Lithium Nickel Cobalt Aluminium Oxide

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 June 2018 to 15 March 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (90-Day (Subchronic) Inhalation Toxicity Study

Version / remarks:

adopted 09 October 2017

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.29 (Sub-Chronic Inhalation Toxicity:90-Day Study)

Version / remarks:

No. L 81

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The analysis of lung burden was not performed in compliance with GLP.

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

aluminium(3+) nickel(2+) λ²-cobalt(2+) lithium(1+) tetraoxidandiide

EC Number:

700-042-6

Cas Number:

177997-13-6

Molecular formula:

LiNiCoAlO2 where stoichiometry of Ni+Co+Al equals 1 and ranges are Li = 0.9 – 1.3, Co = 0.01 – 0.2, Ni = 0.75 – 0.99 and Al = 0.01 – 0.2

IUPAC Name:

aluminium(3+) nickel(2+) λ²-cobalt(2+) lithium(1+) tetraoxidandiide

Test material form:

solid

Details on test material:

Synonyms: NCA

Storage stability: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.

Physical state/ Appearance: solid/ grey to black

Specific details on test material used for the study:

≥ 99.5 g /100 g
Date of production: 12 Mar 2017
Batch identification: K703217-6
Storage stability: 12 Aug 2019

Test animals

Species:

rat

Strain:

Wistar

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
- Females were nulliparous and non-pregnant
- Age at study initiation: about 7 weeks when supplied
- Housing: in groups of 5 per cage in polysulfone cages (about 2065 cm2). Dust-free wooden bedding was used in this study. For enrichtment wooden gnawing blocks were added.
- Diet: ad libitum, except during exposure (mouse and rat maintenance diet, GLP, 12 mm pellets, Granovit AG, Kaiseraugst, Switzerland))
- Water: ad libitum, except during exposure

ACCLIMATISATION
The animals were delivered and subjected immediately to the acclimatization period in which they were adapted to the surroundings.
In order to accustom the animals to exposure they were treated with supply air under conditions comparable to exposure on three days before start of exposure (pre-exposure period).

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

Administration / exposure

Route of administration:

other: inhalation: dust aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: All measurements of particle size resulted in MMADs between 1.45µm and 2.21 µm with GSDs between 1.80 and 2.27.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Generator system: During exposure of the animals, exposure mixtures (inhalation atmospheres: test substance in air) were generated continuously in such a way that they are as homogeneous and as constant as possible.
Used equipment included: Solid particle generators (Glass vessel with porous glass filter), Glass mixing stages, Glass cyclonic separators, Glass dilution tube, Pneumatic vibrator NCT 2
- Generation procedure: The dust aerosol was generated by passing compressed air through a layer of test substance. The generated dust atmosphere was introduced first into a glass mixing stage and then mixed with conditioned air and passed into the inhalation system via cyclonic separator. Different atmospheric dust concentrations were adjusted by varying the air flow of the compressed air. To achieve the low concentration at 0.2 mg/m³, a part of the so generated atmosphere was replaced by conditioned air

EXPOSURE SYSTEM
The inhalation atmosphere was maintained inside aerodynamic exposure systems (INA 60, volume V ≈ 90 L, BASF SE) consisting of a cylindrical inhalation chamber made of stainless steel sheeting and cone-shaped outlets and inlets. The rats were restrained in glass exposure tubes. Their snouts projected into the inhalation chamber and thus they inhaled the aerosol.
The exposure systems were located in exhaust hoods in an air-conditioned room.

A positive pressure was maintained inside the exposure systems by adjusting the air flow of the exhaust air system. This ensured that the aerosol in the breathing zones of the animals was not diluted by laboratory air.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

TEST ATMOSPHERE - Brief description of analytical methods used:
> The nominal concentration was calculated from the study means of the test substance rates and the supply air flows used during exposure to generate the respective concentrations.
> For the analytical determination of concentrations, the concentrations of the inhalation atmospheres were determined by gravimetry. In test group 1, due to the low target concentration the duration of one sample took 4 or 5 consecutive days (5.5 hours per day). In test group 2, only one sample per day (5.5 h/day) was
feasible as the target concentration was only 0.5 mg/m³. In test group 3, three samples per day were taken, based on which daily means were calculated. From the daily mean values of test group 3, mean concentrations and standard deviations for the entire study were derived. In these groups, the constancy of concentrations in each chamber was continuously monitored using scattered light photometers. In the control group (test group 0) no sample was taken.
> Sampling for gravimetrix analyses used the following equipment and sampling technique:
Internal probe diameter: 7 mm
Filter: MN 85/90 BF (d = 4.7 cm)
Vacuum pump (Millipore Corporation)
Balance: Sartorius MSA 6.6S-000-DF (Sartorius AG)
Sampling velocity: 1.25 m/s
Flow rate of sampling: 3 L/min
Sample volumes: Test group 1: 2700 to 4500 L; Test group 2: 990L; Test group 3: 210 L
Sampling site: immediately adjacent to the animals' noses at a separate spare port
Sampling frequency: 1 sample per week for test group 1; 1 sample per day for test group 2; 3 samples per day for test group 3
> In the gravimetrical analysis, a preweighed filter was placed into the filtration equipment. By means of a vacuum compressed air pump a defined volume of the dust aerosol was drawn through the filter. The dust concentration in mg/m³ was calculated from the difference between the weight of the preweighed filter and the weight of the filter after sampling, with reference to the sample volume of the inhalation atmosphere.
> Real time monitoring of constancy of concentrations was carried out to continuously monitor the constancy of concentrations of test substance aerosols in the inhalation systems using scattered light photometers (VisGuard). To this end the inhalation atmosphere was continuously sampled by the measuring devices. The measurements were recorded using line recorders and transferred to the automated measuring system.
> Paricle size analysis was carried out with a cascade impactor using the following equipment and sampling method:
Stack sampler Marple 298 (New Star Environmental, Inc.)
Vacuum compressed air pump (Millipore Corporation)
Limiting orifice 3 L/min (Millipore Corporation)
Sampling probe internal diameter 7 mm
Balance Sartorius MSA-6.6S-000-DF (Sartorius AG)
> In the sampling for particle size analysis, preweighed metal collecting discs and a backup particle filter were placed into the cascade impactor and one sample per week was taken in each concentration at a sampling velocity of 1.25 m/sec. from the breathing zones of the animals.
Sample numbers and volumes were: Test group 1: 2 samples, sample volume 3960 L; Test group 1: 12 samples, sample volume 4950 L; Test group 2: 14 samples, sample volume 1800 L; Test group 3: 14 samples, sample volume 360 L.
> The amount of dust deposited by each stage in mg was calculated from the difference between the weight of metal collecting disc and backup filter before and after sampling.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours per day, on 5 days per week (workdays). The total number of exposures was 65.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations: 0, 0.1, 0.5, 2.0 mg/m3 Basis:nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations: 0, 0.11, 0.50, 2.00 mg/m3 (mean measured) Basis: analytical conc.

No. of animals per sex per dose:

MAIN GROUPS
- 10 males/ 10 females: 0, 0.1, 0.5, 2 mg/m3 (histopathology, clinical chemistry, hematology, lavage (only first 5 animals))
- 5 males: 0, 0.1, 0.5, 2 mg/m3 (organ burden)

RECOVERY GROUP:
- 5 males: 0, 0.1, 0.5, 2 mg/m3 (histopathology, clinical chemistry, hematology, lavage)
- 5 males: 0, 0.1, 0.5, 2 mg/m3 (organ burden)

SATTELITE GROUP:
- 5 males: 0, 2 mg/m3 (histopathology, clinical chemistry, hematology, lavage

Control animals:

yes, concurrent vehicle

Details on study design:

DOSE SELECTION RATIONALE
In a previous 5-day range finding study, male Wistar rats were nose-only exposed to 0.2, 0.5, 2 and 10 mg/m³ test substance for 6 hours a day on 5 consecutive days. Main group animals were sacrificed shortly after the last exposure, and bronchoalveolar lavage, hematology and histopathology were examined. Organ burden was also examined in dedicated animals. Moreover, additional control and high concentration group animals were observed for further 3 weeks to detect potential reversibility or progression of the effect.
Adverse effects occurring at 10 mg/m³ comprised the following treatment-related adverse
effects:
Accelerated respiration on study days 4 and 5,
Reduced body weight gain during exposure period,
Increased hemoglobin and hematocrit values in blood,
Increased absolute and relative neutrophil and relative monocyte counts in blood,
Decreased relative monocyte counts in blood,
Increased total cell and absolute and relative neutrophil, monocyte and eosinophil counts in BAL,
Increased absolute lymphocyte and epithelial cell counts in BAL,
Decreased relative macrophage counts in BAL,
Increased total protein levels, lactate dehydrogenase (LDH), alkaline phosphatase (ALP), β-N-Acetyl glucosaminidase (NAG), and γ-Glutamyl transferase (GGT) activities in BAL.
Lungs:
Statistically significant increase of the mean absolute (229%) and relative (239%) weight,
Inflammation, mixed-cellular: in all animals (slight to severe), alveolar, peribronchial, peribronchiolar, and perivascular (polymorphonuclear granulocytes, lymphocytes, and macrophages),
Hypertrophy / hyperplasia, epithelial in all animals (slight to moderate), in bronchi and bronchioles (including terminal bronchioles),
Hyperplasia, type-II pneumocytes in all animals (slight to severe; with atypia in one out of 5 animals),
Edema, alveolar in 2 out of 5 animals (minimal to moderate).
Trachea:
Hypertrophy / hyperplasia, epithelial in 4 out of 5 animals (minimal to slight).
Some of the local effects were still observed at 2 mg/m³ with less severity. The No Observed Effect concentration in the range finding study was 0.5 mg/m³ for local effects at the respiratory tract, and 2 mg/m³ for systemic toxicity. The effects observed at the high concentration of 10 mg/m³ declined within 3 weeks but did not return to the control level.
Based on these data, 10 mg/m³ was considered too high for exposing the animals for 90-days.
Thus, 2 mg/m³ was selected as the highest concentration causing toxic effects. At this concentration, only mild effects in the lung were observed in the range finding study. The mid and low concentrations were set at 0.5 and 0.1 mg/m³, respectively

RATIONALE FOR ANIMAL ASSIGNMENT:
Based on these data, 10 mg/m³ was considered too high for exposing the animals for 90-days.
Thus, 2 mg/m³ was selected as the highest concentration causing toxic effects. At this concentration, only mild effects in the lung were observed in the range finding study. The mid and low concentrations were set at 0.5 and 0.1 mg/m³, respectively.

RATIONALE FOR INCLUSION OF POST-EXPOSURE RECOVERY (SATELLITE) GROUPS:
Recovery group animals (5 males/group) were installed to assess potential reversibility or progression of the effects about 5 weeks after termination of the exposure. Interim sacrifice was performed after 4 weeks exposure in satellite control and high concentration male animals (5 rats/group) and examined by bronchoalveolarlavage (BALF) and histopathology of the lung.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: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.
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 exposure-free weekends and post exposure observation weekends, no clinical observation was performed. Signs and findings were recorded for each animal

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Main group male animals were subjected to detailed clinical observations outside their cages once before the beginning of the administration period (day 0) once on study day 43 and once on study day 85. DCO was performed in main group females once on study day 0 and once on study day 42 and 83. DCO was performed generally in the morning before exposure. For observation, the animals will therefore be removed from their cages and placed in a standard arena (50 x 37.5 x 25 cm). The scope of examinations and the scoring of the findings that are observed will be based on the current index of findings in PDS ToxData® and includes but is not limited to the following parameters listed: abnormal behavior during handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmus, feces (appearance/consistency) , urine, pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: During the exposure period, 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, twice a week as well as one day prior to gross necropsy. As a rule, the animals were weighed at the same time of the day. During the post-exposure observation period the animals will be weighed weekly. During the exposure period, body weight change of the respective week was calculated as the difference between body weight on the last weighing in comparison to that of the first weighing of the same week. In most cases, it was the difference of Friday to the previous Monday. Group means were derived from the individual differences.
During the post-exposure period, the body weight was determined once a week. The body weight change of the post-exposure period is calculated as the difference of the body weight of the respective week and the weight of the week before.

FOOD CONSUMPTION: Yes
- Time schedule: During exposure period, food consumption was determined weekly (e.g. Monday and Friday), and during the post-exposure period on Mondays.
The animals were maintained in social-housing cages, with 5 animals per cage, during the whole study period. Therefore, the food consumption was determined cage-wise. The food consumption per animal and day was calculated by dividing food consumption of the day of a respective cage by the 5 animals per cage. As the animals of each main group were housed in only two cages per sex, and in the recovery group only one cage per sex no statistical evaluation of food consumption is possible.

WATER CONSUMPTION: No

RECTAL TEMPERATURE: Yes
- Time schedule: Measurement of the rectal temperature were carried out in in all main-group animals (male animals No 1 to 40, female animals No. 61 to 100). Using a digital thermometer (Testo) rectal temperatures were determined prior to exposure on study day -4 (male) and study day -6 (female) and against the end of the exposure period (day 84 for female, day 86 for male) in main-group animals. All measurements were performed in the afternoon (shortly after exposure
on exposure days).

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule: Before the start of the exposure period (day- 7/ -5) the eyes of all main group animals, and at the end of the study (day 84/82) the eyes of the animals of test group 0 (control group), test group 3 (high concentration) were examined for any changes in the refracting media with an ophthalmoscope (HEINE Optotechnik, Herrsching, Germany) after administration of a
mydriatic (Mydrum, Chauvin ankerpharm GmbH, Rudolstadt/Berlin, Germany). Males were examined on study days -5 and 84, female on study days -7 and 82.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: blood was taken from the retro-bulbar venous plexus. Carried out in main group, recovery and satellite group after exposure.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 20/group (main group) +5/group (recovery group) +5/group (satellite group) = 30
- Parameters checked included: Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes (RETA), Prothrombin time (Hepato Quick’s test) (HQT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: blood was taken from the retro-bulbar venous plexus. Carried out in main group, recovery and satellite group after exposure.
- Animals fasted: Yes
- How many animals: 20/group (main group) +5/group (recovery group) +5/group (satellite group) = 30
- Parameters checked included: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT), Sodium (NA), Potassium (K), Chloride (CL), Inorganic phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose
(GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once during the 12th week. On the day of FOB the examined animals were not exposed to test substance. male and female main group animals for each test group. The FOB started with passive observations, without disturbing the animals, followed by removal from home cage, and open field observations in a standard arena. Thereafter, sensorimotor tests and reflex tests were conducted.
- Dose groups that were examined: male and female main group animals for each test group
- Home cage observations: The animals were observed in their closed home cages; any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals. Attention was paid to posture, tremor, convulsions, abnormal movements, impairment of gait, other findings.
- Open field observations: The animals were transferred to a standard arena (50 x 50 cm with sides of 25 cm high) and observed for at least 2 minutes. Examined parameters included behavior when removed from cage, fur, skin, salivation, nasal discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements/ stereotypies, impairment of gait, activity/arousal level, feces (appearance/consistency) within two minutes, urine (amount/color) within two minutes, number of rearings within two minutes, other findings.
- Battery of functions tested:
> Sensorimotor Tests/Reflexes: The animals were removed from the open field and subjected to sensorimotor or reflex tests including approach response, touch response, vision ("visual placing response"), pupillary reflex, pinna reflex, audition ("startle response"), coordination of movements ("righting response"), behavior during "handling", vocalization, pain perception ("tail pinch"), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test, other findings.
> motor activity measurements: Motor activity was measured on the same day and with the same animals as FOB was performed. The measurement was performed in the dark using the TSE Labmaster System (TSE Systems GmbH) with 18 infrared beams per cage. During the measurement the animals were kept in Polycarbonate cages with absorbent material. The animals were put into the cages in a randomized order. The measurements started at about 14:00 p.m. The numbers of beam interrupts were counted over 12 intervals, each lasting
5 minutes. Measurement did not commence at the same instant for all cages; the period of assessment for each animal started when the first beam was interrupted by pushing the cage into the rack (staggered start). Measurements ended exactly 60 minutes thereafter. During the measurements the animals received no food and no water.

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: The animals designated for lung lavage were killed by exsanguination from aorta abdominalis and vena cava under Narcoren anesthesia. The lung was lavaged by two instillations of physiologic saline
- Dose groups that were examined: all
- Number of animals: 10/group (main group) +5/group (recovery group) +5/group (satellite group)
- Parameters checked included Total cell count (TCN_BAL), Macrophages (MPH_BAL), Polymorphonuclear neutrophils (PMN_BAL), Lymphocytes (LY_BAL), Eosinophils (EO_BBAL), Monocytes (MO_BAL), Epithelial (EPI_BAL), γ−Glutamyltransferase (GGT BAL_C), Protein (MTP BAL), Lactate dehydrogenase (LDH BAL), Alkaline phosphatase (ALP BAL), N-acetyl-β-Glucosaminidase (NAG BAL).

LUNG BURDEN: Yes
- Time schedule for analysis: The animals intended for determination of organ burden were killed under pentobarbitone anesthesia by exsanguination from the abdominal aorta and vena cava. Prior to sacrificing, blood were sampled. The following organs and tissues were weighed and packed individually in plastic vessels and stored in a freezer (- 20 °C) until analysis
- Dose groups that were examined: all
- Number of animals: 5/group (main group) + 5/group (recovery group)
- Lungs were examined for content of Al, Li, Co and Ni

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The animals were sacrificed under pentobarbitone anesthesia by exsanguination from the abdominal vessels (aorta and vena cava). Afterwards, the thorax was opened, the right lung lobes were lavaged, whereas the left lung lobe was ligated during lavage. After the lavage procedure, the left and right lung lobes were instilled with formalin for weight assessment and further histopathological assessment. Immediately after lung lavage, the animals were necropsied and assessed by gross pathology.

ORGAN WEIGHTS: YES
The following weights were determined in all animals sacrificed on schedule: Anesthetized animals (terminal body weight), Adrenal glands (fixed), Brain, Epididymides, Heart, Kidneys, Liver, Lungs, Ovaries (fixed), Spleen, Testes, Thymus (fixed), Thyroid glands (with parathyroid glands) (fixed), Uterus with cervix.

ORGAN/TISSUE FIXATION: YES
The following organs or tissues were fixed in 4% neutral buffered formaldehyde solution or in modified Davidson’s solution: All gross lesions, Adrenal glands, Aorta, Bone marrow (femur), Brain with olfactory bulb, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Epididymides, Esophagus, Extraorbital lacrimal gland, Eyes with optic nerve (modified Davidson’s solution), Eyelids, Femur with knee joint, Harderian glands, Heart, Ileum, Jejunum, Kidneys, Larynx, Liver, Lungs, Lymph nodes (tracheobronchial, mediastinal and mesenteric lymph nodes), Mammary gland (male + female), Nose (nasal cavity), Ovaries, Pharynx, Pancreas, Parathyroid glands, Pituitary gland, Prostate, Rectum, Salivary glands (mandibular and sublingual glands), Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Teeth, Testes, Thymus, Thyroid glands, Tongue, Trachea, Ureter, Urethra, Urinary bladder, Uterus, Vagina

HISTOPATHOLOGY: Yes
The organs listed in Table 3-5 were examined by light microscopy.

Statistics:

Means, medians and standard deviations of each test group were calculated for several parameters:
- Body weight, body weight change: Comparison of each group with the control group was performed using DUNNETT test (two-sided) for the hypothesis of equal means.
- Feces, rearing, grip strength length forelimbs, grip strength length hindlimbs, footsplay test, motor activity: Non-parametric one-way analysis using the KRUSKAL-WALLIS test (twosided).If the resulting p-value was equal to or less than 0.05, a pair-wise comparison of each dose group with the
control group was performed using WILCOXON test (two-sided) for the hypothesis of equal medians.
- Blood parameters:
> For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (twosided) for the hypothesis of equal medians
> For parameters with unidirectional changes: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians
- Bronchoalveolar 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:
> Main and recovery groups: Non-parametric one-way analysis using KRUSKAL-WALLIS H test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each test group with the control group 0/10 was performed using WILCOXONtest (two-sided) for the equal medians
> Satellite groups: Pairwise comparison of test group 23 with the control group 20 was performed using the WILCOXON test (two-sided)

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No test material-related adverse effects observed

Mortality:

no mortality observed

Description (incidence):

No test material-related adverse effects observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

See "Details on results" for further information

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No test material-related adverse effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

See "Details on results" for further information

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

See "Details on results" for further information

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

See "Details on results" for further information

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No test material-related adverse effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

See "Details on results" for further information

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

See "Details on results" for further information

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See "Details on results" for further information

Other effects:

effects observed, treatment-related

Description (incidence and severity):

BRONCHOALVEOLAR LAVAGE FLUID (BAL)
See "Details on results" for further information

Details on results:

CLINICAL SIGNS AND MORTALITY
- No deaths were recorded throughout the study.
- During the pre-exposure period, the exposure period and the post-exposure observation period the animals showed no clinical signs and findings different from normal.
- Detailed clinical observations did not reveal any differences between animals exposed to test substances and the control animals.

BODY WEIGHT AND WEIGHT GAIN
- Body weight during the exposure period: The mean body weights of the test substance exposed groups were not statistically significantly different from the control group 0.
- Body weight during the post-exposure observation period: The mean body weights of the test substance exposed groups were not statistically significantly different from the control group 10.
- Body weight change during the exposure period:
The body weight changes of the male animals were statistically significantly different from the control groups 0, 10, 20, 30 and 40 in the following time period:
- Group 3 study day 80 to 83: +8.6 g (control group +5.4 g), p ≤ 0.05
- Group 3 study day 83 to 87: -7.9 g (control group -3.7 g), p ≤ 0.05
- Group 13 study day 62 to 66: -7.1 g (control group -0.6 g), p ≤ 0.01
- Group 23 study day 24 to 26: +5.1 g (control group +9.3 g), p ≤ 0.05
- Group 33 study day 11 to 15: +5.6 g (control group -1.1 g), p ≤ 0.05
- Group 33 study day 50 to 53: +5.3 g (control group +10.0 g), p ≤ 0.05
- Group 41 study day 88 to 90: -1.5 g (control group -5.6 g), p ≤ 0.05
- The body weight changes of the main group female animals were statistically significantly different from the control groups 0 in in the following time period:
- Group 2 study day 25 to 29: -0.2 g (control group -2.4 g), p ≤ 0.05
- Group 3 study day 25 to 29: 0.0 g (control group -2.4 g), p ≤ 0.05
- Group 3 study day 46 to 50: -0.7 g (control group -3.2 g), p ≤ 0.05
- During the recovery period, statistically significant changes were observed in the following group:
- Group 13 study day 118 to 125: +2.2 g (control group + 9.7), p ≤ 0.05
- Some of the above listed deviations were higher than the concurrent control group, some were lower. They were not consistent and of transient nature. Thus, these changes were considered incidental and not biologically relevant.

FOOD CONSUMPTION
No substance-related changes of food consumption were observed during the whole study period.

RECTAL TEMPERATURE
- On study day -4 the mean rectal temperature of male animals of the following groups were lower than the concurrent control group (male, test group 0, 38.3 °C): Test group 1 (37.1, p ≤ 0.01), Test group 2 (37.5, p ≤ 0.01), Test group 3 (37.2, p ≤ 0.01)
- On study day -6, the mean rectal temperature of female animals of test group 3 was higher than the concurrent control group (female, test group 0, 37.9 °C): Test group 3 (38.6 °C, p ≤ 0.05)
- On study day 84, the mean rectal temperature of female animals of test group 3 was higher than the concurrent control group (female, test group 0, 38.2 °C): Test group 3 (38.8 °C, p ≤ 0.01)
- On study day 86, the mean rectal temperature of male animals of test group was lower than the concurrent control group (female, test group 0, 37.8 °C): Test group 1 (37.0 °C, (p ≤ 0.05)
- The changed rectal temperatures on days -4 and -6 were considered incidental, because these values were determined before exposure. The high rectal temperature of female test group 3 animals on study day 84 was considered not biologically relevant, because it was only minimally higher than the mean value of the same animals on study day -6 (38.6 °C) before exposure.

OPHTHALMOSCOPIC EXAMINATION
The ophthalmologic examinations did not show any substance-related impairment of the refracting media. Corneal stippling was observed in several animals of all test groups and the control group without any concentration-response relationship. The individual ophthalmologic findings are archived with the raw data.

FUNCTIONAL OBSERVATION BATTERY
On the day of the performance of the Functional Observation Battery, the animals were not exposed to the test substances.
Deviations from "zero values" were obtained in several animals. However, as most findings were equally distributed between treated groups and controls, were without a dose-response relationship or occurred in single animals only, these observations were considered incidental.
Besides this, the following findings were observed and have to be assessed individually:
Observations on day 77 (females) and 78 (males):
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.

MOTOR ACTIVITY
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, increased values were seen in the male animals of test group 3 at interval 12 on study day 78. No other abnormalities were detected. The higher activity in one isolated interval in only one sex was considered incidental.

HAEMATOLOGY
- No treatment-related changes among hematological parameters were observed
- After the administration period at study day 92, in females of test groups 1, 2 and 3 (0.1, 0.5 and 2 mg/m3) prothrombin time (Hepatoquick’s test, HQT) was significantly prolonged, but the values were within the historical control range (females, HQT 32.5-36.3 sec).
- After the 5-week recovery period at study day 126, in males of test group 13 (2 mg/m3) prothrombin time (HQT) was significantly prolonged, but the mean was within the historical control range (males, HQT 35.0-40.2 sec).
- Therefore, the altered prothrombin time values were regarded as incidental and not treatment related.

CLINICAL CHEMISTRY
- No treatment-related changes among clinical chemistry parameters were observed.
- After the administration period, in males of test group 3 (2 mg/m3) potassium values were significantly decreased. The mean was slightly below the historical control range (males, potassium 4.52-5.04 mmol/L). However, this was the only changed clinical pathology blood parameter among these individuals and therefore, this alteration was regarded as maybe treatment-related but not adverse (ECETOC Technical Report No 85, 2002). In females of test group 2 (0.5 mg/m3) creatinine levels were significantly increased. However, the change was not dose-dependent and therefore, this change was regarded as incidental and not treatment-related.
- After the administration period, in males of test group 3 (2 mg/m3) potassium values were significantly decreased. The mean was slightly below the historical control range (males, potassium 4.52-5.04 mmol/L). However, this was the only changed clinical pathology blood parameter among these individuals and therefore, this alteration was regarded as maybe treatment-related but not adverse (ECETOC Technical Report No 85, 2002). In females of test group 2 (0.5 mg/m3) creatinine levels were significantly increased. However, the change was not dose-dependent and therefore, this change was regarded as incidental and not treatment related.

BRONCHOALVEOLAR LAVAGE FLUID (BAL)
- After a 5-week administration period (study day 27) in the BALF of males in test group 23 (2 mg/m3) a significant increase of the total cell counts occurred, which was mainly due to a significant increase of absolute and relative neutrophil counts and at a lesser degree increased absolute and relative lymphocyte and monocyte counts. These changes were regarded as treatment-related and adverse. Although absolute macrophage counts were statistically significantly increased whereas relative macrophage counts were significantly decreased, these changes were not regarded as toxicological relevant (absolute macrophage counts < 10fold increased).
- At the end of the administration period at study day 92 in the BALF of males in test groups 1, 2 and 3 (0.1, 0.5 and 2 mg/m3), total cell counts as well as absolute neutrophil, lymphocyte and monocyte cell counts were significantly increased (absolute lymphocyte counts not completely dose-dependent). Absolute macrophage counts were significantly higher at test groups 2 and 3. These alterations were regarded as treatment-related and adverse. Relative macrophage counts were significantly decreased whereas relative neutrophil cell, lymphocyte and monocyte counts were significantly increased in test groups 1, 2 and 3. The relative cell counts were not dose-dependently altered due to the very high changes of all absolute cell fractions. However, due to the mentioned absolute cell count changes, the corresponding findings of the relative cell counts were also regarded as treatment-related and adverse.
- After the administration period at study day 92 in females of test group 3 (2 mg/m3) only one BALF cytology could be evaluated, because of bad cell quality on the slides of the other individuals. Therefore, statistical evaluations of test group 3 could not be performed. However, correspondingly to the males, in the BALF of females in test groups 1, 2 and 3 (0.1, 0.5 and 2 mg/m3) total cell counts as well as absolute neutrophil cell, lymphocyte and monocyte counts were increased (absolute lymphocyte counts not dose-dependent). Absolute macrophage cell counts in females were also increased in all mentioned test groups. These changes were regarded as treatment-related and adverse. Relative neutrophil, lymphocyte and monocyte cell counts were correspondingly increased in females of test groups 1, 2 and 3 (relative lymphocyte and monocyte counts not dose-dependently), whereas relative macrophage counts were decreased in the mentioned test groups. As with the BALF of males, adversity of the relative cell counts in females was regarded in combination to the altered absolute cell counts.
- After a 5-week recovery period at study day 126, some relevant alterations among the BALF cytology values in males occurred in comparison to the end of the administration period: absolute neutrophil cell count increases were significantly reduced. After the recovery period they were significantly higher only in test groups 12 and 13 (0.5 and 2 mg/m3) compared to the corresponding control. Total cell and absolute lymphocyte and monocyte counts were still increased after the recovery in test groups 11, 12 and 13 (0.1, 0.5 and 2 mg/m3, monocyte counts in test group 11 not statistically significantly), but the increase of all mentioned cell counts was reduced in test group 11, at the same level for total cell and monocyte counts in test groups 12 and 13, and even higher for lymphocytes in test groups 12 and 13 compared to the corresponding test groups after the administration period. After the recovery, absolute macrophage counts were significantly increased in test groups 11, 12 and 13, which was a higher increase compared to the values after the administration period. The mentioned alterations after the recovery were regarded as treatment-related and adverse. The weakly significant increase of absolute epithelial cell counts in test group 12 (0.5 mg/m3) was regarded as incidental and not treatment-related due to the lack of dose-dependency. After the recovery period, relative lymphocyte and monocyte counts were significantly increased in test groups 12 and 13 (0.5 and 2 mg/m3; relative monocyte counts not dose-dependently), which was regarded as treatment-related and adverse in combination with the corresponding absolute cell count changes
- After a 5-week administration period (study day 27) in the BALF of males in test group 23 (2 mg/m3), total protein values and lactate dehydrogenase (LDH), β-N-Acetyl glucosaminidase (NAG) and γ-Glutamyl transferase (GGT) activities in BALF were significantly increased. These changes were regarded as treatment-related and adverse.
After the administration period (study day 92) in the BALF of males in test groups 1, 2 and 3 (0.1, 0.5 and 2 mg/m3) total protein values and lactate dehydrogenase (LDH), β-N-Acetyl glucosaminidase (NAG) and γ-Glutamyl transferase (GGT) activities in BALF were significantly increased. This was regarded as treatment-related and adverse. Alkaline phosphatase (ALP) activities in BALF were significantly increased in males of test groups 1 and 2 (0.1 and 0.5 mg/m3), but the increase was not dose-dependent and therefore this alteration was regarded as incidental and not treatment-related.
- After the administration period (study day 92) in the BALF of females corresponding changes to those in males were observed: in test groups 1, 2 and 3 (0.1, 0.5 and 2 mg/m3) significant increases of total protein values and lactate dehydrogenase (LDH), β-N-Acetyl glucosaminidase (NAG) and γ-Glutamyl transferase (GGT) activities, which were regarded as treatment-related and adverse, although NAG activities were not completely dose-dependently changed. Alkaline phosphatase (ALP) activities were also significantly changed in all mentioned test groups, but here dose-dependency was clearly lacking, and the increase was marginal (< 3fold). Therefore, the ALP activity increase in females was regarded as incidental and not treatment-related correspondently to the males.

NEUROBEHAVIOUR
Deviations from "zero values" were obtained in several animals. However, as most findings were equally distributed between treated groups and controls, were without a dose-response relationship or occurred in single animals only, these observations were considered incidental.

ORGAN BURDEN:
In 4 of the high concentration animals Al content in lungs was above the limit of the quantification of 1 µg per lung. Li could only be quantified in test group 3 animals.
Considering the fractions of respective metal ion in the test substance (analysis of the test substance, Part III), the average lung burden in test group 1 was calculated to be between 3.8 µg (based on Co) to 9.4 µg (based on Ni). This amount is considerably lower than the expected one. The average lung burdens of test group 2 were between 6.4 and 14.3 µg, based on Co and Ni, respectively. Based on Co, the average lung burden of test group 3 was 26.4 µg per lung, 8.4 µg based on Li and 53.5 µg based on Ni.

ORGAN WEIGHTS (Main group)
> Absolute weights
When compared to the control group 0 (set to 100 %), the mean absolute weights of the lung were significantly increased in all test groups. The mean absolute weights of the adrenal glands was significantly increased in test group 2 and 3 of the females. All other mean absolute weight parameters did not show significant differences when compared to the control group 0.
> Relative organ weights
When compared to the control group 0 (set to 100 %), the mean relative weights of the lung were significantly increased in all test groups. The mean relative weights of the liver was significantly increased in test group 1 of the females. All other mean relative weight parameters did not show significant differences when compared to the control group 0.
The significant increase of the mean absolute and relative weights of the lungs in male and female animals of all treatment groups was concentration-related and were regarded as substance-related. These changes were consistent with histopathological findings.
The significant mean absolute weight increases of the adrenal glands in female animals of test groups 2 and 3 (76.5 and 71.8 mg, respectively) were within the historical control range values (70.500 – 82.000 mg) and the decreased mean relative liver weight in females of test group 1 did not show concentration-response relationship. Therefore, these weight changes were assessed as incidental.

ORGAN WEIGHTS (Recovery group)
> Absolute weights
When compared to the control group 0 (set to 100 %), the mean absolute weights of the lungs were significantly increased in test group 12 and 13 of the males. The mean absolute weights of the thyroid glands were significantly decreased in test group 11 and 12 of the males. All other mean absolute weight parameters did not show significant differences when compared to the control group 10.
> Relative organ weights
When compared with control group 10 (=100%), the mean relative lung weights were significantly increased in all test groups. All other mean relative weight parameters did not show significant differences when compared to the control group 10.
The concentration-related increase of the lungs weights in all treatment groups was considered treatment-related and had a histopathological correlate.
As the changes of the absolute thyroid weight had no concentration-related response, they were assessed as incidental.

ORGAN WEIGHTS (Satellite group)
> Absolute weights
When compared with control group 20 (=100%), the mean absolute weights of the lungs were significantly increased in males.
> Relative organ weights
When compared with control group 20 (=100%), the mean relative weights of the brain, kidney and spleen were significantly decreased in males and the mean relative weights of the lungs was significantly increased in males.
The increased mean absolute and relative lungs weights were considered to be treatment-related.
Because the relative weight decrease of brain, kidneys, and spleen was of low statistical significance and these organs did not show weight changes or histopathological findings in the other sacrifice groups, the decreased relative weight of these organs was regarded as incidental.
All other mean absolute and relative weight parameters did not show significant differences when compared to the control group 20.

GROSS PATHOLOGY (Main group)
The following gross lesions were regarded as treatment-related:
- foci were noted on the lung of animals of all treated test groups
- enlarged tracheobronchial lymph nodes were noted in animals of all test groups.
The foci were white and randomly scattered throughout all lobes (few to more than 10 in number). Their diameter varied from approximately 3 to 6 mm in diameter. All other findings occurred individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment

GROSS PATHOLOGY (Recovery group)
The following gross lesions were regarded as treatment-related:
- Discoloration of the lungs occurred in males in test group 11 and 13
- Foci were noted on the lungs of males in test group 11, 12 and 13.
- Enlarged tracheobronchial lymph nodes were noted in animals in test groups 11, 12 and 13.
The discolorations as well as the foci were white and randomly scattered throughout all lobes. All other findings were single or few observations. They were considered to be incidental or spontaneous in origin and without any relation to treatment

GROSS PATHOLOGY (Satellite group)
There were no treatment-related gross lesions.

HISTOPATHOLOGY (Main group)
Treatment-related findings were observed in the larynx (level I), lungs and tracheobronchial and mediastinal lymph nodes
> Larynx: Epithelial alteration was visualized at the base of the epiglottis and was characterized by a focal loss of cilia and flattening of the cells (three to four stratified layers)
> Lung: The term histiocytosis was used to designate an increased number of alveolar macrophages, which differed from those of the control groups, because they were markedly enlarged with foamy to granulated cytoplasm. They were observed isolated or forming aggregates, or they were recognized as degenerating forms deprived of nuclei leaving their cytoplasmic granular content in the alveoli. This pale eosinophilic granular material was assessed as eosinophilic granular material. Other eosinophilic alveolar contents were differentiated as proteinosis, characterized by bright eosinophilic, round masses (ranging from 20 to 40 µm in dimeter, presumably derived from serum proteins) and cellular debris characterized by thin (2 μm) strands of eosinophilic tissue interpreted as necrotic alveolar septae, sometimes coiled, admixed with polygonal to round eosinophilic cell – shaped debris. Very few black particles, interpreted most likely as test material, were seen free in the alveoli in areas of degenerating macrophages. Two types of multifocal inflammation were noted: mixed-cellular inflammation composed of neutrophilic granulocytes, lymphocytes and macrophages (with an alveolar, peribronchiolar and perivascular localization) without evident tissue damage and granulomatous inflammation with pyogranulomatous features and evident tissue damage. Other accompanying findings indicative of regeneration were: type II pneumocyte hyperplasia: proliferation of cuboidal cells with foamy to granular cytoplasm and hypertrophy/hyperplasia of bronchiolar epithelium, characterized by increase size and number of bronchiolar cells.
- Lymph nodes: Other than in the lungs, a low incidence and grading of findings were noted in the regional lymph nodes. A minimal lympho-reticulocellular hyperplasia of the paracortex and minimal vacuolation in the high endothelial venules (HEV) was detected in the mediastinal and tracheobronchial lymph nodes in males and females. These findings did not always have a concentration-related response. The vacuolation of the high endothelial venules (HEV) was of macrovesicular type and observable in all test groups without concentration-dependent response.
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.

HISTOPATHOLOGY (Recovery group)
Treatment-related findings were observed in the larynx, lungs and in the mediastinal and tracheobronchial lymph nodes of males
> Larynx: The epithelial alteration at the base of the epiglottis (level I) was still present in all test groups but decreased in incidence when compared to the main group and was not concentration dependent.
> Lung: The findings in the lungs resembled those in the main groups, except for the cellular debris and particles which were not observed after the recovery period.
> Lymph nodes: The mediastinal and tracheobronchial lymph nodes showed both a minimal lympho reticulocellaular hyperplasia in test groups 12 and 13. They also displayed a minimal vacuolation in the high endothelial venules (HEV) of macrovesicular type, however without a clear concentration-dependency.

HISTOPATHOLOGY (Satellite group)
Treatment-related findings were observed in the larynx, lungs and mediastinal and tracheobronchial lymph nodes of males.
> Larynx: Epithelial alteration with the same characteristics as in the main groups was visualized at the base of the epiglottis.
> Lung: After 28 days exposure (20 exposures), the cellular debris within the alveoli was more prominent than the eosinophilic granular material and the histiocytosis. Very few particles, mixed cellular inflammation and hypertrophy/hyperplasia of the terminal bronchioles were minimally visualized.
> Lymph nodes: A minimal lympho-reticulocellular hyperplasia of the paracortex, accompanied by minimal vacuolation in the high endothelial venules (HEV) was detected in the mediastinal and tracheobronchial lymph nodes.

Effect levels

1

Key result

Dose descriptor:

NOAEC

Remarks:

systemic toxicity

Effect level:

2 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no systemic toxicity up to the highest dose tested

2

Dose descriptor:

LOAEC

Remarks:

local toxicity

Effect level:

0.1 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: neoplastic

organ weights and organ / body weight ratios

other: BALF

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

0.1 mg/L air

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Any other information on results incl. tables

Tab 6:Changes in mean absolute cell counts in BAL (x-fold of concurrent control) in male rats on study day27 (satellite group, 1 day after last exposure), study day 92 (main groups, 1 day after last exposure) and after a 5-week recovery period on study day 126.

Analyte	Study day 27	Study day 92			Recovery, Study day 126
	Gr.23 2 mg/m^3	Gr.1 0.1 mg/m^3	Gr.2 0.5 mg/m^3	Gr.3 2 mg/m^3	Gr. 11 0.1 mg/m^3	Gr.12 0.5 mg/m^3	Gr.13 2 mg/m^3
Total Cells	10.3**	28.6**	41.7**	49.4**	10.2*	42.9**	90.6**
Macrophages	2.7**	8.0	14.7**	15.7**	11.2*	54.2**	68.5**
Lymphocytes	118.9**	177.0**	260.1**	210.1**	58.9**	401.7**	1219.5**
Neutrophils	986.5**	929.1**	1221.5**	1581.4**	9.0	30.6*	93.1**
Monocytes	118.1**	342.3**	376.9**	626.9**	33.1	491.7**	439.3**
Eosinophils	++	0.0	0.0	240.0	++	++	++
Epithelial cells	18.5	++	++	++	24.7	84.2*	0.0

Tab 7: Changes in mean absolute cell counts in BAL (x-fold of concurrent control) in female rats on study day 92 (main groups, 1 day after last exposure). 

Analyte	Study day 92
	Gr.1 0.1 mg/m^3	Gr.2 0.5 mg/m^3	Gr.3 2 mg/m^3
Total Cells	24.0**	29.1**	29.1**
Macrophages	9.3**	13.2**	10.7#
Lymphocytes	240.5**	191.2**	253.5#
Neutrophils	1210.0**	1387.2**	1723.7#
Monocytes	589.5**	691.4**	189.6#
Eosinophils	++	++	++#
Epithelial cells	50.3	0.0	0.0#

Tab 8:Changes in mean total protein and enzyme levels in BAL (x-fold of concurrent control) in male rats on study day 27 (satellite group, 1 day after last exposure), study day 92 (main groups, 1 day after last exposure) and after a 5-week recovery period on study day 126

Analyte	Study day 27	Study day 92			Recovery, Study day 126
	Gr.23 2 mg/m^3	Gr.1 0.1 mg/m^3	Gr.2 0.5 mg/m^3	Gr.3 2 mg/m^3	Gr. 11 0.1 mg/m^3	Gr.12 0.5 mg/m^3	Gr.13 2 mg/m^3
Total Protein	13.1**	24.0**	27.5**	33.1**	3.4*	9.7**	39.2**
LDH	32.1**	17.8**	22.0**	41.9**	2.5**	7.1**	29.6**
ALP	0.9	2.2**	1.9**	1.2	0.8	1.5	2.0*
NAG	9.1**	3.2**	3.9**	8.3**	2.0	6.2**	10.7**
GGT	10.5**	4.8**	5.9**	14.8**	1.6	3.1**	7.8**

 

Tab 9:Changes in mean total protein and enzyme levels in BAL (x-fold of concurrent control) in female rats on study day 92 (main groups, 1 day after last exposure)

Analyte	Study day 92
	Gr.1 0.1 mg/m^3	Gr.2 0.5 mg/m^3	Gr.3 2 mg/m^3
Total Protein	23.5**	23.0**	33.8**
LDH	16.5**	18.2**	47.8**
ALP	1.7**	1.8**	1.5**
NAG	2.8**	2.6**	7.2**
GGT	6.6**	7.0**	23.1**

Tab 10: Incidence and gradings of histological changes in larynx

Main group
	Male animals				Female animals
Test group (mg/m^3)	0 (0)	1 (0.1)	2 (0.5)	3 (2)	0 (0)	1 (0.1)	2 (0.5)	3 (2)
No. of animals	10	10	10	10	10	10	10	10
Epithelial alterations	1	6	9	19	1	7	7	10
- Grade 1	1	6	9	10	1	7	7	10
Recovery group
	Male animals
Test group (mg/m^3)	10 (0)	11 (0.1)	12 (0.5)	13 (2)
No. of animals	4	5	5	5
Epithelial alterations	0	1	2	1
- Grade 1		1	2	1
Satellite group
	Male animals
Test group (mg/m^3)	20 (0)		23 (2)
No. of animals	5		5
Epithelial alterations	0		3
- Grade 1			3

Tab 11: Incidence and gradings of histological changes in lungs

Main group
	Male animals				Female animals
Test group (mg/m^3)	0 (0)	1 (0.1)	2 (0.5)	3 (2)	0 (0)	1 (0.1)	2 (0.5)	3 (2)
No. of animals	10	10	10	10	10	10	10	10
Histiocytosis, alveolar	2	10	10	10	3	10	10	10
- Grade 1	2	4	3	6	3	3	5	8
- Grade 2		6	6	3		6	3	2
- Grade 3			1	1		1	2
Eosinophilic granular material, alveolar	0	10	10	10	0	10	10	10
- Grade 1		1
- Grade 2		6	4			7	5	4
- Grade 3		3	6	9		3	5	5
- Grade 4				1				1
Proteinosis, alveolar	0	0	2	10	0	0	3	10
- Grade 1			2				3	1
- Grade 2				9				8
- Grade 3				1				1
Cellular debris, alveolar	0	0	0	10	0	0	3	10
- Grade 1				3			3	2
- Grade 2				4				8
- Grade 3				3
Particles, alveolar	0	0	0	10	0	0	0	7
- Grade 1				10				7
Inflammation, mixed-cellular	1	5	9	10	0	6	9	10
- Grade 1	1	5	5	5		6	9	10
- Grade 2			4	5
Inflammation, granulomatous	0	0	3	4	0	0	1	ß
- Grade 1			2	3			1
- Grade 2			1	1
Hyperplasia, type II pneumocytes	0	1	2	3	0	2	1	0
- Grade 1		1	1	3		2	1
- Grade 2			1
Hypertrophy/hyperpl., epithelial, bronchi/ bronchioles	0	0	0	9	0	0	0	10
- Grade 1				7				10
- Grade 2				2
BALT: hyperplasia, lympho-reticulocellular	0	0	0	2	0	0	1	0
- Grade 1				2			1
Recovery group
	Male animals
Test group (mg/m^3)	10 (0)	11 (0.1)	12 (0.5)	13 (2)
No. of animals	5	5	5	5
Histiocytosis, alveolar	1	5	5	5
- Grade 1	1
- Grade 2		2		2
- Grade 3		2	2	1
- Grade 4		1	3	2
Eosinophilic granular material, alveolar	0	4	5	5
- Grade 1		3	2
- Grade 2		1	2
- Grade 4			1	4
- Grade 5				1
Proteinosis, alveolar	0	0	0	5
- Grade 1				5
Inflammation, mixed-cellular	1	4	5	5
- Grade 1	1	4	2	3
- Grade 2			3	2
Inflammation, granulomatous	0	2	2	1
- Grade 1		2
- Grade 2			1	1
- Grade 3			1
Hyperplasia, type II pneumocytes	0	5	4	3
- Grade 1		4	1	3
- Grade 2		1	2
- Grade 3			1
BALT: hyperplasia, lympho-reticulocellular	0	0	0	3
- Grade 1				2
- Grade 2				1
Satellite group
	Male animals
Test group (mg/m^3)	20 (0)		23 (2)
Histiocytosis, alveolar	1		4
- Grade 1	1		4
Eosinophilic granular material, alveolar	0		5
- Grade 1			5
Cellular debris, alveolar	0		5
- Grade 2			5
flammation, mixed-cellular	0		5
- Grade 1			5
Hypertrophy/hyperplasia, epithelial, bronchi/ bronchioles	0		5
- Grade 1			5
Particles, alveolar	0		5
- Grade 1			5

 

Tab 12: Incidence and gradings of histological changes in mediastinal lymph nodes

Main group
	Male animals				Female animals
Test group (mg/m^3)	0 (0)	1 (0.1)	2 (0.5)	3 (2)	0 (0)	1 (0.1)	2 (0.5)	3 (2)
No. of animals	10	10	10	10	10	10	10	10
Hyperplasia, lympho-reticulocellular	0	0	0	3	0	0	1	0
- Grade 1				3			1
Vacuolation, HEV	0	3	3	2	0	6	1	3
- Grade 1		3	3	2		6	1	3
	Recovery group
	Male animals
Test group (mg/m^3)	10 (0)	11 (0.1)	12 (0.5)	13 (2)
No. of animals	5	5	5	5
Hyperplasia, lympho-reticulocellular	0	0	1	2
- Grade 1			1	2
Vacuolation, HEV	0	1	1	0
- Grade 1		1	1
	Satellite group
	Male animals
Test group (mg/m^3)	20 (0)		23 (2)
No. of animals	5		5
Vacuolation, HEV	0		1
- Grade 1			1
Hyperplasia, lympho-reticulocellular	0		2
- Grade 12

Tab 13: Incidence and gradings of histological changes in tracheobronchial lymph nodes

Main group
	Male animals				Female animals
Test group (mg/m^3)	0 (0)	1 (0.1)	2 (0.5)	3 (2)	0 (0)	1 (0.1)	2 (0.5)	3 (2)
No. of animals	10	10	10	10	9	9	10	10
Hyperplasia, lympho-reticulocellular	0	0	2	1	0	1	1	3
- Grade 1			2	1		1	1	3
Vacuolation, HEV	0	1	5	4	0	3	3	1
- Grade 1		1	5	4		3	3	2
	Recovery group
	Male animals
Test group (mg/m^3)	10 (0)	11 (0.1)	12 (0.5)	13 (2)
No. of animals	5	5	5	5
Hyperplasia, lympho-reticulocellular	0	0	3	3
- Grade 1			3	3
Vacuolation, HEV	0	0	1	1
- Grade 1			1	1
	Satellite group
	Male animals
Test group (mg/m^3)	20 (0)		23 (2)
No. of animals	5		5
Vacuolation, HEV	0		2
- Grade 1			1
Hyperplasia, lympho-reticulocellular	0		2
- Grade 1			2

Tab 14: Absolute weight parameters

	Main group
	Male animals			Female animals
Test group (mg/m3)	1 (0.1)	2 (0.5)	3 (2)	1 (0.1)	2 (0.5)	3 (2)
Adrenal glands				105%	118%**	110%*
Lungs	158%**	170%**	192%**	152%**	178%**	200%**
	Recovery group
Test group (mg/m3)	1 (0.1)	2 (0.5)	3 (2)
Lungs	129%	156%**	224%**	-	-	-
Thyroid glands	82%*	80%*	97%	-	-	-
	Satellite group
Test group (mg/m3)	23 (2)	-	-	-	-	-
Lungs	145%**	-	-	-	-	-

* : p <= 0.05, **: p <= 0.01

Tab 15: Relative weight parameters

	Main group
	Male animals			Female animals
Test group (mg/m3)	1 (0.1)	2 (0.5)	3 (2)	1 (0.1)	2 (0.5)	3 (2)
Liver				92%**	95%	97%
Lungs	156%**	170%**	201%**	150%**	167%**	194%**
	Recovery group
Test group (mg/m3)	1 (0.1)	2 (0.5)	3 (2)
Lungs	133%*	163%**	230%**
	Satellite group
Test group (mg/m3)	23 (2)
Brain	95%*
Kidneys	88%*
Lungs	139%**
Spleen	89*

* : p <= 0.05, **: p <= 0.01

Applicant's summary and conclusion

Conclusions:

Inhalation exposure of 0.1, 0.5 and 2 mg/m3 of the test material for 90 days (65 exposures) caused clearly treatment-related changes of lavage parameters and histological changes in lung. The effects were not fully reversible within 5 weeks recovery period. A No Observed Adverse Effect Concentration (NOAEC) could not be established under the current study condition.
Based on the data of clinical observation, body weight, food consumption, clinical chemistry, hematology and histological examinations, there was no indication for any systemic toxicity. Therefore, the NOAEC for systemic toxicity is 2 mg/m³ under the current study condition.

Executive summary:

The subchronic repeated dose toxicity of the test material, via the inhalation route, was determined in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 413, EU Method B.29 and EPA OPPTS 870.3465.

During the study, groups of male and female Wistar rats were exposed nose-only to the dust aerosol of the test item for 6 hours per day, on 5 days per week for 13 weeks (65 exposures). The target concentrations were 0.1, 0.5 and 2 mg/m³ test substance in air. A concurrent control group was exposed to clean air. Recovery group animals (5 males/group) were installed to assess potential reversibility or progression of the effects about 5 weeks after termination of the exposure. For main and recovery groups, 5 male rats per group and time point were used to determine lung burden. Interim sacrifice was performed after 4 weeks exposure in satellite control and high concentration male animals (5 rats/group) and examined by bronchoalveolar-lavage (BALF) and histopathology of the lung. Daily clinical observations were recorded, body weight and food consumption were determined regularly. Ophthalmology, rectal temperature, FOB and MA were performed in main group animals before and against the end of the exposure period. Assessments including hematology, clinical chemistry, bronchoalveolar lavage, lung burden, and histopathology were carried out in main group animals. In satellite animals, bronchoalveolar lavage, clinical chemistry, hematology and histopathology of the lung were performed, as well as in the recovery group animals.

The targeted atmospheric concentrations were maintained well throughout the whole study. During the in-life phase, there were no abnormalities observed during clinical observations and assessment of body weight development, rectal temperature, ophthalmology and functional observation battery and motor activity.

In animals of test group 3 (2 mg/m³, study day 92), adverse effects included: Increased total cell, absolute and relative neutrophil, lymphocyte and monocyte counts as well as absolute macrophage counts in BALF of males and females; Decreased relative macrophage counts in BALF of males and females; Increased total protein values and lactate dehydrogenase (LDH), β-N-Acetyl glucosaminidase (NAG) and γ-Glutamyl transferase (GGT) activities in BALF of males and females; Concerning the lung, the following adverse effects were noted: Statistical significant weight increase (absolute/relative): +92% /+101% in males and +100% / +94% in female; Histiocytosis, alveolar, multifocal: all males and all females (minimal to moderate); Eosinophilic granular material, alveolar, multifocal: all males and females (slight to severe); Proteinosis, alveolar, multifocal: all males and females (minimal to moderate); Cellular debris, alveolar, multifocal: all males and females (minimal to moderate); Inflammation, mixed-cellular, multifocal: all males and females (minimal to slight); Inflammation, granulomatous, multifocal: 4 out of 10 males (minimal to slight); Hyperplasia, type II-pneumocytes, multifocal: 3 out of 10 males (minimal); Hypertrophy/hyperplasia, epithelial, bronchi/bronchioles: 9 out of 10 males and all females (minimal to slight); Hypertrophy/hyperplasia, epithelial, bronchi/bronchioles: 9 out of 10 males and all females (minimal to slight); BALT (bronchial associated lymphoid tissue): hyperplasia, lympho-reticulocellular: 2 out of 10 males (minimal).

In animals of test group 2 (0.5 mg/m³, study day 92), adverse effects included: Increased total cell, absolute and relative neutrophil, lymphocyte and monocyte counts as well as absolute macrophage counts in BALF of males and females; Decreased relative macrophage counts in BALF of males and females; Increased total protein values and lactate dehydrogenase (LDH), β-N-Acetyl glucosaminidase (NAG) and γ-Glutamyl transferase (GGT) activities in BALF of males and females; Concerning the lung, the following adverse effects were noted: Statistical significant weight increase (absolute/relative): +70 % / +70% in males and +78% / +67% in females; Histiocytosis, alveolar, multifocal: all males and females (minimal to moderate); Eosinophilic granular material, alveolar, multifocal: all males and females (slight to moderate); Proteinosis, alveolar, multifocal: 2 out of 10 males and 3 out of 10 females (minimal); Cellular debris, alveolar, multifocal: 3 out of 10 females (minimal); Inflammation, mixed-cellular, multifocal: 9 out of 10 males and 9 out of 10 females (minimal to slight); Inflammation, granulomatous, multifocal: 3 out of 10 males and 1 out of 10 females (minimal to slight); Hyperplasia, type II-pneumocytes, multifocal: 2 out of 10 males and 1 out of 10 females (minimal to slight); BALT (bronchial associated lymphoid tissue): hyperplasia, lympho-reticulocellular: 1 out of 10 females (minimal).

In animals of test group 1 (0.1 mg/m³, study day 92), adverse effects included: Increased total cell, absolute and relative neutrophil, lymphocyte and monocyte counts as well as absolute macrophage counts in BALF of males and females; Decreased relative macrophage counts in BALF of males and females; Increased total protein values and lactate dehydrogenase (LDH), β-N-Acetyl glucosaminidase (NAG) and γ-Glutamyl transferase (GGT) activities in BALF of males and females; Concerning the lung, the following adverse effects were noted: Statistical significant weight increase (absolute/relative): +58 % / +56% in males and +52% / +50% in females; Histiocytosis, alveolar, multifocal: all males and females (minimal to moderate); Eosinophilic granular material, alveolar, multifocal: all males and females (minimal to moderate); Inflammation, mixed-cellular, multifocal: 5 out of 10 males and 6 out of 10 females (minimal); Hyperplasia, type II-pneumocytes, multifocal: 1 out of 10 males and 2 out of 10 females (minimal).

In animals of recovery group 13 (2 mg/m³, study day 126, males), adverse effects included: Increased total cell, absolute and relative lymphocyte and monocyte counts as well as absolute neutrophil and macrophage counts in BALF; Increased total protein values and lactate dehydrogenase (LDH), β-N-Acetyl glucosaminidase (NAG) and γ-Glutamyl transferase (GGT) activities in BALF; Concerning the lung, the following adverse effects were noted: Statistical significant weight increase in males: absolute +124 %, relative +130%; Histiocytosis, alveolar, multifocal: all males (slight to severe); Eosinophilic granular material, alveolar, multifocal: all males (severe to massive); Proteinosis, alveolar, multifocal: all males (minimal); Inflammation, mixed-cellular, multifocal: all males (minimal to slight); Inflammation, granulomatous, multifocal: 1 out of 5 males (slight); Hyperplasia, type II-pneumocytes, multifocal: 3 out of 5 males (minimal); BALT (bronchial associated lymphoid tissue): hyperplasia, lympho-reticulocellular: 3 out of 5 males (minimal to slight).

In animals of recovery group 12 (0.5 mg/m³, study day 126, males), adverse effects included: Increased total cell, absolute and relative lymphocyte and monocyte counts as well as absolute neutrophil and macrophage counts in BALF; Increased total protein values and lactate dehydrogenase (LDH), β-N-Acetyl glucosaminidase (NAG) and γ-Glutamyl transferase (GGT) activities in BALF; Concerning the lung, the following adverse effects were noted: Statistical significant weight increase in males: absolute +56 %, relative, +63%; Histiocytosis, alveolar, multifocal: all males (moderate to severe); Eosinophilic granular material, alveolar, multifocal: all males (minimal to severe); Inflammation, mixed-cellular, multifocal: all males (minimal to slight); Inflammation, granulomatous, multifocal: 2 out of 5 males (slight to moderate); Hyperplasia, type II-pneumocytes, multifocal: 4 out of 5 males (minimal to moderate).

In animals of recovery group 11 (0.1 mg/m³, study day 126, males), adverse effects included: Increased total cell, absolute lymphocyte, monocyte and macrophage counts in BALF; Increased total protein values and lactate dehydrogenase (LDH) activities in BALF; Concerning the lung, the following adverse effects were noted: Statistical significant weight increase in males: absolute +29 %, relative +33%; Histiocytosis, alveolar, multifocal: all males (slight to severe); Eosinophilic granular material, alveolar, multifocal: 4 out of 5 males (minimal to slight); Inflammation, mixed-cellular, multifocal: 4 out of 5 males (minimal); Inflammation granulomatous, multifocal: 2 out of 5 males (minimal); Hyperplasia, type II-pneumocytes, multifocal: all males (minimal to slight).

In animals of the satellite group 23 (2 mg/m³, study day 27, males), adverse effects included: Increased total cell, absolute and relative neutrophil, lymphocyte and monocyte counts in BALF; Increased total protein values and lactate dehydrogenase (LDH), β-N-Acetyl glucosaminidase (NAG) and γ-Glutamyl transferase (GGT) activities in BALF; Concerning the lung, the following adverse effects were noted: Statistical significant weight increase in males: absolute +45 %, relative +39%; Histiocytosis, alveolar, multifocal: 4 out of 5 males (minimal); Eosinophilic granular material, alveolar, multifocal: all males (minimal); Cell debris, alveolar, multifocal: all males (slight); Inflammation, mixed-cellular, multifocal: all males (minimal); Hypertrophy/hyperplasia, epithelial, bronchi/bronchioles: all males (minimal); Particles, alveolar, multifocal: all animals (minimal).

In conclusion, inhalation exposure of 0.1, 0.5 and 2 mg/m3 of the test substance for 90 days (65 exposures) caused clearly treatment-related changes of lavage parameters and histological changes in lung. The effects were not fully reversible within 5 weeks recovery period. A No Observed Adverse Effect Concentration (NOAEC) could not be established under the current study condition. Based on the data of clinical observation, body weight, food consumption, clinical chemistry, hematology and histological examinations, there was no indication for any systemic toxicity. Therefore, the NOAEC for systemic toxicity is 2 mg/m³ under the current study condition.