (HEINE OPTOTECHNIK, Herrsching, Germany) after administration of a mydriatic agent (Mydrum, Dr. Gerhard Mann chem.-pharm. Fabrik GmbH and Bausch & Lomb GmbH, Germany). At the end of the exposure period, only animals selected for examinations according to OECD 413, 10 males and 10 females per group, were subjected to ophthalmological examination. In the first step, only control (test group 0) and high concentration groups (test groups 3, 6, 7 and 8) were examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in the morning
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes
- How many animals: 10 M + 10 F per dose group
-Parameters checked: leukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets, differential blood count, reticulocytes, preparation of blood smears, prothrombin time (PT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in the morning
- Animals fasted: Yes
- How many animals: 10 M + 10 F per dose group
- Parameters checked: alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-glutamyl transpeptidase (GGT), sodium (Na), potassium (K), chloride (CL), Inorganic phosphate (INP), calcium (Ca), urea (UREA), creatinine (CREA), glucose (GLUC), total biluribin (TBIL), total protein (TP), albumin (ALB), globulin (GLB), triglycerides (TRIG), cholesterol (CHOL)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the 90days exposure period
- Dose groups that were examined: 10 M + 10 F per dose group
- Battery of functions tested: sensory activity / grip strength / motor activity / reflexes


IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: Not specified
- Dose groups that were examined: 10 M + 10 F per dose group and recovery groups (highest dose: 5M + 5F)
- Number of animals: 10 M + 10 F per dose group and recovery groups (highest dose: 5M + 5F)
- Parameters checked: Cytological parameters: total cell count, cell differential analysis of cytospin preparations; protein; Enzymes: lactate dehydrogenase, alkaline phosphatase, N-acetyl-beta-D-Glucosaminidase (NAG BAL), gamma−Glutamyltransferase

ORGAN (lung, liver, heart, brain, olfactory bulb) BURDEN: Yes
- Time schedule for analysis: at the end of the exposure period and after the recovery period (45days post exposure)
- Dose groups that were examined: all
- Number of animals: 3 /sex / group
- Parameters checked: Zn content

OTHER: - Electron microscope analysis of particulate matter in organs and tissues: 3 male animals of the highest dose group



Oestrous cyclicity (parental animals):
In all parental females in the premating phase, estrous cycle length and normality were
evaluated by preparing vaginal smears during a minimum of 2 weeks prior to mating and
throughout cohabitation until there is evidence of sperm in the vaginal smear.
Additionally, on the day of scheduled sacrifice, the estrous status was also determined in all
parental female rats.
Sperm parameters (parental animals):
not specified
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum:
On PND 4, the individual litters were standardized in such a way that, whenever possible, each litter contains 5 male and 5 female pups (always the first 5 surviving pups/sex in each litter were taken for further rearing). If individual litters did not have 5 pups/sex, the litters were processed in such a way that the most evenly distributed 10 pups per litter were present for further rearing (e.g., 6 male and 4 female pups). Standardization of litters was not performed in litters with 10 pups or less.

PARAMETERS EXAMINED
The following parameters were examined in offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups, open field observations, motor activity

GROSS EXAMINATION OF DEAD PUPS: yes

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: yes
Necropsy:
On postnatal day 22, 10 animals per sex and group were weighed, subjected to deep
anesthesia (i.p. pentobarbital) and sacrificed by perfusion fixation.
SOERENSEN phosphate buffer was used as the rinsing solution, and neutrally buffered, 4% formaldehyde solution was used as a fixative.
The perfusion fixed animals were necropsied with regard to the question of neuropathology, and the visible organs were assessed by gross pathology as accurately as is possible after a perfusion fixation. The cranial vault was opened and the skin was removed from the head. In this state, the perfused animals were stored in neutrally buffered, 4% formaldehyde solution for at least 48 hours.
Organ weights:
The following weights were determined (the brain was weighed after its removal but before
further preparation):
1. Final body weight
2. Brain (including olfactory bulb)
The final body weights were recorded to calculate the relative organ weights.
Length and width of the brain:
The length and maximum width of the brain were measured in all animals. The length of the brain was measured on a line extending from the rostral end of the frontal lobe to the caudal medulla oblongata of the cerebellum, width: pituitary region.
Organ/Tissue fixation:
The following organs/tissue specimens were carefully removed and processed histotechnically in accordance with the data given in the respective sections of this report:
1. All gross lesions
2. Brain with olfactory bulb
3. Eyes with retina and optic nerve
4. Nose (nasal cavity level III) with olfactory epithelium
5. Pituitary gland
6. Trigeminal ganglia
The animals and the tissue or organ material remaining after trimming was stored in neutrally buffered, 4% formaldehyde solution.
Neurohistopathology:
Morphometry of the brains (PND 22) :
Morphometry was performed in all animals of test groups 0 (control), 3, 6, 7 and 8.
Thickness measurements of major brain layers (neocortex: frontal and parietal cortices, caudate nucleus/putamen, hippocampus, corpus callosum, cerebellum) were performed. Measurements were carried out bilaterally in the left and right halves of the brain except for the corpus callosum and the cerebellum.

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no
Postmortem examinations (parental animals):
SACRIFICE / GROSS NECROPSY / ORGAN WEIGHTS
At the time of sacrifice, adult animals were examined macroscopically for any abnormalities or pathological changes.
The following weights were determined in all animals sacrificed on schedule:
1.Anesthetized animals (final body weight)
2. Adrenal glands (fixed)
3. Brain
4. Epididymides
5. Heart
6. Kidneys
7. Liver
8. Lung
9. Ovaries
10. Prostate (ventral and dorsolateral part together, fixed)
11. Seminal vesicles with coagulating glands (fixed)
12. Spleen
13. Testes
14. Thymus (fixed)
15. Thyroid glands (with parathyroid glands) (fixed)
16. Uterus with cervix
All paired organs were weighed together (left and right).

HISTOPATHOLOGY
Organs and tissues of F0 animals histologically processed:
1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Epididymides
12. Esophagus
13. Eyes with optic nerve
14. Extraorbital lacrimal gland
15. Femur with knee joint
16. Harderian glands
17. Heart
18. Ileum
19. Jejunum
20. Kidneys
21. Larynx (3 levels)
22. Liver
23. Lungs
24. Lymph nodes (tracheobronchial and mediastinal)
25. Lymph nodes (mesenteric)
26. Mammary gland (female)
27. Nasal cavity (4 levels)
28. Olfactory bulb
29. Ovaries
30. Oviducts
31. Pancreas
32. Pharynx
33. Parathyroid glands
34. Peyer’s patches
35. Pituitary gland
36. Prostate
37. Rectum
38. Salivary glands
(mandibular and sublingual glands)
39. Sciatic nerve
40. Seminal vesicles
41. Skeletal muscle
42. Skin
43. Spinal cord
(cervical, thoracic and lumbar cord)
44. Spleen
45. Sternum with marrow
46. Stomach
(forestomach and glandular stomach)
47. Teeth
48. Testes
49. Thymus
50. Thyroid glands
51. Trachea
52. Urinary bladder
53. Uterus
54. Vagina



Postmortem examinations (offspring):
GROSS NECROPSY/ORGAN WEIGHTS:
Five pups per sex and group were sacrificed under pentobarbitone anesthesia by
exsanguination from the abdominal aorta and vena cava. They were necropsied and assessed
by gross pathology.
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (final body weight)
2. Brain
3. Epididymides
4. Heart
5. Kidneys
6. Liver
7. Lungs
8. Ovaries
9. Spleen
10. Testes
11. Thymus (fixed)
12. Uterus with cervix


HISTOPATHOLOGY:
Organs and tissues of PND 22 pups that were histologically processed and
examined by light microscopy.
1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Cecum
6. Cervix
7. Coagulating glands
8. Colon
9. Duodenum
10. Epididymides
11. Esophagus
12. Eyes with optic nerve
13. Extraorbital lacrimal gland
14. Femur with knee joint
15. Harderian glands
16. Heart
17. Ileum
18. Jejunum
19. Kidneys
20. Larynx (level II)
21. Liver
22. Lungs
23. Lung associated lymph nodes
24. Lymph nodes (mesenteric)
25. Mammary gland (female)
26. Nasal cavity (3 levels)
27. Olfactory bulb
28. Ovaries
29. Oviducts
30. Pancreas
31. Pharynx
32. Parathyroid glands
33. Pituitary gland
34. Prostate
35. Rectum
36. Salivary glands
(mandibular and sublingual glands)
37. Sciatic nerve
38. Seminal vesicles
39. Skeletal muscle
40. Skin
41. Spinal cord
(cervical, thoracic and lumbar cord)
42. Spleen
43. Sternum with marrow
44. Stomach
(forestomach and glandular stomach)
45. Teeth
46. Testes
47. Thymus
48. Thyroid glands
49. Trachea
50. Urinary bladder
51. Uterus
52. Vagina
Statistics:
Statistical evaluation for main groups 0 (air control) versus 7 (micro ZnO) as well as 0 versus 8 (Zn sulphate), and recovery groups 20 (air control) versus 23 (T0420), 20 versus 26 (T0421), 20 versus 27 and 20 versus 28
- Food consumption (parental animals), body weight and body weight change (parental animals
and pups (for the pup weights, the litter means were used)), gestation days, anogenital distance,
anogenital index
--> Student's t-test (two-sided)
- Male and female mating indices, male and female fertility indices, gestation index, females mated, females delivering, females with liveborn pups, females with stillborn pups, females with all stillborn pups
--> FISHER'S EXACT test (one-sided)
-Mating days until day 0 pc, %postimplantation loss, pups stillborn, %perinatal loss, nipple development
--> WILCOXON test (one-sided+)
-Implantation sites, pups delivered, pups liveborn, live pups day x, viability Index, lactation index
--> WILCOXON test (one-sided-)
- Rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity
--> KRUSKAL-WALLIS and WILCOXON test (two-sided)
-Number of cycles and Cycle Length
--> KRUSKAL-WALLIS test (two-sided) and WILCOXON test (two-sided)
-Blood parameters
--> For parameters with bidirectional changes: WILCOXON-test (two-sided) for the hypothesis of equal medians
-Broncho-alveolar lavage fluid (BALF)
--> Pairwise comparison of each dose group with the control group using the WILCOXON-test (onesided) for the hypothesis of equal medians
-Weight of the anesthetized animals and absolute and relative organ weights (adult animals and PND 22 pups)
--> WILCOXON test (two-sided)

Statistical evaluation of neuropathological parameters of PND 22 pups (subset II)
-Weight parameters (brain)
-->KRUSKAL-WALLIS test (two-sided)
-Brain width and length
--> WILCOXON test (two-sided) with Bonferroni-Holm-
-Brain morphometry: linear measurements of selected brain regions
--> WILCOXON test (two-sided)
Reproductive indices:
MALES:

Male mating index (%) = (number of males with confirmed mating*/number of males placed with females) x 100

* defined by a female with vaginal sperm or with implants in utero

Male fertility index (%) = (number of males proving their fertility* /number of males placed with females) x 100

* defined by a female with implants in utero

FEMALES:

Female mating index (%) = (number of females mated*/number of females placed with males) x 100

* defined as the number of females with vaginal sperm or with implants in utero


Female fertility index (%) = (number of females pregnant*/number of females mated**)x 100

* defined as the number of females with implants in utero
** defined as the number of females with vaginal sperm or with implants in utero

Gestation index (%) = (number of females with live pups on the day of birth/number of females pregnant*) x 100

* defined as the number of females with implants in utero

Live birth index (%) = (number of liveborn pups at birth/total number of pups born) x 100


Postimplantation loss (%) =[(number of implantations – number of pups delivered)/number of implantations] x 100


Offspring viability indices:
Viability index (%) = (number of live pups on day 4* after birth/ number of live pups on the day of birth) x 100

* before standardization of litters (i.e. before culling)


Lactation index (%) = (number of live pups on day 21 after birth/number of live pups on day 4* after birth) x 100

* after standardization of litters (i.e. after culling)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
During the pre-exposure period and the exposure period the animals showed no clinical signs and findings different from normal.
-Exposure period, control group animals (test groups 0, 10, 20): There were no clinical signs and findings different from normal.
-Exposure period, reference item 2 (test groups 8, 18 and 28):
Eleven of the thirteen male animals of test group 8 showed salivation during exposure period on/or study days 4 - 89. In seven male animals of this group respiration sound was noted in addition. Two male animals (Nos: 136 + 138) of this group sparse fur (study day 79 – 84) were noted in addition. Salivation was also noted in three of the three male animals in test group 18.
Salivation was also noted in four of the five male animals in test group 28 during exposure period on/or study days 4 - 89. Respiration sound was observed in three of the five male rats of this group.
--> During exposure period, salivation and respiration sounds were detected in several male and female animals.
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
No deaths were recorded throughout the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant changes of body weight were determined in male
animals:
- Test group 8: day 18: 329.9g (p< 0.05), whereas the control group was 345.9g
- Test group 8: day 25: 342.5g (p< 0.05), whereas the control group was 363.4g
- Test group 8: day 32: 354.2g (p< 0.01), whereas the control group was 379.4g
- Test group 8: day 39: 364.7g (p< 0.01), whereas the control group was 390.9g
- Test group 8: day 46: 370.4g (p< 0.05), whereas the control group was 394.9g
- Test group 8: day 53: 381.2g (p< 0.05), whereas the control group was 407.9g
- Test group 8: day 60: 387.4g (p< 0.01), whereas the control group was 416.7g
- Test group 8: day 67: 393.1g (p< 0.01), whereas the control group was 424.6g
- Test group 8: day 74: 397.1g (p< 0.01), whereas the control group was 431.2g
- Test group 8: day 81: 406.7g (p< 0.01), whereas the control group was 436.9g
- Test group 8: day 88: 411.8g (p< 0.01), whereas the control group was 442.2g
- Test group 8: day 92: 411.7g (p< 0.01), whereas the control group was 447.0g
- Test group 8: day 102: 383.6g (p< 0.01), whereas the control group was 436.9g
- Test group 8: day 109: 395.7g (p< 0.01), whereas the control group was 444.5g
- Test group 8: day 116: 399.7g (p< 0.01), whereas the control group was 449.3g
- Test group 8: day 123: 402.7g (p< 0.01), whereas the control group was 452.1g
- Test group 8: day 130: 404.4g (p< 0.01), whereas the control group was 459.9g
- Test group 8: day 137: 410.5g (p< 0.01), whereas the control group was 459.4g
- Test group 8: day 144: 410.6g (p< 0.01), whereas the control group was 463.8g
- Test group 8: day 146: 410.8g (p< 0.01), whereas the control group was 466.6g
- Test group 28: day 18: 327.3g (p< 0.05), whereas the control group was 344.0g
- Test group 28: day 25: 339.2g (p< 0.01), whereas the control group was 360.9g
- Test group 28: day 32: 348.7g (p< 0.01), whereas the control group was 375.0g
- Test group 28: day 39: 361.1g (p< 0.01), whereas the control group was 390.9g
- Test group 28: day 46: 371.5g (p< 0.01), whereas the control group was 399.8g
- Test group 28: day 53: 381.5g (p< 0.01), whereas the control group was 411.9g
- Test group 28: day 60: 386.6g (p< 0.01), whereas the control group was 414.4g
- Test group 28: day 67: 392.7g (p< 0.01), whereas the control group was 425.4g
- Test group 28: day 74: 401.5g (p< 0.01), whereas the control group was 429.7g
- Test group 28: day 81: 410.9g (p< 0.01), whereas the control group was 439.5g
- Test group 28: day 88: 417.2g (p< 0.01), whereas the control group was 446.1g
- Test group 28: day 92: 417.9g (p< 0.01), whereas the control group was 449.2g
- Test group 28: day 102: 423.0g (p< 0.05), whereas the control group was 450.4g
- Test group 28: day 109: 434.0g (p< 0.05), whereas the control group was 457.6g
The following statistically significant changes of body weight were determined in female
animals:
- Test group 8: day 93: 238.9g (p< 0.05), whereas the control group was 251.8g
- Test group 28: day 18: 198.4g (p< 0.01), whereas the control group was 213.1g
- Test group 28: day 25: 209.8g (p< 0.05), whereas the control group was 220.9g
- Test group 28: day 32: 212.5g (p< 0.01), whereas the control group was 228.2g
- Test group 28: day 60: 231.2g (p< 0.05), whereas the control group was 245.6g
- Test group 28: day 116: 251.6g (p< 0.05), whereas the control group was 264.2g

The following statistically significant body weight changes were determined in male animals:
- Test group 8: day 0-> 4: 4.7 (p< 0.01), whereas the control group was 10.4g
- Test group 8: day 11-> 18: 17.1 (p< 0.05), whereas the control group was 22.0g
- Test group 8: day 18-> 25: 12.6 (p< 0.01), whereas the control group was 17.5g
- Test group 8: day 25-> 32: 11.7 (p< 0.05), whereas the control group was 16.1g
- Test group 8: day 74-> 81: 9.6 (p< 0.01), whereas the control group was 5.7g
- Test group 8: day 88-> 92: -0.1 (p< 0.01), whereas the control group was 4.8g
- Test group 8: day 102-> 109: 12.1 (p< 0.05), whereas the control group was 7.6g

- Test group 18: day 74-> 81: 7.3 (p< 0.05), whereas the control group was 0.9g

The following statistically significant body weight changes were determined in female animals:
- Test group 28: day 0-> 4: 5.3 (p< 0.01), whereas the control group was 10.3g
- Test group 28: day 11-> 18: 7.3 (p< 0.01), whereas the control group was 17.7g

-> Retarded body weight development in male/female animals as treatment-related, adverse effects

Body weight of F0 females during gestation/lactation of F1 litters:
Significantly lower body weights were noted for animals exposed to reference item 2 (zinc sulphate) at a concentration of 22 mg/m³ at the end of gestation and throughout lactation. The maximum difference to the control was 5.9%. Body weight gain of these females was also lower than control through major parts of gestation (GD 7 – 20) as well as on PND 7 – 13 of lactation.
--> decreased body weights/body weight gain during gestation and lactation of parental female animals as treatment-related, adverse effects
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
The following statistically significant changes of mean food consumption were determined in male animals:
•Test group 8: day 0 - 4: +21.3 g (p≤ 0.01), whereas the control group was +24.4 g
• Test group 8: day 18 - 25: +22.5 g (p≤ 0.05), whereas the control group was +25.2 g
The following statistically significant changes of mean food consumption were determined in female animals:
Test group 8: day 4 - 11: +16.5 g (p≤ 0.01), whereas the control group was +17.6 g
The increased food consumption in female animals was most likely because animals spread out the food from the supply and was considered not adverse. The finding in test group 8 was considered not biologically relevant due to its transient nature.

Food consumption of F0 animals during gestation of F1 litters:
Food consumption of females exposed to 22 mg/m3 reference item 2 (test group 8) was slightly below concurrent control throughout gestation (about 5%), the difference became, however, statistically significant only during GD 14 – 20 (about 6% below control). This effect increased through lactation, average food consumption of the lactating dams in test group 8 was about 8% below control during PND 1 – 13 and was significantly below control during all time segments of this study period (maximum 9.6%).
-->Decreased food consumption during gestation and lactation of parental female animals as treatment-related adverse effects
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
The ophthalmologic examinations did not show any impairment of the refracting media.
Spontaneous findings such as remainders of the pupillary membrane or corneal stippling were
observed in several animals of all test groups and the control group without any concentration response relationship.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The following significant changes were regarded as incidental and not treatment related, because the values were within historical control ranges:
Increased hemoglobin values in males of test group 8 (22 mg/m3 Zinc sulfate)
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of administration in males of test group 8 (22 mg/m3 Zinc sulfate) total bilirubin values were significantly increased, but this was the only relevantly changed clinical chemistry parameter among these individuals. This was regarded if at all treatment related as non-adverse (ECETOC Technical Report No. 85, 2002).

The following significant changes were regarded as incidental and not treatment related because the values were within historical control ranges: increased inorganic phosphate levels in males of test groups 8 (22 mg/m3 Zinc sulfate)
After the 8-week recovery period, in males of test group 28 (10 mg/m3 Zinc oxide 22 mg/m3 Zinc sulfate) creatinine values were significantly lower compared to controls. However, total bilirubin values were within historical control ranges
(males, total bilirubin 1.34-2.07 μmol/L) whereas creatinine values were marginally below this range (males; creatinine 31.8-37.0 μmol/L). Therefore, total bilirubin increase was regarded as incidental and not treatment related whereas creatinine decrease was regarded if at all treatment related as non-adverse (ECETOC Technical Report No. 85, 2002).
Endocrine findings:
no effects observed
Description (incidence and severity):
After the administration period, in parental males and in male and female pups at PND22 of all
test groups, no treatment-related alterations of T4 and TSH levels were observed.
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional observational battery:
Quantitative parameters: no substance-related findings were observed.
Home cage observations: no substance-related findings were observed.
Open field observations: no substance-related findings were observed.
Sensorimotor tests/reflexes: no substance-related findings were observed.

Overall motor activity (summation of all intervals):
there were no statistically significant deviations from the control group 0.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings were observed in in the larynx, lungs, nasal cavity and the tracheobronchial lymph nodes. These are further described in the details on results section

Reference item 2 (Zinc sulfate monohydrate)
Test group 8 (22 mg/m³):
Erosion/ulcer of the laryngeal epithelium at the base of the epiglottis in 1 female
• Minimal to slight squamous metaplasia of the laryngeal epithelium at the base of the
epiglottis in all males and all females
• Minimal to slight inflammatory cell infiltrates of the laryngeal epithelium in 1 male and 9
females
• Minimal to severe numbers of foamy macrophages in the lungs in all male and all female
animals
• Minimal to moderate cellular debris in the lungs in all male and all female animals
• Minimal to slight infiltration of neutrophils of alveoli of the lungs in all male and all female
animals
• Minimal to slight hyperplasia of type II pneumocytes in 6 males and 8 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes
(exemplarily) in 5 males and 5 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes
(exemplarily) in 8 males and 8 females
• Minimal to moderate degeneration/regeneration of the olfactory epithelium (nasal
cavity, level IV, exemplarily) in all males and all females

Test group 28 (Recovery group (Zinc sulfate monohydrate):
Minimal squamous metaplasia of the laryngeal epithelium at the base of the epiglottis
in 1 male and 1 female animal
• Minimal to slight inflammatory cell infiltrates in the laryngeal epithelium in 4 males and
3 females
• Minimal to slight numbers of foamy macrophages in the lungs in 2 male and 4 female
animals
• Minimal hyperplasia of type II pneumocytes in 3 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes in 1
male and 1 female animal
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes in
4 males and 4 females
• Minimal degeneration/regeneration of the olfactory epithelium (nasal cavity, level IV,
exemplarily) in 1 male and 1 female
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
BAL
Main group (F0)
The following treatment-related, adverse effects were observed:
Reference item 2 (Zinc sulfate monohydrate)
Test group 8 (22 mg/m³)

• Increased total cell counts as well as absolute and relative lymphocyte, neutrophil cell
and monocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute macrophage and eosinophil cell counts in BAL of males
• Increased total protein levels lactate dehydrogenase (LDH), alkaline phosphatase
(ALP) and γ-Glutamyl-transferase (GGT) activities in BAL of both sexes

Test group 28 (Recovery group Zinc sulfate monohydrate)
• No treatment-related adverse findings in lavage
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Estrous cycle data, generated during the last 3 weeks prior to mating to produce the F1 litter, revealed regular cycles in the females of all test groups. The mean estrous cycle duration was comparable: 3.9 / 3.9 / 4.0, 4.0, 3.9, 3.9, 4.0 and 4.0 days in test groups 1 – 6 as well as 7 and 8.
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
Description (incidence and severity):
MALE REPRODUCTION DATA:
For all F0 parental males of all test groups, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100% in all test groups.

Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter. However, two males (No. 105, 112) of test group 6 (test item T0421, 10 mg/m3) did not generate F1 pups.

Thus, the male fertility index was 87.5% in test group 6 and 100% in all other groups. This reflects the normal range of biological variation inherent in the strain of rats used for this study.

FEMALE REPRODUCTION DATA AND DELIVERY DATA:
The female mating index was 100% in all test groups. The mean duration until copulation was detected (GD 0) varied between 1.9 and 2.8 days without any relation to test item and concentration.

All female rats delivered pups or had implants in utero with the following exception:
• Test group 6 (test item T0421)
female No. 305 (mated with male No. 105) did not become pregnant
female No. 312 (mated with male No. 112) did not become pregnant

The female fertility index was 87.5% in test group 6 and 100% in all other groups. This reflects the normal range of biological variation inherent in the strain of rats used for this study.

The gestation index was 100% in in all test groups. The mean duration of gestation was comparable in all test groups (i.e. between 21.9 and 22.4 days).

Litter and delivery parameters:
Implantation was not affected by the treatment since the mean number of implantation sites was comparable between the test substance-treated group and the control (13.2 and 12.1 implants/dam in test groups 0 and 8). All values are well within the historical control range (HCD: 11.1 - 13.9).

Post-implantation loss was 2.8 and 6.5 mean% in test groups 0 and 8, respectively. Both values are well within the historical control range (HCD: 2.4 - 17.7).

Also, the mean number of F1 pups delivered per dam remained unaffected in the exposure group (12.9 and 11.3 pups/dam in test groups 0 and 8, respectively). While the control was above, numbers of F1 pups delivered per dam in the exposure group are well within the historical control range (HCD: 10.3 - 12.7).

The rate of liveborn pups was not affected by the test substance, as indicated by live birth indices of 100% and 99.4% in control and exposure group, respectively. Moreover, the rate of stillborn pups was not significantly different between the groups and within the historical control range (0 – 5.5%).
CLINICAL SIGNS AND MORTALITY:
Mortality:
No deaths were recorded throughout the study.
Clinical observations:

During the pre-exposure period and the exposure period the animals showed no clinical signs and findings different from normal.
-Exposure period, control group animals (test groups 0, 10, 20): There were no clinical signs and findings different from normal.
-Exposure period, reference item 2 (test groups 8, 18 and 28):
Eleven of the thirteen male animals of test group 8 showed salivation during exposure period on/or study days 4 - 89. In seven male animals of this group respiration sound was noted in addition. Two male animals (Nos: 136 + 138) of this group sparse fur (study day 79 – 84) were noted in addition. Salivation was also noted in three of the three male animals in test group 18.
Salivation was also noted in four of the five male animals in test group 28 during exposure period on/or study days 4 - 89. Respiration sound was observed in three of the five male rats of this group.
--> During exposure period, salivation and respiration sounds were detected in several male and female animals.


BODY WEIGHT AND WEIGHT GAIN
The following statistically significant changes of body weight were determined in male
animals:
- Test group 8: day 18: 329.9g (p< 0.05), whereas the control group was 345.9g
- Test group 8: day 25: 342.5g (p< 0.05), whereas the control group was 363.4g
- Test group 8: day 32: 354.2g (p< 0.01), whereas the control group was 379.4g
- Test group 8: day 39: 364.7g (p< 0.01), whereas the control group was 390.9g
- Test group 8: day 46: 370.4g (p< 0.05), whereas the control group was 394.9g
- Test group 8: day 53: 381.2g (p< 0.05), whereas the control group was 407.9g
- Test group 8: day 60: 387.4g (p< 0.01), whereas the control group was 416.7g
- Test group 8: day 67: 393.1g (p< 0.01), whereas the control group was 424.6g
- Test group 8: day 74: 397.1g (p< 0.01), whereas the control group was 431.2g
- Test group 8: day 81: 406.7g (p< 0.01), whereas the control group was 436.9g
- Test group 8: day 88: 411.8g (p< 0.01), whereas the control group was 442.2g
- Test group 8: day 92: 411.7g (p< 0.01), whereas the control group was 447.0g
- Test group 8: day 102: 383.6g (p< 0.01), whereas the control group was 436.9g
- Test group 8: day 109: 395.7g (p< 0.01), whereas the control group was 444.5g
- Test group 8: day 116: 399.7g (p< 0.01), whereas the control group was 449.3g
- Test group 8: day 123: 402.7g (p< 0.01), whereas the control group was 452.1g
- Test group 8: day 130: 404.4g (p< 0.01), whereas the control group was 459.9g
- Test group 8: day 137: 410.5g (p< 0.01), whereas the control group was 459.4g
- Test group 8: day 144: 410.6g (p< 0.01), whereas the control group was 463.8g
- Test group 8: day 146: 410.8g (p< 0.01), whereas the control group was 466.6g
- Test group 28: day 18: 327.3g (p< 0.05), whereas the control group was 344.0g
- Test group 28: day 25: 339.2g (p< 0.01), whereas the control group was 360.9g
- Test group 28: day 32: 348.7g (p< 0.01), whereas the control group was 375.0g
- Test group 28: day 39: 361.1g (p< 0.01), whereas the control group was 390.9g
- Test group 28: day 46: 371.5g (p< 0.01), whereas the control group was 399.8g
- Test group 28: day 53: 381.5g (p< 0.01), whereas the control group was 411.9g
- Test group 28: day 60: 386.6g (p< 0.01), whereas the control group was 414.4g
- Test group 28: day 67: 392.7g (p< 0.01), whereas the control group was 425.4g
- Test group 28: day 74: 401.5g (p< 0.01), whereas the control group was 429.7g
- Test group 28: day 81: 410.9g (p< 0.01), whereas the control group was 439.5g
- Test group 28: day 88: 417.2g (p< 0.01), whereas the control group was 446.1g
- Test group 28: day 92: 417.9g (p< 0.01), whereas the control group was 449.2g
- Test group 28: day 102: 423.0g (p< 0.05), whereas the control group was 450.4g
- Test group 28: day 109: 434.0g (p< 0.05), whereas the control group was 457.6g
The following statistically significant changes of body weight were determined in female
animals:
- Test group 8: day 93: 238.9g (p< 0.05), whereas the control group was 251.8g
- Test group 28: day 18: 198.4g (p< 0.01), whereas the control group was 213.1g
- Test group 28: day 25: 209.8g (p< 0.05), whereas the control group was 220.9g
- Test group 28: day 32: 212.5g (p< 0.01), whereas the control group was 228.2g
- Test group 28: day 60: 231.2g (p< 0.05), whereas the control group was 245.6g
- Test group 28: day 116: 251.6g (p< 0.05), whereas the control group was 264.2g

The following statistically significant body weight changes were determined in male animals:
- Test group 8: day 0-> 4: 4.7 (p< 0.01), whereas the control group was 10.4g
- Test group 8: day 11-> 18: 17.1 (p< 0.05), whereas the control group was 22.0g
- Test group 8: day 18-> 25: 12.6 (p< 0.01), whereas the control group was 17.5g
- Test group 8: day 25-> 32: 11.7 (p< 0.05), whereas the control group was 16.1g
- Test group 8: day 74-> 81: 9.6 (p< 0.01), whereas the control group was 5.7g
- Test group 8: day 88-> 92: -0.1 (p< 0.01), whereas the control group was 4.8g
- Test group 8: day 102-> 109: 12.1 (p< 0.05), whereas the control group was 7.6g

- Test group 18: day 74-> 81: 7.3 (p< 0.05), whereas the control group was 0.9g

The following statistically significant body weight changes were determined in female animals:
- Test group 28: day 0-> 4: 5.3 (p< 0.01), whereas the control group was 10.3g
- Test group 28: day 11-> 18: 7.3 (p< 0.01), whereas the control group was 17.7g

-> Retarded body weight development in male/female animals as treatment-related, adverse effects

Body weight of F0 females during gestation/lactation of F1 litters:
Significantly lower body weights were noted for animals exposed to reference item 2 (zinc sulphate) at a concentration of 22 mg/m³ at the end of gestation and throughout lactation. The maximum difference to the control was 5.9%. Body weight gain of these females was also lower than control through major parts of gestation (GD 7 – 20) as well as on PND 7 – 13 of lactation.
--> decreased body weights/body weight gain during gestation and lactation of parental female animals as treatment-related, adverse effects


FOOD CONSUMPTION
The following statistically significant changes of mean food consumption were determined in male animals:
•Test group 8: day 0 - 4: +21.3 g (p≤ 0.01), whereas the control group was +24.4 g
• Test group 8: day 18 - 25: +22.5 g (p≤ 0.05), whereas the control group was +25.2 g
The following statistically significant changes of mean food consumption were determined in female animals:
Test group 8: day 4 - 11: +16.5 g (p≤ 0.01), whereas the control group was +17.6 g
The increased food consumption in female animals was most likely because animals spread out the food from the supply and was considered not adverse. The finding in test group 8 was considered not biologically relevant due to its transient nature.

Food consumption of F0 animals during gestation of F1 litters:
Food consumption of females exposed to 22 mg/m3 reference item 2 (test group 8) was slightly below concurrent control throughout gestation (about 5%), the difference became, however, statistically significant only during GD 14 – 20 (about 6% below control). This effect increased through lactation, average food consumption of the lactating dams in test group 8 was about 8% below control during PND 1 – 13 and was significantly below control during all time segments of this study period (maximum 9.6%).
-->Decreased food consumption during gestation and lactation of parental female animals as treatment-related adverse effects

HAEMATOLOGICAL FINDINGS:
The following significant changes were regarded as incidental and not treatment related, because the values were within historical control ranges:
Increased hemoglobin values in males of test group 8 (22 mg/m3 Zinc sulfate)

CLINICAL CHEMISTRY:
At the end of administration in males of test group 8 (22 mg/m3 Zinc sulfate) total bilirubin values were significantly increased, but this was the only relevantly changed clinical chemistry parameter among these individuals. This was regarded if at all treatment related as non-adverse (ECETOC Technical Report No. 85, 2002).

The following significant changes were regarded as incidental and not treatment related because the values were within historical control ranges: increased inorganic phosphate levels in males of test groups 8 (22 mg/m3 Zinc sulfate)
After the 8-week recovery period, in males of test group 28 (10 mg/m3 Zinc oxide 22 mg/m3 Zinc sulfate) creatinine values were significantly lower compared to controls. However, total bilirubin values were within historical control ranges
(males, total bilirubin 1.34-2.07 μmol/L) whereas creatinine values were marginally below this range (males; creatinine 31.8-37.0 μmol/L). Therefore, total bilirubin increase was regarded as incidental and not treatment related whereas creatinine decrease was regarded if at all treatment related as non-adverse (ECETOC Technical Report No. 85, 2002).

NEUROBEHAVIOUR:
Quantitative parameters: no substance-related findings were observed.
Home cage observations: no substance-related findings were observed.
Open field observations: no substance-related findings were observed.
Sensorimotor tests/reflexes: no substance-related findings were observed.
Overall motor activity (summation of all intervals):
there were no statistically significant deviations from the control group 0.

Single intervals:
Comparing the single intervals with the control group, the following statistically significant
deviations were seen:
Decrease of activity in the female animals of test group 7 (10 mg/m³, reference item 1)
at interval 5 on day 87 (p ≤ 0.05).
-->No other abnormalities were detected.
These changes were considered incidental because they were of transient nature and the
overall motor activity was not changed in the respective group.

ORGAN WEIGHTS
When compared with control group 0 (=100%),
Reference item 2- Test group 8 (22mg/m3) (Zinc sulfate) :
Increase of absolute/relative lung weights in males (125%/138%) and females
(114%/119%)
--> These effects were observed as treatment-related, adverse effects

GROSS PATHOLOGY

Reference item 2 (Zinc sulfate monohydrate)
Test group 8 (22 mg/m³)
•Macroscopically observed white foci in the lungs of 3 males and 7 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial,
highest number is given) in 5 males and 8 females--> These effects were observed as treatment-related, adverse effects
Test group 28 (Recovery group , Zinc sulfate monohydrate)
• Macroscopically observed white foci in the lungs of 2 males and 2 females
• Macroscopically enlarged draining lymph nodes (mediastinal) in 1 male and 1 female

HISTOPATHOLOGICAL FINDINGS: NON-NEOPLASTIC:

Larynx (level I):
In the larynx, the most severe findings were observed in level I, therefore only findings in level I of the larynx are given

Parental animals:
One female animal of test group 8 (reference item 2, 22 mg/m³) revealed an erosion/ulcer at the base of the epiglottis. All male and all female animals of the same test group revealed squamous metaplasia of the respiratory epithelium, mainly in the region of the base of the epiglottis. This finding is characterized by flattening of cells, increase of cellular layers, and keratinization on the surface. Furthermore, one male and nine females showed mixed (macrophages, neutrophils, lymphocytes) inflammatory cell infiltrates in this region. These findings were regarded to be treatment-related.
Recovery animals:
In the larynx mainly males and females of the reference test groups were affected. The same findings as described for the main group animals were still observed in the recovery animals.
These findings were regarded to be treatment-related.


Lungs:
Mainly, high dose group males and females (test group 3 and 6 [test item 1 and 2, 2 mg/m³]) were affected. Within alveoli, mainly in the bronchio-alveolar transition region, a multifocal accumulation of alveolar macrophages with vacuolar (foamy) cytoplasm was seen. The alveolar macrophages often revealed nuclei of increased size and occasionally multiple nuclei.
Intermingled with the foamy macrophages, cellular debris of presumable fragmented
macrophages and neutrophils were observed. In the region of these cellular accumulations, proliferation (hyperplasia) of type II pneumocytes was observed.
Males of test group 2 and 4 (test item 1 and 2, 2 mg/m³) revealed also an accumulation of foamy macrophages, only.

In males and females of the two reference items, similar findings were observed as described for test group 3 and 6 (test item 1 and 2, 2 mg/m³). Only the severity was slightly higher when compared with the other test items, especially in test group 7 animals.

Recovery animals:
The same findings as described for the main group animals were observed in the recovery animals. These findings were regarded to be treatment-related.

Lymph nodes (mediastinal):
Parental animals:
The mediastinal and tracheobronchial lymph nodes revealed comparable findings.
In general, the high dose group males and females (test group 3 and 6 [test item 1 and 2, 2 mg/m³]) were more severely affected. A lympho-reticular cell hyperplasia was observed, which can be explained by an activation of the draining lymph nodes of the lungs. Furthermore, aggregates of macrophages were seen within the lymph nodes. These findings were considered as treatment-related.
Single animals of test group 1, 2 (test item 1, 0.5 and 2 mg/m³), and test group 5 (test item 2, 2 mg/m³) revealed similar findings.

The males and females exposed to the reference items showed similar findings as the animals exposed to the test substances.

Recovery animals:
The same findings as described for the main group animals were observed in the recovery animals. These findings were regarded to be treatment-related.


Nasal cavity:

Parental animals:
The nasal cavity was investigated in four levels. The most severely affected levels were level III and IV
Females of test group 7 (reference item 1, 10 mg/m³) and males and females of test group 8 (reference item 2, 22 mg/m³) revealed the same findings in the nasal cavity.

Recovery animals:
Findings occurred either individually or were biologically equally distributed over
control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Trachea
In the trachea, two male animals of test group 8 (reference item 2, 22 mg/m³) revealed a flattening of the respiratory epithelium at the carina. This finding was considered to be treatment-related.
All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.



BRONCHOALVEOLAR LAVAGE FLUID (BALF):
Cytology:
Parental animals:
After the administration period in BAL of males and females in test group 8 (22 mg/m3 Zinc sulfate) total cell counts as well as absolute and relative lymphocyte, neutrophil cell and monocyte counts were significantly increased whereas relative macrophage counts were significantly decreased. Additionally, in BAL of males in this test group absolute macrophage and eosinophil cell (not significantly) counts were significantly increased. These alterations were regarded as treatment related and adverse.
Recovery animals:
After the 8-week recovery period, in BAL cytology of males and females of test group 28 (22 mg/m3 Zinc sulfate) no changes were observed.

Proteins/enzymes:

Parental animals:
After the administration period, in BAL of males and females of test group 8 (22 mg/m3 Zinc sulfate) total protein levels as well as lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) activity were moderately, significantly increased whereas γ -Glutamyl-transferase (GGT) activity was only marginally but also significantly increased. These alterations were regarded as treatment related and adverse. In BAL of males of test group 8 (22 mg/m3 Zinc sulfate) β -N-Acetyl glucosaminidase (NAG) activity was marginally, significantly increased in males of this test group, but the change was below 2fold. Therefore, this alteration was regarded as maybe treatment related but nonadverse.
Recovery animals:
After the 8-week recovery period, no changes of total protein and enzyme activities in BAL of males and females in test group 28 (22 mg/m3 Zinc sulfate) were observed.


OTHER FINDINGS
- Electron microscopy:
Dose descriptor:
LOAEC
Remarks:
local toxicity
Effect level:
21.92 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: changes in lung, lung-draining lymph nodes, larynx and nasal cavity at the target concentration of 22 mg/m³
Dose descriptor:
NOAEC
Remarks:
systemic toxicity
Effect level:
21.92 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology
clinical biochemistry
histopathology: non-neoplastic
Remarks on result:
other: No systemic toxicity was observed in hematology, clinical chemistry and histopathology at the target conc of 22 mg/m3
Critical effects observed:
yes
Lowest effective dose / conc.:
21.92 other: mg/m3 air (analytical)
System:
respiratory system: lower respiratory tract
Organ:
lungs
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Clinical signs:
no effects observed
Description (incidence and severity):
No test or reference item related adverse clinical signs were observed in any of the F1 generation pups of the different test groups. Individual findings in few pups, like dehydrated appearance or gasping, were noted in several groups including control. They were most likely related to the technical procedure of inhalation exposure rather than any test or reference item.
Dermal irritation (if dermal study):
not specified
Mortality / viability:
no mortality observed
Description (incidence and severity):
The viability index indicating pup survival during early lactation (PND 0 - 4) varied between 99.5% / 99.0% / 100% / 100% / 95.7% / 97.9% / 100% / 99.6% and 99.5% in test groups 0 - 8 without showing significant differences between the groups. All values were within the historical control range (94 – 100%).

The lactation index indicating pup survival on PND 4 - 21 was 98.8% in test group 5 and 100% in all remaining test groups. All values were within the historical control range (95.7 – 100%).
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weights of all male and female pups in all test and reference item-treated groups 1 – 8 were comparable to the concurrent control values throughout the entire study.

The statistically significantly higher body weights in male pups and in both sexes combined in test group 4 on PND 4 were considered to be spontaneous in nature.

Calculation of body weight change resulted in a number of statistical changes in various groups, sometimes higher, sometimes lower than the concurrent control:

Increased:
- test group 3 males and both sexes combined on PND 1
- test group 4 males, females and both sexes combined on PND 1
- test group 7 males, females and both sexes combined on PND 1

Decreased:
- test group 5 females and both sexes combined on PND 4
- test group 7 males, females and both sexes combined on PND 13
- test group 8 males, females and both sexes combined on PND 4
- test group 8 males, females and both sexes combined on PND 13

None of these apparent changes is considered to be associated with the respective test or reference items.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not specified
Sexual maturation:
not specified
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
The anogenital distance and anogenital index of all treated male and female pups was comparable to the concurrent control values.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
The apparent number and percentage of male pups having areolae was not influenced by the test item when examined on PND 13. Likewise, no test item-related effect was detected in any of the test groups during the re-examination on PND 20.
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings were observed in in the lungs and nasal cavity.

The following treatment-related, adverse effects were observed:
PUPS (F1)
Reference item 2 (Zinc sulfate monohydrate)
Test group 8 (22 mg/m³)
• Minimal cellular debris in the lungs in 2 male and 1 female animals
• Minimal infiltration of neutrophils of lung alveoli in 2 male and 1 female animals
• Minimal degeneration/regeneration of the olfactory epithelium (nasal cavity, level III) in
1 male and 1 female
Other effects:
no effects observed
Description (incidence and severity):
OPEN FIELD OBSERVATIONS (OFO):
None of the animals in all test groups showed abnormalities attributable to the exposure to the test substance.

MOTOR ACTIVITY MEASUREMENT (MA):
Motor activity of male and female F1 offspring was not influenced by the test item at all concentration levels and at any of the testing dates PND 13, 17 and 21. Overall activity levels and habituation to the test environment corresponded to the age of these animals at the specific testing date, if usual biological variation inherent in rats used for this type of experiment was considered.

Across the test groups, there were a number of statistically significant changes in either the number of beam interrupts or in the number of rearings, in single intervals on various testing dates, sometimes higher, sometimes lower than the concurrent control:

Test item ZnSO4
Decreased:
- test group 8 males, beam interrupts, interval 3, 9 and 1-12, PND 13
- test group 8 males, beam interrupts, interval 3 and 5, PND 17
- test group 8 males, rearings, interval 3, PND 13
- test group 8 males, rearings, interval 2, 3, 5, 6 and 1-12, PND 17
- test group 8 males, rearings, interval 3, 4, and 5, PND 21
- test group 8 females, beam interrupts, interval 5, PND 13
- test group 8 females, beam interrupts, interval 3, PND 17
- test group 8 females, beam interrupts, interval 4 and 1-12, PND 21
- test group 8 females, rearings, interval 3 and 1-12, PND 17
- test group 8 females, rearings, interval 4, PND 21

None of these apparent changes is considered to be associated with the respective test or reference items.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Regarding neuropathology, no treatment-related findings were seen in pups of PND 22: Neuropathology, brain weight determination, necropsy, gross measurements of the brain,
neuropathology examination by light microscopy and morphometry did not reveal any
neuropathological, treatment-related findings.
--> There was no developmental neurotoxicity in all examined pups.
Developmental immunotoxicity:
not examined
Dose descriptor:
LOAEC
Remarks:
local toxicity
Generation:
F1
Effect level:
21.92 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: minimal cellular debris, neutrophilic infiltration in the lungs and minimal degeneration/regeneration of the olfactory epithelium in nasal cavity in 1 male and 1 female animal at the target concentration of 22 mg/m³
Dose descriptor:
NOAEC
Remarks:
systemic toxicity
Generation:
F1
Effect level:
21.92 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology
clinical biochemistry
histopathology: non-neoplastic
Remarks on result:
other: No systemic toxicity was observed in hematology, clinical chemistry and histopathology the target concentration of
Critical effects observed:
yes
Lowest effective dose / conc.:
21.92 other: mg/m3 air (analytical)
System:
respiratory system: lower respiratory tract
Organ:
lungs
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Reproductive effects observed:
no
Conclusions:
Overall assessment for adult animals:

With regards to systemic toxicity, none of the test or reference substances caused any systemic toxicity that were not triggered by the local toxicity.

Comparing the local effects of the two nano Zinc oxide materials, the overall finding in the lungs, mediastinal lymph nodes, in the nasal cavity were comparable at the tested concentrations, as well as the changes of lavage parameters. The small differences are considered biological variations. There were no considerable differences between the effects caused by zinc oxide nanoparticles and those caused by micron-size zinc oxide particle.

For reference substance 2 (zinc sulfate monohydrate), lower incidence and severity was found in the lungs than in the other zinc oxide treated groups, but higher incidence and severity in nasal cavity and larynx. This difference is considered being related to the different deposition pattern, caused by the different aerodynamic diameter. The aerodynamic diameter of zinc sulfate monohydrate was larger than the different types of zinc oxide. The mean MMAD of zinc sulfate monohydrate was with 2.3 µm considerably higher than those measured at the high concentrations of the test items 1 (1.19 µm) and 2 (0.97 µm). The deposited dose at the upper respiratory tract was higher, while those deposited in the lung was lower.

After the recovery period, all parameters in lavage fluid returned to the control level in all animals, irrespective of the exposed test and reference substance. With regards of histological findings in the respiratory tract, all changes reduced greatly in incidence and severity. Only single animals showed still some mild effects.

Overall assessment for PND 22 animals:
With regards to effects observed in PND 22 pups that were exposed whole-body to zinc oxide nanomaterials from PND 4 to PND 21, the findings were limited to lungs and nasal cavities. The effects observed in the parental animals showed much higher incidence and severity than in the pups exposed at the same concentration. This could be explained by the duration of the exposure, because pups were only exposed for 17 days, while the adult animals were exposed for 90-days.

Comparing the toxicity of the two nano Zinc oxide materials in pups of PND 22, the overall finding in the lungs were comparable at the high concentration of 10 mg/m³ for test items 1 and 2. However, lesions were also observed in nasal cavity in pups exposed to test item 2. While lesions in nasal cavity were still observed at 0.5 mg/m³ test item 2, there were no effect observed in animals exposed to 2 mg/m³ and 0.5 mg/m³ test item 1.

Comparing the toxicity caused by the two nano zinc oxide materials with those caused by the microscale zinc oxide material, or by the soluble zinc sulfate monohydrate, the changes in lungs were comparable in incidence and severity in exposed pups on PND 22. However, no nasal cavity lesions were observed in animals exposed to test item 1, while similar lesions were seen in those exposed to test item 2 and the reference items.

None of the substances cause any systemic toxicity, nor were there any developmental neurotoxicity in exposed pups. .
Executive summary:

This study was a 90-Day Study (OECD test guideline (TG) 413) combined with the Reproduction/ Developmental Toxicity Screening Test (OECD TG 421) in rat with neurotoxicity and developmental (neuro)toxicity evaluation, including detailed clinical observations addressing potential neurobehavioral effects, histological and morphological evaluations of the brains of the pups on post-natal day 22.


To compare the toxicity of uncoated and coated nano Zinc oxide, these two materials (Zinc oxide T0420 was uncoated, Zinc oxide T0421 was coated) were tested at each three concentrations. In addition, micronsize Zinc oxide T0242 and a soluble salt zinc sulfate monohydrate was tested as reference items. 


Groups of male and female Wistar rats were whole-body exposed to the aerosols of ZnO nano materials, Zinc oxide T0420 and Zinc oxide T0421, for 6 hours daily, at least 90 days. Zinc oxide T0420 was uncoated, Zinc oxide T0421 was coated.


The target concentrations for Zinc oxide T0420 and T0421 were 0.5, 2 and 10 mg/m³ referring to the non-volatile fraction. For the reference item 1 microscale Zinc oxide T0242, 10 mg/m³ was tested. For the reference item 2, Zinc sulfate monohydrate a target concentration of 22 mg/m³ was tested because this is equimolar to zinc ion of the ZnO materials. Concurrent control groups were exposed to humidified air (control group 0, 10 and 20).


All animals were exposed to the respective concentrations of test substance for 6 hours a day according to the time schedule (exception: no exposure on the day of FOB/MA and parental females from GD20 – PND 3)). Control animals were exposed to conditioned air. Male and female rats aged about 6 or 7 weeks when supplied, were used as F0 generation parental animals. The animals were exposed for 43 days before mating. The mating period were maximal 2 weeks. After the mating period, the exposure of all male F0 animals were continued until they are exposed for total minimal 90 days. After the mating period, the female F0 animals were exposed further until gestation day 19. To allow them to deliver and rearing their pups (F1 generation), they were not exposed from gestation day 20 to postnatal day (PND) 3. From PND 4 through to PND 21, the dams were exposed with their pups in exposure cages containing beddings. During the exposure food was withdrawn. Water was provided in form of hydrogel pads from PND 14 to 16 onward. The first parental female animals were in gestation stage already after the first few mating days, therefore, the post-weaning period were adjusted in such a way, that a total of minimum 90 exposure will be achieved for females.


Daily clinical observations, body weights, food consumption, ophthalmology, detailed clinical observation and FOB/MA were recorded. Moreover, male and female fertility were determined. Additional assessments including hematology and clinical chemistry in blood, bronchoalveolar lavage, and histopathology according to the referenced guidelines were carried out at the termination of exposure period. In addition, recovery groups of male and female animals were included; after an exposure period of about 90 days, these animals were kept for an additional period of ca. 60 days without exposure (control group 20, and test groups 23, 26, 27 and 28, respectively).


To assess the reproductive/developmental toxicity of the test substances (incl. reference substances), estrus cycles, male and female reproduction, delivery data were collected. In the pups, open field observations were performed on PND 13 and 21, motor activity measurements were performed on PND 13, 17 and 21. On PND 22, thyroid hormones, brain weights, neuropathology, general histopathology were examined in separate subsets of animals.


The following treatment-related, adverse effects were observed:
Main group (F0)
Test item 1 (Zinc oxide T0420)
Test group 3 (10 mg/m³)


• Decreased food consumption during gestation and lactation of parental females
• Increased total cell counts as well as absolute and relative lymphocyte, neutrophil cell and monocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute eosinophil cell counts in males in BAL
• Increased total protein levels lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and γ-Glutamyl-transferase (GGT) activities in BAL of both sexes
• Increased β-N-Acetyl glucosaminidase (NAG) activity in BAL of males
• Increase of absolute/relative lung weights in males (140%/150%) and females (128%/130%)
• Macroscopically observed white foci in the lungs of 5 males and 8 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial, highest number is given) in 6 males and 9 females
• Slight to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to severe cellular debris in the lungs in all male and all female animals
• Minimal to moderate infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 9 males and all females
• Minimal to slight degeneration/regeneration of the olfactory epithelium 



Test group 23 (Recovery group R1, 10 mg/m³)
• Macroscopically observed white foci in the lungs of 1 male and 2 females
• Macroscopically enlarged draining lymph nodes (mediastinal) in 1 female
• Minimal to moderate numbers of foamy macrophages in the lungs in 3 male and 4 female animals
• Minimal cellular debris in the lungs in 1 male and 2 female animals
• Minimal infiltration of neutrophils of alveoli of the lungs in 1 male and 2 female animals
• Minimal hyperplasia of type II pneumocytes in 3 females


Test group 2 (2 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium in one male animal


Test group 1 (0.5 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium in 1 female



Conclusion for adult animals exposed to test item 1 (Zinc oxide T0420):
Inhalation exposure to Zinc oxide T0420 caused changes in lung, lung-draining lymph nodes and nasal cavity at the high concentration of 10 mg/m³. These findings were almost, though not completely resolved during the post-exposure observation period. At 2 mg/m³, minimal degeneration/regeneration in the nasal cavity was noted in one male animal, and at 0.5 mg/m³ in one female animal. Due to findings in nasal cavity, the NOAEC for local toxicity at the respiratory tract was 0.5 mg/m³ for male rats. a No Observed Adverse Effect Concentration (NOAEC) for local toxicity for females could not be unequivocally determined.


No systemic toxicity was observed in hematology, clinical chemistry and histopathology the NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity was 10 mg/m³ for Zinc oxide T0420.



Test item 2 (Zinc oxide T0421)
Test group 6 (10 mg/m³)
• Decreased food consumption during gestation and lactation of parental females
• Decreased body weights/body weight gain during gestation and lactation of parental females
• Increased total white blood cell (WBC) as well as absolute neutrophil and lymphocyte counts in blood of males
• Slightly increased total cell counts as well as absolute and relative lymphocyte, neutrophil cell and monocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute eosinophil cell counts in males in BAL
• Increased total protein levels lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) activities in BAL of both sexes
• Increased  γ-Glutamyl-transferase (GGT) activity in BAL of males
• Increase of absolute/relative lung weights in males (136%/143%) and females (131%/137%)
• Macroscopically observed white foci in the lungs of 6 males and 8 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial, highest number is given) in 10 males and 5 females
• Minimal to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to severe cellular debris in the lungs in all male and all female animals
• Minimal to slight infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 8 males and 9 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes (exemplarily) in 8 males and 3 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes (exemplarily) in 4 males and 2 females
• Minimal to slight degeneration/regeneration of the olfactory epithelium 

Test group 26 (Recovery group R1, 10 mg/m³)
• No treatment-related adverse findings in lavage and histopathology

Test group 5 (2 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium 

Test group 4 (0.5 mg/m³)
No treatment-related adverse findings


Conclusion for adult animals exposed to test item 2 (Zinc oxide T0421):
Inhalation exposure to Zinc oxide T0421 caused changes several lavage parameters, as well as histological changes in lung, lung-draining lymph nodes and nasal cavity at the highest tested concentration of 10 mg/m³. All these effects were completely resolved after the postexposure observation period. In blood, increased neutrophils and lymphocyte was notice at the concentration of 10 mg/m³, which is considered secondary to the inflammation in the lung.
At the mid concentration of 2 mg/m³, histological findings were still observed in the nasal cavity of three male and two female rats. Thus, the No Observed Adverse Effect Concentration (NOAEC) for local toxicity was 0.5 mg/m³ under the current study conditions. 
Besides the increased neutrophils and lymphocytes in blood, no other changes were observed in hematology, clinical chemistry. No histopathological changes were observed in any organs and tissues that are not part of the respiratory tract. The NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity, that were not attributed to the local effect, was 10 mg/m³ for Zinc oxide T0421.



Reference item 1 (Zinc oxide T0242)
Test group 7 (10 mg/m³)
• Retarded body weight development in male animals
• Increased total cell counts as well as absolute and relative neutrophil cell and monocyte counts in BAL of both sexes
• Increased absolute lymphocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute macrophage and eosinophil cell counts in BAL of males
• Increased total protein levels lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and γ-Glutamyl-transferase (GGT) activities in BAL of both sexes
• Increased β-N-Acetyl glucosaminidase (NAG) activity in BAL of males
• Increase of absolute/relative lung weights in males (130%/141%) and females (137%/141%)
• Macroscopically observed white foci in the lungs of 5 males and 8 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial, highest number is given) in 7 males and 10 females
• Minimal to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to severe cellular debris in the lungs in all male and all female animals
• Minimal to moderate infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 6 males and 8 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes (exemplarily) in 4 males and 6 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes (exemplarily) in 6 males and 8 females
• Minimal degeneration/regeneration of the olfactory epithelium 


Test group 27 (Recovery group R1, 10 mg/m³)
• Macroscopically observed white foci in the lungs of 3 males and 3 females
• Minimal to slight numbers of foamy macrophages in the lungs in 2 male and 4 female animals
• Minimal cellular debris in the lungs in 1 male animal
• Minimal infiltration of neutrophils of lung alveoli in 1 male animal
• Minimal hyperplasia of type II pneumocytes in 4 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes in 2 males and 3 females


Conclusion for adult animals exposed to reference item 1 (Zinc oxide T0242):
Inhalation exposure to Zinc oxide T0242 caused changes in lung, lung-draining lymph nodes and nasal cavity at the highest tested concentration of 10 mg/m³. These findings were greatly, though not completely, resolved during the post-exposure observation period.
No systemic toxicity was observed in hematology, clinical chemistry and histopathology the NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity was 10 mg/m³ for Zinc oxide T0242.


Reference item 2 (Zinc sulfate monohydrate)
Test group 8 (22 mg/m³)
• During exposure period, salivation and respiration sounds were detected in several male and female animals.
• Retarded body weight development in all male and female animals. 
• Decreased food consumption during gestation and lactation of parental female animals
• Recreased body weights/body weight gain during gestation and lactation of parental female animals
• Increased total cell counts as well as absolute and relative lymphocyte, neutrophil cell and monocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute macrophage and eosinophil cell counts in BAL of males
• Increased total protein levels lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and γ-Glutamyl-transferase (GGT) activities in BAL of both sexes
• Increase of absolute/relative lung weights in males (125%/138%) and females (114%/119%)
• Macroscopically observed white foci in the lungs of 3 males and 7 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial, highest number is given) in 5 males and 8 females
• Erosion/ulcer of the laryngeal epithelium at the base of the epiglottis in 1 female
• Minimal to slight squamous metaplasia of the laryngeal epithelium at the base of the epiglottis in all males and all females
• Minimal to slight inflammatory cell infiltrates of the laryngeal epithelium in 1 male and 9 females
• Minimal to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to moderate cellular debris in the lungs in all male and all female animals
• Minimal to slight infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 6 males and 8 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes (exemplarily) in 5 males and 5 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes (exemplarily) in 8 males and 8 females
• Minimal to moderate degeneration/regeneration of the olfactory epithelium in all males and all females

Test group 28 (Recovery group R1: 22 mg/m³)
• Macroscopically observed white foci in the lungs of 2 males and 2 females
• Macroscopically enlarged draining lymph nodes (mediastinal) in 1 male and 1 female
• Minimal squamous metaplasia of the laryngeal epithelium at the base of the epiglottis in 1 male and 1 female animal
• Minimal to slight inflammatory cell infiltrates in the laryngeal epithelium in 4 males and 3 females
• Minimal to slight numbers of foamy macrophages in the lungs in 2 male and 4 female animals
• Minimal hyperplasia of type II pneumocytes in 3 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes in 1 male and 1 female animal
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes in 4 males and 4 females
• Minimal degeneration/regeneration of the olfactory epithelium in 1 male and 1 female 


Conclusion for adult animals exposed to reference item 2 (Zinc sulfate monohydrate):
Inhalation exposure to Zinc sulfate monohydrate caused changes in lung, lung-draining lymph nodes, larynx and nasal cavity at the highest tested concentration of 22 mg/m³. These findings were partly resolved during the post-exposure observation period.
No systemic toxicity was observed in hematology, clinical chemistry and histopathology the NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity was 22 mg/m³ for Zinc sulfate monohydrate.


Overall assessment for adult animals:


With regards to systemic toxicity, none of the test or reference substances caused any systemic toxicity that were not triggered by the local toxicity.


Comparing the local effects of the two nano Zinc oxide materials, the overall finding in the lungs, mediastinal lymph nodes, in the nasal cavity were comparable at the tested concentrations, as well as the changes of lavage parameters. The small differences are considered biological variations. There were no considerable differences between the effects caused by zinc oxide nanoparticles and those caused by micron-size zinc oxide particle.


For reference substance 2 (zinc sulfate monohydrate), lower incidence and severity was found in the lungs than in the other zinc oxide treated groups, but higher incidence and severity in nasal cavity and larynx. This difference is considered being related to the different deposition pattern, caused by the different aerodynamic diameter. The aerodynamic diameter of zinc sulfate monohydrate was larger than the different types of zinc oxide. The mean MMAD of zinc sulfate monohydrate was with 2.3 µm considerably higher than those measured at the high concentrations of the test items 1 (1.19 µm) and 2 (0.97 µm). The deposited dose at the upper respiratory tract was higher, while those deposited in the lung was lower.


After the recovery period, all parameters in lavage fluid returned to the control level in all animals, irrespective of the exposed test and reference substance. With regards of histological findings in the respiratory tract, all changes reduced greatly in incidence and severity. Only single animals showed still some mild effects.


PUPS on PND 22 
Test item 1 (Zinc oxide T0420)
Test group 3 (10 mg/m³)
Minimal cellular debris in the lungs in 2 male and 4 female animals
• Minimal infiltration of neutrophils of alveoli of the lungs in 1 male and 2 female animals
Test group 2 (2 mg/m³) and test group 1 (0.5 mg/m³)
No treatment-related adverse findings observed
Conclusion for pups exposed to test item 1 (Zinc oxide T0420) sacrificed on PND 22:
Inhalation exposure to Zinc oxide T0420 caused minimal cellular debris and neutrophilic infiltration in a few male and female animals at 10 mg/m³. These findings were considered treatment-related and adverse. The NOAEC for local toxicity was 2 mg/m³. 


There were no test-item related adverse findings in clinical examination/OFO/MA and gross necropsy up to the highest tested concentration of 10 mg/m³. No systemic toxicity was observed in hematology, clinical chemistry, no changes of thyroid hormones. There were no histopathological changes in organs and tissues other than those of the respiratory tract.



Test item 2 (Zinc oxide T0421)
Test group 6 (10 mg/m³)
• Minimal cellular debris in the lungs in 1 male and 4 female animals
• Minimal infiltration of neutrophils of alveoli of the lungs in 1 male and 1 female animals
• Minimal to moderate degeneration/regeneration of the olfactory epithelium (nasal cavity, level III) in 4 male and 2 female animals
Test group 5 (2 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium (nasal cavity, level III) in 2 female animals
Test group 4 (0.5 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium (nasal cavity, level III) in 1 male animal 


Conclusion for pups exposed to test item 2 (Zinc oxide T0421) sacrificed on PND 22:
Inhalation exposure to Zinc oxide T0421 caused minimal cellular debris, neutrophilic infiltration in the lungs and minimal to moderate degeneration/regeneration of the olfactory epithelium in nasal cavity in a few male and female animals at 10 mg/m³. The changes in nasal cavity was still observed at the mid concentration of 2 mg/m³. This findings were considered treatmentrelated and adverse. The NOAEC for local toxicity was 0.5 mg/m³.


There were no test-item related adverse findings in clinical examination/OFO/MA and gross necropsy up to the highest tested concentration of 10 mg/m³. No systemic toxicity was observed in hematology, clinical chemistry, no changes in thyroid hormones. There were no histopathological changes in organs and tissues other than those of the respiratory tract.



Reference item 1 (Zinc oxide T0242)
Test group 7 (10 mg/m³)
• Minimal cellular debris in the lungs in 5 male and 5 female animals
• Minimal infiltration of neutrophils of lung alveoli in 2 male and 3 female animals
• Minimal degeneration/regeneration of the olfactory epithelium (nasal cavity, level III) in 1 male and 1 female


Conclusion for pups exposed to reference item 1 (Zinc oxide T0242) sacrificed on PND 22:
Inhalation exposure to Zinc oxide T0242 caused minimal cellular debris, neutrophilic infiltration in the lungs and minimal degeneration/regeneration of the olfactory epithelium in nasal cavity in a one male and one female animals at 10 mg/m³.


There were no test-item related adverse findings in clinical examination/OFO/MA and gross necropsy up to the highest tested concentration of 10 mg/m³. No systemic toxicity was observed in hematology, clinical chemistry, no changes in thyroid hormones. There were no histopathological changes in organs and tissues other than those of the respiratory tract.


Reference item 2 (Zinc sulfate monohydrate)
Test group 8 (22 mg/m³)
• Minimal cellular debris in the lungs in 2 male and 1 female animals
• Minimal infiltration of neutrophils of lung alveoli in 2 male and 1 female animals
• Minimal degeneration/regeneration of the olfactory epithelium (nasal cavity, level III) in 1 male and 1 female


Conclusion for pups exposed to reference item 2 (zinc sulfate monohydrate) sacrificed on PND 22:
Inhalation exposure to zinc sulfate monhydrate caused minimal cellular debris, neutrophilic infiltration in the lungs and minimal degeneration/regeneration of the olfactory epithelium in nasal cavity in a few male and female animals at 22 mg/m³.


There were no test-item related adverse findings in clinical examination/OFO/MA and gross necropsy up to the highest tested concentration of 10 mg/m³. No systemic toxicity was observed in hematology, clinical chemistry, no changes in thyroid hormones. There were no histopathological changes in organs and tissues other than those of the respiratory tract.


Overall assessment for PND 22 animals:
With regards to effects observed in PND 22 pups that were exposed whole-body to zinc oxide nanomaterials from PND 4 to PND 21, the findings were limited to lungs and nasal cavities. The effects were observed also in the parental animals with much higher incidence and severity than in the pups exposed at the same concentration. This could be explained by the duration of the exposure, because pups were only exposed for 17 days, while the adult animals were exposed for 90-days.


Comparing the toxicity of the two nano Zinc oxide materials in pups of PND 22, the overall finding in the lungs were comparable at the high concentration of 10 mg/m³ for test items 1 and 2. However, lesions were also observed in nasal cavity in pups exposed to test item 2. While lesions in nasal cavity were still observed at 0.5 mg/m³ test item 2, there were no effect observed in animals exposed to 2 mg/m³ and 0.5 mg/m³ test item 1.


Comparing the toxicity caused by the two nano zinc oxide materials with those caused by the microscale zinc oxide material, or by the soluble zinc sulfate monohydrate, the changes in lungs were comparable in incidence and severity in exposed pups on PND 22. However, no nasal cavity lesions were observed in animals exposed to test item 1, while similar lesions were seen in those exposed to test item 2 and the reference items.


None of the substances cause any systemic toxicity, nor were there any developmental neurotoxicity in exposed pups.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016-07-29
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 426 (Developmental Neurotoxicity Study)
Version / remarks:
2007-10-16
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
not applicable

Test material

Constituent 1
Chemical structure
Reference substance name:
Zinc oxide
EC Number:
215-222-5
EC Name:
Zinc oxide
Cas Number:
1314-13-2
Molecular formula:
OZn
IUPAC Name:
oxozinc
Test material form:
solid: nanoform
Details on test material:
SOURCE OF TEST MATERIAL

- Purity, including information on contaminants, isomers, etc.: 98.2% for T0420
- Test substance No.: 20/0050-1 for T0420
- Batch identification: T0420

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at room temperature. The stability under the storage condition over the exposure period is guaranteed by the sponsor, and the sponsor holds this responsibility. Expiry date of the test substance: May 2022 for T0420

Test substance preparation:
- Generation procedure: For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage; mixed with conditioned dilution air and passed into the inhalation system.

OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added: homogenous, white solid.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL

- Purity, including information on contaminants, isomers, etc.: 98.2% for T0420
- Test substance No.: 20/0050-1 for T0420
- Batch identification: T0420

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at room temperature. The stability under the storage condition over the exposure period is guaranteed by the sponsor, and the sponsor holds this responsibility. Expiry date of the test substance: May 2022 for T0420.

INFORMATION ON NANOMATERIALS
- Chemical Composition:
- Density:
- Particle size & distribution:
- Specific surface area:
- Isoelectric point:
- Dissolution (rate):

Test substance preparation:
- Generation procedure: For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage; mixed with conditioned dilution air and passed into the inhalation system.

OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added: homogenous, white solid.

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Wistar rats, Crl:WI(Han) Rats were selected since this rodent species is recommended in the respective test guidelines. Wistar rats were selected since there is extensive experience available in the laboratory with this strain of rats.
Details on species / strain selection:
not specified
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
- Females nulliparous and non-pregnant: yes
- Age at study initiation: about 7 weeks (female), about 8 weeks (male)
- Weight at study initiation: The weight variation of the animals used did not exceed +/- 20 percent of the mean weight of each sex.
- Fasting period before study: The animals did not have access to food or water during exposure.
- Housing:
From delivery until mating and male animals after mating: Typ 2000P: ca. 2065 cm2 (polysulfone cages) / up to 5 animals
During mating: type III polycarbonate cages, 1 male/1 female per cage
During rearing: up to PND 22: type III polycarbonate cages, 1 dam with her litter
After weaning the females from study day 90 after exposure onward until sacrifice: Typ 2000P: ca. 2065 cm2 (polysulfone cages) / up to 5 animals. Remaining females with litters will be
maintained in type III cages until weaning.
For Motor Activity Measurement: Typ III polycarbonate cages (floor area about 800 cm²) / 1 animal
During Exposure: Wire cages, type DK III / up to 2 animals Females from PND 4 until study day 94 (and females without litter from the same time period onwards): perforated polycarbonate cages type II. From study day 95 onward wire cages, type DK III
- Diet (ad libitum): mouse and rat maintenance diet, GLP, 12 mm pellets, Granovit AG, Kaiseraugst, Switzerland before and after exposure. Food was withdrawn during exposure.
- Water (ad libitum): tap water
- Acclimation period: 11 days

DETAILS OF FOOD AND WATER QUALITY: The food used in the study was assayed for chemical as well as for microbiological contaminants. In view of the aim and duration of the study, the contaminants occurring in commercial food should not influence the results. The drinking water is regularly assayed for chemical contaminants both by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring of BASF SE as well as for bacteria by a contract laboratory. The Drinking Water Regulation will serve as the guideline for maximum tolerable contaminants. In view of the aim and duration of the study, there are no special requirements exceeding the specification of drinking water.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 45 - 65%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: beginning of experiment To: end of experiment

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure (if applicable):
whole body
Remarks:
whole-body exposure for the reasons explained see IUCLID section 13.2 'Human health requirements Final Decision: protocol deviations and rationale'
Mass median aerodynamic diameter (MMAD):
>= 0.52 - <= 2.01 µm
Remarks on MMAD:
MMAD / GSD: MMAD = 0.52-2.01 μm (geometric standard deviation = 4.04--2.28)
Vehicle:
unchanged (no vehicle)
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Generation of the inhalation atmospheres via a solid particle generators (brush-generator; BASF SE, Ludwigshafen, Germany) & Aerosol mixing tube (stainless steel; BASF SE, Ludwigshafen, Germany). Whole body exposure systems were used. The animals were kept singly in wire cages located in a glass steel inhalation chamber, volume of 1.1 m³ (BASF SE).
- Method of holding animals in test chamber: Whole body exposure systems. The animals were kept singly in wire cages located in a glass steel inhalation chamber, volume of 1.1 m³ (BASF SE). The chambers were located in exhaust hoods in an air conditioned room.
- Source and rate of air: Conditioned air from the central air conditioning system, compressed and exhaust air. Compressed air was produced by an oil-free compressor (HT 6, Josef Mehrer GmbH & Co KG, Germany). For this purpose, air is filtered by an inlet air strainer and introduced into the compressor. After passing through an second ultra filter (SMF 5/3, 108 mm, Donalson), the compressed air (15 bar) is stored in a storage of 1500 or 5000 L. The compressed air is conducted to the laboratories via pipes, where the pressure is reduced to 5 - 6 bar. In the laboratory, the compressed air can be taken as required.
- Method of conditioning air: Conditioned air from the central air conditioning system provides cold air of about 15°C. This cold air passes through an activated charcoal filter, is adjusted to room temperature of 20 to 24°C and passes through a second particle filter (H13 (HEPA) Camfil Farr, Germany). The so generated conditioned air was used to generate inhalation atmospheres.
- System of generating particulates/aerosols: The particles/aerosol was generated via a solid particle generator (brush-generator; BASF SE, Ludwigshafen, Germany) and an aerosol mixing tube (stainless steel; BASF SE, Ludwigshafen, Germany), according to the following method: For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage; mixed with conditioned dilution air and passed into the inhalation system.
- Temperature, humidity, pressure in air chamber: Daily mean relative humidities in the inhalation systems ranged between 41.6 and 60.8 %. Daily mean temperatures in the inhalation systems ranged between 21.4 and 23.7°C. They are within the range suggested by the respective testing guidelines.
- Air flow rate: The air flows were constantly maintained in the desired range.
- Air change rate: An air change of about 24 to 25 times per hour can be calculated by dividing the supply air flow through the volume of each inhalation system.
- Method of particle size determination: The particle size analysis was carried out with a cascade impactor.Equipment for particle size analysis: Stack sampler Marple 298 (New Star Environmental, Inc., Roswell, Georgia 30075, USA) ; Vacuum compressed air pump (Millipore Corporation, Billerica, MA 01821, USA) ; Limiting orifice 3 L/min (Millipore Corporation, Billerica, MA 01821, USA) ; Sampling probe internal diameter 7 mm ; Balance Sartorius MSA 6.6S-000-DF (Sartorius AG, Göttingen, Germany). The calculation of the particle size distribution was carried out in the Laboratory for Inhalation Toxicology of the Experimental Toxicology and Ecology of BASF SE on the basis of mathematical methods for evaluating particle measurements (OECD guidance document No. 39). Particle Size distribution of the test atmosphere were determined also with the Aerodynamic Particle Spectrometer APS 3321 (TSI, USA). MMAD and GSD is obtained directly by the piece of equipment used APS 3321. Frequency: On two days during the exposure period, with 3 repeats on each day. To determine the particle size distribution in the submicrometer range, each test atmosphere was measured with the Scanning Mobility Particle Sizer (SMPS; Grimm Aerosol Technik GmbH & Co KG, Ainring, Germany). The SMPS system comprises an Electrostatic Classifier (Model Vienna U-DMA) which separates the particles into known size fractions, and a Condensation Particle Counter (CPC) which measures particle count concentrations. The DMA was equipped with Am-241 neutralizer. The instrument measures particles in the size range from 0.011 to 1.083 µm. Using a conductive sample hose, the SMPS sampled at 0.3 liters per minute (LPM) with a sheath flow of 3 LPM. At this setting the single-stage, inertial impactor incorporated into the inlet of the SMPS to remove larger particles had a 50% cut size of 1.082 µm according to the software calculation. The sampling duration was about 7 minutes. As a rule 10 repeats were measured for each exposure concentration.
- Treatment of exhaust air: Exhaust air was filtered and conducted into the exhaust air of the building.

TEST ATMOSPHERE
- Brief description of analytical method used: The concentrations of the inhalation atmospheres were determined by gravimetrical measurements of filter samples in all test groups. Control group was not sampled. This analytical method was judged to be valid because the test substances did not possess an appreciable vapor pressure.
- Samples taken from breathing zone: yes
Details on mating procedure:
Mating of the F0 generation parental animals
After 44 days premating period, the male and female parental animals were mated overnight in a 1:1 ratio until there was evidence of copulation or the maximum period of 14 days has elapsed. Throughout the mating period, each female was mated with a predetermined male.

Normally, the female was placed into the cage of her male partner about 16:00 h and separated from the male between 06:30 and 09:00 h, the following morning. Deviations from the specified times are possible on Saturdays, Sundays and public holidays and were documented in the raw data.

A vaginal smear was prepared for each pair after each mating and examined for sperm. If sperm was detected, mating of the pair was discontinued. The day on which sperm were detected, was referred to as gestation day (GD) 0 and the following day as GD 1.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
The nominal concentration was calculated from the study means of the test-substance flow and the supply air flows used during exposure to generate the respective concentrations. The concentrations of the inhalation atmospheres were determined by gravimetrical measurements of filter samples in all test groups. Control group was not sampled. This analytical method was judged to be valid because the test substances did not possess an appreciable vapor pressure.
Duration of treatment / exposure:
The animals were exposed for 44 days before mating. The mating period were
maximal 2 weeks. After the mating period, the exposure of all male F0 animals were continued
until they are exposed for total minimal 90 days. After the mating period, the female F0 animals
were exposed further until gestation day 19. To allow them deliver and rearing their pups (F1
generation), they were not exposed from gestation day 20 to postnatal day (PND) 3. From
PND 4 through to PND 21, the dams were exposed with their pups in exposure cages
containing beddings. The first parental female animals were in gestation stage already after
the first few mating days, therefore, the post-weaning period were adjusted in such a way, that
a total of minimum 90 exposure will be achieved for females.
Frequency of treatment:
7 consecutive days per week, 6 hours per day
from PND4 through PND21
Details on study schedule:
not specified
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/m³ air
Remarks:
Test Group 0 (>Parental animals F0) - air control
Dose / conc.:
0.52 mg/m³ air (analytical)
Remarks:
SD: 0.10 mg/m3; target concentration: 0.5 mg/m³: Test Group 1 (>Parental animals F0)
Dose / conc.:
2 mg/m³ air (analytical)
Remarks:
SD: 0.20 mg/m3; target concentration: 2.0 mg/m³: Test Group 2 (>Parental animals F0)
Dose / conc.:
9.97 mg/m³ air (analytical)
Remarks:
SD: 1.23 mg/m3, target concentration: 10 mg/m³: Test Group 3 (>Parental animals F0)
No. of animals per sex per dose:
16/sex/dose group (parental animals)
5/sex at the high dose (recovery animals)
3 males at the high dose (for particle detection)

Subset Number of pups selected Day of examination Examination
I 10/sex/group PND 22 Measurement of thyroid hormones
II 10/sex/group PND 22 Perfusion fixation, brain weights
and neuropathology
III 5/sex/group PND 22 Histopathological examination and
organ burden
IV 10/sex/group PND 13 and 21 Open field observation
PND 13, 17 and 21 Motor activity
V 3 males/group PND 22 Perfusion fixation and electron
(highest dose) microscopic for particle detection
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of the 14-day range finding study (BASF study no 36I0050/20I005 - Ma- Hock 2021), upon approval of the sponsor, nominal aerosol concentrations of 0.5, 2.0 and 10.0 mg/m³ were used for the test substance in the low, mid and high dose groups, respectively.
- Rationale for animal assignment:
Prior to the pre-exposure period, the animals were distributed according to weight among the
individual test groups, separated by sex. The weight variation of the animals used did not
exceed ± 20 percent of the mean weight of each sex. The list of randomization instructions
was compiled with a computer.
For each neurofunctional test and motor activity measurement, separate randomization lists
were created. The list of randomization instructions were compiled with a computer (Laboratory
data processing, Experimental Toxicology and Ecology, BASF SE).
- Fasting period before blood sampling for clinical biochemistry: not specified
- Post-exposure recovery period in satellite groups: 45 days recovery period
Positive control:
none

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The clinical observation was performed on each animal at least three times (before, during and after exposure) on exposure days and once a day during pre-exposure and post exposure observation days. On non-exposure days a cage-side examination will be conducted at least once daily for any signs of morbidity, pertinent behavioral changes and/or signs of overall toxicity.

MORTALITY: The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) on working days and once a day (in the morning) on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS: YES
- Time schedule: All parental animals and recovery group animals were subjected to detailed clinical observations (DCO) outside their cages once before the beginning of the administration period and once during the first two weeks of the exposure, once monthly thereafter. DCO was performed in the morning before exposure. For observation, the animals were removed from their cages and placed in a standard arena (50 x 37.5 cm with a lateral border of 25 cm) for at least 20 seconds/animal.

BODY WEIGHT: Yes
- Time schedule for examinations:
The body weight of the animals was determined at the start of the pre-exposure, at the start of
the exposure period and then, as a rule, once a week as well as prior to gross necropsy. The
body weight of the recovery animals were determined at the start of the recovery period, and
once a week during the recovery period.
The following exceptions were notable for the female parental animals:
• During the mating period, the females were weighed on the day of positive evidence of
sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day after parturition (PND1) and on PND 4, 7, 14, and 21.
• In the females without positive evidence of sperm, body weight was determined once a week during mating and gestation periods and in the females without litter during lactation period.

As a rule, the animals were weighed at the same time of the day (in the morning).

Body weight change was calculated as the difference between body weight on the respective exposure day and body weight and the weight of previous weighing. Group means were derived from the individual differences.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was determined weekly and calculated as mean food consumption in grams
per animal and day.
Generally, food consumption was determined once a week for the male and female animals
and post mating period (males), with the following exceptions:
• Food consumption was not determined during the mating period (male and female
parental animals).
• Food consumption of the females with evidence of sperm was determined for GD 0-7, 7-
14 and 14-20.
• Food consumption of the females which gave birth to a litter was determined for PND 1-
4, 4-7, 7-13.
During recovery period, food consumption was determined in the animals of test groups 20 –
28 of the recovery animals. It was determined at the start of the recovery period and once a
week during the recovery period.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OPHTHALMOSCOPIC EXAMINATION: YES
Before the beginning of exposure, the eyes of all parental animals were examined with an ophthalmoscope (HEINE OPTOTECHNIK, Herrsching, Germany) after administration of a mydriatic agent (Mydrum, Dr. Gerhard Mann chem.-pharm. Fabrik GmbH and Bausch & Lomb GmbH, Germany). At the end of the exposure period, only animals selected for examinations according to OECD 413, 10 males and 10 females per group, were subjected to ophthalmological examination. In the first step, only control (test group 0) and high concentration groups (test groups 3, 6, 7 and 8) were examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in the morning
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes
- How many animals: 10 M + 10 F per dose group
-Parameters checked: leukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets, differential blood count, reticulocytes, preparation of blood smears, prothrombin time (PT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in the morning
- Animals fasted: Yes
- How many animals: 10 M + 10 F per dose group
- Parameters checked: alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-glutamyl transpeptidase (GGT), sodium (Na), potassium (K), chloride (CL), Inorganic phosphate (INP), calcium (Ca), urea (UREA), creatinine (CREA), glucose (GLUC), total biluribin (TBIL), total protein (TP), albumin (ALB), globulin (GLB), triglycerides (TRIG), cholesterol (CHOL)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the 90days exposure period
- Dose groups that were examined: 10 M + 10 F per dose group
- Battery of functions tested: sensory activity / grip strength / motor activity / reflexes


IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: Not specified
- Dose groups that were examined: 10 M + 10 F per dose group and recovery groups (highest dose: 5M + 5F)
- Number of animals: 10 M + 10 F per dose group and recovery groups (highest dose: 5M + 5F)
- Parameters checked: Cytological parameters: total cell count, cell differential analysis of cytospin preparations; protein; Enzymes: lactate dehydrogenase, alkaline phosphatase, N-acetyl-beta-D-Glucosaminidase (NAG BAL), gamma−Glutamyltransferase

ORGAN (lung, liver, heart, brain, olfactory bulb) BURDEN: Yes
- Time schedule for analysis: at the end of the exposure period and after the recovery period (45days post exposure)
- Dose groups that were examined: all
- Number of animals: 3 /sex / group
- Parameters checked: Zn content

OTHER: - Electron microscope analysis of particulate matter in organs and tissues: 3 male animals of the highest dose group



Oestrous cyclicity (parental animals):
In all parental females in the premating phase, estrous cycle length and normality were
evaluated by preparing vaginal smears during a minimum of 2 weeks prior to mating and
throughout cohabitation until there is evidence of sperm in the vaginal smear.
Additionally, on the day of scheduled sacrifice, the estrous status was also determined in all
parental female rats.
Sperm parameters (parental animals):
not specified
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum:
On PND 4, the individual litters were standardized in such a way that, whenever possible, each litter contains 5 male and 5 female pups (always the first 5 surviving pups/sex in each litter were taken for further rearing). If individual litters did not have 5 pups/sex, the litters were processed in such a way that the most evenly distributed 10 pups per litter were present for further rearing (e.g., 6 male and 4 female pups). Standardization of litters was not performed in litters with 10 pups or less.

PARAMETERS EXAMINED
The following parameters were examined in offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups, open field observations, motor activity

GROSS EXAMINATION OF DEAD PUPS: yes

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: yes
Necropsy:
On postnatal day 22, 10 animals per sex and group were weighed, subjected to deep
anesthesia (i.p. pentobarbital) and sacrificed by perfusion fixation.
SOERENSEN phosphate buffer was used as the rinsing solution, and neutrally buffered, 4% formaldehyde solution was used as a fixative.
The perfusion fixed animals were necropsied with regard to the question of neuropathology, and the visible organs were assessed by gross pathology as accurately as is possible after a perfusion fixation. The cranial vault was opened and the skin was removed from the head. In this state, the perfused animals were stored in neutrally buffered, 4% formaldehyde solution for at least 48 hours.
Organ weights:
The following weights were determined (the brain was weighed after its removal but before
further preparation):
1. Final body weight
2. Brain (including olfactory bulb)
The final body weights were recorded to calculate the relative organ weights.
Length and width of the brain:
The length and maximum width of the brain were measured in all animals. The length of the brain was measured on a line extending from the rostral end of the frontal lobe to the caudal medulla oblongata of the cerebellum, width: pituitary region.
Organ/Tissue fixation:
The following organs/tissue specimens were carefully removed and processed histotechnically in accordance with the data given in the respective sections of this report:
1. All gross lesions
2. Brain with olfactory bulb
3. Eyes with retina and optic nerve
4. Nose (nasal cavity level III) with olfactory epithelium
5. Pituitary gland
6. Trigeminal ganglia
The animals and the tissue or organ material remaining after trimming was stored in neutrally buffered, 4% formaldehyde solution.
Neurohistopathology:
Morphometry of the brains (PND 22) :
Morphometry was performed in all animals of test groups 0 (control), 3, 6, 7 and 8.
Thickness measurements of major brain layers (neocortex: frontal and parietal cortices, caudate nucleus/putamen, hippocampus, corpus callosum, cerebellum) were performed. Measurements were carried out bilaterally in the left and right halves of the brain except for the corpus callosum and the cerebellum.

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no
Postmortem examinations (parental animals):
SACRIFICE / GROSS NECROPSY / ORGAN WEIGHTS
At the time of sacrifice, adult animals were examined macroscopically for any abnormalities or pathological changes.
The following weights were determined in all animals sacrificed on schedule:
1.Anesthetized animals (final body weight)
2. Adrenal glands (fixed)
3. Brain
4. Epididymides
5. Heart
6. Kidneys
7. Liver
8. Lung
9. Ovaries
10. Prostate (ventral and dorsolateral part together, fixed)
11. Seminal vesicles with coagulating glands (fixed)
12. Spleen
13. Testes
14. Thymus (fixed)
15. Thyroid glands (with parathyroid glands) (fixed)
16. Uterus with cervix
All paired organs were weighed together (left and right).

HISTOPATHOLOGY
Organs and tissues of F0 animals histologically processed:
1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Epididymides
12. Esophagus
13. Eyes with optic nerve
14. Extraorbital lacrimal gland
15. Femur with knee joint
16. Harderian glands
17. Heart
18. Ileum
19. Jejunum
20. Kidneys
21. Larynx (3 levels)
22. Liver
23. Lungs
24. Lymph nodes (tracheobronchial and mediastinal)
25. Lymph nodes (mesenteric)
26. Mammary gland (female)
27. Nasal cavity (4 levels)
28. Olfactory bulb
29. Ovaries
30. Oviducts
31. Pancreas
32. Pharynx
33. Parathyroid glands
34. Peyer’s patches
35. Pituitary gland
36. Prostate
37. Rectum
38. Salivary glands
(mandibular and sublingual glands)
39. Sciatic nerve
40. Seminal vesicles
41. Skeletal muscle
42. Skin
43. Spinal cord
(cervical, thoracic and lumbar cord)
44. Spleen
45. Sternum with marrow
46. Stomach
(forestomach and glandular stomach)
47. Teeth
48. Testes
49. Thymus
50. Thyroid glands
51. Trachea
52. Urinary bladder
53. Uterus
54. Vagina



Postmortem examinations (offspring):
GROSS NECROPSY/ORGAN WEIGHTS:
Five pups per sex and group were sacrificed under pentobarbitone anesthesia by
exsanguination from the abdominal aorta and vena cava. They were necropsied and assessed
by gross pathology.
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (final body weight)
2. Brain
3. Epididymides
4. Heart
5. Kidneys
6. Liver
7. Lungs
8. Ovaries
9. Spleen
10. Testes
11. Thymus (fixed)
12. Uterus with cervix


HISTOPATHOLOGY:
Organs and tissues of PND 22 pups that were histologically processed and
examined by light microscopy.
1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Cecum
6. Cervix
7. Coagulating glands
8. Colon
9. Duodenum
10. Epididymides
11. Esophagus
12. Eyes with optic nerve
13. Extraorbital lacrimal gland
14. Femur with knee joint
15. Harderian glands
16. Heart
17. Ileum
18. Jejunum
19. Kidneys
20. Larynx (level II)
21. Liver
22. Lungs
23. Lung associated lymph nodes
24. Lymph nodes (mesenteric)
25. Mammary gland (female)
26. Nasal cavity (3 levels)
27. Olfactory bulb
28. Ovaries
29. Oviducts
30. Pancreas
31. Pharynx
32. Parathyroid glands
33. Pituitary gland
34. Prostate
35. Rectum
36. Salivary glands
(mandibular and sublingual glands)
37. Sciatic nerve
38. Seminal vesicles
39. Skeletal muscle
40. Skin
41. Spinal cord
(cervical, thoracic and lumbar cord)
42. Spleen
43. Sternum with marrow
44. Stomach
(forestomach and glandular stomach)
45. Teeth
46. Testes
47. Thymus
48. Thyroid glands
49. Trachea
50. Urinary bladder
51. Uterus
52. Vagina
Statistics:
Statistical evaluation for test groups low, mid, high in comparison with air control group
- Food consumption (parental animals), body weight and body weight change (parental animals
and pups (for the pup weights, the litter means were used)), gestation days, anogenital distance,
anogenital index
--> DUNNETT test (two-sided)
- Male and female mating indices, male and female fertility indices, gestation index, females mated, females delivering, females with liveborn pups, females with stillborn pups, females with all stillborn pups
--> FISHER'S EXACT test (one-sided)
-Mating days until day 0 pc, %postimplantation loss, pups stillborn, %perinatal loss, nipple development
--> WILCOXON test (one-sided+) with BONFERRONI-HOLM
-Implantation sites, pups delivered, pups liveborn, live pups day x, viability Index, lactation index
--> WILCOXON test (one sided-) with BONFERRONI-HOLM
-% live male day x, %live female day x
--> WILCOXON test (two-sided)
- Rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity
--> KRUSKAL-WALLIS and WILCOXON test (two-sided)
-Number of cycles and Cycle Length
--> KRUSKAL-WALLIS test (two-sided) and WILCOXON test (two-sided)
-Blood parameters
--> For parameters with bidirectional changes: WILCOXON-test (two-sided)
For parameters with unidirectional changes: WILCOXON-test (one-sided)
-Broncho-alveolar lavage fluid (BALF)
--> Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians
-Weight parameters in pathology (adult animals and PND 22 pups)
-->Non-parametric one-way analysis using KRUSKAL-WALLIS H test (two-sided).

Statistical evaluation of neuropathological parameters of PND 22 pups (subset II)
-Weight parameters (brain)
-->KRUSKAL-WALLIS test (two-sided)
-Brain width and length
--> WILCOXON test (two-sided) with Bonferroni-Holm-
-Brain morphometry: linear measurements of selected brain regions
--> WILCOXON test (two-sided)
Reproductive indices:
MALES:

Male mating index (%) = (number of males with confirmed mating*/number of males placed with females) x 100

* defined by a female with vaginal sperm or with implants in utero

Male fertility index (%) = (number of males proving their fertility* /number of males placed with females) x 100

* defined by a female with implants in utero

FEMALES:

Female mating index (%) = (number of females mated*/number of females placed with males) x 100

* defined as the number of females with vaginal sperm or with implants in utero


Female fertility index (%) = (number of females pregnant*/number of females mated**)x 100

* defined as the number of females with implants in utero
** defined as the number of females with vaginal sperm or with implants in utero

Gestation index (%) = (number of females with live pups on the day of birth/number of females pregnant*) x 100

* defined as the number of females with implants in utero

Live birth index (%) = (number of liveborn pups at birth/total number of pups born) x 100


Postimplantation loss (%) =[(number of implantations – number of pups delivered)/number of implantations] x 100


Offspring viability indices:

Viability index (%) = (number of live pups on day 4* after birth/ number of live pups on the day of birth) x 100

* before standardization of litters (i.e. before culling)


Lactation index (%) = (number of live pups on day 21 after birth/number of live pups on day 4* after birth) x 100

* after standardization of litters (i.e. after culling)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
-During the pre-exposure period and the exposure period the animals showed no clinical signs and findings different from normal.
-Exposure period, control group animals (test groups 0, 10, 20):
There were no clinical signs and findings different from normal.
Exposure period, test item 1 (test groups 1, 2, 3, 13, and 23):
No clinical signs of toxicity were observed in male and female animals.
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
No deaths were recorded throughout the study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The following statistically significant body weight changes were determined in male animals:
- Test group 1: day 74 -> 81: 9.9g (p< 0.01), whereas the control group was 5.7g
- Test group 1: day 92 -> 93: -5.1g (p< 0.05), whereas the control group was 2.3g
- Test group 1: day 93 -> 94: 6.7g (p< 0.05), whereas the control group was -5.3g
- Test group 3: day 11 -> 18: 17.8g (p< 0.05), whereas the control group was 22.0g
- Test group 3: day 25 -> 32: 11.0g (p< 0.05), whereas the control group was 16.1g
- Test group 3: day 94 -> 95: -10.7g (p< 0.05), whereas the control group was 2.0g
- Test group 13: day 18 -> 25: 9.9g (p< 0.01), whereas the control group was 5.7g
- Test group 13: day 74 -> 81: 7.5g (p< 0.01), whereas the control group was 0.9g
- Test group 23: day 0 -> 4: 4.5g (p< 0.01), whereas the control group was 12.6g
- Test group 23: day 18 -> 25: 9.7g (p< 0.01), whereas the control group was 16.9g
- Test group 23: day 53 -> 60: 9.5g (p< 0.05), whereas the control group was 2.6g
- Test group 23: day 109 -> 116: 9.6g (p< 0.01), whereas the control group was 2.2g
The following statistically significant body weight changes were determined in female
animals:
- Test group 23: day 60 -> 67: 12.1g (p< 0.05), whereas the control group was 1.0g
- Test group 23 day 102 -> 109: 3.5g (p< 0.05), whereas the control group was 9.4g

Although the deviations in body weight changes were statistically significant, they did not show any trend with the exposure-duration, as some of the means were higher than the control, on the other days lower, indicating that they were rather biological variations than substance-related changes. Moreover, the mean body weight (as well as the final body weight) did not significantly change, when compared with the concurrent control. These deviations from the control were considered not biologically relevant.

Body weight of F0 females during gestation/lactation of F1 litters:
no adverse effects on body weights/body weight gain at concentrations of 0.5, 2 and 10 mg/m3 during gestation and lactation.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant changes of mean food consumption were determined in
female animals:
• Test group 1: day 11 - 18: +19.4 g (p≤ 0.05), whereas the control group was +17.6 g
• Test group 1: day 25 - 32: +19.1 g (p≤ 0.05), whereas the control group was +17.5 g
• Test group 1: day 32 - 39: +19.3 g (p≤ 0.05), whereas the control group was +17.8 g
The increased food consumption in female animals was most likely because animals spread out the food from the supply and was considered not adverse.

Food consumption of F0 animals during gestation of F1 litters:
Food consumption of females exposed to 10 mg/m3 (test group 3) was consistently below concurrent control throughout gestation (about 7%), the difference became, however, statistically significant only during GD 7 – 14 (about 13% below control). This effect continued through lactation, average food consumption of the lactating dams in test group 3 was about 9% below control during PND 1 – 13.
--> at 10mg/m3: decreased food consumption during gestation and lactation as treatment related adverse effects

Food consumption of test groups 1 and 2 (0.5 and 2 mg/m3) was comparable to the concurrent control.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
The ophthalmologic examinations did not show any impairment of the refracting media.
Spontaneous findings such as remainders of the pupillary membrane or corneal stippling were
observed in several animals of all test groups and the control group without any concentration response relationship.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In females of test group 3 (10 mg/m3 Zinc oxide T0420) absolute and relative neutrophil cell
counts were significantly increased whereas relative lymphocyte counts were significantly
decreased. However, total white blood cell counts were not altered among these individuals,
and absolute neutrophil counts were within the historical control range (females, absolute
neutrophils 0.60-0.96 giga/L). Therefore, these changes were regarded as incidental and not
treatment related.

The following significant changes were regarded as incidental and not treatment related,
because the values were within historical control ranges: decreased relative eosinophil cell
counts in males of test groups 2 and 3 (2 and 10 mg/m3 Zinc oxide T0420) prolonged prothrombin time (HQT, i.e., Hepatoquick’s test) in females of test group 3 (10 mg/m3 Zinc oxide T0420)( males, relative eosinophils 1.4-3.1 %; relative basophils 0.1-0.4 %; hemoglobin 8.6-9.3 mmol/L; females, absolute monocytes 0.05-0.11 Giga/L; relative monocytes 1.8-2.8 %; RBC 7.55-8.84 Tera/L; MCV 50.7-55.1 fL; MCH 1.10-1.21 fmol; HQT 34.0-40.2 sec).
The following significant changes were regarded as incidental and not treatment related,
because the alteration was not dose dependent: decreased absolute and relative monocyte counts in females of test group 2 (2 mg/m3 Zinc oxide T0420) as well as absolute monocyte counts in females of test group 3 (10 mg/m3 Zinc oxide T0420).

In females of test group 23 (10 mg/m3 Zinc oxide T0420) absolute monocyte counts were
significantly decreased, but the values were within the historical control range (females,
absolute monocytes 0.05-0.07 Giga/L). Therefore this change was regarded as incidental and
not treatment related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The following significant changes were regarded as incidental and not treatment related
because the values were within historical control ranges: increased inorganic phosphate levels
in males of test groups 3 (10 mg/m3 Zinc oxide T0420 )
Endocrine findings:
no effects observed
Description (incidence and severity):
After the administration period, in parental males and in male and female pups at PND22 of all
test groups, no treatment-related alterations of T4 and TSH levels were observed.
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional observational battery:
Quantitative parameters: no substance-related findings were observed.
Home cage observations: no substance-related findings were observed.
Open field observations: no substance-related findings were observed.
Sensorimotor tests/reflexes: no substance-related findings were observed.
Overall motor activity (summation of all intervals):
Test item T0420 (Test groups 1, 2 and 3):
there were no statistically significant deviations from the control group 0.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings were observed in in the larynx, lungs, nasal cavity and the tracheobronchial lymph nodes. These are further described in details on results section below

The following treatment-related, adverse effects were observed:
Main group (F0)
Test item 1 (Zinc oxide T0420)
Test group 3 (10 mg/m³)
• Slight to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to severe cellular debris in the lungs in all male and all female animals
• Minimal to moderate infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 9 males and all females
• Minimal to slight degeneration/regeneration of the olfactory epithelium (nasal cavity, level IV, exemplarily) in 9 males and 10 females

Test group 23 (Recovery group R1, 10 mg/m³)
• Minimal to moderate numbers of foamy macrophages in the lungs in 3 male and 4
female animals
• Minimal cellular debris in the lungs in 1 male and 2 female animals
• Minimal infiltration of neutrophils of alveoli of the lungs in 1 male and 2 female animals
• Minimal hyperplasia of type II pneumocytes in 3 females

Test group 2 (2 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium in one male animal
Test group 1 (0.5 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium (nasal cavity, level IV, exemplarily) in 1 female
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
BAL
Main group (F0)
Test item 1 (Zinc oxide T0420)
The following treatment-related, adverse effects were observed:
Test group 3 (10 mg/m³):
• Increased total cell counts as well as absolute and relative lymphocyte, neutrophil cell and monocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute eosinophil cell counts in males in BAL
• Increased total protein levels lactate dehydrogenase (LDH), alkaline phosphatase
(ALP) and γ -Glutamyl-transferase (GGT) activities in BAL of both sexes
• Increased β-N-Acetyl glucosaminidase (NAG) activity in BAL of males

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Estrous cycle data, generated during the last 3 weeks prior to mating to produce the F1 litter, revealed regular cycles in the females of all test groups. The mean estrous cycle duration was comparable: 3.9 / 3.9 / 4.0, 4.0, 3.9, 3.9, 4.0 and 4.0 days in test groups 1 – 6 as well as 7 and 8.
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
Description (incidence and severity):
MALE REPRODUCTION DATA:
For all F0 parental males of all test groups, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100% in all test groups.

Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter. However, two males (No. 105, 112) of test group 6 (test item T0421, 10 mg/m3) did not generate F1 pups.

Thus, the male fertility index was 87.5% in test group 6 and 100% in all other groups. This reflects the normal range of biological variation inherent in the strain of rats used for this study.

FEMALE REPRODUCTION DATA AND DELIVERY DATA:
The female mating index was 100% in all test groups. The mean duration until copulation was detected (GD 0) varied between 1.9 and 2.8 days without any relation to test item and concentration.

All female rats delivered pups or had implants in utero with the following exception:
• Test group 6 (test item T0421)
female No. 305 (mated with male No. 105) did not become pregnant
female No. 312 (mated with male No. 112) did not become pregnant

The female fertility index was 87.5% in test group 6 and 100% in all other groups. This reflects the normal range of biological variation inherent in the strain of rats used for this study.

The gestation index was 100% in in all test groups. The mean duration of gestation was comparable in all test groups (i.e. between 21.9 and 22.4 days).

Litter and delivery parameters:
Implantation was not affected by the treatment since the mean number of implantation sites was comparable between the test substance-treated groups and the control, taking normal biological variation into account (13.2 / 12.0 / 12.8 and 12.2 implants/dam in test groups 0 - 3, respectively). All values are well within the historical control range (HCD: 11.1 - 13.9).

Post-implantation loss was 2.8 / 3.6 / 6.5 and 11.5* ([*p<=0.05] mean% in test groups 0 - 3, respectively. The slightly, but statistically significantly, higher post-implantation loss in test group 3 exposed to 10 mg/m3 was well within the historical control range and thus considered spontaneous in nature and not treatment related (HCD: 2.4 - 17.7).

Consequently, the mean number of F1 pups delivered per dam was also lower in test group 3 while it remained unaffected in the other exposure groups (12.9 / 11.6* / 11.9 and 10.9* pups/dam in test groups 0 - 3, respectively). While the control was above, the statistically significantly decreased mean numbers of F1 pups delivered per dam in test groups 1 and 3 are well within the historical control range (HCD: 10.3 - 12.7). Thus, these apparent effects are considered spontaneous in nature and not treatment related.

The rate of liveborn pups was not affected by the test substance, as indicated by live birth indices of 100% / 100% / 98.4% and 98.9% in test groups 0 - 3. Moreover, the rate of stillborn pups was not significantly different between the groups and within the historical control range (0 – 5.5%).



Details on results (P0)

CLINICAL SIGNS AND MORTALITY:
Mortality:
No deaths were recorded throughout the study.
Clinical observations:
During pre-exposure period, none of the male and female rats showed any clinical signs and findings different from normal.
Exposure period, control group animals (test groups 0, 10, 20):
There were no clinical signs and findings different from normal.
Exposure period, test item 1 (test groups 1, 2, 3, 13, and 23):
No clinical signs of toxicity were observed in male and female animals.

BODY WEIGHT AND WEIGHT GAIN
Body weight change:
The following statistically significant body weight changes were determined in male animals:
- Test group 1: day 74 -> 81: 9.9g (p< 0.01), whereas the control group was 5.7g
- Test group 1: day 92 -> 93: -5.1g (p< 0.05), whereas the control group was 2.3g
- Test group 1: day 93 -> 94: 6.7g (p< 0.05), whereas the control group was -5.3g
- Test group 3: day 11 -> 18: 17.8g (p< 0.05), whereas the control group was 22.0g
- Test group 3: day 25 -> 32: 11.0g (p< 0.05), whereas the control group was 16.1g
- Test group 3: day 94 -> 95: -10.7g (p< 0.05), whereas the control group was 2.0g
- Test group 13: day 18 -> 25: 9.9g (p< 0.01), whereas the control group was 5.7g
- Test group 13: day 74 -> 81: 7.5g (p< 0.01), whereas the control group was 0.9g
- Test group 23: day 0 -> 4: 4.5g (p< 0.01), whereas the control group was 12.6g
- Test group 23: day 18 -> 25: 9.7g (p< 0.01), whereas the control group was 16.9g
- Test group 23: day 53 -> 60: 9.5g (p< 0.05), whereas the control group was 2.6g
- Test group 23: day 109 -> 116: 9.6g (p< 0.01), whereas the control group was 2.2g
The following statistically significant body weight changes were determined in female
animals:
- Test group 23: day 60 -> 67: 12.1g (p< 0.05), whereas the control group was 1.0g
- Test group 23 day 102 -> 109: 3.5g (p< 0.05), whereas the control group was 9.4g
Although the deviations in body weight changes were statistically significant, they did not show any trend with the exposure-duration, as some of the means were higher than the control, on the other days lower, indicating that they were rather biological variations than substance-related changes. Moreover, the mean body weight (as well as the final body weight) did not significantly change, when compared with the concurrent control. These deviations from the control were considered not biologically relevant.

FOOD CONSUMPTION
The following statistically significant changes of mean food consumption were determined in
female animals:
• Test group 1: day 11 - 18: +19.4 g (p≤ 0.05), whereas the control group was +17.6 g
• Test group 1: day 25 - 32: +19.1 g (p≤ 0.05), whereas the control group was +17.5 g
• Test group 1: day 32 - 39: +19.3 g (p≤ 0.05), whereas the control group was +17.8 g
The increased food consumption in female animals was most likely because animals spread out the food from the supply and was considered not adverse.

Food consumption of F0 animals during gestation of F1 litters:
Food consumption of females exposed to 10 mg/m3 (test group 3) was consistently below concurrent control throughout gestation (about 7%), the difference became, however, statistically significant only during GD 7 – 14 (about 13% below control). This effect continued through lactation, average food consumption of the lactating dams in test group 3 was about 9% below control during PND 1 – 13.
--> at 10mg/m3: decreased food consumption during gestation and lactation as treatment related adverse effects

Food consumption of test groups 1 and 2 (0.5 and 2 mg/m3) was comparable to the concurrent control.

HAEMATOLOGICAL FINDINGS:
In females of test group 3 (10 mg/m3 Zinc oxide T0420) absolute and relative neutrophil cell
counts were significantly increased whereas relative lymphocyte counts were significantly
decreased. However, total white blood cell counts were not altered among these individuals,
and absolute neutrophil counts were within the historical control range (females, absolute
neutrophils 0.60-0.96 giga/L). Therefore, these changes were regarded as incidental and not
treatment related.

The following significant changes were regarded as incidental and not treatment related,
because the values were within historical control ranges: decreased relative eosinophil cell
counts in males of test groups 2 and 3 (2 and 10 mg/m3 Zinc oxide T0420) prolonged prothrombin time (HQT, i.e., Hepatoquick’s test) in females of test group 3 (10 mg/m3 Zinc oxide T0420)( males, relative eosinophils 1.4-3.1 %; relative basophils 0.1-0.4 %; hemoglobin 8.6-9.3 mmol/L; females, absolute monocytes 0.05-0.11 Giga/L; relative monocytes 1.8-2.8 %; RBC 7.55-8.84 Tera/L; MCV 50.7-55.1 fL; MCH 1.10-1.21 fmol; HQT 34.0-40.2 sec).
The following significant changes were regarded as incidental and not treatment related,
because the alteration was not dose dependent: decreased absolute and relative monocyte counts in females of test group 2 (2 mg/m3 Zinc oxide T0420) as well as absolute monocyte counts in females of test group 3 (10 mg/m3 Zinc oxide T0420).

In females of test group 23 (10 mg/m3 Zinc oxide T0420) absolute monocyte counts were
significantly decreased, but the values were within the historical control range (females,
absolute monocytes 0.05-0.07 Giga/L). Therefore this change was regarded as incidental and
not treatment related.

CLINICAL CHEMISTRY:
The following significant changes were regarded as incidental and not treatment related
because the values were within historical control ranges: increased inorganic phosphate levels
in males of test groups 3 (10 mg/m3 Zinc oxide T0420 )

NEUROBEHAVIOUR:
Functional observational battery:
Quantitative parameters: no substance-related findings were observed.
Home cage observations: no substance-related findings were observed.
Open field observations: no substance-related findings were observed.
Sensorimotor tests/reflexes: no substance-related findings were observed.
Overall motor activity (summation of all intervals):
Test item T0420 (Test groups 1, 2 and 3):
there were no statistically significant deviations from the control group 0.

ORGAN WEIGHTS
When compared with control group 0 (=100%), Test item 1 (Zinc oxide T0420):
Test group 3 (10 mg/m³): Increase of absolute/relative lung weights in males (140%/150%) and females (128%/130%)

GROSS PATHOLOGY
Test item 1 (Zinc oxide T0420):
Test group 3 (10 mg/m³):
•Macroscopically observed white foci in the lungs of 5 males and 8 females.
•Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial,
highest number is given) in 6 males and 9 females
Test group 23 (Recovery group R1, 10 mg/m³)
• Macroscopically observed white foci in the lungs of 1 male and 2 females
• Macroscopically enlarged draining lymph nodes (mediastinal) in 1 female
--> The foci observed in the lungs of males and females of test group 23 (test item 1, 10 mg/m³)
and test group 26 (test item 2, 10 mg/m³) were considered to be treatment-related as similar
findings were observed in the respective main groups. The same comes true for the
enlargement of the mediastinal lymph nodes in one female of test group 23 (test item 1,
10 mg/m³) and one male of test group 26 (test item 2, 10 mg/m³). These findings were regarded
to be treatment-related.


HISTOPATHOLOGICAL FINDINGS: NON-NEOPLASTIC:

Larynx (level I):
In the larynx, the most severe findings were observed in level I, therefore only findings in level I of the larynx are given

Parental animals:
Minimal epithelial alteration was observed in several test groups treated with test item 1 or test item 2 as well as in control animals. This finding is characterized by an increase of cell layers and replacement of respiratory epithelium by squamous epithelial cells, which may exhibit slight nuclear polymorphism and cellular atypia. The site most susceptible for this lesion, is the base of the epiglottis as it was observed in the present study. This finding was regarded to be treatment-related (inhalation).

Recovery animals: no findings observed

Lungs:

Parental animals:
Mainly, high dose group males and females (test group 3 and 6 [test item 1 and 2, 2 mg/m³]) were affected. Within alveoli, mainly in the bronchio-alveolar transition region, a multifocal accumulation of alveolar macrophages with vacuolar (foamy) cytoplasm was seen. The alveolar macrophages often revealed nuclei of increased size and occasionally multiple nuclei.
Intermingled with the foamy macrophages, cellular debris of presumable fragmented
macrophages and neutrophils were observed. In the region of these cellular accumulations, proliferation (hyperplasia) of type II pneumocytes was observed.
Males of test group 2 and 4 (test item 1 and 2, 2 mg/m³) revealed also an accumulation of foamy macrophages, only.

Recovery animals:
The same findings as described for the main group animals were observed in the recovery animals. These findings were regarded to be treatment-related.

Lymph nodes (mediastinal):
Parental animals:
The mediastinal and tracheobronchial lymph nodes revealed comparable findings.
In general, the high dose group males and females (test group 3 and 6 [test item 1 and 2, 2 mg/m³]) were more severely affected. A lympho-reticular cell hyperplasia was observed, which can be explained by an activation of the draining lymph nodes of the lungs. Furthermore, aggregates of macrophages were seen within the lymph nodes. These findings were considered as treatment-related.
Single animals of test group 1, 2 (test item 1, 0.5 and 2 mg/m³), and test group 5 (test item 2, 2 mg/m³) revealed similar findings.

Recovery animals:
The same findings as described for the main group animals were observed in the recovery animals. These findings were regarded to be treatment-related.

Nasal cavity:

Parental animals:
The nasal cavity was investigated in four levels. The most severely affected levels were level III and IV
In general, the high dose group males and females (test group 3 and 6 [test item 1 and 2, 2 mg/m³]) were affected. The finding was characterized by loss of olfactory epithelial cells and occasionally regeneration. Mainly the dorsal meatus and areas on the nasal septum were affected. This finding was regarded to be treatment-related.
One female of test group 1 (test item 1, 0.5 mg/m³) and three males and one female of test group 5 (test item 2, 2 mg/m³) showed minimal to slight degeneration of the olfactory epithelium. As this finding normally does not occur as a background lesion, it was assumed to have been most likely caused by the test substances.

Recovery animals:
Findings occurred either individually or were biologically equally distributed over
control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Trachea
In the trachea, two male animals of test group 8 (reference item 2, 22 mg/m³) revealed a flattening of the respiratory epithelium at the carina. This finding was considered to be treatment-related.
All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.



BRONCHOALVEOLAR LAVAGE FLUID (BALF):
Cytology:
After the administration period, in the bronchoalveolar lavage (BAL) of males and females of
test group 3 (10 mg/m3 Zinc oxide T0420) total cell counts, as well as absolute and relative
lymphocyte, neutrophil cell (PMN) and monocyte counts as well as absolute eosinophil cell
counts (not significantly) were increased. Relative macrophage counts were significantly
decreased. These alterations were regarded as treatment related and adverse.
In the BAL of males of test group 2 (2 mg/m3 Zinc oxide T0420) absolute and relative lymphocyte, neutrophil cell and monocyte counts were already significantly increased whereas
relative macrophage counts were significantly decreased. In males of test group 1 (0.5 mg/m3
Zinc oxide T0420) absolute and relative lymphocyte counts were significantly increased. In
females of test group 2 absolute and relative monocyte counts as well as relative neutrophil
counts were significantly increased whereas relative macrophage counts were significantly
decreased. However, in the BAL of both sexes of test group 2 as well as in BAL of males of
test group 1 total cell counts were not altered, and the differential cell counts were only
marginally changed (below 10fold). Therefore, the cell count changes in BAL of both sexes in
test group 2 and in BAL of males of test group 1 were regarded as treatment related but non adverse.
After the 8-week recovery period, no significant changes in BAL cytology were observed in
BAL of both sexes of test group 23 (10 mg/m3 Zinc oxide T0420).
Proteins/enzymes:
After the administration period, in BAL of males and females of test group 3 (10 mg/m3 Zinc
oxide T0420) total protein levels as well as lactate dehydrogenase (LDH) and alkaline
phosphatase (ALP) activity were moderately, significantly increased whereas β-N-Acetyl
glucosaminidase (NAG) in males of this test group and γ-Glutamyl-transferase (GGT) activity
in both sexes were marginally but also significantly increased. These alterations were regarded
as treatment related and adverse.
Additionally, in BAL of females of test group 3 (10 mg/m3 Zinc oxide T0420) NAG activity was
significantly increased, and in BAL of males of test group 2 (2 mg/m3 Zinc oxide T0420) LDH,
ALP and GGT activities and in females of this test group ALP and GGT activities were
significantly increased. However, the changes were marginally (below 2fold). Therefore, these
alterations were regarded as treatment related but non-adverse.
After the 8-week recovery period, in BAL of males and females of test group 23 (10 mg/m3
Zinc oxide T0420) no protein level and enzyme activity changes were observed.


OTHER FINDINGS
- Electron microscopy:

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEC
Remarks:
local toxicity
Effect level:
0.52 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: At the target mid concentration of 2 mg/m³, minimal degeneration/regeneration in the nasal cavity was noted in one male animal
Dose descriptor:
LOAEC
Effect level:
0.52 mg/m³ air (analytical)
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: At the target low concentration of 0.5 mg/m³, minimal degeneration/regeneration in the nasal cavity was noted in one female animal
Dose descriptor:
NOAEC
Remarks:
systemic toxicity
Effect level:
9.97 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology
clinical biochemistry
histopathology: non-neoplastic
Remarks on result:
other: No systemic toxicity was observed in hematology, clinical chemistry and histopathology
Dose descriptor:
NOEC
Remarks:
fertility and reproductive performance
Effect level:
9.97 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:
yes
Lowest effective dose / conc.:
2 other: mg/m3 air (analytical)
System:
respiratory system: lower respiratory tract
Organ:
lungs
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
No test or reference item related adverse clinical signs were observed in any of the F1 generation pups of the different test groups. Individual findings in few pups, like dehydrated appearance or gasping, were noted in several groups including control. They were most likely related to the technical procedure of inhalation exposure rather than any test or reference item.
Dermal irritation (if dermal study):
not specified
Mortality / viability:
no mortality observed
Description (incidence and severity):
The viability index indicating pup survival during early lactation (PND 0 - 4) varied between 99.5% / 99.0% / 100% / 100% / 95.7% / 97.9% / 100% / 99.6% and 99.5% in test groups 0 - 8 without showing significant differences between the groups. All values were within the historical control range (94 – 100%).

The lactation index indicating pup survival on PND 4 - 21 was 98.8% in test group 5 and 100% in all remaining test groups. All values were within the historical control range (95.7 – 100%).
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weights of all male and female pups in all test and reference item-treated groups 1 – 8 were comparable to the concurrent control values throughout the entire study.

The statistically significantly higher body weights in male pups and in both sexes combined in test group 4 on PND 4 were considered to be spontaneous in nature.

Calculation of body weight change resulted in a number of statistical changes in various groups, sometimes higher, sometimes lower than the concurrent control:

Increased:
- test group 3 males and both sexes combined on PND 1
- test group 4 males, females and both sexes combined on PND 1
- test group 7 males, females and both sexes combined on PND 1

Decreased:
- test group 5 females and both sexes combined on PND 4
- test group 7 males, females and both sexes combined on PND 13
- test group 8 males, females and both sexes combined on PND 4
- test group 8 males, females and both sexes combined on PND 13

None of these apparent changes is considered to be associated with the respective test or reference items.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not specified
Sexual maturation:
not specified
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
The anogenital distance and anogenital index of all treated male and female pups was comparable to the concurrent control values.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
The apparent number and percentage of male pups having areolae was not influenced by the test item when examined on PND 13. Likewise, no test item-related effect was detected in any of the test groups during the re-examination on PND 20.
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings were observed in in the lungs and nasal cavity.

The following treatment-related, adverse effects were observed:
PUPS (F1)
Test item 1 (Zinc oxide T0420)
Test group 3 (10 mg/m³)
• Minimal cellular debris in the lungs in 2 male and 4 female animals
• Minimal infiltration of neutrophils of alveoli of the lungs in 1 male and 2 female animals
Test group 2 (2 mg/m³) and test group 1 (0.5 mg/m³)
No treatment-related adverse findings observed
Other effects:
no effects observed
Description (incidence and severity):
OPEN FIELD OBSERVATIONS (OFO):
None of the animals in all test groups showed abnormalities attributable to the exposure to the test substance.

MOTOR ACTIVITY MEASUREMENT (MA):
Motor activity of male and female F1 offspring was not influenced by the test item at all concentration levels and at any of the testing dates PND 13, 17 and 21. Overall activity levels and habituation to the test environment corresponded to the age of these animals at the specific testing date, if usual biological variation inherent in rats used for this type of experiment was considered.

Across the test groups, there were a number of statistically significant changes in either the number of beam interrupts or in the number of rearings, in single intervals on various testing dates, sometimes higher, sometimes lower than the concurrent control:

Test item T0420
Increased:
- test group 2 females, beam interrupts, interval 1, PND 13
- test group 2 females, beam interrupts, interval 8, PND 21
- test group 3 females, beam interrupts, interval 8, PND 21
- test group 2 females, rearings, interval 1, PND 13

Decreased:
- test group 1 males, beam interrupts, interval 8, PND 13
- test group 1 males, rearings, interval 8, PND 13
- test group 1 males, rearings, interval 5, PND 21
- test group 2 males, rearings, interval 5, PND 13
- test group 1 females, beam interrupts, interval 4, PND 21
- test group 2 females, beam interrupts, interval 4, PND 21
- test group 1 females, rearings, interval 4 and 12, PND 13
- test group 3 females, rearings, interval 1 and 1-12, PND 13
- test group 2 females, rearings, interval 3, PND 17
- test group 3 females, rearings, interval 3, PND 17

None of these apparent changes is considered to be associated with the respective test or reference items.


Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Regarding neuropathology, no treatment-related findings were seen in pups of PND 22: Neuropathology, brain weight determination, necropsy, gross measurements of the brain,
neuropathology examination by light microscopy and morphometry did not reveal any
neuropathological, treatment-related findings.
--> There was no developmental neurotoxicity in all examined pups.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

open allclose all
Dose descriptor:
NOAEC
Remarks:
local toxicity
Generation:
F1
Effect level:
2 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: minimal cellular debris and neutrophilic infiltration in a few male and female animals at 10 mg/m³.
Dose descriptor:
NOAEC
Remarks:
systemic toxicity
Generation:
F1
Effect level:
9.97 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology
clinical biochemistry
histopathology: non-neoplastic
Remarks on result:
other: No systemic toxicity was observed in hematology, clinical chemistry and histopathology
Dose descriptor:
NOEC
Remarks:
developmental toxicity and developmental neurotoxicity
Generation:
F1
Effect level:
9.97 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
developmental neurotoxicity

Target system / organ toxicity (F1)

Critical effects observed:
yes
Lowest effective dose / conc.:
9.97 other: mg/m3 air (analytical)
System:
respiratory system: lower respiratory tract
Organ:
lungs
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Overall assessment for adult animals:

With regards to systemic toxicity, none of the test or reference substances caused any systemic toxicity that were not triggered by the local toxicity.

Comparing the local effects of the two nano Zinc oxide materials, the overall finding in the lungs, mediastinal lymph nodes, in the nasal cavity were comparable at the tested concentrations, as well as the changes of lavage parameters. The small differences are considered biological variations. There were no considerable differences between the effects caused by zinc oxide nanoparticles and those caused by micron-size zinc oxide particle.

For reference substance 2 (zinc sulfate monohydrate), lower incidence and severity was found in the lungs than in the other zinc oxide treated groups, but higher incidence and severity in nasal cavity and larynx. This difference is considered being related to the different deposition pattern, caused by the different aerodynamic diameter. The aerodynamic diameter of zinc sulfate monohydrate was larger than the different types of zinc oxide. The mean MMAD of zinc sulfate monohydrate was with 2.3 µm considerably higher than those measured at the high concentrations of the test items 1 (1.19 µm) and 2 (0.97 µm). The deposited dose at the upper respiratory tract was higher, while those deposited in the lung was lower.

After the recovery period, all parameters in lavage fluid returned to the control level in all animals, irrespective of the exposed test and reference substance. With regards of histological findings in the respiratory tract, all changes reduced greatly in incidence and severity. Only single animals showed still some mild effects.

Overall assessment for PND 22 animals:
With regards to effects observed in PND 22 pups that were exposed whole-body to zinc oxide nanomaterials from PND 4 to PND 21, the findings were limited to lungs and nasal cavities. The effects observed in the parental animals showed much higher incidence and severity than in the pups exposed at the same concentration. This could be explained by the duration of the exposure, because pups were only exposed for 17 days, while the adult animals were exposed for 90-days.

Comparing the toxicity of the two nano Zinc oxide materials in pups of PND 22, the overall finding in the lungs were comparable at the high concentration of 10 mg/m³ for test items 1 and 2. However, lesions were also observed in nasal cavity in pups exposed to test item 2. While lesions in nasal cavity were still observed at 0.5 mg/m³ test item 2, there were no effect observed in animals exposed to 2 mg/m³ and 0.5 mg/m³ test item 1.

Comparing the toxicity caused by the two nano zinc oxide materials with those caused by the microscale zinc oxide material, or by the soluble zinc sulfate monohydrate, the changes in lungs were comparable in incidence and severity in exposed pups on PND 22. However, no nasal cavity lesions were observed in animals exposed to test item 1, while similar lesions were seen in those exposed to test item 2 and the reference items.

None of the substances cause any systemic toxicity, nor were there any developmental neurotoxicity in exposed pups.
Executive summary:

This study was a 90-Day Study (OECD test guideline (TG) 413) combined with the Reproduction/ Developmental Toxicity Screening Test (OECD TG 421) in rat with neurotoxicity and developmental (neuro)toxicity evaluation, including detailed clinical observations addressing potential neurobehavioral effects, histological and morphological evaluations of the brains of the pups on post-natal day 22.


To compare the toxicity of uncoated and coated nano Zinc oxide, these two materials (Zinc oxide T0420 was uncoated, Zinc oxide T0421 was coated) were tested at each three concentrations. In addition, micronsize Zinc oxide T0242 and a soluble salt zinc sulfate monohydrate was tested as reference items. 


Groups of male and female Wistar rats were whole-body exposed to the aerosols of ZnO nano materials, Zinc oxide T0420 and Zinc oxide T0421, for 6 hours daily, at least 90 days. Zinc oxide T0420 was uncoated, Zinc oxide T0421 was coated.


The target concentrations for Zinc oxide T0420 and T0421 were 0.5, 2 and 10 mg/m³ referring to the non-volatile fraction. For the reference item 1 microscale Zinc oxide T0242, 10 mg/m³ was tested. For the reference item 2, Zinc sulfate monohydrate a target concentration of 22 mg/m³ was tested because this is equimolar to zinc ion of the ZnO materials. Concurrent control groups were exposed to humidified air (control group 0, 10 and 20).


All animals were exposed to the respective concentrations of test substance for 6 hours a day according to the time schedule (exception: no exposure on the day of FOB/MA and parental females from GD20 – PND 3)). Control animals were exposed to conditioned air. Male and female rats aged about 6 or 7 weeks when supplied, were used as F0 generation parental animals. The animals were exposed for 43 days before mating. The mating period were maximal 2 weeks. After the mating period, the exposure of all male F0 animals were continued until they are exposed for total minimal 90 days. After the mating period, the female F0 animals were exposed further until gestation day 19. To allow them to deliver and rearing their pups (F1 generation), they were not exposed from gestation day 20 to postnatal day (PND) 3. From PND 4 through to PND 21, the dams were exposed with their pups in exposure cages containing beddings. During the exposure food was withdrawn. Water was provided in form of hydrogel pads from PND 14 to 16 onward. The first parental female animals were in gestation stage already after the first few mating days, therefore, the post-weaning period were adjusted in such a way, that a total of minimum 90 exposure will be achieved for females.


Daily clinical observations, body weights, food consumption, ophthalmology, detailed clinical observation and FOB/MA were recorded. Moreover, male and female fertility were determined. Additional assessments including hematology and clinical chemistry in blood, bronchoalveolar lavage, and histopathology according to the referenced guidelines were carried out at the termination of exposure period. In addition, recovery groups of male and female animals were included; after an exposure period of about 90 days, these animals were kept for an additional period of ca. 60 days without exposure (control group 20, and test groups 23, 26, 27 and 28, respectively).


To assess the reproductive/developmental toxicity of the test substances (incl. reference substances), estrus cycles, male and female reproduction, delivery data were collected. In the pups, open field observations were performed on PND 13 and 21, motor activity measurements were performed on PND 13, 17 and 21. On PND 22, thyroid hormones, brain weights, neuropathology, general histopathology were examined in separate subsets of animals.


The following treatment-related, adverse effects were observed:
Main group (F0)
Test item 1 (Zinc oxide T0420)
Test group 3 (10 mg/m³)


• Decreased food consumption during gestation and lactation of parental females
• Increased total cell counts as well as absolute and relative lymphocyte, neutrophil cell and monocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute eosinophil cell counts in males in BAL
• Increased total protein levels lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and γ-Glutamyl-transferase (GGT) activities in BAL of both sexes
• Increased β-N-Acetyl glucosaminidase (NAG) activity in BAL of males
• Increase of absolute/relative lung weights in males (140%/150%) and females (128%/130%)
• Macroscopically observed white foci in the lungs of 5 males and 8 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial, highest number is given) in 6 males and 9 females
• Slight to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to severe cellular debris in the lungs in all male and all female animals
• Minimal to moderate infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 9 males and all females
• Minimal to slight degeneration/regeneration of the olfactory epithelium 



Test group 23 (Recovery group R1, 10 mg/m³)
• Macroscopically observed white foci in the lungs of 1 male and 2 females
• Macroscopically enlarged draining lymph nodes (mediastinal) in 1 female
• Minimal to moderate numbers of foamy macrophages in the lungs in 3 male and 4 female animals
• Minimal cellular debris in the lungs in 1 male and 2 female animals
• Minimal infiltration of neutrophils of alveoli of the lungs in 1 male and 2 female animals
• Minimal hyperplasia of type II pneumocytes in 3 females


Test group 2 (2 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium in one male animal


Test group 1 (0.5 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium in 1 female



Conclusion for adult animals exposed to test item 1 (Zinc oxide T0420):
Inhalation exposure to Zinc oxide T0420 caused changes in lung, lung-draining lymph nodes and nasal cavity at the high concentration of 10 mg/m³. These findings were almost, though not completely resolved during the post-exposure observation period. At 2 mg/m³, minimal degeneration/regeneration in the nasal cavity was noted in one male animal, and at 0.5 mg/m³ in one female animal. Due to findings in nasal cavity, the NOAEC for local toxicity at the respiratory tract was 0.5 mg/m³ for male rats. a No Observed Adverse Effect Concentration (NOAEC) for local toxicity for females could not be unequivocally determined.


No systemic toxicity was observed in hematology, clinical chemistry and histopathology the NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity was 10 mg/m³ for Zinc oxide T0420.



Test item 2 (Zinc oxide T0421)
Test group 6 (10 mg/m³)
• Decreased food consumption during gestation and lactation of parental females
• Decreased body weights/body weight gain during gestation and lactation of parental females
• Increased total white blood cell (WBC) as well as absolute neutrophil and lymphocyte counts in blood of males
• Slightly increased total cell counts as well as absolute and relative lymphocyte, neutrophil cell and monocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute eosinophil cell counts in males in BAL
• Increased total protein levels lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) activities in BAL of both sexes
• Increased  γ-Glutamyl-transferase (GGT) activity in BAL of males
• Increase of absolute/relative lung weights in males (136%/143%) and females (131%/137%)
• Macroscopically observed white foci in the lungs of 6 males and 8 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial, highest number is given) in 10 males and 5 females
• Minimal to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to severe cellular debris in the lungs in all male and all female animals
• Minimal to slight infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 8 males and 9 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes (exemplarily) in 8 males and 3 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes (exemplarily) in 4 males and 2 females
• Minimal to slight degeneration/regeneration of the olfactory epithelium 

Test group 26 (Recovery group R1, 10 mg/m³)
• No treatment-related adverse findings in lavage and histopathology

Test group 5 (2 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium 

Test group 4 (0.5 mg/m³)
No treatment-related adverse findings


Conclusion for adult animals exposed to test item 2 (Zinc oxide T0421):
Inhalation exposure to Zinc oxide T0421 caused changes several lavage parameters, as well as histological changes in lung, lung-draining lymph nodes and nasal cavity at the highest tested concentration of 10 mg/m³. All these effects were completely resolved after the postexposure observation period. In blood, increased neutrophils and lymphocyte was notice at the concentration of 10 mg/m³, which is considered secondary to the inflammation in the lung.
At the mid concentration of 2 mg/m³, histological findings were still observed in the nasal cavity of three male and two female rats. Thus, the No Observed Adverse Effect Concentration (NOAEC) for local toxicity was 0.5 mg/m³ under the current study conditions. 
Besides the increased neutrophils and lymphocytes in blood, no other changes were observed in hematology, clinical chemistry. No histopathological changes were observed in any organs and tissues that are not part of the respiratory tract. The NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity, that were not attributed to the local effect, was 10 mg/m³ for Zinc oxide T0421.



Reference item 1 (Zinc oxide T0242)
Test group 7 (10 mg/m³)
• Retarded body weight development in male animals
• Increased total cell counts as well as absolute and relative neutrophil cell and monocyte counts in BAL of both sexes
• Increased absolute lymphocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute macrophage and eosinophil cell counts in BAL of males
• Increased total protein levels lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and γ-Glutamyl-transferase (GGT) activities in BAL of both sexes
• Increased β-N-Acetyl glucosaminidase (NAG) activity in BAL of males
• Increase of absolute/relative lung weights in males (130%/141%) and females (137%/141%)
• Macroscopically observed white foci in the lungs of 5 males and 8 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial, highest number is given) in 7 males and 10 females
• Minimal to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to severe cellular debris in the lungs in all male and all female animals
• Minimal to moderate infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 6 males and 8 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes (exemplarily) in 4 males and 6 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes (exemplarily) in 6 males and 8 females
• Minimal degeneration/regeneration of the olfactory epithelium 


Test group 27 (Recovery group R1, 10 mg/m³)
• Macroscopically observed white foci in the lungs of 3 males and 3 females
• Minimal to slight numbers of foamy macrophages in the lungs in 2 male and 4 female animals
• Minimal cellular debris in the lungs in 1 male animal
• Minimal infiltration of neutrophils of lung alveoli in 1 male animal
• Minimal hyperplasia of type II pneumocytes in 4 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes in 2 males and 3 females


Conclusion for adult animals exposed to reference item 1 (Zinc oxide T0242):
Inhalation exposure to Zinc oxide T0242 caused changes in lung, lung-draining lymph nodes and nasal cavity at the highest tested concentration of 10 mg/m³. These findings were greatly, though not completely, resolved during the post-exposure observation period.
No systemic toxicity was observed in hematology, clinical chemistry and histopathology the NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity was 10 mg/m³ for Zinc oxide T0242.


Reference item 2 (Zinc sulfate monohydrate)
Test group 8 (22 mg/m³)
• During exposure period, salivation and respiration sounds were detected in several male and female animals.
• Retarded body weight development in all male and female animals. 
• Decreased food consumption during gestation and lactation of parental female animals
• Recreased body weights/body weight gain during gestation and lactation of parental female animals
• Increased total cell counts as well as absolute and relative lymphocyte, neutrophil cell and monocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute macrophage and eosinophil cell counts in BAL of males
• Increased total protein levels lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and γ-Glutamyl-transferase (GGT) activities in BAL of both sexes
• Increase of absolute/relative lung weights in males (125%/138%) and females (114%/119%)
• Macroscopically observed white foci in the lungs of 3 males and 7 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial, highest number is given) in 5 males and 8 females
• Erosion/ulcer of the laryngeal epithelium at the base of the epiglottis in 1 female
• Minimal to slight squamous metaplasia of the laryngeal epithelium at the base of the epiglottis in all males and all females
• Minimal to slight inflammatory cell infiltrates of the laryngeal epithelium in 1 male and 9 females
• Minimal to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to moderate cellular debris in the lungs in all male and all female animals
• Minimal to slight infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 6 males and 8 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes (exemplarily) in 5 males and 5 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes (exemplarily) in 8 males and 8 females
• Minimal to moderate degeneration/regeneration of the olfactory epithelium in all males and all females

Test group 28 (Recovery group R1: 22 mg/m³)
• Macroscopically observed white foci in the lungs of 2 males and 2 females
• Macroscopically enlarged draining lymph nodes (mediastinal) in 1 male and 1 female
• Minimal squamous metaplasia of the laryngeal epithelium at the base of the epiglottis in 1 male and 1 female animal
• Minimal to slight inflammatory cell infiltrates in the laryngeal epithelium in 4 males and 3 females
• Minimal to slight numbers of foamy macrophages in the lungs in 2 male and 4 female animals
• Minimal hyperplasia of type II pneumocytes in 3 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes in 1 male and 1 female animal
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes in 4 males and 4 females
• Minimal degeneration/regeneration of the olfactory epithelium in 1 male and 1 female 


Conclusion for adult animals exposed to reference item 2 (Zinc sulfate monohydrate):
Inhalation exposure to Zinc sulfate monohydrate caused changes in lung, lung-draining lymph nodes, larynx and nasal cavity at the highest tested concentration of 22 mg/m³. These findings were partly resolved during the post-exposure observation period.
No systemic toxicity was observed in hematology, clinical chemistry and histopathology the NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity was 22 mg/m³ for Zinc sulfate monohydrate.


Overall assessment for adult animals:


With regards to systemic toxicity, none of the test or reference substances caused any systemic toxicity that were not triggered by the local toxicity.


Comparing the local effects of the two nano Zinc oxide materials, the overall finding in the lungs, mediastinal lymph nodes, in the nasal cavity were comparable at the tested concentrations, as well as the changes of lavage parameters. The small differences are considered biological variations. There were no considerable differences between the effects caused by zinc oxide nanoparticles and those caused by micron-size zinc oxide particle.


For reference substance 2 (zinc sulfate monohydrate), lower incidence and severity was found in the lungs than in the other zinc oxide treated groups, but higher incidence and severity in nasal cavity and larynx. This difference is considered being related to the different deposition pattern, caused by the different aerodynamic diameter. The aerodynamic diameter of zinc sulfate monohydrate was larger than the different types of zinc oxide. The mean MMAD of zinc sulfate monohydrate was with 2.3 µm considerably higher than those measured at the high concentrations of the test items 1 (1.19 µm) and 2 (0.97 µm). The deposited dose at the upper respiratory tract was higher, while those deposited in the lung was lower.


After the recovery period, all parameters in lavage fluid returned to the control level in all animals, irrespective of the exposed test and reference substance. With regards of histological findings in the respiratory tract, all changes reduced greatly in incidence and severity. Only single animals showed still some mild effects.


PUPS on PND 22 
Test item 1 (Zinc oxide T0420)
Test group 3 (10 mg/m³)
Minimal cellular debris in the lungs in 2 male and 4 female animals
• Minimal infiltration of neutrophils of alveoli of the lungs in 1 male and 2 female animals
Test group 2 (2 mg/m³) and test group 1 (0.5 mg/m³)
No treatment-related adverse findings observed
Conclusion for pups exposed to test item 1 (Zinc oxide T0420) sacrificed on PND 22:
Inhalation exposure to Zinc oxide T0420 caused minimal cellular debris and neutrophilic infiltration in a few male and female animals at 10 mg/m³. These findings were considered treatment-related and adverse. The NOAEC for local toxicity was 2 mg/m³. 


There were no test-item related adverse findings in clinical examination/OFO/MA and gross necropsy up to the highest tested concentration of 10 mg/m³. No systemic toxicity was observed in hematology, clinical chemistry, no changes of thyroid hormones. There were no histopathological changes in organs and tissues other than those of the respiratory tract.



Test item 2 (Zinc oxide T0421)
Test group 6 (10 mg/m³)
• Minimal cellular debris in the lungs in 1 male and 4 female animals
• Minimal infiltration of neutrophils of alveoli of the lungs in 1 male and 1 female animals
• Minimal to moderate degeneration/regeneration of the olfactory epithelium (nasal cavity, level III) in 4 male and 2 female animals
Test group 5 (2 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium (nasal cavity, level III) in 2 female animals
Test group 4 (0.5 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium (nasal cavity, level III) in 1 male animal 


Conclusion for pups exposed to test item 2 (Zinc oxide T0421) sacrificed on PND 22:
Inhalation exposure to Zinc oxide T0421 caused minimal cellular debris, neutrophilic infiltration in the lungs and minimal to moderate degeneration/regeneration of the olfactory epithelium in nasal cavity in a few male and female animals at 10 mg/m³. The changes in nasal cavity was still observed at the mid concentration of 2 mg/m³. This findings were considered treatmentrelated and adverse. The NOAEC for local toxicity was 0.5 mg/m³.


There were no test-item related adverse findings in clinical examination/OFO/MA and gross necropsy up to the highest tested concentration of 10 mg/m³. No systemic toxicity was observed in hematology, clinical chemistry, no changes in thyroid hormones. There were no histopathological changes in organs and tissues other than those of the respiratory tract.



Reference item 1 (Zinc oxide T0242)
Test group 7 (10 mg/m³)
• Minimal cellular debris in the lungs in 5 male and 5 female animals
• Minimal infiltration of neutrophils of lung alveoli in 2 male and 3 female animals
• Minimal degeneration/regeneration of the olfactory epithelium (nasal cavity, level III) in 1 male and 1 female


Conclusion for pups exposed to reference item 1 (Zinc oxide T0242) sacrificed on PND 22:
Inhalation exposure to Zinc oxide T0242 caused minimal cellular debris, neutrophilic infiltration in the lungs and minimal degeneration/regeneration of the olfactory epithelium in nasal cavity in a one male and one female animals at 10 mg/m³.


There were no test-item related adverse findings in clinical examination/OFO/MA and gross necropsy up to the highest tested concentration of 10 mg/m³. No systemic toxicity was observed in hematology, clinical chemistry, no changes in thyroid hormones. There were no histopathological changes in organs and tissues other than those of the respiratory tract.


Reference item 2 (Zinc sulfate monohydrate)
Test group 8 (22 mg/m³)
• Minimal cellular debris in the lungs in 2 male and 1 female animals
• Minimal infiltration of neutrophils of lung alveoli in 2 male and 1 female animals
• Minimal degeneration/regeneration of the olfactory epithelium (nasal cavity, level III) in 1 male and 1 female


Conclusion for pups exposed to reference item 2 (zinc sulfate monohydrate) sacrificed on PND 22:
Inhalation exposure to zinc sulfate monhydrate caused minimal cellular debris, neutrophilic infiltration in the lungs and minimal degeneration/regeneration of the olfactory epithelium in nasal cavity in a few male and female animals at 22 mg/m³.


There were no test-item related adverse findings in clinical examination/OFO/MA and gross necropsy up to the highest tested concentration of 10 mg/m³. No systemic toxicity was observed in hematology, clinical chemistry, no changes in thyroid hormones. There were no histopathological changes in organs and tissues other than those of the respiratory tract.


Overall assessment for PND 22 animals:
With regards to effects observed in PND 22 pups that were exposed whole-body to zinc oxide nanomaterials from PND 4 to PND 21, the findings were limited to lungs and nasal cavities. The effects were observed also in the parental animals with much higher incidence and severity than in the pups exposed at the same concentration. This could be explained by the duration of the exposure, because pups were only exposed for 17 days, while the adult animals were exposed for 90-days.


Comparing the toxicity of the two nano Zinc oxide materials in pups of PND 22, the overall finding in the lungs were comparable at the high concentration of 10 mg/m³ for test items 1 and 2. However, lesions were also observed in nasal cavity in pups exposed to test item 2. While lesions in nasal cavity were still observed at 0.5 mg/m³ test item 2, there were no effect observed in animals exposed to 2 mg/m³ and 0.5 mg/m³ test item 1.


Comparing the toxicity caused by the two nano zinc oxide materials with those caused by the microscale zinc oxide material, or by the soluble zinc sulfate monohydrate, the changes in lungs were comparable in incidence and severity in exposed pups on PND 22. However, no nasal cavity lesions were observed in animals exposed to test item 1, while similar lesions were seen in those exposed to test item 2 and the reference items.


None of the substances cause any systemic toxicity, nor were there any developmental neurotoxicity in exposed pups.