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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 March 2012 to 30 January 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to valid guidelines and the study was conducted under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.29 (Sub-Chronic Inhalation Toxicity:90-Day Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: milled solid
Details on test material:
- Name of test material (as cited in study report): Cobalt Lithium Manganese Nickel oxide
- Appearance: black solid
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Crl:WI(Han)
- Age: approximately 7 weeks (when supplied)
- Weight at study initiation: Males: 258.9 - 265.2 g (group means); females 172.2 - 180.1 g (group means)
- Housing: in groups of 5 per cage in Polysulfone cages (floor area ~ 2065 cm²). Lignocel fibres, dust-free wooden bedding was used in this study. For enrichment, wooden gnawing blocks were added.
- Diet: ad libitum (except during exposure)
- Water: ad libitum (except during exposure)

ACCLIMATISATION
The animals were delivered and subjected immediately to the acclimatisation 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 two days before start of exposure (pre-exposure period).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C; 21.0 - 23.8 °C in the inhalation systems
- Humidity (%): 30 - 70 % (relative); 34.0 - 63.7 % in the inhalation systems
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light (06:00 to 18:00)

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 2.0 and 5.7 µm with GSDs between 1.9 and 4.4.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Generator systems: Solid particle generators (brush-generator), Mixing tube
- Generation procedure: The test material was used unchanged. For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing tube mixed with conditioned dilution air and passed into the inhalation system. Conditioned supply air is activated charcoal filtered air conditioned to about 50 ± 20 % relative humidity and 22 ± 2 °C. Compressed air is filtered air pressurised to about 6 bar (see Table 2).

EXPOSURE SYSTEM
The inhalation atmosphere was maintained inside aerodynamic exposure 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 (see Figure 1).
Two inhalation systems were used. Prior to exposure, to adjust the animals to the exposure conditions, INA 120 (V ≈ 155 L, BASF SE) was used. During substance exposure, INA 60 (V ≈ 90 L, BASF SE) was used for test groups 1 to 3, and INA 120 for the controls.
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 method used:
> In test groups 2 and 3, the nominal concentration was calculated from the study means of the substance flow and the supply air flows used during exposure to generate the respective concentrations. In test group 1 nominal concentration was not determined because a dilution system was used to achieve the low target concentration of 0.2 mg/m³. The nominal concentration in this group was not comparable to those determined in the other groups and did not reflect the generation efficiency. As the nominal concentration is not of significance to the analytically measured one, it was not determined for this group.
> The concentrations of the inhalation atmospheres were analysed by gravimetry in test groups 1 - 3. In these groups, the constancy of concentrations in the inhalation systems were continuously monitored using scattered light photometers.
> Sampling for gravimetric 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 M3P-000V001
Sampling velocity: 1.25 m/s
Flow rate of sampling: 3 L/min
Sample volumes: Test group 1: 180 L; Test group 2: 180 L; test group 3: 90 or 180 L
Sampling site: Immediately adjacent to the animals' noses at a separate spare port
Sampling frequency: Three samples per exposure and concentration group
> In the gravimetrical analysis, a pre-weighed filter was placed into the filter holder. By means of a vacuum compressed air pump a defined volume of the dust was drawn through the filter. The dust concentration in mg/m³ was calculated from the difference between the weight of the pre-weighed 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 dusts in the inhalation systems using scattered light photometers (VisGuard) in test groups 1-3.The data were recorded by an automated measuring system.
> Particle 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 M3P-000V001 and LC 1201S-00MS
> In the sampling for particle size analyses, pre-weighed metal collecting discs and a backup particle filter were placed into the cascade impactor. One sample was taken in each concentration per week at a sampling velocity of 1.25 m/s from the breathing zones of the animals.
Sample numbers and volumes for particle size measurement were: Test group 1: 15 samples, sample volume 720 or 990 L; test group 2: 15 samples, sample volume 720 L; test group 3: 15 samples, sample volume 720 or 6300 L.
> The amount of dust deposited by each stage in mg was calculated from the difference between the weight of the metal collecting and backup filter before and after sampling. The deposits in the probe and the wall losses in the impactor were also determined as the difference of the total mass increase of the impactor and the sum of masses on the collecting discs and backup filter.
> Particle size distribution in the test atmospheres was measured by Aerodynamic Particle Sizer (APS) TSI APS 3321. The Mass Median Aerodynamic Diameter (MMAD) and Geometric Standard Deviation (GSD) were obtained directly by the equipment used. The frequency was 3 repeats per test group.
Duration of treatment / exposure:
3 months (90 days)
Frequency of treatment:
6 hours per day on 5 consecutive days per week (workdays). The total number of exposures was 65.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 0.2, 1 and 5 mg/m³
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 0.25, 1.42 and 5.10 mg/m³ (mean measured)
Basis:
analytical conc.
No. of animals per sex per dose:
10 males and 10 females per dose group (main groups)
5 additional males and 5 additional females were exposed to 0 and 5 mg/m³ (recovery groups)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The concentrations to be tested in this study were selected based on the results of a 5-day range finding study.
In the range finding study, inhalation exposure to 50 mg/m³ test material caused the death of 16 out of 22 rats after 2 exposures. Clinical signs of toxicity comprised accelerated and intermittent respiration, piloerection, pale skin and hypothermia. Two animals that showed abdominal position, poor general state / convulsion were sacrificed moribund. They were counted as deaths. Histopathological examination of the dead animals revealed severe alveolar damage as the cause of death. In the surviving animals of this group, which were sacrificed after a 3-week recovery period, a minimal bronchial epithelial degeneration/regeneration was observed. Clinically, the surviving animals recovered very fast and were free of clinical signs of toxicity 4 days after termination of the exposure. The body weights of survivors were significantly lower than the controls on study day 4 but reached the control level on study day 6. The recovery of the surviving animals and the minimal morphological change in the histopathology demonstrated the reversibility of the effects on the bronchial epithelium. Focal necrosis and squamous metaplasia in the olfactory epithelium, bronchial epithelial degeneration/ regeneration, lympho-reticular hyperplasia in mediastinal and tracheobronchial lymph nodes, inflammation of the trachea, and squamous metaplasia of the larynx were also noted in animals exposed to 50 mg/m³ test material.
At 2 and 10 mg/m³, no clinical signs of toxicity were noted; mean body weight was comparable with the controls. In the lavage fluid, counts of neutrophils and lymphocytes were significantly increased, accompanied by increased total protein and activity of several enzymes, indicative of inflammatory responses. All these changes were almost fully reversible within the recovery period of 3 weeks. Histologically, concentration-related changes were observed in nasal cavity, lymph nodes (mediastinal and tracheobronchial) and trachea. Focal necrosis in the olfactory epithelium and bronchial epithelial degeneration/regeneration were still present at the low concentration of 2 mg/m³, though with less incidence and severity than at 10 mg/m³.

- Rationale for animal assignment (if not random): Prior to the pre-exposure period, the animals were distributed according to weight among the individual test groups, separated by sex. The weight variation of the animals used did not exceed ± 20 % of the mean weight of each sex. The list of randomisation instructions was compiled with a computer. For each neurofunctional test and motor activity measurement, separate randomisation lists were created.

- Rationale for inclusion of post-exposure recovery (satellite) groups: In order to check for any reversibility, progression or delay of toxic effects the recovery groups were used. The recovery group animals were observed for 4 weeks after the exposure period.

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 condition of the test animals was recorded once daily during the pre-exposure period and on post-exposure observation days and at least 3 times per day (before, during and after exposure) on exposure days. During exposure only a group wise examination was possible.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were subjected to detailed clinical observations outside their cages once before the beginning of the administration period (day 0), at midterm and again at the end of the exposure period. For observation, the animals were removed from their cages and placed in a standard arena (50 x 37.5 x 25 cm).
- Parameters checked included: abnormal behaviour during handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmos, faeces (appearance/consistency), urine and pupil size.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of the animals was determined at the start of the pre-exposure, at the start of the exposure period then once a week, as well as prior to gross necropsy.
Body weight change was calculated as the difference between body weight on Monday and Friday of the same week. Group means were derived from the individual differences.

FOOD CONSUMPTION: Yes
- Time schedule: Food consumption was determined weekly and calculated as mean food consumption in grams per animal and day.
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 test group 1 and 2 were housed in only two cages per sex, no statistical evaluation of food consumption was possible.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before the start of the exposure period (day -2) the eyes of all main group animals, and at the end of the study (day 83) the eyes of the animals of test group 0 (control group) and test group 3 (high concentration) were examined for any changes in the refracting media with an ophthalmoscope after administration of a mydriatic.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the administration period (main group animals); at the end of the recovery period (recovery group animals). Blood was taken from the retro-bulbar venous plexus.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked included: leukocyte count (WBC), erythrocyte count (RBC), haemoglobin (HGB), haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelet count (PLT), differential blood count, reticulocytes (RET) and prothrombin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the administration period (main group animals); at the end of the recovery period (recovery group animals). Blood was taken from the retro-bulbar venous plexus.
- Animals fasted: Yes
- How many animals: All animals
- 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) and cholesterol (CHOL).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes (on day 90)
At least one hour before the start of the FOB the animals were transferred singly to Polycarbonate cages (floor area about 800 cm²). The cages were placed in the racks in a randomised order. 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. Drinking water was provided ad libitum during the measurements. On the day of the FOB, animals were not exposed to the test material.
> 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 behaviour of the animals. Attention was paid to: posture, tremor, convulsions, abnormal movements and impairment of gait.
> Open field observations: The animals were transferred to a standard arena (50 x 50 cm with sides 25 cm high) and observed for at least 2 minutes. The following parameters were examined: behaviour 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, faeces (number of faecal pellets/appearance/consistency) within two minutes, urine (amount/colour) within two minutes and number of rearings within two minutes.
- Battery of functions tested:
> Sensorimotor tests/ Reflexes
The animals were removed from the open field and subjected to the following sensorimotor or reflex tests: approach response, touch response, vision ("visual placing response"), pupillary reflex, pinna reflex, audition ("startle response"), coordination of movements ("righting response"), behaviour during "handling", vocalisation, pain perception ("tail pinch"), grip strength of forelimbs, grip strength of hindlimbs and landing foot-splay test.
> Motor activity measurements
Motor activity was measured on the same day that theFOB was performed. The measurement was performed in the dark using the TSE Labmaster System with 18 infrared beams per cage. During the measurement the animals were kept in Polycarbonate cages with wire covers (floor area about 800 cm²) and small amounts of absorbent material. The animals were put into the cages in a randomised order.
The numbers of beam interrupts were counted over 12 intervals, each lasting 5 minutes. 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.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
The animals were sacrificed under pentobarbitone anaesthesia by exsanguination from the abdominal aorta and vena cava. The exsanguinated animals were necropsied and assessed by gross pathology.

ORGAN WEIGHTS: Yes
The following weights were determined in all animals sacrificed on schedule: anaesthetised animals, adrenal glands, brain, epididymides, heart, kidneys, liver, lung, ovaries, spleen, testes, thymus, thyroid glands and uterus.

ORGAN/TISSUE FIXATION: Yes
The following organs or tissues were fixed in 4 % buffered formaldehyde solution or in modified Davidson’s solution: All gross lesions, adrenal glands, aorta, bone marrow (femur), brain with olfactory bulb, cecum, colon, duodenum, epididymides, extraorbital lacrimal gland, eyes with optic nerve and eyelid, femur with knee joint, harderian glands, heart, ileum, jejunum, kidneys, larynx, liver, lung, lymph nodes (tracheobronchial, mediastinal and mesenteric lymph nodes), mammary gland (male & female), nose (nasal cavity), oesophagus, 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 (modified Davidson’s solution), thymus, thyroid glands, tongue, trachea, ureter, urethra, urinary bladder and uterus.

HISTOPATHOLOGY: Yes
The organs listed in Table 3 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 and food consumption: A comparison of each group with the control group was performed using Dunnett's test (two-sided) for the hypothesis of equal means.
- Faeces, rearing, grip strength length (forelimbs and hindlimbs), footsplay, motor activity and blood parameters (with bidirectional changes): Non-parametric one-way analysis using Kruskal-Wallis test (two-sided). If the resulting p-value was equal to or less than 0.05, a pairwise comparison of each dose group with the control group was performed using Wilcoxon-test (two-sided) for the equal medians.
- Blood parameters (with unidirectional changes): Pairwise comparison of each dose group with the control group using the Wilcoxon test (one-sided) with Bonferroni-Holm adjustment for the hypothesis of equal medians.
- Weight of the anaesthetised animals and absolute and relative organ weights: Non-parametric one-way analysis using Kruskal-Wallis test (two-sided). If the resulting p-value was equal to or less than 0.05, a pairwise comparison of each dose group with the control group was performed using Wilcoxon-test (two-sided) for the equal medians.

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:
no effects observed
Description (incidence and severity):
No test material-related adverse effects observed
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:
no effects observed
Description (incidence and severity):
No test material-related adverse effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
No test material-related adverse effects observed
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No test material-related adverse effects observed
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
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
No deaths were recorded throughout the study.
All male animals did not show any clinical signs of toxicity throughout the study. In female animals, reduced fur care was observed in individuals of test group 2 (2 of the 10 females) and group 3 (3 of the 15 females).
The detailed clinical observations did not reveal any abnormalities in all male and female animals of test groups.

BODY WEIGHT AND WEIGHT GAIN
The mean body weights of the male and female animals exposed to the test material were not statistically significantly different from the control group 0 throughout the study.
The body weight changes of all exposed female animals were comparable to the controls throughout the study.
The following statistically significant body weight changes were observed in exposed male animals:
- From study day -2 to 0: group 2
- From study day 0 to 2: group 1, group 2 and group 3
- From study day 12 to 16: group 3
- From study day 54 to 58: group 2
- From study day 61 to 65: group 2
- From study day 82 to 86: group 2 and 3
The reduced body weight gain in test group 2 from study day -2 to 0 is clearly not test material-related, because the exposure did not start at the measurement time point. From study day 0 to 2 body weight gains of all exposed groups were reduced when compared with the control. As this change is transient and there is no concentration-response relationship, this finding is considered to be incidental.
During the exposure period, the body weight gains of test groups 2 and 3 were marginally lower than those of the control, though not statistically significant in most cases. Therefore, the significances in body weight gain of test groups 2 and 3 might be related to the exposure. The findings are considered to be not adverse because there was no concentration-response relationship, and the reduction (maximum -2.3 %) was so minimal.

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

OPHTHALMOSCOPIC EXAMINATION
Spontaneous findings such as remainders of the pupillary membrane or corneal stippling were observed in several animals of all test groups and the control group without any concentration-response relationship.

HAEMATOLOGY
No treatment-related, adverse changes among haematological parameters were observed.
After the 4-week recovery period, in males of test group 3 (5 mg/m³), haemoglobin and haematocrit values, and in females of this dose group red blood cell (RBC) counts, were higher compared to controls. However, all values were within historical control ranges (males: haemoglobin 9.0-9.8 mmol/L, haematocrit 0.396-0.448 L/L; females: RBC 7.28-8.25 Tera/L) and therefore, the alterations were regarded as incidental and not treatment-related.
After the recovery period in females of test group 3 (5 mg/m³), absolute and relative neutrophil counts were increased and correspondingly, relative lymphocyte counts were decreased. Total white blood cell counts were not changed in this group and there was no change in the differential blood cell counts during the 3 month administration period. Therefore, the higher neutrophil counts after the recovery period were regarded as possibly treatment-related, but not adverse.

CLINICAL CHEMISTRY
No treatment-related, adverse changes among clinical chemistry parameters were observed.
After the recovery period in males of test group 3 (5 mg/m³), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were higher compared to controls. The ALT mean was at the border of the historical control range and the AST mean was slightly above this range (ALT 0.46-0.94 μkat/L; AST 1.37-2.29 μkat/L). The AST mean in test group 3 was 36 % higher compared to that of the study control, which was regarded as a marginal elevation. Neither any change of liver enzyme activities nor any alteration of other clinical chemistry parameters occurred during the administration phase. Even after the recovery period, no other liver parameter (total proteins, albumin, cholesterol, triglycerides, prothrombin time and bilirubin), apart from both mentioned liver enzymes, was altered. Therefore, these higher liver enzyme activities after the recovery period were regarded as treatment-related, but not adverse.
After the recovery period in males of test group 3 (5 mg/m³), potassium levels were higher compared to controls, but the values were within the historical control range (potassium 4.35-4.82 mmol/L) and therefore, this alteration was regarded as incidental and not treatment-related.

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 to have been incidental.

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. 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. All other mean relative weight parameters did not show significant differences when compared to the control group 0.
The increased lung weights in males and females (absolute and relative) of all treated test groups were regarded to be treatment-related.

ORGAN WEIGHTS (Recovery group)
> Absolute weights
When compared to the control group 0 (set to 100 %), the mean absolute weights of the lung were significantly increased in test group 3.
> Relative organ weights
When compared to the control group 0 (set to 100 %), the mean relative weights of the lung were significantly increased in test group 3. All other mean relative weight parameters did not show significant differences when compared to the control group 0.

The increased lung weights in males and females (absolute and relative) of test group 3 were regarded as being treatment-related.

GROSS PATHOLOGY (Main group)
Treatment-related foci were noted on the lung of animals of all treated test groups.
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)
Treatment-related findings were noted in the lung of recovery animals.
All other findings occurred individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

HISTOPATHOLOGY (Main group)
Target organs were the larynx, level I and the lung.
> Larynx, level I: Treatment-related findings in the larynx were epithelial alteration characterised by slight modification of epithelial cells (i.e., three to four cell layers, focally flattened and stratified, indicating the beginning of metaplastic transformation) and minimal squamous metaplasia (characterised by three to four cell layers of flattened, stratified epithelium with no signs of keratinisation and only affecting the epiglottis).
> Lung: There were numerous treatment–related findings in alveoli and bronchi (as detailed below).
- Alveoli (cellular debris, histiocytosis, inflammation (multifocal), infiltrates (inflammatory cells), mucus, regeneration of alveolar epithelium and emphysema.
- Bronchi: reduced number of clara cells, degeneration of bronchial epithelial cells, metaplasia (mucoid, bronchial), bronchial obliteration and test material in bronchioles.
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)
> Larynx, level I: Laryngeal epithelial alteration was only present in 2/5 female animals recovering from 5 mg/m³, indicating recovery from this finding in most animals.
> Lung: Findings in the lung alveoli persisted in recovery animals; notably, there was a higher grade of alveolar histiocytosis in animals recovering from 5 mg/m³. Findings in bronchi were no longer detected after the recovery period.

Effect levels

Dose descriptor:
NOAEC
Remarks:
(systemic)
Effect level:
5 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No test material-related signs of systemic toxicity were noted during the study.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 4: Incidence and grading of histological findings in the larynx

Larynx, level I

Male animals

Female animals

Test group (mg/m³)

0 (0)

1 (0.2)

2 (1)

3 (5)

0 (0)

1 (0.2)

2 (1)

3 (5)

Main Group Animals

No. of animals/group

10

10

10

10

10

10

10

10

Epithelial alteration

 

6

9

8

1

3

8

8

Grade 1

 

6

9

8

1

3

8

8

Metaplasia, squamous

 

 

1

1

 

 

 

 

Grade 1

 

 

1

1

 

 

 

 

Recovery Group Animals

No. of animals/group

5

-

-

5

5

-

-

5

Epithelial alteration

 

 

 

 

 

 

 

2

Grade 1

 

 

 

 

 

 

 

2

 

Table 5: Incidence and grading of histological findings in the lung

Lung

Male animals

Female animals

Test group (mg/m³)

0 (0)

1 (0.2)

2 (1)

3 (5)

0 (0)

1 (0.2)

2 (1)

3 (5)

Main Group Animals

No. of animals/group

10

10

10

10

10

10

10

10

Cellular debris, alveolar

 

10

10

10

 

10

10

10

Grade 1

 

4

2

 

 

3

2

 

Grade 2

 

6

5

6

 

6

8

6

Grade 3

 

 

3

4

 

1

 

3

Grade 4

 

 

 

 

 

 

 

1

Histiocytosis, alveolar

 

10

10

10

 

10

10

10

Grade 1

 

 

10

5

 

4

10

6

Grade 2

 

9

 

5

 

5

 

4

Grade 3

 

1

 

 

 

1

 

 

Inflammation, (m)f

 

9

7

10

 

2

9

1

Grade 1

 

8

7

2

 

1

9

1

Grade 2

 

1

 

7

 

1

 

 

Grade 3

 

 

 

1

 

 

 

 

Infiltrates, inflammatory cell

2

10

10

10

 

10

10

10

Grade 1

2

10

10

7

 

7

10

10

Grade 2

 

 

 

3

 

3

 

 

Mucus

 

10

10

10

 

10

10

10

Grade 1

 

 

1

1

 

5

1

6

Grade 2

 

6

2

9

 

5

6

2

Grade 3

 

4

6

 

 

 

3

1

Grade 4

 

 

1

 

 

 

 

1

Regeneration, alveolar epithelium

 

5

 

5

 

6

4

6

Grade 1

 

5

 

4

 

5

4

6

Grade 2

 

 

 

1

 

 

 

 

Grade 3

 

 

 

 

 

1

 

 

Emphysema

 

1

 

2

 

 

3

 

Present

 

1

 

2

 

 

3

 

Reduced no. of clara cells

 

4

10

10

 

 

10

10

Grade 1

 

3

2

 

 

 

2

 

Grade 2

 

1

 

 

 

 

2

2

Grade 3

 

 

8

 

 

 

4

2

Grade 4

 

 

 

10

 

 

2

6

Degeneration, bronchial epithelial cells

 

3

10

10

 

 

10

10

Grade 1

 

3

10

10

 

 

10

10

Metaplasia, mucoid, bronchial

 

 

 

 

 

4

2

5

Grade 1

 

 

 

 

 

3

2

3

Grade 2

 

 

 

 

 

1

 

2

Obliteration, bronchial

 

 

 

 

 

 

 

2

Grade 1

 

 

 

 

 

 

 

1

Grade 2

 

 

 

 

 

 

 

1

Test material, bronchial

 

 

 

1

 

 

 

1

Present

 

 

 

1

 

 

 

1

Recovery Group Animals

No. of animals/group

5

-

-

5

5

-

-

5

Cellular debris, alveolar

 

 

 

5

 

 

 

5

Grade 1

 

 

 

2

 

 

 

1

Grade 2

 

 

 

2

 

 

 

4

Grade 3

 

 

 

1

 

 

 

 

Histiocytosis, alveolar

 

 

 

5

 

 

 

5

Grade 3

 

 

 

5

 

 

 

5

Inflammation, (m)f

 

 

 

5

 

 

 

5

Grade 1

 

 

 

3

 

 

 

2

Grade 2

 

 

 

2

 

 

 

3

Infiltrates, inflammatory cell

 

 

 

5

1

 

 

5

Grade 1

 

 

 

3

1

 

 

3

Grade 2

 

 

 

2

 

 

 

2

Mucus

 

 

 

5

 

 

 

5

Grade 1

 

 

 

1

 

 

 

1

Grade 2

 

 

 

 

 

 

 

3

Grade 3

 

 

 

2

 

 

 

1

Grade 4

 

 

 

2

 

 

 

 

Regeneration, alveolar epithelium

 

 

 

5

 

 

 

5

Grade 1

 

 

 

3

 

 

 

2

Grade 2

 

 

 

1

 

 

 

3

Grade 3

 

 

 

1

 

 

 

 

Emphysema

 

 

 

 

1

 

 

 

Present

 

 

 

 

1

 

 

 

Applicant's summary and conclusion

Conclusions:
Inhalation exposure of 0.2, 1 and 5 mg/m³ of the test material for 90 days (65 exposures) caused treatment-related histological changes in the lung. The effects were not fully reversible within a 4 week recovery period. A No Observed Adverse Effect Concentration (NOAEC) could not be established under the conditions of this study.
Based on the data of clinical observation, body weight, food consumption, clinical chemistry, haematology and histological examinations, there was no indication of any systemic toxicity. Therefore, under the conditions of the study, the NOAEC for systemic toxicity was determined to be 5 mg/m³.
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 ten male and ten female Wistar rats per test group were exposed in a nose-only fashion to a dynamic atmosphere of the test material for 6 hours per day on 5 consecutive days per week for 3 months (90-day study). To enable dust atmosphere generation, the test material was milled prior to the study. The target concentrations were 0.2, 1 and 5 mg/m³. A concurrent control group was exposed to conditioned air. To detect reversibility of the effects, recovery group animals (5 male and 5 female animals per group) were exposed to conditioned air and the high concentration. The recovery period was 4 weeks.

Clinical observations were performed at least three times on exposure days and once a day during the other days. Body weight and food consumption were determined for all animals. Detailed clinical observations were performed three times (prior to the start, at mid-term and again at the end of the exposure). Ophthalmological examinations were performed for the main group animals prior to exposure and towards the end of the exposure. A functional observation battery and motor activity test were performed on study day 90.

After the last exposure or at the end of the 4 week recovery period, blood was sampled and haematology and clinical chemistry parameters were determined. The animals were then subjected to gross necropsy (including macroscopic examination of the major internal organs and collection of organ weight data). Histological examinations were performed according to respective test guidelines.

 

The targeted atmospheric concentrations were maintained well throughout the study. No treatment-related effects were noted concerning clinical observations, body weight development and clinical pathology at test concentrations up to 5 mg/m³.

In main group animals treated at a concentration of 5 mg/m³, the following treatment-related adverse effects were noted: increased absolute and relative lung weights in males and females; alveolar cellular debris in all male and female animals (graded slight to moderate in males and slight to marked in females); alveolar histiocytosis in all male and female animals (graded minimal to slight); multifocal inflammation in all male animals (graded minimal to moderate) and 1 female animal (graded minimal); inflammatory cell infiltrates in all male (graded minimal to slight) and female animals (graded minimal); mucus in all male and female animals (graded minimal to slight in males and minimal to severe in females); alveolar epithelium regeneration in 5/10 males and 6/10 females (graded minimal to slight in males and minimal in females); emphysema in 2/10 males; a reduced number of clara cells in all male and female animals (graded severe in male animals and slight to severe in female animals); bronchial epithelial cell degeneration in all male and female animals (graded minimal); mucoid bronchial metaplasia in 5/10 female animals (graded minimal to slight); and bronchial obliteration in 2/10 female animals (graded minimal to slight).

 

In recovery group animals exposed to the test material at 5 mg/m³ the following adverse effects were noted: increased absolute and relative lung weights in males and females; alveolar cellular debris in all male and female animals (graded minimal to moderate in males and minimal to slight in females); alveolar histiocytosis in all male and female animals (graded moderate in both sexes); multifocal inflammation in all male and female animals (graded minimal to slight); inflammatory cell infiltrates in all male and female animals (graded minimal to slight); mucus in all male and female animals (graded minimal to severe in males and minimal to moderate in females); alveolar epithelium regeneration in all male and female animals (graded minimal to moderate in males and minimal to slight in females).

 

In animals of test group 2 (1 mg/m³), adverse effects included: increased absolute and relative lung weights in males and females; alveolar cellular debris in all male and female animals (graded minimal to moderate in males and minimal to slight in females); alveolar histiocytosis in all male and female animals (graded minimal); multifocal inflammation in 7/10 male animals and 9/10 female animals (graded minimal); inflammatory cell infiltrates in all male and female animals (graded minimal); mucus in all male and female animals (graded minimal to severe in males and minimal to moderate in females); alveolar epithelium regeneration in 4/10 females (graded minimal); emphysema in 3/10 females; a reduced number of clara cells in all male and female animals (graded minimal or moderate in males and minimal to severe in females); bronchial epithelial cell degeneration in all male and female animals (graded minimal); and bronchial mucoid metaplasia in 2/10 female animals (graded minimal).

 

In animals of test group 1, exposed to the test material at 0.2 mg/m³, test material-related adverse effects included: increased absolute and relative lung weights in males and females; alveolar cellular debris in all male and female animals (graded minimal to slight in males and minimal to moderate in females); alveolar histiocytosis in all male and female animals (graded slight to moderate in males and minimal to moderate in females); multifocal inflammation in 9/10 male animals and 2/10 female animals (graded minimal to slight); inflammatory cell infiltrates in all male and female animals (graded minimal in males and minimal to slight in females); mucus in all male and female animals (graded slight to moderate in males and minimal to slight in females); alveolar epithelium regeneration in 5/10 males and 6/10 females (graded minimal in males and minimal or moderate in females); emphysema in 1/10 males; a reduced number of clara cells in 4/10 male animals (graded minimal to slight); bronchial epithelial cell degeneration in 3/10 male animals (graded minimal); and bronchial mucoid metaplasia in 4/10 female animals (graded minimal to slight).

 

In conclusion, inhalation exposure of 0.2, 1 and 5 mg/m³ of the test material for 90 days (65 exposures) caused treatment-related histological changes in the lung. The effects were not fully reversible within a 4 week recovery period. A No Observed Adverse Effect Concentration (NOAEC) could not be established under the conditions of the study.

Based on the data on clinical observations, body weight, food consumption, clinical chemistry, haematology and histological examinations, there was no indication for any systemic toxicity. Therefore, under the conditions of the study the NOAEC for systemic toxicity was determined to be 5 mg/m³.