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Acute Toxicity: inhalation

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Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6 September 2011 to December 2011
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was conducted under GLP conditions and largely in accordance with standardised guidelines. Although the test animals received multiple exposures to the test material it is possible to derive an LC50 value from the results of the study for use in hazard classification. However, since a standard acute inhalation study was not performed the data have been assigned a reliability score of 2.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
yes
Remarks:
(see below)
Qualifier:
equivalent or similar to
Guideline:
EU Method B.2 (Acute Toxicity (Inhalation))
Deviations:
yes
Remarks:
(see below)
Principles of method if other than guideline:
The purpose of the study was to determine the pulmonary toxicity of the test material in rats using a short-term bioassay including bronchoalveolar lavage with clinico-chemical and cytological evaluation of lavage fluid and pathological examination of the respiratory tract.
The exposure period was 5 days rather than the single acute exposure stipulated in the guidelines. As effects were seen following a single exposure to the test material, the results of the study are considered to be suitable for assessing the acute toxicity of the test material via the inhalation route.
GLP compliance:
yes (incl. certificate)
Test type:
other: Short-term (5 day) inhalation study
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
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Wistar, Crl:WI(Han)
- Age: approximately 7 weeks (when supplied)
- Weight at study initiation: 257.8 - 258.8 g (mean group weights)
- Housing: in groups of 5 in Polysulfone cages (floor area ca. 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; 21.4 - 22.2 °C in the inhalation systems
- Humidity (%): 30 – 70 % (relative); 41.9 - 59.9 % 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
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) was used. During substance exposure, INA 60 (V ≈ 90 L) 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.

TEST ATMOSPHERE
- Brief description of analytical method used:
> 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.
> 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.
> The particle size distribution of the sample was measured using a cascade impactor.
> The particle size distribution in the test atmospheres was measured by Aerodynamic Particle Sizer (APS)
- Samples taken from breathing zone: yes

TEST ATMOSPHERE
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): 3.2 - 4.9 µm / 2.2 - 3.8 µm
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
5 d
Concentrations:
0, 2, 10 and 50 mg/m³ (target concentrations)
0, 2.2, 10.0 and 54.5 mg/m³ (analysed concentrations, mean values)
No. of animals per sex per dose:
22 males per dose
Control animals:
yes
Details on study design:
OBSERVATIONS AND EXAMINATIONS PERFORMED AND FREQUENCY

CAGE SIDE OBSERVATIONS
- 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.

CLINICAL OBSERVATIONS
- Time schedule: The clinical condition of the test animals was recorded once during the pre-exposure period and on post-exposure observation days and at least 3 times (before, during and after exposure) on exposure days.
During exposure only a group wise examination was possible.

BODY WEIGHT
- Time schedule for examinations: The body weight of the animals was determined at the start of the pre-exposure (day -4), at the start of the exposure period (day 0), on study days 2, 3 (group 3 only), 4, 7, 11, 14, 18, 21, 25 and 28 (test groups 0, 1 and 2).
Due to clinical signs of toxicity and lethality observed in test group 3, body weights were determined additionally on study days 2 and 3 in surviving animals of this group.

RECTAL TEMPERATURE
- Time schedule: On study day 2, rectal temperature was determined in all surviving animals. For animals exposed on this day, the measurement was performed after exposure.
On study day 3, rectal temperature was determined in all surviving animals of test group 3.
On study day 4, rectal temperature was determined in all surviving animals of test group 3 and in recovery group animals (n = 5) designated for lavage (test group 0, 1 and 2). For animals exposed on this day, the measurement was performed after exposure.

HAEMATOLOGY
- Time schedule for collection of blood: Blood samples were taken in rats of test groups 1 and 2 (2 and 10 mg/m³) at study day 7 and after a 3 week recovery period at study day 28. In rats of test group 3 (50 mg/m³) venipuncture was performed at study day 2 and again after a 3 week recovery period at study day 25.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5 per group
- Parameters measured 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 and prothrombin time.

CLINICAL CHEMISTRY
- Time schedule for collection of blood: Blood samples were taken in rats of test groups 1 and 2 (2 and 10 mg/m³) at study day 7 and after a 3 week recovery period at study day 28. In rats of test group 3 (50 mg/m³) venipuncture was performed at study day 2 and again after a 3 week recovery period at study day 25.
- Animals fasted: Yes
- How many animals: 5 per group
- Parameters measured included: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), γ-glutamyltransferase (GGT), sodium (Na), potassium (K), chloride (Cl), inorganic phosphate (INP), calcium (Ca), urea (UREA), creatinine (CREA), glucose (GLUC), total bilirubin (TBIL), total protein (TPROT), albumin (ALB), globulins (GLOB), triglycerides (TRIG), cholesterol (CHOL) and magnesium (Mg).

URINALYSIS
- How many animals: Due to premature death in test group 3, urine samples were collected in 4 surviving animals (No. 50 to 53) from study day 2 to 3, as well as in one concurrent control animal (No. 46). Animal No. 52 died during the night, but still provided a small amount of urine for analysis. Urine samples were collected in recovery animals in control (animal No. 76 to 80) and test group 2 animals (animal No. 106 to 110) assigned for lavage.
- Time schedule for collection of urine: After the last exposure and respective clinical observation, the animals were transferred into metabolism cages (with access to water). Urine was collected over night for a total of approximately 18 hours. The whole procedure was repeated again starting from study day 17 until study day 18.
- Parameters measured included: metal ions (Li, Mn, Co, Ni) to determine their urinary excretion, and creatinine.

ACUTE PHASE PROTEINS IN SERUM
- Time schedule for collection of blood: Blood samples were taken in rats of test groups 1 and 2 (2 and 10 mg/m³) at study day 7 and after a 3 week recovery period at study day 28. In rats of test group 3 (50 mg/m³) venipuncture was performed at study day 2 and again after a 3 week recovery period at study day 25.
- Animals fasted: Yes
- How many animals: 5 per group
- Investigations included the measurement of rat α2-macroglobulin and rat haptoglobin.

BRONCHOALVEOLAR LAVAGE FLUID (BAL)
- Time schedule and number of animals: Broncho-alveolar lavage fluid was sampled in 5 rats each from test groups 1 and 2 (2 and 10 mg/m³), only. Sampling days were study days 7 and 28.
The animals designated for lung lavage were killed by exsanguination from the aorta abdominalis and vena cava under Narcoren anaesthesia. The lung was lavaged by two instillations of physiologic saline.
The following examinations were carried out in 5 male animals per test group:
> Cytology in BAL
- Parameters measured included: total cell count (BALTCN), macrophages (BALMPH), polymorphonuclear neutrophils (BALPMN), lymphocytes (BALLY), eosinophils (BALEO), monocytes (BALMO) and atypical cells (BALATY).
> Total Protein and enzymes in BAL
- Parameters measured included: γ -glutamyltransferase (GGT), protein (MTP), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), N-acetyl-ß-glucosaminidase (NAG).

LUNG BURDEN
- Time schedule:
> Study day 2 - Animal No. 9 (Group 0), Animal Numbers 47-49, 55 (Group 3)
> Study day 25 - Animal No 84 (Group 0), Animal Numbers 99-101 (Group 1) and Animal Numbers 114-116 (Group 2)
Due to the premature death of group 3 animals, the lungs of 4 dead animals were examined for metal ions. Other organs were not preserved. No further animals of test group 3 were examined for organ burden. All samples were stored at -80 °C until analysis.
With exception of test group 3 animals, the animals intended for determination of organ burden were sacrificed under Narcoren (pentobarbital sodium) anaesthesia by exsanguination from the abdominal aorta and vena cava. The following organs and tissues were stored at -80 °C.
- Organs examined: lungs, lymph nodes (mediastinal & hilar), liver, spleen, kidney and basal brain with olfactory bulb.
The extra-pulmonary organs liver, kidney and spleen were only examined after the 3 week recovery period, in order to examine the accumulation potency of the metal ions. All other samples were stored in the freezer (-80 °C) and were not analysed.

SACRIFICE AND PATHOLOGY
The animals were sacrificed under Narcoren (pentobarbital sodium) anaesthesia by exsanguination from the abdominal aorta and vena cava. The exsanguinated animals were necropsied and assessed by gross pathology. Animals that died intercurrently or were sacrificed moribund were necropsied and assessed by gross pathology as soon as possible after their death.

GROSS PATHOLOGY
- How many animals:
> Animal Numbers 6-8 and 81-83 (Group 0)
> Animal Numbers 21-23 and 96-98 (Group 1)
> Animal Numbers 36-38 and 111-113 (Group 2)
> Animal No 46, 50-53, 121-129 and 131 (Group 3)

ORGAN WEIGHTS
The weights of the following were recorded: anaesthetised animals, adrenal glands, brain, epididymides, heart, kidneys, liver, lungs, spleen, testes, thymus and thyroid glands.
- How many animals:
> Animal Numbers 6-8 and 81-83 (Group 0)
> Animal Numbers 21-23 and 96-98 (Group 1)
> Animal Numbers 36-38 and 111-113 (Group 2)
> Animal Numbers 46, 50, 51, 53, 123 and 129 (Group 3)

HISTOPATHOLOGY
The following organs/tissues were preserved: all gross lesions, adrenal glands, brain with olfactory bulb, epididymides*, heart, kidneys, larynx, liver, lung, lymph nodes (tracheobronchial, mediastinal and mesenteric), nose (nasal cavity), pharynx, spleen, testes*, thymus and trachea.
All organs and tissues were fixed in 4 % buffered formaldehyde, with the exception of those marked with * which were fixed in modified Davidson’s solution.
From the liver, one slice each of the Lobus dexter medialis and the Lobus sinster lateralis was fixed in Carnoy’s solution and embedded in paraplast.
The liver, testes and epididymides of animals that died or had to be sacrificed intercurrently were fixed in 4 % buffered formaldehyde solution.
- How many animals:
> Animal Numbers 6-8 (Group 0)
> Animal Numbers 21-23 (Group 1)
> Animal Numbers 36-38 (Group 2)
> Animal Numbers 51, 53, 121, 122, 124-128 and 131 (Group 3).

Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings for the following organs/tissues in all animals per group: nasal cavity (4 levels), larynx (3 levels), liver, trachea, lungs (5 lobes), lymph nodes (tracheobronchial and mediastinal). Additionally, gross lesions of all animals affected per group were processed and examined in the same way.
Statistics:
> Body weight, body weight change, rectal temperature: A comparison of each group with the control group was performed using DUNNETT's test (two-sided) for the hypothesis of equal means.
> Blood parameters: For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. 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 hypothesis of equal medians. For parameters with unidirectional changes: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.
> Broncho-alveolar lavage fluid (BALF) and lung tissue parameters: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.

Results and discussion

Effect levelsopen allclose all
Sex:
male
Dose descriptor:
other: no NOAEC could be derived
Remarks on result:
other: Due to findings in haematology, lavage fluid and histology of the respiratory tract.
Sex:
male
Dose descriptor:
LC50
Effect level:
0.07 mg/L air
Based on:
test mat.
Mortality:
Lethality was observed only in test group 3 animals after 2 exposures. A total of 11 animals were found dead on study day 2, a further 3 animals were found dead on study day 3, and 2 animals were sacrificed in a moribund state on study day 3.
A total of 16 of the 22 animals were found dead or sacrificed in a moribund state within the first 3 days of the study.
Clinical signs:
other: Clinical signs of toxicity were only observed in test group 3 animals. On study day 2, all surviving animals of group 3 showed accelerated and intermittent respiration. Most were in a poor general condition; pale skin and piloerection were observed. A few
Body weight:
The mean body weights of the group 1 and 2 animals were not statistically significantly different from the control group throughout the study.
In test group 3 (50 mg/m³) the mean body weights were significantly lower than the control on study day 2. The mean body weights of the surviving animals were significantly lower on study day 4. This effect was not observed from study day 7 onward.
The body weight changes of test group 1 and 2 animals were comparable to the controls throughout the study. The reduced body weight gain in test group 3 from study day 0 to 4 was considered to be treatment-related, because lethality occurred and severe clinical signs of toxicity were observed. From study day 14 to 18, the mean body weight change of test group 1 and 3 was higher than the concurrent control group. This effect was considered to be incidental.
Gross pathology:
- Main group animals
One animal (No. 52) of test group 3 (50 mg/m³) showed a red discoloration of all lung lobes. One animal of test group 2 showed an enlarged mediastinal lymph node.

- Recovery group animals
No abnormalities were detected.

- Premature decedents
All animals showed a red discoloration of all lung lobes accompanied in one animal each by thoracic effusion or oedema.
Other findings:
RECTAL TEMPERATURE
The mean rectal temperatures of group 1 and 2 animals were comparable to the concurrent control group on both measurement days (study day 2 and 4). On study day 2 for group 3 animals, the mean value of rectal temperature of the surviving animals was significantly lower than the concurrent control. This effect was not observed on study days 3 and 4.

URINARY EXCRETION OF METAL IONS
Urine samples were collected and examined for metal ions. Li, Co and Ni were detected in urine in group 2 animals after 5 exposures, and in group 3 animals after 2 exposures. The amount of Mn was below the detection limit of 0.3 μg Mn which is in the range < 0.005 μg/μmol creatinine. After the recovery period, the detected metal ions were in the control range.

LUNG BURDEN
The amounts of metal ions analysed in the lung were recorded. Considering the fractions of respective metal ions in the test material, a lung burden was calculated to be between 14 μg (based on Li) to 30 μg (based on Co or Ni). This amount is considerably lower than expected.

CLINICAL CHEMISTRY
At study day 7, in rats of test group 2 (10 mg/m³) alkaline phosphatase activities were increased.
At study day 2, in rats of test group 3 (50 mg/m³) sodium levels were higher, but inorganic phosphate levels were lower compared to controls. The means of both parameters were within historical control ranges (sodium: 139.1-146.0 mmol/L; inorganic phosphate: 1.73-2.39 mmol/L) and therefore these alterations were regarded as incidental and not treatment-related.
After the recovery period at study day 25, in rats of test group 3 (50 mg/m³) γ-glutamyltransferase (GGT) were lower compared to controls. This alteration cannot be correlated to any patho-physiological finding and therefore it was regarded as incidental and not treatment-related.

HAEMATOLOGY
At study day 7, in rats of test groups 1 and 2 (2 and 10 mg/m³) red blood cell (RBC) counts, haemoglobin and haematocrit values were higher compared to controls.
At study day 7, in rats of test group 1 and 2 relative reticulocyte counts were lower compared to controls, and in rats of test group 2 prothrombin time (HQT) was prolonged. However, the means of these parameters were within historical controls (reticulocyte counts: 1.1-3.1 %; prothrombin time: 33.2-39.6 seconds), whereas for the study control, the reticulocyte count was above, and the prothrombin time mean was below the historical control ranges. Moreover, reticulocyte counts were not changed dose-dependently. Therefore, the mentioned alterations of reticulocyte counts and prothrombin time were regarded as incidental and not treatment-related.
At study day 7 in rats of test group 1 (2 mg/m³) relative neutrophil counts were higher and relative lymphocyte counts were lower compared to controls, without any changes of the absolute cell counts. Both parameters were not changed dose-dependently and therefore the alterations were regarded as incidental and not treatment-related.
After the 3-week-recovery period, in rats of test group 3 (50 mg/m³) relative reticulocyte counts and relative basophil counts, as well as absolute large unstained cell (LUC) counts were lower compared to controls. All values were within historical control ranges (relative reticulocyte counts 1.1-3.1 %; relative basophil counts 0-0.9 %, absolute LUC counts 0.01-0.05 Giga/L) and therefore, these changes were regarded as incidental and not treatment-related.

ACUTE PHASE PROTEINS
No treatment-related changes of acute phase protein levels were measured after the administration as well as after the recovery period.

BRONCHOALVEOLAR LAVAGE FLUID (BAL)
> Cytology in BAL
At study day 7, in bronchoalveolar lavage fluid (BALF) of rats in test group 1 and 2 (2 and 10 mg/m³) total cell counts were increased, although not dose-dependently. This was mainly due to an increase of absolute and relative polymorphonuclear neutrophil (PMN) and lymphocyte counts. Absolute and relative eosinophil, monocyte and atypical cell counts were moderately increased.
Absolute macrophage counts were marginally higher compared to controls (not statistically significant in test group 2), resulting in a decrease of relative macrophage counts. The changes of the macrophage counts were regarded as not adverse.
After the 3-week-recovery period, cell counts in BALF of rats of both dose groups were no longer different from those of controls.

> Total protein and enzymes in BAL
At study day 7, in BALF of rats in test groups 1 and 2 (2 and 10 mg/m³) total protein, γ-glutamyltransferase (GGT), and lactate dehydrogenase (LDH) activities were moderately increased. Alkaline phosphatase (ALP) and β-N-acetyl glucosaminidase (NAG) activities were marginally increased in rats of test group 1 (2 mg/m³) and moderately increased in those of test group 2 (10 mg/m³).
After the 3-week-recovery period, all mentioned parameter values were within the normal range. GGT activities in rats of test group 2 (10 mg/m³) were statistically increased, but the mean was only slightly above the border of the historical control range (GGT: 0-41 nkat/L) and therefore this alteration was regarded as not treatment-related.


SACRIFICE AND PATHOLOGY
- ORGAN WEIGHTS
> Main group animals: When compared to control group 0 (set to 100 %), the mean absolute, and relative, weights of the lungs were increased in a dose-responsive way.
> Recovery group animals: None of the weight parameters in the recovery group showed a clear dose response.

- HISTOPATHOLOGY
> Main group animals:
- Lungs: Bronchial epithelial degeneration and regeneration was noted in all treated animals, peribronchial inflammation was seen in all test group 2 (10 mg/m³) animals.
- Mediastinal lymph node: In the mediastinal lymph node, lympho-reticulocellular hyperplasia was observed in all treated animals of test group 1 (2 mg/m³) and 2 (10 mg/m³).
- Nasal cavity, level IV: Necrosis of the olfactory epithelium was noted in animals of test group 1 (2 mg/m³) and 2 (10 mg/m³).
- Trachea: Degeneration/regeneration of the respiratory epithelium was seen in the trachea in animals of test group 1 (2 mg/m³) and 2 (10 mg/m³).
- Tracheobronchial lymph node: In the tracheobronchial lymph node, lympho-reticulocellular hyperplasia was observed in all treated animals of test group 1 (2 mg/m³) and 2 (10 mg/m³).

> Recovery group animals
The two animals of test group 3 (50 mg/m³) showed minimal degeneration and regeneration of the bronchial epithelium after a 2 week recovery period, while none of the other organs examined showed treatment – related findings.

> Premature decedents
Not all organs could be examined for all animals as some were autolytic and not possible to evaluate.
- Larynx, level I and II: Squamous metaplasia was seen in the larynx in 8 animals of group 3.
- Lungs: The following findings were present in the lungs, varying with survival time. Alveolar fibrin formed hyaline membranes over alveolar septae. Inflammation consisted of varying amounts of neutrophils, macrophages and eosinophils, sometimes eosinophil granules were free in alveolar spaces. Degeneration/regeneration of the bronchial epithelium was characterised by sloughed epithelial cells (degeneration) and mitotic figures in the epithelium (regeneration). Diffuse hyperaemia correlated with reddened lungs at necropsy.
- Mediastinal lymph node: In the mediastinal lymph node, lympho-reticulocellular hyperplasia was observed in 6 animals of test group 3 (50 mg/m³).
- Nasal cavity: Degeneration of the olfactory epithelium was noted in some animals in level III of the nasal cavity, while necrosis of the olfactory epithelium and squamous metaplasia of the olfactory epithelium were seen in level IV of the nasal cavity. Some findings in the nasal cavity varied with survival time.
- Trachea: Multifocal, minimal inflammation was seen in all animals, characterised by a lymphocytic infiltrate and degeneration/ regeneration of the epithelium was seen in one animal.
- Tracheobronchial lymph node: In the tracheobronchial lymph node, lympho-reticulocellular hyperplasia was observed in 7 animals of group 3 (50 mg/m³).
- Comparison of findings varying with survival time: Animals 121,125,126,127 and 128 died on day 2 (directly after the 2nd exposure) and showed diffuse peracute alveolar damage with alveolar septal hyperaemia and alveolar hyaline membranes (fibrin) and also eosinophil degranulation in the lungs.
Animal 131 died on day 3 (later in the day after the 2nd exposure). Hyperaemia, alveolar hyaline membranes, beginning type II pneumocyte proliferation, inflammation and also eosinophil degranulation was seen in the lungs. In the nasal cavity, level IV, there was squamous metaplasia of the olfactory epithelium.
Animals 122 and 124 died on day 3 (later in the day after the 2nd exposure).Type II pneumocyte proliferation and inflammation were noted in the lungs. In the nasal cavity, level IV, there was squamous metaplasia of the olfactory epithelium.
Other findings did not show a relation to time.

Any other information on results incl. tables

Table 3: Bronchoalveolar lavage fluid - cytology

Analyte

Study day 7

Study day 28

Group 1

(2 mg/m³)

Group 2

(10 mg/m³)

Group 1

(2 mg/m³)

Group 2

(10 mg/m³)

Total cells

4.6**

3.9**

1.3

1.7

Eosinophils

13.4*

22.9**

0.6

0.0

Lymphocytes

54.8**

28.7**

1.4

3.9

Macrophages

1.7*

1.7

1.3

1.7

Neutrophils

219.7**

156.9**

0.7

1.7

Monocytes

+**

+**

0.0

2.6

Atypical cells

+*

+*

0.0

3.9

* p   0.05, ** p 0.01 (Two-sided Wilcoxon test)

+ Increase could not be calculated due to zero activity in controls

Table 4: Bronchoalveolar lavage fluid - total protein and enzymes

Analyte

Study day 7

Study day 28

Group 1

(2 mg/m³)

Group 2

(10 mg/m³)

Group 1

(2 mg/m³)

Group 2

(10 mg/m³)

Total protein

7.9**

14.6**

1.0

1.4

GGT

8.6**

25.2**

1.0

1.9*

LDH

14.7**

22.6**

0.9

1.4

ALP

2.3**

4.6**

0.5

0.5

NAG

2.9**

9.6**

0.8

1.3

 * p   0.05, ** p 0.01 (Two-sided Wilcoxon test)

GGT = γ-Glutamyltransferse; LDH = Lactate dehydrogenase; ALP = Alkaline phosphatase; NAG = β-N-Acetyl glucosaminidase.

Table 5: Lung weights

Test group (mg/m³)

1 (2)

2 (10)

3 (50)

Relative increase of absolute lung weight (%)

111

127

N/A (no survivors)

Relative increase of relative lung weight (%)

101

129

N/A (no survivors)

Table 6: Incidence of histopathology findings - Main group animals

Test group (mg/m³)

0 (0)

1 (2)

2 (10)

3 (50)

No. of animals

3

3

3

0

Histological findings in lungs

Degeneration/regeneration bronchial epithelium

 

3

3

 

Grade 1

 

1

 

Grade 2

 

2

3

 

Inflammation, peribronchial

 

 

3

 

Grade 2

 

 

3

 

Histological findings in mediastinal lymph node

Hyperplasia, lympho-reticulocellular

 

3

3

 

Grade 1

 

3

2

 

Grade 2

 

 

1

 

Histological findings in nasal cavity - level IV

Necrosis, olfactory epithelium

 

2

3

 

Grade 1

 

2

3

 

Histological findings in trachea

Degeneration/regeneration epithelium

 

3

3

 

Grade 1

 

3

3

 

Histological findings in tracheobronchial lymph node

Hyperplasia, lympho-reticulocellular

 

2

3

 

Grade 1

 

2

 

 

Grade 2

 

 

3

 

Table 7: Incidence of histological findings in premature decedents

Test group (mg/m³)

3 (50)

Histological findings in the larynx - level I

No. of animals

8

Squamous metaplasia

2

Grade 1

1

Grade 2

1

Histological findings in the larynx - level II

No. of animals

8

Squamous metaplasia

8

Grade 1

4

Grade 2

3

Grade 3

1

Histological findings in the lungs

No. of animals

8

Fibrin, alveolar

6

Grade 2

3

Grade 3

3

Inflammation, multifocal

8

Grade 2

3

Grade 3

3

Grade 4

2

Hyperaemia, diffuse

8

Grade 2

2

Grade 3

6

Type II pneumocyte proliferation

3

Grade 3

1

Grade 4

2

Eosinophil degranulation

4

Grade 1

2

Grade 2

2

Degeneration/regeneration bronchial epithelium

4

Grade 2

4

Histological findings in mediastinal lymph node

No. of animals

6

Hyperplasia, lympho-reticulocellular

6

Grade 2

4

Grade 3

2

Histological findings in nasal cavity

No. of animals

5

Nasal cavity level III

Degeneration, olfactory epithelium

2

Grade 1

2

Nasal cavity - Level IV

Necrosis olfactory epithelium

5

Grade 1

4

Grade 2

1

Squamous metaplasia, olfactory epithelium

4

Grade 1

4

Histological findings in the trachea

No. of animals

8

Inflammation, multifocal

8

Grade 1

8

Degeneration/regeneration epithelial

1

Grade 1

1

Histological findings in the tracheobronchial lymph node

No. of animals

7

Hyperplasia, lympho-reticulocellular

7

Grade 1

1

Grade 2

5

Grade 3

1

 

Applicant's summary and conclusion

Interpretation of results:
other: Category 2
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Inhalation exposure of 50 mg/m³ test material caused lethality after only two exposures; after which, the exposure was terminated in this group. The surviving animals showed transient body weight loss, laboured and intermittent respiration as well as several unspecific clinical signs of toxicity. Histological examination of the premature decedents showed lesions throughout the respiratory tract. Apart from minimal degeneration and regeneration of the bronchial epithelia, no other morphological changes were observed in surviving animals of test group 3 sacrificed on study day 25. At 2 and 10 mg/m³ clearly treatment-related changes were observed in lavage fluid, haematology and in histology of the respiratory tract. The effects in haematology and lavage fluid were fully reversible within the 3 week recovery period.
Due to these findings, A No Observed Adverse Effect Concentration (NOAEC) could not be established. The LC50 was determined to be 0.07 mg/L.
Executive summary:

The purpose of this study was to determine the pulmonary toxicity in rats using a short-term bioassay including bronchoalveolar lavage with clinico-chemical and cytological evaluation of lavage fluid and pathological examination of the respiratory tract.

The inhalation study was conducted over 5 days to inform on a suitable dosing regimen for a 90-day inhalation study. The study was conducted under GLP conditions and in accordance with the principles of standardised guidelines OECD 403 and EU Method B.2.

 

During the study, groups of male Wistar rats were exposed in a nose-only fashion to a dust aerosol of milled test material for 6 hours per day on 5 consecutive days with target concentrations of 2, 10 and 50 mg/m³. A concurrent control group was exposed to clean air. Daily clinical observations and body weight measurements were recorded. Additional assessments including clinical chemistry, haematology, and histopathology were carried out at the termination of the study. Additionally, animals from each dose group were assessed after a 3-week exposure-free recovery period.

Gravimetric analysis showed that targeted atmospheric concentrations were well maintained throughout the study.

In the main group animals exposed to 50 mg/m³ (group 3), 16 of the 22 animals were found dead or were sacrificed moribund on study day 2 or 3 after two exposures. Clinical signs of toxicity comprised accelerated respiration, ruffled fur, hypothermia, poor general condition and reduced body weight. Animals dying prematurely were observed to display signs of acute alveolar damage (fibrinous hyaline alveolar membranes, eosinophil degranulation and type II pneumocyte proliferation, inflammation) in the lungs; degeneration/ regeneration of the bronchial epithelium of the lungs; degeneration of the olfactory epithelium level III of the nasal cavity; necrosis of the olfactory epithelium and squamous metaplasia of the olfactory epithelium in level IV of the nasal cavity; minimal to moderate squamous metaplasia in the larynx, level I and II; and degeneration/regeneration of the respiratory epithelium in the trachea.

Recovery animals of test group 3 had minimal bronchial epithelial degeneration and regeneration in the lungs.

In the main group animals, exposed to 10 mg/m³ (group 2), treatment-related adverse effects included: increased red blood cell (RBC) counts, haemoglobin and haematocrit values; increased alkaline phosphatase (ALP) activities in blood; increased total cell counts in BALF as well as absolute and relative neutrophil, lymphocyte monocyte, eosinophil and atypical cell counts; increased total protein in BALF as well as alkaline phosphatase (ALP), lactate dehydrogenase (LDH),γ-glutamyl transferase (GGT) and β-N-acetyl glucosaminidase (NAG) activities; minimal to slight bronchial epithelial degeneration and regeneration and peribronchial inflammation in the lungs in all animals; necrosis of the olfactory epithelium in all animals in level IV of the nasal cavity; and degeneration/regeneration of the respiratory epithelium in the trachea in all animals.

There were no adverse effects noted in group 2 animals in the recovery group.

In the main group animals exposed to 2 mg/m³ (group 1), treatment-related adverse signs included: increased red blood cell (RBC) counts, haemoglobin and haematocrit values; increased total cell counts in BALF as well as absolute and relative neutrophil, lymphocyte monocyte, eosinophil and atypical cell counts; increased total protein in BALF as well as alkaline phosphatase (ALP), lactate dehydrogenase (LDH),γ-glutamyl transferase (GGT) and β-N-acetyl glucosaminidase(NAG) activities; minimal to slight bronchial epithelial degeneration and regeneration in the lungs in all animals; necrosis of the olfactory epithelium of the nasal cavity, level IV in 2/3 animals; and degeneration/regeneration of the respiratory epithelium in the trachea in all animals.

No adverse effects were observed in the recovery animals of test group 1.

 

In summary, inhalation exposure of 50 mg/m³ test material caused lethality after only two exposures; after which, the exposure was terminated in this group. The surviving animals showed transient body weight loss, laboured and intermittent respiration as well as several unspecific clinical signs of toxicity. Histological examination of the premature decedents showed lesions throughout the respiratory tract. Apart from minimal degeneration and regeneration of the bronchial epithelia, no other morphological changes were observed in surviving animals of test group 3 sacrificed on study day 25. At 2 and 10 mg/m³ clearly treatment-related changes were observed in lavage fluid, haematology and in histology of the respiratory tract. The effects in haematology and lavage fluid were fully reversible within the 3 week recovery period.

Due to these findings, A No Observed Adverse Effect Concentration (NOAEC) could not be established. The LC50 was determined to be 0.07 mg/L. In accordance with the EU criteria, the test material therefore requires classification as Category 2 for acute inhalation toxicity.