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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:
2007-02-23 to 2007-12-11
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: According to ECHA Practical Guide 6 the maximum score for read across is rel. 2

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Version / remarks:
(1981)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Crl:WI(Han)
- Source: Charles River Laboratories, Sulzfeld, Germany
- Age at study initiation: 7 weeks at arrival, 9 weeks at first exposure
- Weight at study initiation: females approx. 176 +/- 9 g; males approx. 215 +/- 10 g
- Housing: singly in makrolon-wire cages type MD III, Becker & Co., Castrop-Rauxel, Germany (floor area about 800 cm²)
- Diet and water: ad libitum
- Acclimation period: no data (3 days preflow , i.e. exposure to supply air for 6 hours/day)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
nose/head only
Vehicle:
other: conditioned air
Remarks on MMAD:
MMAD / GSD: Three samples were taken at each concentration level from the breathing zones of the animals. Sampled volumes were 450, 90, and 18 liters for low, mid and high-dose exposure, respectively. The particle sizes of the aerosol in the inhalation atmosphere were well within the respirable range:
3 mg/m³: 1.3 – 1.4 µm / GSD 2.1
15 mg/m³: 1.2 – 1.3 µm / GSD 2.0 - 2.1
75 mg/m³: 1.2 – 1.4 µm / GSD 2.1 – 2.2
The calculated mass fractions of particles below 3 μm aerodynamic size ranged between 84.1 and 89.5 %.
Details on inhalation exposure:
- Type or preparation of particles:
Generation procedure: The test substnce was used unchanged and stirred in its container before a sample for dust generation was taken. Dust aerosols were produced at target concentrations by dry dispersion of powder pellets with a brush dust generator. To prevent possible electrostatic charging of the aerosol particles, the dust generator was equipped with a brush made of stainless steel and the exposure units were well grounded. The test substance powder was used as delivered (see above). The aerosol was generated with compressed air in a mixing tube, mixed with conditioned dilution air and passed via a cyclone into a head-nose inhalation system.
- Air flow rate: An air change in the inhalation system of about 67 times per hour was calculated.
- Temperature: Mean temperatures in the inhalation systems ranged between 22.0 and 23.7 °C.

- Head-nose exposure system:
The inhalation atmosphere was maintained inside aerodynamic exposure systems (INA 60, volume V ≈ 90 L, BASF SE) consisting of a cylindrical inhalation chamber made of stainless steel sheeting and cone-shaped outlets and inlets. The rats were restrained in glass exposure tubes. Their snouts projected into the inhalation chamber and thus they inhaled the aerosol. The exposure systems were located in exhaust hoods in an air conditioned room.
- Exposures
The head-nose exposure technique was preferably selected for this aerosol inhalation study to minimize fur contamination of the animals with the substance, which cannot be avoided during whole-body exposure. Fur contamination may lead to an additional dermal and oral uptake (animals preen as their fur becomes contaminated). Thus an estimation of an nominal dose, taken up by the animals and its correlation to a toxic effect becomes more difficult. Furthermore, by using the dynamic mode of operation with a low-volume chamber, the equilibrium characteristic of this exposure technique is favorable: t99 (the time to reach 99 % of the final target concentration) is shorter as compared to whole-body chambers with a higher chamber volume. 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. In order to accustom the animals to exposure they were treated with supply air under conditions comparable to exposure on three days before start of exposure (preflow period). Then all test groups were exposed for 6 hours on each workday over a time period suitable to reach 65 exposures. The animals did not have access to water or feed during the exposure.

- Measurements of the exposure conditions
No surveillance of the oxygen content in the inhalation system was performed. The air change within the inhalation systems was judged to be sufficient to prevent oxygen depletion by the breathing of the animals and the concentrations of the test substance used could not have a substantial influence on oxygen partial pressure.
Principles of recording with the automated measuring system:
Each parameter was measured at appropriate measuring points using suitable measuring equipment (sensors, orifice plates etc.). The measurements were standardized (0 - 20 or 4 - 20 mA) and transferred to instrumentation consoles. There, the measured values were displayed in an analogous way (where this is provided for) and some were used as actual value for regulating the specific parameter.
In addition, the measured values were scanned every 10 seconds, converted from analog to digital, transferred to a personal computer, displayed on its screen, and saved on hard disk. The computer checked the arriving values against preset threshold values, displayed warnings if violations of thresholds occurred and recorded the start and the end of threshold violations for each measured parameter affected. After the end of each exposure all data gathered during this exposure were backed up on optical media.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Gravimetrical measurement of 2 samples per exposure and test concentration (130 measurements per concentration): A defined volume of dust aerosol was drawn through a pre-weighed filter and the increase in weight was determined.
The constancy of the aerosol concentrations was determined continuously by scatterled light photometers. Constancy was generally proved.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours/day, 5 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
3 mg/m³ air (nominal)
Remarks:
2.9 mg/m³ air (analytical)
Dose / conc.:
15 mg/L air (nominal)
Remarks:
15.0 mg/m³ air (analytical)
Dose / conc.:
75 mg/m³ air (nominal)
Remarks:
75.0 mg/m³ air (analytical)
No. of animals per sex per dose:
10
Additional 10 animals for recovery groups (High-dose and control)
Additional 5 males for bronchoalveolar lavage groups
Control animals:
yes, concurrent vehicle
Details on study design:
Satellite groups of five male rats per exposure level (including control) were lavaged three days after termination of exposure (duration of exposure reduced by 4 days for these groups).
In addition, a recovery group of 10 rats per sex treated with the high target concentration (75 mg/m³) and air control for 90 days were observed for 28 days for reversibility of effects.

Examinations

Observations and examinations performed and frequency:
- Clinical signs: before, during and after each exposure; once each day during post exposure period
- Mortality: morning and afternoon Monday through Friday; morning only on Saturdays, Sundays and public holidays
- Body weight: weekly
- Food consumption: weekly
- Rectal temperature: prior to, at start, at mid-term and at end of the exposure period = 4 times; shortly after exposure (where applicable)
- Functional observation: 5 animals per test group prior to the start and one week before termination of exposure (non-exposure days)
- Ophthalmoscopic examination: One week each before first and last exposures; first examination: all main-group animals; terminal examination: main-group animals of control and high-dose
- Hematology: At sacrifice according to guideline: 10 animals per dose and sex; Leukocyte count, Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Platelet count, Differential blood count, Reticulocytes, Prothrombin time
- Biochemistry: At sacrifice according to guideline: 10 animals per dose and sex; Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, γ-Glutamyltransferase, Sodium, Potassium, Chloride, Inorganic phosphate, Calcium, Urea, Creatinine, Glucose, Total bilirubin, Total protein, Albumin, Globulins, Triglycerides, Cholesterol, Magnesium
Sacrifice and pathology:
10 animals per sex and dose group were sacrificed and underwent gross macroscopic examination on the day after the last exposure. The recovery animals were examinated likewise at the end of the recovery period.
- Weights of: anesthetized animals, lung, liver, kidneys, adrenals, testes, epididymides, ovaries, uterus, thymus, spleen, brain, heart
- Organ / tissue preservation: All gross lesions, Brain, Spinal cord (cervical, thoracic and lumbar cord), Sciatic nerve, Gastrocnemius muscle, Pituitary gland, Salivary glands (mandibular gland, sublingual gland), Thyroids/parathyroids, Adrenal glands, Testes/ovaries, Epididymides/oviducts, Prostate/seminal vesicle/coagulation gland, Uterus/vagina, Female mammary gland, Thymus, Lymph nodes (axillar, mediastinal and mesenteric), Spleen, Trachea, Lungs, Heart, Aorta, Liver, Pancreas, Kidneys, Esophagus, Stomach (fore- and glandular stomach), Duodenum, jejunum, ileum, Cecum, colon, rectum, Urinary bladder, Sternum with sternal marrow, Bone marrow (femur), Femur with joint, Eyes, Lacrimal gland, Skin, Head, Larynx, Pharynx.
- Histotechnical processing / evaluation by light microscopy:
Examinations in all high-dose and control group rats of the main test and in selected low- and mid-dose animals: Nasal cavities (4 level), Larynx (3 level), Oropharynx, Trachea (longitudinal, with carina), Lungs (5 lobes), Mediastinal lymph nodes, Liver, Kidneys, Spleen, Adrenal glands, Heart, Brain, Spinal cord (cervical, thoracic, lumbar), Sciatic nerve, Thyroid glands/ parathyroid glands, Testes, Epididymides, Ovaries, oviducts, Uterus, vagina, Prostate gland, seminal vesicles, coagulation glands, Female mammary gland, Thymus, Mesenterial lymph nodes, Stomach (fore- and glandular stomach), Duodenum, jejunum, ileum, Cecum, colon, rectum, Urinary bladder, Bone marrow (femur), all gross lesions (affected animals only)
Examinations in recovery rats: Lungs (5 lobes), Mediastinal lymph nodes, Larynx (level I only), Adrenal glands (females only)
From 3 animals per sex each of the main and recovery groups, the lungs were stained with Hart-Masson-Goldner modified stain to prove the content of collagen fibers in the interstitium of the lung parenchyma of control and treated animals.
Other examinations:
Sacrifice and lavage of lungs of 5 additional males per exposure level (including control). Analysis of bronchoalveolar lavage fluid (BALF; 2 x 7 or 8 mL per animal, corresponding to body weight) for: Total cell count, Macrophages, Polymorphonuclear neutrophils, Lymphocytes, Eosinophils, Monocytes, Atypical cells; γ-Glutamyltransferase, Protein, Lactate dehydrogenase, Alkaline phosphatase, N-acetyl-β-Glucosaminidase.
Statistics:
- Body weight, body weight change, food consumption, food efficiency: DUNNETT's test (two-sided) for the hypothesis of equal means
- Feces, rearing, grip strength length forelimbs, grip strength length hindlimbs, footsplay test, motor activity; Clinical pathology parameters of the main groups: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If p <=0.05: Wilcoxon-test (two-sided)
- Clinical pathology parameters of the recovery groups: Wilcoxon-test (two-sided)
- Organ weight parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If p <=0.05: Wilcoxon-test

Results and discussion

Results of examinations

Details on results:
High dose group (75.0 ± 3.6 mg/m³)
- Increased white blood cell counts (significant in males only: +31 %, p <= 0.01) and neutrophil counts in both sexes, still present after the recovery:
males: relative neutrophils +56 % (p <= 0.01); absolute neutrophils +99 % (p <= 0.01)
females: relative neutrophils +53 % (p <= 0.01); absolute neutrophils +131 % (p <= 0.01)
- Decreased relative lymphocyte counts (males -16 %, p < 0.01; females -9 %, p < 0.05), no reversal during recovery period
- Increased absolute eosinophil counts in both sexes (males + 45 %,p < 0.01; females +33 %, p < 0.05) as well as monocyte counts in males (+ 50 %, p < 0.01); not present after the recovery period any more
- Increase of inorganic phosphate levels (males +16 %, p <= 0.01; females +18 %, p <= 0.05); reversal complete in males, incomplete in females
- Slightly decreased creatinine levels in males (-3.7 %, p <= 0.05)
- Decreased albumin levels in both sexes (males -4.0 %, p <= 0.01; females -1.2 %, not statistically significant), significant (p <= 0.05) in females only after the recovery period
- In the BALF of the males:
o Increased total cell counts mainly due to increased neutrophil and lymphocyte counts and accompanied by increased monocyte and atypical cell counts
o Increased total protein concentration
o Increased enzyme activities, above all of the cell damage indicating enzymes (LDH, GGT)
- Lung weight increase in both sexes (absolute: males +76 %, females +81 %; relative males +75 %, females +85 %, all p <= 0.01), not reversed during recovery period
- Gross pathology: The only effects considered to be neither incidental nor spontaneous were enlargments (9 of 10 males, 8 of 10 females) and discolorations of the mediastinal lymph nodes. The enlargements were reduced during the reversibility period. Enlargements were more frequent in both mid-dose and recovery animals than in high-dose rats. They correspond to an up to severe accumulation of macrophage aggregates histomorphologically.
- Lungs:
o Bronchiolo-alveolar hyperplasia in both sexes, no reversibility within the 4-week recovery period
o Inflammatory cell infiltrates in both sexes, no reversibility within the 4-week recovery period
- Mediastinal lymph nodes
o Accumulation of macrophage aggregates, no recovery observed
- Larynx
o Epithelial alteration of the larnyngeal epithelium, reversibility observed after recovery period

Mid dose group (15.0 ± 2.3 mg/m3)
- Decreased albumin levels in both sexes (males -3.1 %, p <= 0.01; females -1.7 %, not statistically significant)
- Slightly decreased creatinine levels in males (-4.4 %, p <= 0.05)
- Increase of inorganic phosphate levels in females (+20 %, p <= 0.01)
- In the BALF of the males:
o Increased neutrophil and lymphocyte counts accompanied by increased monocyte counts
o Slightly increased LDH activity
- Lung weight increase in females (absolute +7.9 %, relative +5.7 %; both p <= 0.05)
- Gross pathology: see high-dose group
- Lungs:
o Bronchiolo-alveolar hyperplasia in both sexes
- Mediastinal lymph nodes
o Accumulation of macrophage aggregates

Low dose group (2.9 ± 0.7 mg/m3)
- No treatment-related adverse effect. Occasional differences of blood parameters from control with statistical significance showed no reasonable dose-response relationships.

Findings of uncertain relevance:
- Mortality: One female animal (no. 115) exposed to the high concentration (75 mg/m³) died during the exposure period on study day 41 (before exposure).
- Clinical signs: No clinical signs and findings different from normal were observed except one high-dose female animal showing injury (forelimbs) from day 114 to the end of the study.
- Food consumption: Deviations from control were noted only in main group female rats and considered to be incidental:
High-dose on study day 56 (- 8 %)
Mid-dose on study days 42 to 49 (about + 9.2 to + 11.3 %) and from study day 63 to the end of the study period (about + 7.9 to + 14.7 %)
Low-dose on study days 63 to 77 (+ 8.4 % to + 9.9 %)
- Food efficiency: An isolated statistically significant deviation from control was observed in high-dose females on study day 84 (-84 %, p <= 0.05) and considered to be incidental.
- Rectal temperature: Statistically significant deviations from control were observed only in female rats. These were slight increases before exposure (low-dose and high-dose: + 0.7 °C each) and on day 44 (mid-dose: +0.7 °C), which were considered to be not treatment-related.

Effect levels

Dose descriptor:
NOAEC
Effect level:
2.9 mg/m³ air
Sex:
male/female
Basis for effect level:
other: based on pulmonary irritation as indicated by biochemical and cytological parameters in BALF, increased lung weights and corresponding histological findings at next higher concentration (15 mg/m³)

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Executive summary:

In a 90 day inhalation study, 10 male and 10 female Wistar rats per test group were head-nose exposed to respirable dust of IPDI homopolymer on 6 hours per day and 5 consecutive days per week for 13 weeks (65 exposures). The target concentrations were 3, 15, and 75 mg/m³. A concurrent control group was exposed to conditioned air. One additional high target concentration group and one control group per sex were kept for further 28 days to detect recovery or persistence of the toxic effects. Additional satellite groups of five male rats per exposure concentration were used for BALF preparation two days after termination of exposure.

 

Clinical examination was performed before, during and after exposure on each exposure day and once on each working day during the post exposure period. Body weight and food consumption of the animals were determined weekly. Rectal temperature was determined four times during the exposure period. Functional observation was determined in 5 animals per test group prior to the start and against the end of the exposure. The main group animals (10 animals per sex per group) were sacrificed and underwent gross macroscopic examination on the day after the last exposure. Blood was examined for a range of clinical chemical parameters as indicated in the guideline. Selected organs were collected for gravimetrical and histopathological examinations. The lavage fluids from the satellite groups were examined for cytological and biochemical parameters indicating pulmonary irritation.

 

The targeted aerosol concentrations were maintained well throughout the whole study and the particle sizes of the aerosol in the inhalation atmosphere were well within the respirable range. Filter samples from the beginning of the exposure period, from the mid term and from the end were retained and analysed by IR spectroscopy, which confirmed the identity as IPDI homopolymer.

 

The inhalation to the test substance led to the following treatment-related, adverse findings:

 

High dose group (75.0±3.6 mg/m³)

•Increased white blood cell counts, and absolute neutrophil counts in both sexes, still present after the recovery

•Increased absolute eosinophil counts in both sexes as well as monocyte counts in the males which was not present after the recovery period any more

•Decreased albumin levels in both sexes still significant in the females after the recovery

•In the BALF of the males:

o Increased total cell counts mainly due to increased neutrophil and lymphocyte counts and accompanied by increased monocyte and atypical cell counts

o Increased total protein concentration

o Increased enzyme activities, above all of the cell damage indicating enzymes (LDH, GGT)

•Lung weight increase in both sexes

•Lungs:

o Bronchiolo-alveolar hyperplasia in both sexes, no reversibility within the 4-week recovery period

o Inflammatory cell infiltrates in both sexes, no reversibility within the 4-week recovery period

 

Mid dose group (15.0±2.3 mg/m³)

•Decreased albumin levels in both sexes

•In the BALF of the males:

o Increased neutrophil and lymphocyte counts accompanied by increased monocyte counts

o Slightly increased LDH activity

•Lung weight increase in females

•Lungs:

o Bronchiolo-alveolar hyperplasia in both sexes

 

Low dose group (2.9±0.7 mg/m³)

•No treatment-related adverse effect

Summarizing, the exposure of rats to the test substance caused concentration dependent pulmonary irritation as indicated by biochemical and cytological parameters in BALF, increased lung weights and corresponding histological findings in lungs and mediastinal lymph nodes. Within the 4-weeks recovery period several effects were not reversible for the high concentration group (75 mg/m³) due to the low lung clearance rate for poorly soluble particles. The lowest tested concentration of 2.9 mg/m³ is the NOAEL for the test conditions applied.