Registration Dossier
Registration Dossier
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EC number: 231-592-0 | CAS number: 7646-85-7
Repeated dose toxicity: inhalation
Administrative data
- Endpoint:
- short-term repeated dose toxicity: inhalation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 09 June 2020 - .. September 2021
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Remarks:
- The study was not performed under GLP conditions. Only male rats were used.
Cross-reference
- Reason / purpose for cross-reference:
- reference to same study
Reference
- Endpoint:
- short-term repeated dose toxicity: inhalation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 09 June 2020 - .. September 2021
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Remarks:
- The study was not performed under GLP condtions. Only male rats were used.
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 412 (28-Day (Subacute) Inhalation Toxicity Study
- Version / remarks:
- This study is a dose-range finding study
- Deviations:
- yes
- Remarks:
- only male rats were used
- Principles of method if other than guideline:
- No testing guidelines exist for this type of study, which is a dose range finding study. The results of this study should facilitate the selection of appropriate concentration for the following 90-day inhalation study.
The study was conducted based on the above test guidelines pertaining to inhalation repeated dose inhalation toxicity studies. - GLP compliance:
- no
- Limit test:
- no
- 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
- Details on species / strain selection:
- 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. - 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
- 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: rats were housed together (5 animals per cage) in Typ 2000P ca. 2065 cm2 (polysulfone cages) supplied by TECNIPLAST, Germany. Bedding in the Polycarbonate cages were dust-free bedding. Dust-free wooden bedding was used in this study (the present supplier is documented in the raw data). For enrichment wooden Play Tunnel, large (Art. 14153); PLEXX b.v., Elst, Netherlands were added. Wooden gnawing blocks (SAFE® block large) J. Rettenmaier & Söhne GmbH + Co KG, Rosenberg, Germany.
- Diet (ad libitum): mouse and rat maintenance diet, GLP, 12 mm pellets, Granovit AG, Kaiseraugst, Switzerland
- Water (ad libitum): tap water
- Acclimation period: 13 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. 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:
- whole body
- Vehicle:
- other: unchanged (no vehicle)
- Mass median aerodynamic diameter (MMAD):
- >= 1.77 - <= 1.99 µm
- Geometric standard deviation (GSD):
- 2.08
- Remarks on MMAD:
- MMAD / GSD: MMAD = 1.77-1.99 μm (geometric standard deviation = 1.97-2.26)
- Details on inhalation 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 - Analytical verification of doses or concentrations:
- yes
- 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:
- 14 days
- Frequency of treatment:
- Exposure was over 14 days, at a rate of 6 hours per day for 5 days per week. The target concentrations were 12 and 24 mg/m³ for each of the test items. A concurrent control group was exposed to clean air.
- Dose / conc.:
- 10.9 mg/m³ air
- Remarks:
- Test Group 1
(T 0420, 12 mg/m³) - Dose / conc.:
- 20.7 mg/m³ air
- Remarks:
- Test Group 2
(T 0420, 24 mg/m³) - Dose / conc.:
- 10.8 mg/m³ air
- Remarks:
- Test Group 3
(T 0421, 12 mg/m³) - Dose / conc.:
- 21.3 mg/m³ air
- Remarks:
- Test Group 4
(T 0421, 24 mg/m³) - Dose / conc.:
- 0 mg/m³ air
- Remarks:
- Control group
- No. of animals per sex per dose:
- 5 animals were used per group (total of 5 groups and total of 25 animals).
- Control animals:
- yes
- Details on study design:
- - Dose selection rationale: The doses were chosen based on available data and upon approval of the sponsor. This is a dose range finding study which should facilitate the selection of appropriate concentration for a following 90-day inhalation study.
- Rationale for animal assignment (if not random): /
- Fasting period before blood sampling for clinical biochemistry: not specified
- Rationale for selecting satellite groups: /
- Post-exposure recovery period in satellite groups: /
- Section schedule rationale (if not random): /
- Other: / - Positive control:
- None
- Observations and examinations performed and frequency:
- 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 exposure-free weekends and post exposure observation weekends, no clinical observation was performed. Signs and findings were recorded for each animal. During exposure only a group wise examination was possible.
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: Not specified
BODY WEIGHT: Yes
- Time schedule for examinations: The animals were weighed prior to the pre-exposure period, at the start of the exposure period (day 0) and twice weekly (Monday and Friday) thereafter until one day prior to gross necropsy. The body weight change was calculated as the difference of actual body weights and the weights of last weighing. Those of the weekends will be calculated as the difference of Monday to the previous Friday.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified
Food consumption was determined weekly, from Monday to Friday and from Friday to Monday. Food consumption was calculated as mean food consumption in grams per animal and day.
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: Not specified
HAEMATOLOGY: Yes
- Time schedule for collection of blood: Not specified
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: all
CLINICAL CHEMISTRY: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No
BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: Not specified
- Dose groups that were examined: all
- Number of animals: 25
LUNG BURDEN: No
OTHER: / - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes - Statistics:
- No information provided
- 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.
- Mortality:
- no mortality observed
- Description (incidence):
- No deaths were recorded throughout the study.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- The mean body weights of the test substance exposed groups were not statistically significantly different from the control group 0.
The body weight change was statistically significantly decreased in the following animals:
- Test group 2 (24 mg/m³ test item 1) from study day 0 to day 1: -7.9 g (p≤ 0.05, concurrent control was -0.4 g)
- Test group 4 (24 mg/m³ test item 4) from study day 0 to day 1: -6.7 g (p≤ 0.01, concurrent control was -0.4 g)
- Test group 4 (24 mg/m³ test item 4) from study day 4 to day 8: -1.7 g (p≤ 0.01, concurrent control was 5.5 g) - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- Due to social housing, there was only once cage (with 5 animals) per test group. No statistical evaluation was possible.
Food consumption of all exposed animals were slightly lower than the concurrent control group in a concentration-related manner. In the high concentrations, the food consumption of test group 2 (test item 1) animals was about 24 % lower than the control, while that of test group 4 animals (test item 2) was 32 % lower than in the controls. At low concentration, the food consumption was 10 % and 20 % lower than the control in test group 2 (test item 1) and test group 4 (test item 2) animals, respectively. - Food efficiency:
- not specified
- Description (incidence and severity):
- /
- Water consumption and compound intake (if drinking water study):
- not specified
- Description (incidence and severity):
- /
- Ophthalmological findings:
- not specified
- Description (incidence and severity):
- /
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- At study day 14, in males of test group 4 (T0421, 24 mg/m3) absolute and relative neutrophil cell counts were slightly, but significantly increased, whereas relative lymphocyte counts were significantly decreased. These changes were regarded as treatment related and adverse.
Absolute and relative neutrophil cell counts were already significantly increased in rats of test group 3 (T0421, 12 mg/m3) The absolute neutrophil mean was marginally above the historical control range, whereas the mean of neutrophil relative counts was within this range (males, absolute neutrophils 0.53-1.09 Giga/L; relative neutrophils 8.5-19.0 %). Because the change was marginal and no other differential blood cell counts were altered, this change was regarded as treatment-related, but non-adverse (ECETOC Technical Report No. 85, 2002).
In rats of test groups 1 and 2 (T0420, 12 and 24 mg/m3) absolute neutrophil cell counts were significantly increased, and in rats of test group 1 absolute eosinophil cell counts were significantly increased. However, the mentioned alterations were not dose dependent and therefore, they were regarded as incidental and not treatment related.
In rats of test group 4 (T0421, 24 mg/m3) relative, large unstained cell (LUC) counts were significantly decreased and hemoglobin values were significantly increased. Both parameters were within historical control ranges (males, relative LUC 0.2-0.7 %, hemoglobin 8.6-9.6 mmol/L). In males of test group 4 absolute reticulocyte counts were significantly decreased. The mean was below the historical control range (males, absolute reticulocytes 112.7-190.0 Giga/L). However, because of slightly higher hemoglobin and hematocrit values as well as red blood cell (RBC) counts compared to the controls lower reticulocyte counts were a consequence because the bone marrow wanted to adjust the circulating red blood cell counts. This effect is regarded as a physiological feedback regulation and was therefore regarded as treatment related, but non adverse. - Clinical biochemistry findings:
- not specified
- Description (incidence and severity):
- /
- Endocrine findings:
- not specified
- Description (incidence and severity):
- /
- Urinalysis findings:
- not specified
- Description (incidence and severity):
- /
- Behaviour (functional findings):
- not specified
- Description (incidence and severity):
- /
- Immunological findings:
- not specified
- Description (incidence and severity):
- /
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Absolute weights
When compared to control group 0 (=100%), the mean absolute weights of following organs were significantly changed.
Relative changes of absolute lung weights:
Lung weights were increased in test group 1 with test item T0420 (113%) and were statistically significantly increased (p <= 0.01) in test groups 2 (135%) with test item T0420, 3 (114%) and 4 (141%) with test item T0421. All other mean absolute weight parameters did not show significant differences when compared to the control group 0.
Relative organ weights
When compared to control group 0 (=100%), the mean relative weights of following organs were significantly changed:
Relative changes of relative lung weights
Lung weights were increased in test group 1 with test item T0420 (117%) and were statistically significantly increased (p <= 0.01) in test groups 2 (138%) with test item T0420, 3 (119%) and 4 (154%) with test item T0421. All other mean absolute weight parameters did not show significant differences when compared to the control group 0.
All other mean absolute and relative weight parameters did not show significant differences when compared to the control group 0.
The statistical significantly increased lung weight in test group 2, 3, and 4 was regarded to be treatment-related. - Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Enlarged, gray mottled lungs with enlarged and gray coloured lung-associated lymph nodes (high dose Days 28 and 42), enlarged lungs and the lung associated lymph nodes (mid dose) and enlarged lung-associated lymph nodes
(low dose).
Incidence of gross lesions observed during necropsy
None of the animals in the control group nor in Test group 3 showed any gross lesions in the tracheobronchial lymph nodes. 2 males animals of the Test group 1 (12mg/m3) showed enlarged heobronchial lymph nodes, 1 male animal of the Test group 2 (24mg/m3) and 2 male animals of the test group 4 (24mg/m3).
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. - Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Treatment-related findings were observed in organs listed in the larynx, lungs, nasal cavity and the tracheobronchial lymph nodes. These are further described in the ADDITIONAL RESULTS section below with incidences and grading
- Histopathological findings: neoplastic:
- not specified
- Other effects:
- effects observed, treatment-related
- Description (incidence and severity):
- BAL
At study day 14, rats in high concentration groups of both test items (test groups 2 and 4, T0420 and T0421, in each group 24 mg/m3) showed equivalent cell count increases in the bronchoalveolar lavage fluid (BALF): about 9fold significant increase of the total cell counts; highly, significantly increased absolute and relative neutrophil counts (about 700 fold absolute neutrophil increase) and absolute and relative monocyte counts (about 70 to 90 fold absolute monocyte increase) as well as moderately, significantly increased absolute lymphocyte counts (about 7 fold absolute lymphocyte increase). Relative macrophage cell counts were significantly decreased in both mentioned test groups.
Whereas the low concentration test group of T0420 (test group 1, 12 mg/m3) showed nearly the same changes as the high test item concentration groups, low concentration test group of T0421 (test group 3, 12 mg/m3) had considerable lower values: in test group 1 (T420) about 7 fold significant increase of total cell counts, about 600 fold, significant increase of absolute neutrophil cell counts, about 100 fold significant increase of absolute monocyte cell counts and 5 fold significant increase of absolute lymphocyte counts; in test group 3 (T0421) about 5 fold significant increase of total cell counts, about 300 fold significant increase of absolute neutrophil cell counts, about 50 fold significant increase of absolute monocyte counts and about 4 fold significant increase of absolute monocyte counts. Relative neutrophil and monocyte counts were also significantly increased in the mentioned test groups whereas relative macrophage counts were significantly decreased.
At study day 14, total protein levels as well as enzyme activities of -Glutamyl-transferase (GGT), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP) and -N-Acetyl glucosaminidase (NAG) in BALF were significantly increased in all test groups. Quantitatively, the same trend could be observed as described in the BALF cytology. High concentration test groups of both test items (test groups 2 and 4, T0420 and T0421, each 24 mg/m3) showed equivalent changes: about 18 fold increase in total protein levels, 24 to 27 fold increase of ALP, 11 fold increase of LDH, 4 fold increase of GGT and 3 fold increase of NAG activities.
The low concentration test group of T0420 (test group 1, 12 mg/m3) showed nearly the same alterations in BALF compared to the high concentration test groups: 14 fold increase of total protein levels, 17 fold increase of ALP, 11 fold increase of LDH, 5 fold increase of GGT and 3 fold increase of NAG activities.
In the low concentration test group of T0421 (test group 3 (12 mg/m3) considerable lower changes could be observed: 5 fold increase of total protein levels, 7 fold increase of ALP, 5 fold increase of LDH, 3 fold increase of GGT and 2 fold increase of NAG activities. - Details on results:
- In this study, male Wistar rats were whole-body exposed to dust aerosols of T0420 and T0421 at target concentrations of 12 and 24 mg/m³ for 6 hours daily on 5 consecutive days for 14 days. A concurrent control group was exposed to conditioned air. To examine the influence of coating, two substances T0420 and T0421 were tested at comparable concentrations. After the last exposure, animals designated for bronchoalveolar lavage, histological examinations.
The tested atmospheric concentrations were slightly lower than the target concentration. They were maintained throughout the study. Cascade impactor measurement of both substances showed particle sizes that were close to each other. The MMADs ranged from 1.77 to 1.99 µm, which were well within the respirable range. The fraction of particles < 3 µm MMAD was higher than 70 % in all test groups. With regard to particle size distribution, there were no difference between the two substances.
During the exposure period, no clinical signs of toxicity were observed. The body weight, body weight gain was slightly lower than in the concurrent control group, although statistical significance was only in body weight change of test group 4 on single days. Although statistical evaluation could not be performed due to social housing, food consumption was apparently lower in animals exposed to both test substances than in the controls. Overall, the retarded body weight development and food consumption were slightly more severe in animals exposed to test item 2 (T0421) than those exposed to test item 1 (T0420).
Regarding clinical pathology, alterations of bronchoalveolar lavage (BAL) parameters were similar in the high concentration test groups of T0420 and T0421 (test groups 2 and 4, in each group 24 mg/m3): moderately, significantly increased total cell counts (about 9 fold), highly increased neutrophil and monocyte counts (absolute neutrophils about 700 fold, absolute monocyte about 70 to 90 fold), and slightly increased lymphocyte counts (absolute lymphocytes 7 fold). Correspondingly to the neutrophil cell count increase, alkaline phosphatase (ALP) activities were moderately, significantly increased in both test groups (24 to 27-fold). Lactate dehydrogenase (LDH) activities indicating general cell destruction were also moderately, significantly increased (about 11-fold), whereas gamma-Glutamyl-transferase (GGT) and beta-N-Acetyl glucosaminidase (NAG) activities were only marginally, but also significantly increased (GGT about 4 fold, NAG about 3 fold).
BAL values in the low concentration test group of T420 (test group 1, 12 mg/m3) were changed in the same magnitude as in the high concentration test groups: 7 fold significant increase of total cell counts, about 600 fold, significant increase of absolute neutrophil cell counts, about 100 fold significant increase of absolute monocyte cell counts and 5 fold significant increase of absolute lymphocyte counts, 14 fold increase of total protein levels, 17 fold increase of ALP, 11 fold increase of LDH, 5 fold increase of GGT and 3 fold increase of NAG activities
In contrast BAL values of the low concentration group of T0421 (test group 3, 12 mg/m3) showed considerable lower changes compared to controls: about 5 fold significant increase of total cell counts, about 300 fold significant increase of absolute neutrophil cell counts, about 50 fold significant increase of absolute monocyte counts and about 4 fold significant increase of absolute monocyte counts, 5 fold increase of total protein levels, 7 fold increase of ALP, 5 fold increase of LDH, 3 fold increase of GGT and 2 fold increase of NAG activities.
In addition to the mentioned local changes in the BAL, in the high concentration group of T0421 (test group 4, 24 mg/m3), a marginal increase of the absolute and relative blood neutrophil cell counts coupled with a decrease of the relative lymphocyte counts indicated a systemic acute-phase reaction.
Regarding pathology, the lungs and nasal cavity were the target organs.
In the lungs a disseminated infiltration of granulocytes and alveolar histiocytes was observed. This resulted also in an increase of the lung weight in most test groups. Some histiocytes were assumed to be destroyed. This inflammatory reaction was seen as consequence to the inhalation of the ZnO and was considered to be adverse.
In the nasal cavity in all levels, but most severely in level IV, there was degeneration and/or regeneration of the olfactory epithelium seen. This finding was regarded to be treatment-related and adverse.
The increased size of tracheobronchial lymph nodes was caused by alveolar histiocytes, transporting the phagocytosed ZnO particles from the lungs to the regional lymph nodes. As no other findings were observed, this was regarded to be treatment-related but not adverse.
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. - Dose descriptor:
- other: Dose range finding study - no conclusion on NOAEL or LOAEL at this stage.
- Effect level:
- mg/m³ air
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Dose range finding study - no conclusion on NOAEL or LOAEL at this stage.
- Remarks on result:
- other: Dose range finding study - no conclusion on NOAEL or LOAEL at this stage.
- Critical effects observed:
- not specified
- Conclusions:
- Both Zinc oxide T420 and T421 caused lesions in nasal cavity and lung already at the low targeted concentration of 12 mg/m³ (measured concentration about 11 mg/m³). No systemic effect was observed. At comparable atmospheric concentrations, Zinc oxide T421 seemed to cause more severe effects than those caused by T420.
- Executive summary:
Groups of male Wistar rats were exposed whole-body to the dust aerosols of Zinc oxide T0420 and Zinc oxide T0421 for 6 hours per day on 5 days per week for two weeks. The target concentrations were 12 and 24 mg/m³ for each of the test items. A concurrent control group was exposed to clean air. Daily clinical observation and body weight were recorded. Animals were sacrificed, assessments including hematology, bronchoalveolar lavage and histopathology (control and high concentration only) of the respiratory tract were carried out.
The following is a summary of the most relevant results:
Test group 4 (T0421, 24 mg/m3)
- Impaired body weight development, reduced food consumption
- Increased absolute and relative neutrophil cell counts in blood
- Decrease relative lymphocyte counts in blood
- Increased total cell counts, absolute and relative neutrophil and monocyte counts as well as absolute lymphocyte counts in BAL
- Decreased relative macrophage counts in BALF
- Increased total protein levels as well as g-Glutamyl-transferase (GGT), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP) and b-N-Acetyl glucosaminidase (NAG) activities in BALF
- Increase of absolute and relative lung weight (141%/154%)
- Minimal to slight infiltration of neutrophilic granulocytes and moderate to severe infiltration of alveolar histiocytes in the lungs in all animals
- Slight to severe degeneration/regeneration of the olfactory epithelium in the nasal cavity of all animals
Test group 3 (T0421 12 mg/m3)
- Increased total cell counts, absolute and relative neutrophil and monocyte counts as well as absolute lymphocyte counts in BAL
- Decreased relative macrophage counts in BALF
- Increased total protein levels as well as g-Glutamyl-transferase (GGT), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP) and b-N-Acetyl glucosaminidase (NAG) activities in BALF
- Increase of absolute and relative lung weight (114%/119%)
- Minimal to slight infiltration of neutrophilic granulocytes and minimal to moderate infiltration of alveolar histiocytes in the lungs in all animals
- Minimal to moderate degeneration/regeneration of the olfactory epithelium in the nasal cavity of all animals
Test group 2 (T0420, 24 mg/m3)
- Increased total cell counts, absolute and relative neutrophil and monocyte counts as well as absolute lymphocyte counts in BAL
- Decreased relative macrophage counts in BALF
- Increased total protein levels as well as g-Glutamyl-transferase (GGT), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP) and b-N-Acetyl glucosaminidase (NAG) activities in BALF
- Increase of absolute and relative lung weight (135%/138%)
- Slight infiltration of neutrophilic granulocytes and moderate infiltration of alveolar histiocytes in the lungs in all animals
- Minimal to moderate degeneration/regeneration of the olfactory epithelium in the nasal cavity of all animals
Test group 1 (T0420, 12 mg/m3)
- Increased total cell counts, absolute and relative neutrophil and monocyte counts as well as absolute lymphocyte counts in BAL
- Decreased relative macrophage counts in BALF
- Increased total protein levels as well as g-Glutamyl-transferase (GGT), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP) and b-N-Acetyl glucosaminidase (NAG) activities in BALF
- Minimal to slight infiltration of neutrophilic granulocytes and slight to moderate infiltration of alveolar histiocytes in the lungs in all animals
Minimal to slight degeneration/regeneration of the olfactory epithelium in the nasal cavity of all animals
Concentration measurements in the exposure system
Study means and standard deviations of test substance concentrations:
Test group | Target concentration | Measured concentration (mg/m³) | Nominal concentration (mg/m³) | Effectiveness dust generation | |
Mean | SD | ||||
1 | 12 | 10.94 | 0.41 | 18 | 59.9 % |
2 | 24 | 20.67 | 0.89 | 28 | 74.7 % |
3 | 12 | 10.76 | 0.37 | 19 | 56.7 % |
4 | 24 | 21.28 | 1.02 | 42 | 50.3 % |
Results of the particle size analyses
All measurements of particle size resulted in MMADs between 1.77 and 1.99 µm with GSDs between 1.97 and 2.26. The calculated mass fractions of particles below 3 µm aerodynamic size ranged between 74.2 % and 76.5 % for test item 1 (T0420) and between 71.7 % to 78.2 % for test item 2 (T0421). These values were well within the requirement of the test guidelines and showed that the generated test atmospheres contained high fraction of respirable particles. Moreover, all the measured values were close to each other, there were no significant difference between the two test items at comparable concentrations.
Cascade impactor measurements:
T 420 | T 0421 | ||||
| MMAD / GSD | % |
| MMAD / GSD | % |
Test group 1 | 1.90 µm / 2.03 | 74.2 % | Test group 3 | 1.99 µm / 1.99 | 72.4 % |
Test group 1 | 1.79 µm / 2.04 | 76.5 % | Test group 3 | 1.92 µm / 2.17 | 71.7 % |
Test group 2 | 1.85 µm / 2.02 | 75.3 % | Test group 4 | 1.77 µm / 1.97 | 78.2 % |
Test group 2 | 1.77 µm / 2.17 | 75.2 % | Test group 4 | 1.79 µm / 2.26 | 73.5 % |
In the following table the data of APS measurements were presented. The APS measurement showed slightly higher MMAD. The difference between the two devices are to be explained by the mechanisms the measurements based on.
Particle size distribution measured by APS:
| Measurement date | MMAD / GSD |
| Measurement date | MMAD / GSD |
Test group 1 | 26 Jun 20 | 2.47 µm/2.24 | Test group 3 | 26 Jun 20 | 3.23 µm/1.87 |
| 2.39 µm/2.22 |
| 3.38 µm/1.98 | ||
| 2.47 µm/2.34 |
| 3.33 µm/1.91 | ||
02 Jul 20 | 2.28 µm/2.37 | 02 Jul 20 | 2.93 µm/1.97 | ||
| 2.13 µm/2.25 |
| 3.11 µm/2.21 | ||
| 2.10 µm/2.23 |
| 3.46 µm/2.39 | ||
Test group 2 | 25 Jun 20 | 1.94 µm/2.66 | Test group 4 | 25 Jun 20 | 2.41 µm/2.16 |
| 1.82 µm/2.54 |
| 2.40 µm/2.18 | ||
| 1.82 µm/2.52 |
| 2.29 µm/2.09 | ||
03 Jul 20 | 2.41 µm/2.46 | 03 Jul 20 | 2.76 µm/2.25 | ||
| 2.50 µm/2.37 |
| 2.67 µm/2.22 | ||
| 2.50 µm/2.55 |
| 2.59 µm/2.17 |
BAL RESULTS
Changes in mean absolute cell counts in BAL (x-fold of concurrent control) of male rats on study day 14 (1 day after last exposure):
Analyte | Gr. 1 T0420 12 mg/m3 | Gr. 2 T0420 24 mg/m3 | Gr. 3 T0421 12 mg/m3 | Gr. 4 T0421 24 mg/m3 |
Total Cells | 7.4* | 8.9** | 4.8** | 8.6** |
Eosinophils | 4.1 | 16.0 | 6.7 | 11.0 |
Lymphocytes | 4.9* | 7.4** | 4.0* | 7.5** |
Macrophages | 0.6 | 1.1 | 1.1 | 0.7 |
Neutrophils | 620.8* | 722.3** | 333.3** | 734.0** |
Monocytes | 105.2* | 91.2** | 48.0** | 71.4** |
Epithelial cells | + | + | + | + |
One-sided Wilcoxon-test: * : p £ 0.05; ** : p £ 0.01
+ increase could not be calculated because of zero activity in controls
Changes in median total protein and enzyme levels in BAL (x-fold of concurrent control) of male rats on study day 14 (1 day after last exposure). Medians instead of means were used because of high individual variation of the values with great impact on the mean values:
Analyte | Gr. 1 T0420 12 mg/m3 | Gr. 2 T0420 24 mg/m3 | Gr. 3 T0421 12 mg/m3 | Gr. 4 T0421 24 mg/m3 |
Total Protein | 14.4** | 18.6** | 5.4** | 17.5** |
GGT | 4.6** | 4.1** | 2.9** | 4.3** |
LDH | 11.0** | 11.2** | 4.6** | 11.7** |
ALP | 16.6** | 23.5** | 7.0** | 27.4** |
NAG | 2.7* | 2.7* | 1.7** | 2.8** |
GGT = g-Glutamyl-transferase; LDH = Lactate dehydrogenase; ALP = Alkaline phosphatase;
NAG = b-N-Acetyl glucosaminidase, One-sided Wilcoxon-test: * : p £ 0.05; ** : p £ 0.01
Histopathology
Treatment-related findings were observed in organs listed in the tables below with incidences and grading:
Incidence and grading of histological findings in larynx:
Larynx (Level I) | Male animals | ||||
Control | T0420 | T0421 | |||
Test group (mg/m³) | 0 (0) | 1 (12) | 2 (24) | 3 (12) | 4 (24) |
Epithelial alteration | 1 | 0 | 4 | 1 | 2 |
· Grade 1 | 1 |
| 4 | 1 | 2 |
The finding epithelial alteration is a common observation in inhalation studies and in most cases, the base of the epiglottis is affected. It shows a minimal flattening of the epithelial cells and loss of cilia. It was considered to be treatment-related but with the minimal grading it was not regarded to be adverse (Kaufmann et al., 2009).
Incidence and grading of histological findings in lungs
Lungs | Male animals | ||||
Control | T0420 | T0421 | |||
Test group (mg/m³) | 0 (0) | 1 (12) | 2 (24) | 3 (12) | 4 (24) |
Infiltration, granulocytic | 0 | 5 | 5 | 5 | 5 |
· Grade 1 |
| 4 |
| 3 | 2 |
· Grade 2 |
| 1 | 5 | 2 | 3 |
Histiocytosis, alveolar | 0 | 5 | 5 | 5 | 5 |
· Grade 1 |
|
|
| 1 |
|
· Grade 2 |
| 4 |
| 2 |
|
· Grade 3 |
| 1 | 5 | 2 | 3 |
· Grade 4 |
|
|
|
| 2 |
In general, Lob. cran. dexter, Lob. medius dexter, and Lob. accessorius of the lungs were slightly less severely affected compared to Pulmo sinister and Lob. caudalis dexter. The finding was characterized by disseminated intra-alveolar inflammatory cells (mainly histiocytes and less number of granulocytes), intermingled by foamy roundish structures, containing occasionally a faint, bluish, roundish structures inside (interpreted as nucleus) or finely granular, eosinophilic material inside the alveoli. These structures were considered to be destroyed alveolar macrophages. These findings were regarded to be treatment-related.
Incidence and grading of histological findings in the nasal cavity:
Nasal cavity (Level IV) | Male animals | ||||
Control | T0420 | T0421 | |||
Test group (mg/m³) | 0 (0) | 1 (12) | 2 (24) | 3 (12) | 4 (24) |
Degeneration/regeneration, olfactory epithelium | 0 | 5 | 5 | 5 | 5 |
· Grade 1 |
| 3 |
|
|
|
· Grade 2 |
| 2 |
| 3 |
|
· Grade 3 |
|
| 5 | 2 | 1 |
· Grade 4 |
|
|
|
| 4 |
Level IV of the nasal cavity was the most severely affected level and was here taken representatively for findings in the nasal cavity. There was loss, irregularity or flattening of the olfactory epithelium (interpreted as degeneration). The most affected areas were the septum, the dorsal meatus and the tips of the conchae. In some areas there was in addition an increase of nuclear size and basophilia, mainly of basal cells (interpreted as regeneration). These findings were considered as treatment-related.
Incidence and grading of histological findings in the tracheobronchial lymph nodes:
Tracheobronchial lymph nodes | Male animals | ||||
Control | T0420 | T0421 | |||
Test group (mg/m³) | 0 (0) | 1 (12) | 2 (24) | 3 (12) | 4 (24) |
Histiocytosis (m)focal | 0 | 3 | 3 | 4 | 5 |
· Grade 1 |
|
|
| 2 | 1 |
· Grade 2 |
| 2 | 3 | 2 | 4 |
· Grade 3 |
| 1 |
|
|
|
Histiocytes with intracytoplasmic material similar to those seen in the lungs, were found in tracheobronchial lymph nodes. This finding is regarded as treatment-related, but not adverse.
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.
Pathology
Weight parameters
Absolute weights
When compared to control group 0 (=100%), the mean absolute weights of following organs were significantly changed (statistically significant changes printed in bold):
Relative changes of absolute lung weights:
| Male animals | |||
| T0420 | T0421 | ||
Test group (mg/m³) | 1 (12) | 2 (24) | 3 (12) | 4 (24) |
Lungs | 113% | 135%** | 114%** | 141%** |
*p <= 0.05; **p <= 0.01
All other mean absolute weight parameters did not show significant differences when compared to the control group 0.
Relative organ weights
When compared to control group 0 (=100%), the mean relative weights of following organs were significantly changed (statistically significant changes printed in bold):
Relative changes of relative lung weights:
| Male animals | |||
| T0420 | T0421 | ||
Test group (mg/m³) | 1 (12) | 2 (24) | 3 (12) | 4 (24) |
Lungs | 117% | 138%** | 119%** | 154%** |
*p <= 0.05; **p <= 0.01
All other mean absolute and relative weight parameters did not show significant differences when compared to the control group 0.
The statistical significantly increased lung weight in test group 2, 3, and 4 was regarded to be treatment-related.
Gross lesions
In some animals, the tracheobronchial lymph nodes were enlarged. This was considered as treatment-related.
Incidence of gross lesions observed during necropsy:
Tracheobronchial lymph nodes | Male animals | ||||
Control | T0420 | T0421 | |||
Test group (mg/m³) | 0 (0) | 1 (12) | 2 (24) | 3 (12) | 4 (24) |
Enlarged | 0 | 2 | 1 | 0 | 2 |
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.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 021
Materials and methods
Test guideline
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 412 (28-Day (Subacute) Inhalation Toxicity Study
- Version / remarks:
- This study is a dose-range finding study
- Deviations:
- yes
- Remarks:
- only male rats were used
- Principles of method if other than guideline:
- No testing guidelines exist for this type of study, which is a dose range finding study. The results of this study should facilitate the selection of appropriate concentration for the following 90-day inhalation study.
The study was conducted based on the above test guidelines pertaining to inhalation repeated dose inhalation toxicity studies. - GLP compliance:
- no
- Limit test:
- no
Test material
- Reference substance name:
- Zinc oxide
- EC Number:
- 215-222-5
- EC Name:
- Zinc oxide
- Cas Number:
- 1314-13-2
- Molecular formula:
- ZnO
- 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.
Constituent 1
- 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
- Details on species / strain selection:
- 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. - 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
- 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: rats were housed together (5 animals per cage) in Typ 2000P ca. 2065 cm2 (polysulfone cages) supplied by TECNIPLAST, Germany. Bedding in the Polycarbonate cages were dust-free bedding. Dust-free wooden bedding was used in this study (the present supplier is documented in the raw data). For enrichment wooden Play Tunnel, large (Art. 14153); PLEXX b.v., Elst, Netherlands were added. Wooden gnawing blocks (SAFE® block large) J. Rettenmaier & Söhne GmbH + Co KG, Rosenberg, Germany.
- Diet (ad libitum): mouse and rat maintenance diet, GLP, 12 mm pellets, Granovit AG, Kaiseraugst, Switzerland
- Water (ad libitum): tap water
- Acclimation period: 13 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. 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:
- whole body
- Vehicle:
- other: unchanged (no vehicle)
- Mass median aerodynamic diameter (MMAD):
- >= 1.77 - <= 1.99 µm
- Geometric standard deviation (GSD):
- 2.08
- Remarks on MMAD:
- MMAD / GSD: MMAD = 1.77-1.99 μm (geometric standard deviation = 1.97-2.26)
- Details on inhalation 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 - Analytical verification of doses or concentrations:
- yes
- 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:
- 14 days
- Frequency of treatment:
- Exposure was over 14 days, at a rate of 6 hours per day for 5 days per week. The target concentrations were 12 and 24 mg/m³ for each of the test items. A concurrent control group was exposed to clean air.
Doses / concentrationsopen allclose all
- Dose / conc.:
- 10.9 mg/m³ air
- Remarks:
- Test Group 1
(T 0420, 12 mg/m³)
- Dose / conc.:
- 20.7 mg/m³ air
- Remarks:
- Test Group 2
(T 0420, 24 mg/m³)
- Dose / conc.:
- 10.8 mg/m³ air
- Remarks:
- Test Group 3
(T 0421, 12 mg/m³)
- Dose / conc.:
- 21.3 mg/m³ air
- Remarks:
- Test Group 4
(T 0421, 24 mg/m³)
- Dose / conc.:
- 0 mg/m³ air
- Remarks:
- Control group
- No. of animals per sex per dose:
- 5 animals were used per group (total of 5 groups and total of 25 animals).
- Control animals:
- yes
- Details on study design:
- - Dose selection rationale: The doses were chosen based on available data and upon approval of the sponsor. This is a dose range finding study which should facilitate the selection of appropriate concentration for a following 90-day inhalation study.
- Rationale for animal assignment (if not random): /
- Fasting period before blood sampling for clinical biochemistry: not specified
- Rationale for selecting satellite groups: /
- Post-exposure recovery period in satellite groups: /
- Section schedule rationale (if not random): /
- Other: / - Positive control:
- None
Examinations
- Observations and examinations performed and frequency:
- 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 exposure-free weekends and post exposure observation weekends, no clinical observation was performed. Signs and findings were recorded for each animal. During exposure only a group wise examination was possible.
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: Not specified
BODY WEIGHT: Yes
- Time schedule for examinations: The animals were weighed prior to the pre-exposure period, at the start of the exposure period (day 0) and twice weekly (Monday and Friday) thereafter until one day prior to gross necropsy. The body weight change was calculated as the difference of actual body weights and the weights of last weighing. Those of the weekends will be calculated as the difference of Monday to the previous Friday.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified
Food consumption was determined weekly, from Monday to Friday and from Friday to Monday. Food consumption was calculated as mean food consumption in grams per animal and day.
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: Not specified
HAEMATOLOGY: Yes
- Time schedule for collection of blood: Not specified
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: all
CLINICAL CHEMISTRY: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No
BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: Not specified
- Dose groups that were examined: all
- Number of animals: 25
LUNG BURDEN: No
OTHER: / - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes - Statistics:
- No information provided
Results and discussion
Results of examinations
- 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.
- Mortality:
- no mortality observed
- Description (incidence):
- No deaths were recorded throughout the study.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- The mean body weights of the test substance exposed groups were not statistically significantly different from the control group 0.
The body weight change was statistically significantly decreased in the following animals:
- Test group 2 (24 mg/m³ test item 1) from study day 0 to day 1: -7.9 g (p≤ 0.05, concurrent control was -0.4 g)
- Test group 4 (24 mg/m³ test item 4) from study day 0 to day 1: -6.7 g (p≤ 0.01, concurrent control was -0.4 g)
- Test group 4 (24 mg/m³ test item 4) from study day 4 to day 8: -1.7 g (p≤ 0.01, concurrent control was 5.5 g) - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- Due to social housing, there was only once cage (with 5 animals) per test group. No statistical evaluation was possible.
Food consumption of all exposed animals were slightly lower than the concurrent control group in a concentration-related manner. In the high concentrations, the food consumption of test group 2 (test item 1) animals was about 24 % lower than the control, while that of test group 4 animals (test item 2) was 32 % lower than in the controls. At low concentration, the food consumption was 10 % and 20 % lower than the control in test group 2 (test item 1) and test group 4 (test item 2) animals, respectively. - Food efficiency:
- not specified
- Description (incidence and severity):
- /
- Water consumption and compound intake (if drinking water study):
- not specified
- Description (incidence and severity):
- /
- Ophthalmological findings:
- not specified
- Description (incidence and severity):
- /
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- At study day 14, in males of test group 4 (T0421, 24 mg/m3) absolute and relative neutrophil cell counts were slightly, but significantly increased, whereas relative lymphocyte counts were significantly decreased. These changes were regarded as treatment related and adverse.
Absolute and relative neutrophil cell counts were already significantly increased in rats of test group 3 (T0421, 12 mg/m3) The absolute neutrophil mean was marginally above the historical control range, whereas the mean of neutrophil relative counts was within this range (males, absolute neutrophils 0.53-1.09 Giga/L; relative neutrophils 8.5-19.0 %). Because the change was marginal and no other differential blood cell counts were altered, this change was regarded as treatment-related, but non-adverse (ECETOC Technical Report No. 85, 2002).
In rats of test groups 1 and 2 (T0420, 12 and 24 mg/m3) absolute neutrophil cell counts were significantly increased, and in rats of test group 1 absolute eosinophil cell counts were significantly increased. However, the mentioned alterations were not dose dependent and therefore, they were regarded as incidental and not treatment related.
In rats of test group 4 (T0421, 24 mg/m3) relative, large unstained cell (LUC) counts were significantly decreased and hemoglobin values were significantly increased. Both parameters were within historical control ranges (males, relative LUC 0.2-0.7 %, hemoglobin 8.6-9.6 mmol/L). In males of test group 4 absolute reticulocyte counts were significantly decreased. The mean was below the historical control range (males, absolute reticulocytes 112.7-190.0 Giga/L). However, because of slightly higher hemoglobin and hematocrit values as well as red blood cell (RBC) counts compared to the controls lower reticulocyte counts were a consequence because the bone marrow wanted to adjust the circulating red blood cell counts. This effect is regarded as a physiological feedback regulation and was therefore regarded as treatment related, but non adverse. - Clinical biochemistry findings:
- not specified
- Description (incidence and severity):
- /
- Endocrine findings:
- not specified
- Description (incidence and severity):
- /
- Urinalysis findings:
- not specified
- Description (incidence and severity):
- /
- Behaviour (functional findings):
- not specified
- Description (incidence and severity):
- /
- Immunological findings:
- not specified
- Description (incidence and severity):
- /
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Absolute weights
When compared to control group 0 (=100%), the mean absolute weights of following organs were significantly changed.
Relative changes of absolute lung weights:
Lung weights were increased in test group 1 with test item T0420 (113%) and were statistically significantly increased (p <= 0.01) in test groups 2 (135%) with test item T0420, 3 (114%) and 4 (141%) with test item T0421. All other mean absolute weight parameters did not show significant differences when compared to the control group 0.
Relative organ weights
When compared to control group 0 (=100%), the mean relative weights of following organs were significantly changed:
Relative changes of relative lung weights
Lung weights were increased in test group 1 with test item T0420 (117%) and were statistically significantly increased (p <= 0.01) in test groups 2 (138%) with test item T0420, 3 (119%) and 4 (154%) with test item T0421. All other mean absolute weight parameters did not show significant differences when compared to the control group 0.
All other mean absolute and relative weight parameters did not show significant differences when compared to the control group 0.
The statistical significantly increased lung weight in test group 2, 3, and 4 was regarded to be treatment-related. - Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Enlarged, gray mottled lungs with enlarged and gray coloured lung-associated lymph nodes (high dose Days 28 and 42), enlarged lungs and the lung associated lymph nodes (mid dose) and enlarged lung-associated lymph nodes
(low dose).
Incidence of gross lesions observed during necropsy
None of the animals in the control group nor in Test group 3 showed any gross lesions in the tracheobronchial lymph nodes. 2 males animals of the Test group 1 (12mg/m3) showed enlarged heobronchial lymph nodes, 1 male animal of the Test group 2 (24mg/m3) and 2 male animals of the test group 4 (24mg/m3).
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. - Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Treatment-related findings were observed in organs listed in the larynx, lungs, nasal cavity and the tracheobronchial lymph nodes. These are further described in the ADDITIONAL RESULTS section below with incidences and grading
- Histopathological findings: neoplastic:
- not specified
- Other effects:
- effects observed, treatment-related
- Description (incidence and severity):
- BAL
At study day 14, rats in high concentration groups of both test items (test groups 2 and 4, T0420 and T0421, in each group 24 mg/m3) showed equivalent cell count increases in the bronchoalveolar lavage fluid (BALF): about 9fold significant increase of the total cell counts; highly, significantly increased absolute and relative neutrophil counts (about 700 fold absolute neutrophil increase) and absolute and relative monocyte counts (about 70 to 90 fold absolute monocyte increase) as well as moderately, significantly increased absolute lymphocyte counts (about 7 fold absolute lymphocyte increase). Relative macrophage cell counts were significantly decreased in both mentioned test groups.
Whereas the low concentration test group of T0420 (test group 1, 12 mg/m3) showed nearly the same changes as the high test item concentration groups, low concentration test group of T0421 (test group 3, 12 mg/m3) had considerable lower values: in test group 1 (T420) about 7 fold significant increase of total cell counts, about 600 fold, significant increase of absolute neutrophil cell counts, about 100 fold significant increase of absolute monocyte cell counts and 5 fold significant increase of absolute lymphocyte counts; in test group 3 (T0421) about 5 fold significant increase of total cell counts, about 300 fold significant increase of absolute neutrophil cell counts, about 50 fold significant increase of absolute monocyte counts and about 4 fold significant increase of absolute monocyte counts. Relative neutrophil and monocyte counts were also significantly increased in the mentioned test groups whereas relative macrophage counts were significantly decreased.
At study day 14, total protein levels as well as enzyme activities of -Glutamyl-transferase (GGT), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP) and -N-Acetyl glucosaminidase (NAG) in BALF were significantly increased in all test groups. Quantitatively, the same trend could be observed as described in the BALF cytology. High concentration test groups of both test items (test groups 2 and 4, T0420 and T0421, each 24 mg/m3) showed equivalent changes: about 18 fold increase in total protein levels, 24 to 27 fold increase of ALP, 11 fold increase of LDH, 4 fold increase of GGT and 3 fold increase of NAG activities.
The low concentration test group of T0420 (test group 1, 12 mg/m3) showed nearly the same alterations in BALF compared to the high concentration test groups: 14 fold increase of total protein levels, 17 fold increase of ALP, 11 fold increase of LDH, 5 fold increase of GGT and 3 fold increase of NAG activities.
In the low concentration test group of T0421 (test group 3 (12 mg/m3) considerable lower changes could be observed: 5 fold increase of total protein levels, 7 fold increase of ALP, 5 fold increase of LDH, 3 fold increase of GGT and 2 fold increase of NAG activities. - Details on results:
- In this study, male Wistar rats were whole-body exposed to dust aerosols of T0420 and T0421 at target concentrations of 12 and 24 mg/m³ for 6 hours daily on 5 consecutive days for 14 days. A concurrent control group was exposed to conditioned air. To examine the influence of coating, two substances T0420 and T0421 were tested at comparable concentrations. After the last exposure, animals designated for bronchoalveolar lavage, histological examinations,.
The tested atmospheric concentrations were slightly lower than the target concentration. They were maintained throughout the study. Cascade impactor measurement of both substances showed particle sizes that were close to each other. The MMADs ranged from 1.77 to 1.99 µm, which were well within the respirable range. The fraction of particles < 3 µm MMAD was higher than 70 % in all test groups. With regard to particle size distribution, there were no difference between the two substances.
During the exposure period, no clinical signs of toxicity were observed. The body weight, body weight gain was slightly lower than in the concurrent control group, although statistical significance was only in body weight change of test group 4 on single days. Although statistical evaluation could not be performed due to social housing, food consumption was apparently lower in animals exposed to both test substances than in the controls. Overall, the retarded body weight development and food consumption were slightly more severe in animals exposed to test item 2 (T0421) than those exposed to test item 1 (T0420).
Regarding clinical pathology, alterations of bronchoalveolar lavage (BAL) parameters were similar in the high concentration test groups of T0420 and T0421 (test groups 2 and 4, in each group 24 mg/m3): moderately, significantly increased total cell counts (about 9 fold), highly increased neutrophil and monocyte counts (absolute neutrophils about 700 fold, absolute monocyte about 70 to 90 fold), and slightly increased lymphocyte counts (absolute lymphocytes 7 fold). Correspondingly to the neutrophil cell count increase, alkaline phosphatase (ALP) activities were moderately, significantly increased in both test groups (24 to 27-fold). Lactate dehydrogenase (LDH) activities indicating general cell destruction were also moderately, significantly increased (about 11-fold), whereas gamma-Glutamyl-transferase (GGT) and beta-N-Acetyl glucosaminidase (NAG) activities were only marginally, but also significantly increased (GGT about 4 fold, NAG about 3 fold).
BAL values in the low concentration test group of T420 (test group 1, 12 mg/m3) were changed in the same magnitude as in the high concentration test groups: 7 fold significant increase of total cell counts, about 600 fold, significant increase of absolute neutrophil cell counts, about 100 fold significant increase of absolute monocyte cell counts and 5 fold significant increase of absolute lymphocyte counts, 14 fold increase of total protein levels, 17 fold increase of ALP, 11 fold increase of LDH, 5 fold increase of GGT and 3 fold increase of NAG activities
In contrast BAL values of the low concentration group of T0421 (test group 3, 12 mg/m3) showed considerable lower changes compared to controls: about 5 fold significant increase of total cell counts, about 300 fold significant increase of absolute neutrophil cell counts, about 50 fold significant increase of absolute monocyte counts and about 4 fold significant increase of absolute monocyte counts, 5 fold increase of total protein levels, 7 fold increase of ALP, 5 fold increase of LDH, 3 fold increase of GGT and 2 fold increase of NAG activities.
In addition to the mentioned local changes in the BAL, in the high concentration group of T0421 (test group 4, 24 mg/m3), a marginal increase of the absolute and relative blood neutrophil cell counts coupled with a decrease of the relative lymphocyte counts indicated a systemic acute-phase reaction.
Regarding pathology, the lungs and nasal cavity were the target organs.
In the lungs a disseminated infiltration of granulocytes and alveolar histiocytes was observed. This resulted also in an increase of the lung weight in most test groups. Some histiocytes were assumed to be destroyed. This inflammatory reaction was seen as consequence to the inhalation of the ZnO and was considered to be adverse.
In the nasal cavity in all levels, but most severely in level IV, there was degeneration and/or regeneration of the olfactory epithelium seen. This finding was regarded to be treatment-related and adverse.
The increased size of tracheobronchial lymph nodes was caused by alveolar histiocytes, transporting the phagocytosed ZnO particles from the lungs to the regional lymph nodes. As no other findings were observed, this was regarded to be treatment-related but not adverse.
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.
Effect levels
- Dose descriptor:
- other: Dose range finding study - no conclusion on NOAEL or LOAEL at this stage.
- Effect level:
- mg/m³ air
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Dose range finding study - no conclusion on NOAEL or LOAEL at this stage.
- Remarks on result:
- other: Dose range finding study - no conclusion on NOAEL or LOAEL at this stage.
Target system / organ toxicity
- Critical effects observed:
- not specified
Any other information on results incl. tables
Concentration measurements in the exposure system
Study means and standard deviations of test substance concentrations:
Test group | Target concentration | Measured concentration (mg/m³) | Nominal concentration (mg/m³) | Effectiveness dust generation | |
Mean | SD | ||||
1 | 12 | 10.94 | 0.41 | 18 | 59.9 % |
2 | 24 | 20.67 | 0.89 | 28 | 74.7 % |
3 | 12 | 10.76 | 0.37 | 19 | 56.7 % |
4 | 24 | 21.28 | 1.02 | 42 | 50.3 % |
Results of the particle size analyses
All measurements of particle size resulted in MMADs between 1.77 and 1.99 µm with GSDs between 1.97 and 2.26. The calculated mass fractions of particles below 3 µm aerodynamic size ranged between 74.2 % and 76.5 % for test item 1 (T0420) and between 71.7 % to 78.2 % for test item 2 (T0421). These values were well within the requirement of the test guidelines and showed that the generated test atmospheres contained high fraction of respirable particles. Moreover, all the measured values were close to each other, there were no significant difference between the two test items at comparable concentrations.
Cascade impactor measurements:
T 420 | T 0421 | ||||
| MMAD / GSD | % |
| MMAD / GSD | % |
Test group 1 | 1.90 µm / 2.03 | 74.2 % | Test group 3 | 1.99 µm / 1.99 | 72.4 % |
Test group 1 | 1.79 µm / 2.04 | 76.5 % | Test group 3 | 1.92 µm / 2.17 | 71.7 % |
Test group 2 | 1.85 µm / 2.02 | 75.3 % | Test group 4 | 1.77 µm / 1.97 | 78.2 % |
Test group 2 | 1.77 µm / 2.17 | 75.2 % | Test group 4 | 1.79 µm / 2.26 | 73.5 % |
In the following table the data of APS measurements were presented. The APS measurement showed slightly higher MMAD. The difference between the two devices are to be explained by the mechanisms the measurements based on.
Particle size distribution measured by APS:
| Measurement date | MMAD / GSD |
| Measurement date | MMAD / GSD |
Test group 1 | 26 Jun 20 | 2.47 µm/2.24 | Test group 3 | 26 Jun 20 | 3.23 µm/1.87 |
| 2.39 µm/2.22 |
| 3.38 µm/1.98 | ||
| 2.47 µm/2.34 |
| 3.33 µm/1.91 | ||
02 Jul 20 | 2.28 µm/2.37 | 02 Jul 20 | 2.93 µm/1.97 | ||
| 2.13 µm/2.25 |
| 3.11 µm/2.21 | ||
| 2.10 µm/2.23 |
| 3.46 µm/2.39 | ||
Test group 2 | 25 Jun 20 | 1.94 µm/2.66 | Test group 4 | 25 Jun 20 | 2.41 µm/2.16 |
| 1.82 µm/2.54 |
| 2.40 µm/2.18 | ||
| 1.82 µm/2.52 |
| 2.29 µm/2.09 | ||
03 Jul 20 | 2.41 µm/2.46 | 03 Jul 20 | 2.76 µm/2.25 | ||
| 2.50 µm/2.37 |
| 2.67 µm/2.22 | ||
| 2.50 µm/2.55 |
| 2.59 µm/2.17 |
BAL RESULTS
Changes in mean absolute cell counts in BAL (x-fold of concurrent control) of male rats on study day 14 (1 day after last exposure):
Analyte | Gr. 1 T0420 12 mg/m3 | Gr. 2 T0420 24 mg/m3 | Gr. 3 T0421 12 mg/m3 | Gr. 4 T0421 24 mg/m3 |
Total Cells | 7.4* | 8.9** | 4.8** | 8.6** |
Eosinophils | 4.1 | 16.0 | 6.7 | 11.0 |
Lymphocytes | 4.9* | 7.4** | 4.0* | 7.5** |
Macrophages | 0.6 | 1.1 | 1.1 | 0.7 |
Neutrophils | 620.8* | 722.3** | 333.3** | 734.0** |
Monocytes | 105.2* | 91.2** | 48.0** | 71.4** |
Epithelial cells | + | + | + | + |
One-sided Wilcoxon-test: * : p £ 0.05; ** : p £ 0.01
+ increase could not be calculated because of zero activity in controls
Changes in median total protein and enzyme levels in BAL (x-fold of concurrent control) of male rats on study day 14 (1 day after last exposure). Medians instead of means were used because of high individual variation of the values with great impact on the mean values:
Analyte | Gr. 1 T0420 12 mg/m3 | Gr. 2 T0420 24 mg/m3 | Gr. 3 T0421 12 mg/m3 | Gr. 4 T0421 24 mg/m3 |
Total Protein | 14.4** | 18.6** | 5.4** | 17.5** |
GGT | 4.6** | 4.1** | 2.9** | 4.3** |
LDH | 11.0** | 11.2** | 4.6** | 11.7** |
ALP | 16.6** | 23.5** | 7.0** | 27.4** |
NAG | 2.7* | 2.7* | 1.7** | 2.8** |
GGT = g-Glutamyl-transferase; LDH = Lactate dehydrogenase; ALP = Alkaline phosphatase;
NAG = b-N-Acetyl glucosaminidase, One-sided Wilcoxon-test: * : p £ 0.05; ** : p £ 0.01
Histopathology
Treatment-related findings were observed in organs listed in the tables below with incidences and grading:
Incidence and grading of histological findings in larynx:
Larynx (Level I) | Male animals | ||||
Control | T0420 | T0421 | |||
Test group (mg/m³) | 0 (0) | 1 (12) | 2 (24) | 3 (12) | 4 (24) |
Epithelial alteration | 1 | 0 | 4 | 1 | 2 |
· Grade 1 | 1 |
| 4 | 1 | 2 |
The finding epithelial alteration is a common observation in inhalation studies and in most cases, the base of the epiglottis is affected. It shows a minimal flattening of the epithelial cells and loss of cilia. It was considered to be treatment-related but with the minimal grading it was not regarded to be adverse (Kaufmann et al., 2009).
Incidence and grading of histological findings in lungs
Lungs | Male animals | ||||
Control | T0420 | T0421 | |||
Test group (mg/m³) | 0 (0) | 1 (12) | 2 (24) | 3 (12) | 4 (24) |
Infiltration, granulocytic | 0 | 5 | 5 | 5 | 5 |
· Grade 1 |
| 4 |
| 3 | 2 |
· Grade 2 |
| 1 | 5 | 2 | 3 |
Histiocytosis, alveolar | 0 | 5 | 5 | 5 | 5 |
· Grade 1 |
|
|
| 1 |
|
· Grade 2 |
| 4 |
| 2 |
|
· Grade 3 |
| 1 | 5 | 2 | 3 |
· Grade 4 |
|
|
|
| 2 |
In general, Lob. cran. dexter, Lob. medius dexter, and Lob. accessorius of the lungs were slightly less severely affected compared to Pulmo sinister and Lob. caudalis dexter. The finding was characterized by disseminated intra-alveolar inflammatory cells (mainly histiocytes and less number of granulocytes), intermingled by foamy roundish structures, containing occasionally a faint, bluish, roundish structures inside (interpreted as nucleus) or finely granular, eosinophilic material inside the alveoli. These structures were considered to be destroyed alveolar macrophages. These findings were regarded to be treatment-related.
Incidence and grading of histological findings in the nasal cavity:
Nasal cavity (Level IV) | Male animals | ||||
Control | T0420 | T0421 | |||
Test group (mg/m³) | 0 (0) | 1 (12) | 2 (24) | 3 (12) | 4 (24) |
Degeneration/regeneration, olfactory epithelium | 0 | 5 | 5 | 5 | 5 |
· Grade 1 |
| 3 |
|
|
|
· Grade 2 |
| 2 |
| 3 |
|
· Grade 3 |
|
| 5 | 2 | 1 |
· Grade 4 |
|
|
|
| 4 |
Level IV of the nasal cavity was the most severely affected level and was here taken representatively for findings in the nasal cavity. There was loss, irregularity or flattening of the olfactory epithelium (interpreted as degeneration). The most affected areas were the septum, the dorsal meatus and the tips of the conchae. In some areas there was in addition an increase of nuclear size and basophilia, mainly of basal cells (interpreted as regeneration). These findings were considered as treatment-related.
Incidence and grading of histological findings in the tracheobronchial lymph nodes:
Tracheobronchial lymph nodes | Male animals | ||||
Control | T0420 | T0421 | |||
Test group (mg/m³) | 0 (0) | 1 (12) | 2 (24) | 3 (12) | 4 (24) |
Histiocytosis (m)focal | 0 | 3 | 3 | 4 | 5 |
· Grade 1 |
|
|
| 2 | 1 |
· Grade 2 |
| 2 | 3 | 2 | 4 |
· Grade 3 |
| 1 |
|
|
|
Histiocytes with intracytoplasmic material similar to those seen in the lungs, were found in tracheobronchial lymph nodes. This finding is regarded as treatment-related, but not adverse.
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.
Pathology
Weight parameters
Absolute weights
When compared to control group 0 (=100%), the mean absolute weights of following organs were significantly changed (statistically significant changes printed in bold):
Relative changes of absolute lung weights:
| Male animals | |||
| T0420 | T0421 | ||
Test group (mg/m³) | 1 (12) | 2 (24) | 3 (12) | 4 (24) |
Lungs | 113% | 135%** | 114%** | 141%** |
*p <= 0.05; **p <= 0.01
All other mean absolute weight parameters did not show significant differences when compared to the control group 0.
Relative organ weights
When compared to control group 0 (=100%), the mean relative weights of following organs were significantly changed (statistically significant changes printed in bold):
Relative changes of relative lung weights:
| Male animals | |||
| T0420 | T0421 | ||
Test group (mg/m³) | 1 (12) | 2 (24) | 3 (12) | 4 (24) |
Lungs | 117% | 138%** | 119%** | 154%** |
*p <= 0.05; **p <= 0.01
All other mean absolute and relative weight parameters did not show significant differences when compared to the control group 0.
The statistical significantly increased lung weight in test group 2, 3, and 4 was regarded to be treatment-related.
Gross lesions
In some animals, the tracheobronchial lymph nodes were enlarged. This was considered as treatment-related.
Incidence of gross lesions observed during necropsy:
Tracheobronchial lymph nodes | Male animals | ||||
Control | T0420 | T0421 | |||
Test group (mg/m³) | 0 (0) | 1 (12) | 2 (24) | 3 (12) | 4 (24) |
Enlarged | 0 | 2 | 1 | 0 | 2 |
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.
Applicant's summary and conclusion
- Conclusions:
- Both Zinc oxide T420 and T421 caused lesions in nasal cavity and lung already at the low targeted concentration of 12 mg/m³ (measured concentration about 11 mg/m³). No systemic effect was observed. At comparable atmospheric concentrations, Zinc oxide T421 seemed to cause more severe effects than those caused by T420.
- Executive summary:
Groups of male Wistar rats were exposed whole-body to the dust aerosols of Zinc oxide T0420 and Zinc oxide T0421 for 6 hours per day on 5 days per week for two weeks. The target concentrations were 12 and 24 mg/m³ for each of the test items. A concurrent control group was exposed to clean air. Daily clinical observation and body weight were recorded. Animals were sacrificed, assessments including hematology, bronchoalveolar lavage and histopathology (control and high concentration only) of the respiratory tract were carried out.
The following is a summary of the most relevant results:
Test group 4 (T0421, 24 mg/m3)
- Impaired body weight development, reduced food consumption
- Increased absolute and relative neutrophil cell counts in blood
- Decrease relative lymphocyte counts in blood
- Increased total cell counts, absolute and relative neutrophil and monocyte counts as well as absolute lymphocyte counts in BAL
- Decreased relative macrophage counts in BALF
- Increased total protein levels as well as g-Glutamyl-transferase (GGT), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP) and b-N-Acetyl glucosaminidase (NAG) activities in BALF
- Increase of absolute and relative lung weight (141%/154%)
- Minimal to slight infiltration of neutrophilic granulocytes and moderate to severe infiltration of alveolar histiocytes in the lungs in all animals
- Slight to severe degeneration/regeneration of the olfactory epithelium in the nasal cavity of all animals
Test group 3 (T0421 12 mg/m3)
- Increased total cell counts, absolute and relative neutrophil and monocyte counts as well as absolute lymphocyte counts in BAL
- Decreased relative macrophage counts in BALF
- Increased total protein levels as well as g-Glutamyl-transferase (GGT), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP) and b-N-Acetyl glucosaminidase (NAG) activities in BALF
- Increase of absolute and relative lung weight (114%/119%)
- Minimal to slight infiltration of neutrophilic granulocytes and minimal to moderate infiltration of alveolar histiocytes in the lungs in all animals
- Minimal to moderate degeneration/regeneration of the olfactory epithelium in the nasal cavity of all animals
Test group 2 (T0420, 24 mg/m3)
- Increased total cell counts, absolute and relative neutrophil and monocyte counts as well as absolute lymphocyte counts in BAL
- Decreased relative macrophage counts in BALF
- Increased total protein levels as well as g-Glutamyl-transferase (GGT), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP) and b-N-Acetyl glucosaminidase (NAG) activities in BALF
- Increase of absolute and relative lung weight (135%/138%)
- Slight infiltration of neutrophilic granulocytes and moderate infiltration of alveolar histiocytes in the lungs in all animals
- Minimal to moderate degeneration/regeneration of the olfactory epithelium in the nasal cavity of all animals
Test group 1 (T0420, 12 mg/m3)
- Increased total cell counts, absolute and relative neutrophil and monocyte counts as well as absolute lymphocyte counts in BAL
- Decreased relative macrophage counts in BALF
- Increased total protein levels as well as g-Glutamyl-transferase (GGT), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP) and b-N-Acetyl glucosaminidase (NAG) activities in BALF
- Minimal to slight infiltration of neutrophilic granulocytes and slight to moderate infiltration of alveolar histiocytes in the lungs in all animals
Minimal to slight degeneration/regeneration of the olfactory epithelium in the nasal cavity of all animals
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