3-Isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate homopolymer, isocyanurate type

Endpoint summary

Description of key information

No repeated-dose toxicity tests are available for the oral and dermal route of exposure. Data waiver are claimed. A 14-day range finding inhalation study (5, 25, and 125 mg/m3; 6 hours/day, 5 days/week; OECD TG 412) with male Wistar rats indicates the respiratory tract to be the target organ. No adverse systemic effects were observed. The exposure of rats to the test substance caused concentration dependent pulmonary irritation as indicated by biochemical and cytological parameters in BALF, the increased weights of lung and the histomorphology of the lungs. The lowest tested concentration of 5 mg/m3 is the NOAEC under the current test conditions. The 90 day inhalation exposure to 3, 15, and 75 mg test item/m3 (male and female Wistar rats; OECD TG 413) did not lead to any substance related clinical signs of toxicity. The exposure of rats to the test substance caused concentration dependent pulmonary irritation as indicated by biochemical and cytological parameters in BALF, increased weights of lung and corresponding histological findings of the lung and the mediastinal lymph nodes. The lowest tested concentration of 2.9 mg/m3 is the No Observed Adverse Effect Level (NOAEC) under the current test conditions.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007-02-23 - 2007-12-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(1981)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(1988)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)

Version / remarks:

(1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Charles River Laboratories Sulzfeld (Germany)
- Age: 7 weeks at arrival, 9 weeks at first exposure; 5 / 6 days before first exposure: 3 days preflow , i.e. exposure to vehicle for 6 hours/day
- Weight at study initiation: females approx. 176 +- 9 g; males approx. 215 +- 10 g

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose/head only

Vehicle:

other: conditioned air

Mass median aerodynamic diameter (MMAD):

> 1.1 - < 1.8 µm

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 electostatic 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:

5 days/week x 6 hours/day

Dose / conc.:

3 mg/m³ air (analytical)

Remarks:

2.9 +/- 0.7 mg/m³

Dose / conc.:

15 mg/m³ air (analytical)

Remarks:

15.0 +/-2.3 mg/m³

Dose / conc.:

75 mg/m³ air (analytical)

Remarks:

75.0 +/- 3.6 mg/m³

No. of animals per sex per dose:

15 male (10 main and 5 satellite) and 10 female animals per exposure level
Additional groups for recovery study: High-dose + control with 10 animal/sex each

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 the termination of the 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) for 90 days were observed for potential reversibility for 28 days.

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 males per exposure level (including control) of satellite groups. 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

Clinical signs:

no effects observed

Description (incidence and severity):

During the whole study period, the animals showed no clinical signs and findings different from normal.

Mortality:

no mortality observed

Description (incidence):

No deaths were recorded throughout the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weights and the mean body-weight changes of the test substance exposed groups were not statistically significantly different from the control group 0.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Protein and enzymes in bronchoalveolar lavage fluid
Increases in the protein concentration in the 125 mg/m3 dose group as well as the enzyme activities beginning in the 25 mg/m3 dose group were assessed. No effects were noted in animals exposed to 5 mg/m3 test substance.

Cytology of broncho-alveolar lavage fluid
Statistically significant effects were observed in mid- and high-concentration animals.
Moderate increases in the total cell counts were present in these animals. These increases were due to the high polymorphonuclear cell counts of the respective groups (Figure 006a + b) and were accompanied by elevated numbers of lymphocytes and monocytes. Moreover, slight, but significantly (p ≤ 0.05) increased atypical cell and eosinophil counts were observed in the 25 mg/m3 dose group, only. Although the mean atypical cell and eosinophil counts of the 125 mg/m3 dose group were the highest, the difference compared to the controls was not statistically significant because of a great interindividual variance. Therefore, the increase of these cell counts was regarded not to be toxicological relevant.

Assessment of findings from clinical pathology of lung lavage fluid
In test groups 2 and 3, the inhalation of 25 or 125 mg/m³ of respirable dust of the test substances produced changes in total protein, enzyme activities and cellcounts of bronchoalveolar lavage fluid indicating an inflammatory process in the lungs of the treated animals.
In test group 1 (5 mg/m3), no treatment related effect was observed.

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute organ weights
The lungs in the high concentration group show a significantly increased weight. This increase is regarded to be treatment-related.
The weakly but significantly increased weight of the mediastinal lymph nodes represents rather a high inter individual variability among animals of that age than a true compoundrelated effect. In addition, no histomorphological correlate could be found that would indicate a compound- or treatment-related effect.

Relative organ weights
The relative organ weights were calculated by deviding the organ weight by the body weight of the respective animal.
The lungs in the high concentration group show a significantly increased weight. This increase is regarded to be treatment-related.
The significant liver weight increase in only the low concentration group is regarded to be incidental and of no biological significance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No gross lesions were reported.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Regarding pathology, treatment-related findings were observed in the lungs. In high and midconcentration males, a slight to moderate (grade 2 to 3) macrophage accumulation was noted multifocally in various examined lung lobes. Additionally, a minimal to slight bronchioloalveolar hyperplasia was observed multifocally in various examined lung lobes as well. The findings were slightly more pronounced in the high concentration group, indicating a concentration-dependent effect. No such findings were recorded for the low concentration group and controls. One animal of the mid concentration group showed a hypertrophy of the bronchial epithelium in addition that may be incidental but could also represent an adaptive effect on treatment with the test article.
In the larynx (level I), two males each of mid and high concentration groups and one male from the low concentration group showed a minimal (grade 1) and focal epithelial alteration at the base of epiglottis (below the ventral glands) what was not observed in the controls. It can not be excluded that this finding represents a first adaptive reaction on a slight irritative effect due to treatment, although those little alterations may be observed in control animals as well.
However, as no dysfunction of the larynx is to be expected, this minimal change is regarded as non-adverse in any case. In addition, Osimitz et al. (2007)* regard those findings as not
being relevant for human risk assessment.
All other findings noted are considered to be spontaneous or incidental in origin and not related to treatment.

Other effects:

no effects observed

Description (incidence and severity):

Rectal temperature
Rectal temperatures of the treated animals were not different to those of the controls during the whole study period

Details on results:

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.

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.

Key result

Dose descriptor:

NOAEC

Effect level:

2.9 mg/m³ air

Sex:

male/female

Basis for effect level:

other: No treatment-related adverse effects were observed in the low dose group.

Dose descriptor:

LOAEC

Effect level:

15 mg/m³ air

Sex:

male/female

Basis for effect level:

other: pulmonary irritation as indicated by biochemical and cytological parameters in BALF, increased weights of lung and corresponding histological findings of the lung and the mediastinal lymph nodes.

Critical effects observed:

not specified

no remarks

Conclusions:

The exposure of rats to the test substance caused concentration dependent pulmonary irritation as indicated by biochemical and cytological parameters in BALF, increased weights of lung and corresponding histological findings of the lung and the mediastinal lymph nodes. Overall, at the high concentration of 75.0 mg/m3 several effects were not reversible within 4 weeks recovery period due to the expected low lung clearance rate for poorly soluble particles. The lowest tested concentration of 2.9 mg/m3 is the No Observed Adverse Effect Level (NOAEL) under the current test conditions.

Executive summary:

In a 90 day inhalation study, 10 male and 10 female Wistar rats per test group were head-nose exposed to respirable dusts of IPDI homopolymer on 6 hours per day, on 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. Satellite groups of five male rats per exposure concentration were lavaged two days after the termination of the 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

Reference 2

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2006-08-01 to 2007-07-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study; GLP study without deviations

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

(1981)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

(1992)

Deviations:

yes

Remarks:

Reduced duration

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

- Source: Charles River Laboratories, Sulzfeld (Germany)
- Age: approx. 7 weeks when supplied; acclimatization 13 days including 2 days preflow (conditioned air in exposure device)
- Weight at study initiation: approx. 215 +- 10 g

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose/head only

Vehicle:

other: conditioned air

Remarks on MMAD:

MMAD / GSD: The particle sizes determined with an eight-stage Marple cascade impactor were well within the respirable range:
5 mg/m3: 1.1 - 1.2 µm / GSD 2.1 - 2.4
25 mg/m3: 1.3 - 1.8 µm / GSD 2.1 - 2.4
125 mg/m3: 1.1 µm / GSD 2.1 - 2.4
The calculated mass fractions of particles below 3 μm aerodynamic size ranged between 75.2 % and 90.5 %.

Details on inhalation exposure:

- Type or preparation of particles: 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 20.1 and 21.7°C.
- Post exposure period: 3 days (5 of 10 animals per dose group)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Gravimetrical measurement of 3 samples per exposure and test concentration (30 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 scattered light photometers. Constancy was generally proved. Some oscillations occurred in the high concentration during three exposures. The cause could be identified and eliminated. Because of their short duration, the impact of the oscillations on the study results was considered to be negligible.

Duration of treatment / exposure:

2 weeks

Frequency of treatment:

5 days/week x 6 hours/day

Remarks:

Doses / Concentrations:
5; 25; 125 mg/m3
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
5.0 +- 0.8; 26.3 +- 3.7; 122.1 +- 17.1 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

- Mortality: Morning and late afternoon of exposure days, once daily on other days
- Clinical signs: once during preflow period; before, during and after each exposure; each working day during post exposure period
- Body weight: days -4 (begin of preflow), 0 (first exposure), 7 and 11
- Rectal temperature: before "exposure" on day -4; after exposure on days 0, 7, 11
- Hematology: total cell count, macrophages, polymorphonuclear neutrophils, lymphocytes, eosinophils, monocytes, atypical cells.

Sacrifice and pathology:

Sacrifice of 5 animals per dose group three days after last exposure (= day 14).
- Weights of anesthetized animals, lung, Mediastinal lymph nodes, liver, kidneys, adrenals, thymus, spleen, brain (absolute and relative)
- Organ / tissue preservation: all gross lesions, head, larynx, trachea, lungs, mediastinal lymph nodes, liver, kidneys, adrenals, thymus, spleen, brain
- Histological processing of respiratory tract, examination by light microscopy: nasal cavity (4 levels), larynx (3 levels), oropharynx, trachea longitudinal with bifurcation, lungs (5 lobes), mediastinal lymph nodes (all animals); all gross lesions (affected animals only)

Other examinations:

Sacrifice and lavage of lungs of 5 animals per dose group after last exposure (= day 11). Analysis of bronchoalveolar lavage fluid (BALF; 2 x 5 ml per animal) for markers indicative for injury of the bronchoalveolar region: protein, lactate dehydrogenase, alkaline phosphatase, N-acetyl-β-D-glucosaminidase, γ-glutamyltransferase

Statistics:

- Body weight, body weight change: DUNNETT's test (two-sided)
- Clinical Pathology, weight parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If p <= 0.05: pairwise comparison of each dose group with the control group using Wilcoxon-test (two-sided) for the hypothesis of equal medians

Details on results:

Rectal temperatures: No effects

High concentration (125 mg/m3):
- increased protein concentration (+180 %, p ≤ 0.01) in bronchoalveolar lavage fluid (BALF)
- increased enzyme acitivities of lactate dehydrogenase (+366 %), alkaline phosphatase (+119 %), N-acetyl-β-D-glucosaminidase (+179 %) and γ-glutamyltransferase (+590 %) in BALF (p ≤ 0.01)
- Increased total cell count in BALF (p ≤ 0.01) driven by increased polymorphonuclear neutrophile granulocytes, monocytes and lymphocytes (p ≤ 0.01)
- Lung weight increase (absolute +43 %, p≤ 0.01; relative +41 %, p≤ 0.01)
- Lungs: slight to moderate (grade 2 to 3) macrophage accumulation, bronchiolo-alveolar hyperplasia

Mid concentration (25 mg/m³):
- increased enzyme acitivities of lactate dehydrogenase (+76 %), alkaline phosphatase (+74 %), N-acetyl-β-D-glucosaminidase (+84 %) and γ-glutamyltransferase (+460 %) in BALF (p ≤ 0.05)
- Increased total cell count in BALF (p ≤ 0.05) driven by increased polymorphonuclear neutrophile granulocytes (p ≤ 0.01) and lymphocytes (p ≤ 0.05)
- Lungs: slight to moderate (grade 2 to 3) macrophage accumulation, bronchiolo-alveolar hyperplasia

Low concentration (5 mg/m³):
- No compound-related adverse findings

Additional observations:
- A weak but significant increase in weight (only absolute, not relative) of the mediastinal lymph nodes (25 mg/m3: +114 %, p ≤ 0.05; 125 mg/m3: +159 %, p ≤ 0.05) was assigned to high inter individual variability among animals of that age rather than to treatment with the test substance. No histomorphological correlate could be found.
- A significant relative liver weight increase in only the low concentration group (+8.2 %, p ≤ 0.01) is regarded to be incidental and of no biological significance.
- In the larynx, minimal (grade 1) and focal epithelial alteration at the base of epiglottis (below the ventral glands) was observed in each 2/5 males of the mid and high dose groups. It might be a first adaptive reaction on a slight irritative effect due to treatment and is in any case considered as non-adverse.

Key result

Dose descriptor:

NOAEC

Effect level:

5 mg/m³ air

Sex:

male

Dose descriptor:

LOAEC

Effect level:

25 mg/m³ air

Sex:

male

Critical effects observed:

not specified

no remarks

Conclusions:

The exposure of rats to the test substance caused concentration dependent pulmonary irritation as indicated by biochemical and cytological
parameters in BALF, the increased weights of lung and the histomorphology of the lungs. The lowest tested concentration of 5 mg/m3 is the NOAEC
under the current test conditions.

Executive summary:

In a 14-day range finding study, ten male Wistar rats per test group were head-nose exposed to respirable dusts of IPDI homopolymer on 6 hours per day, on 5 consecutive days per week for 2 weeks (10 exposures). The target concentrations were 5, 25 and 125 mg/m3. A concurrent control group was exposed to conditioned air.

 

On each exposure day clinical examination was performed before, during and after exposure. Body weight and rectal temperature of the animals were determined in suitable intervals. Five animals per group were sacrificed on study day 11 (after the last exposure), the lungs were lavaged, and the bronchoalveolar lavage fluid (BALF) was analyzed for markers indicative for injury of the bronchoalveolar region. On study day 14 (3rd day after exposure) the remaining animals were sacrificed, the respiratory tract was histologically processed and examined by light microscopy.

 

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

of 5th and 10th exposure 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 concentration (125 mg/m³):

•increased protein concentration (p≤0.01) in broncho alveolar fluide (BALF)

•increased enzyme acitivities of lactate dehydrogenase, alkaline phosphatase, N-acetyl-β-D-glucosaminidase andγ-glutamyltransferase in BALF (p≤0.01)

•Increased total cell count in BALF (p≤0.01) driven by increased polymorphonuclear neutrophile granulocytes, monocytes and lymphocytes (p≤0.01)

•Lung weight increase

•Lungs: bronchiolo-alveolar hyperplasia

 

Mid concentration (25 mg/m³):

•increased enzyme acitivities of lactate dehydrogenase, alkaline phosphatase, N-acetyl-β-D-glucosaminidase andγ-glutamyltransferase in BALF (p≤0.05)

•Increased total cell count in BALF (p≤0.05) driven by increased polymorphonuclear neutrophile granulocytes (p≤0.01) and lymphocytes (p≤0.05)

•Lungs: bronchiolo-alveolar hyperplasia

 

Low concentration (5 mg/m³)

•No compound-related adverse findings

Therefore the lowest tested concentration of 5 mg/m3 is the NOAEC under the current test conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

subchronic

Species:

rat

Quality of whole database:

The study is valid without restriction (Klimisch score 1).

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007-02-23 - 2007-12-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(1981)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(1988)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)

Version / remarks:

(1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Charles River Laboratories Sulzfeld (Germany)
- Age: 7 weeks at arrival, 9 weeks at first exposure; 5 / 6 days before first exposure: 3 days preflow , i.e. exposure to vehicle for 6 hours/day
- Weight at study initiation: females approx. 176 +- 9 g; males approx. 215 +- 10 g

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose/head only

Vehicle:

other: conditioned air

Mass median aerodynamic diameter (MMAD):

> 1.1 - < 1.8 µm

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 electostatic 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:

5 days/week x 6 hours/day

Dose / conc.:

3 mg/m³ air (analytical)

Remarks:

2.9 +/- 0.7 mg/m³

Dose / conc.:

15 mg/m³ air (analytical)

Remarks:

15.0 +/-2.3 mg/m³

Dose / conc.:

75 mg/m³ air (analytical)

Remarks:

75.0 +/- 3.6 mg/m³

No. of animals per sex per dose:

15 male (10 main and 5 satellite) and 10 female animals per exposure level
Additional groups for recovery study: High-dose + control with 10 animal/sex each

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 the termination of the 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) for 90 days were observed for potential reversibility for 28 days.

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 males per exposure level (including control) of satellite groups. 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

Clinical signs:

no effects observed

Description (incidence and severity):

During the whole study period, the animals showed no clinical signs and findings different from normal.

Mortality:

no mortality observed

Description (incidence):

No deaths were recorded throughout the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weights and the mean body-weight changes of the test substance exposed groups were not statistically significantly different from the control group 0.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Protein and enzymes in bronchoalveolar lavage fluid
Increases in the protein concentration in the 125 mg/m3 dose group as well as the enzyme activities beginning in the 25 mg/m3 dose group were assessed. No effects were noted in animals exposed to 5 mg/m3 test substance.

Cytology of broncho-alveolar lavage fluid
Statistically significant effects were observed in mid- and high-concentration animals.
Moderate increases in the total cell counts were present in these animals. These increases were due to the high polymorphonuclear cell counts of the respective groups (Figure 006a + b) and were accompanied by elevated numbers of lymphocytes and monocytes. Moreover, slight, but significantly (p ≤ 0.05) increased atypical cell and eosinophil counts were observed in the 25 mg/m3 dose group, only. Although the mean atypical cell and eosinophil counts of the 125 mg/m3 dose group were the highest, the difference compared to the controls was not statistically significant because of a great interindividual variance. Therefore, the increase of these cell counts was regarded not to be toxicological relevant.

Assessment of findings from clinical pathology of lung lavage fluid
In test groups 2 and 3, the inhalation of 25 or 125 mg/m³ of respirable dust of the test substances produced changes in total protein, enzyme activities and cellcounts of bronchoalveolar lavage fluid indicating an inflammatory process in the lungs of the treated animals.
In test group 1 (5 mg/m3), no treatment related effect was observed.

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute organ weights
The lungs in the high concentration group show a significantly increased weight. This increase is regarded to be treatment-related.
The weakly but significantly increased weight of the mediastinal lymph nodes represents rather a high inter individual variability among animals of that age than a true compoundrelated effect. In addition, no histomorphological correlate could be found that would indicate a compound- or treatment-related effect.

Relative organ weights
The relative organ weights were calculated by deviding the organ weight by the body weight of the respective animal.
The lungs in the high concentration group show a significantly increased weight. This increase is regarded to be treatment-related.
The significant liver weight increase in only the low concentration group is regarded to be incidental and of no biological significance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No gross lesions were reported.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Regarding pathology, treatment-related findings were observed in the lungs. In high and midconcentration males, a slight to moderate (grade 2 to 3) macrophage accumulation was noted multifocally in various examined lung lobes. Additionally, a minimal to slight bronchioloalveolar hyperplasia was observed multifocally in various examined lung lobes as well. The findings were slightly more pronounced in the high concentration group, indicating a concentration-dependent effect. No such findings were recorded for the low concentration group and controls. One animal of the mid concentration group showed a hypertrophy of the bronchial epithelium in addition that may be incidental but could also represent an adaptive effect on treatment with the test article.
In the larynx (level I), two males each of mid and high concentration groups and one male from the low concentration group showed a minimal (grade 1) and focal epithelial alteration at the base of epiglottis (below the ventral glands) what was not observed in the controls. It can not be excluded that this finding represents a first adaptive reaction on a slight irritative effect due to treatment, although those little alterations may be observed in control animals as well.
However, as no dysfunction of the larynx is to be expected, this minimal change is regarded as non-adverse in any case. In addition, Osimitz et al. (2007)* regard those findings as not
being relevant for human risk assessment.
All other findings noted are considered to be spontaneous or incidental in origin and not related to treatment.

Other effects:

no effects observed

Description (incidence and severity):

Rectal temperature
Rectal temperatures of the treated animals were not different to those of the controls during the whole study period

Details on results:

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.

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.

Key result

Dose descriptor:

NOAEC

Effect level:

2.9 mg/m³ air

Sex:

male/female

Basis for effect level:

other: No treatment-related adverse effects were observed in the low dose group.

Dose descriptor:

LOAEC

Effect level:

15 mg/m³ air

Sex:

male/female

Basis for effect level:

other: pulmonary irritation as indicated by biochemical and cytological parameters in BALF, increased weights of lung and corresponding histological findings of the lung and the mediastinal lymph nodes.

Critical effects observed:

not specified

no remarks

Conclusions:

The exposure of rats to the test substance caused concentration dependent pulmonary irritation as indicated by biochemical and cytological parameters in BALF, increased weights of lung and corresponding histological findings of the lung and the mediastinal lymph nodes. Overall, at the high concentration of 75.0 mg/m3 several effects were not reversible within 4 weeks recovery period due to the expected low lung clearance rate for poorly soluble particles. The lowest tested concentration of 2.9 mg/m3 is the No Observed Adverse Effect Level (NOAEL) under the current test conditions.

Executive summary:

In a 90 day inhalation study, 10 male and 10 female Wistar rats per test group were head-nose exposed to respirable dusts of IPDI homopolymer on 6 hours per day, on 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. Satellite groups of five male rats per exposure concentration were lavaged two days after the termination of the 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

Reference 2

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2006-08-01 to 2007-07-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study; GLP study without deviations

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

(1981)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

(1992)

Deviations:

yes

Remarks:

Reduced duration

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

- Source: Charles River Laboratories, Sulzfeld (Germany)
- Age: approx. 7 weeks when supplied; acclimatization 13 days including 2 days preflow (conditioned air in exposure device)
- Weight at study initiation: approx. 215 +- 10 g

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose/head only

Vehicle:

other: conditioned air

Remarks on MMAD:

MMAD / GSD: The particle sizes determined with an eight-stage Marple cascade impactor were well within the respirable range:
5 mg/m3: 1.1 - 1.2 µm / GSD 2.1 - 2.4
25 mg/m3: 1.3 - 1.8 µm / GSD 2.1 - 2.4
125 mg/m3: 1.1 µm / GSD 2.1 - 2.4
The calculated mass fractions of particles below 3 μm aerodynamic size ranged between 75.2 % and 90.5 %.

Details on inhalation exposure:

- Type or preparation of particles: 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 20.1 and 21.7°C.
- Post exposure period: 3 days (5 of 10 animals per dose group)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Gravimetrical measurement of 3 samples per exposure and test concentration (30 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 scattered light photometers. Constancy was generally proved. Some oscillations occurred in the high concentration during three exposures. The cause could be identified and eliminated. Because of their short duration, the impact of the oscillations on the study results was considered to be negligible.

Duration of treatment / exposure:

2 weeks

Frequency of treatment:

5 days/week x 6 hours/day

Remarks:

Doses / Concentrations:
5; 25; 125 mg/m3
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
5.0 +- 0.8; 26.3 +- 3.7; 122.1 +- 17.1 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

- Mortality: Morning and late afternoon of exposure days, once daily on other days
- Clinical signs: once during preflow period; before, during and after each exposure; each working day during post exposure period
- Body weight: days -4 (begin of preflow), 0 (first exposure), 7 and 11
- Rectal temperature: before "exposure" on day -4; after exposure on days 0, 7, 11
- Hematology: total cell count, macrophages, polymorphonuclear neutrophils, lymphocytes, eosinophils, monocytes, atypical cells.

Sacrifice and pathology:

Sacrifice of 5 animals per dose group three days after last exposure (= day 14).
- Weights of anesthetized animals, lung, Mediastinal lymph nodes, liver, kidneys, adrenals, thymus, spleen, brain (absolute and relative)
- Organ / tissue preservation: all gross lesions, head, larynx, trachea, lungs, mediastinal lymph nodes, liver, kidneys, adrenals, thymus, spleen, brain
- Histological processing of respiratory tract, examination by light microscopy: nasal cavity (4 levels), larynx (3 levels), oropharynx, trachea longitudinal with bifurcation, lungs (5 lobes), mediastinal lymph nodes (all animals); all gross lesions (affected animals only)

Other examinations:

Sacrifice and lavage of lungs of 5 animals per dose group after last exposure (= day 11). Analysis of bronchoalveolar lavage fluid (BALF; 2 x 5 ml per animal) for markers indicative for injury of the bronchoalveolar region: protein, lactate dehydrogenase, alkaline phosphatase, N-acetyl-β-D-glucosaminidase, γ-glutamyltransferase

Statistics:

- Body weight, body weight change: DUNNETT's test (two-sided)
- Clinical Pathology, weight parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If p <= 0.05: pairwise comparison of each dose group with the control group using Wilcoxon-test (two-sided) for the hypothesis of equal medians

Details on results:

Rectal temperatures: No effects

High concentration (125 mg/m3):
- increased protein concentration (+180 %, p ≤ 0.01) in bronchoalveolar lavage fluid (BALF)
- increased enzyme acitivities of lactate dehydrogenase (+366 %), alkaline phosphatase (+119 %), N-acetyl-β-D-glucosaminidase (+179 %) and γ-glutamyltransferase (+590 %) in BALF (p ≤ 0.01)
- Increased total cell count in BALF (p ≤ 0.01) driven by increased polymorphonuclear neutrophile granulocytes, monocytes and lymphocytes (p ≤ 0.01)
- Lung weight increase (absolute +43 %, p≤ 0.01; relative +41 %, p≤ 0.01)
- Lungs: slight to moderate (grade 2 to 3) macrophage accumulation, bronchiolo-alveolar hyperplasia

Mid concentration (25 mg/m³):
- increased enzyme acitivities of lactate dehydrogenase (+76 %), alkaline phosphatase (+74 %), N-acetyl-β-D-glucosaminidase (+84 %) and γ-glutamyltransferase (+460 %) in BALF (p ≤ 0.05)
- Increased total cell count in BALF (p ≤ 0.05) driven by increased polymorphonuclear neutrophile granulocytes (p ≤ 0.01) and lymphocytes (p ≤ 0.05)
- Lungs: slight to moderate (grade 2 to 3) macrophage accumulation, bronchiolo-alveolar hyperplasia

Low concentration (5 mg/m³):
- No compound-related adverse findings

Additional observations:
- A weak but significant increase in weight (only absolute, not relative) of the mediastinal lymph nodes (25 mg/m3: +114 %, p ≤ 0.05; 125 mg/m3: +159 %, p ≤ 0.05) was assigned to high inter individual variability among animals of that age rather than to treatment with the test substance. No histomorphological correlate could be found.
- A significant relative liver weight increase in only the low concentration group (+8.2 %, p ≤ 0.01) is regarded to be incidental and of no biological significance.
- In the larynx, minimal (grade 1) and focal epithelial alteration at the base of epiglottis (below the ventral glands) was observed in each 2/5 males of the mid and high dose groups. It might be a first adaptive reaction on a slight irritative effect due to treatment and is in any case considered as non-adverse.

Key result

Dose descriptor:

NOAEC

Effect level:

5 mg/m³ air

Sex:

male

Dose descriptor:

LOAEC

Effect level:

25 mg/m³ air

Sex:

male

Critical effects observed:

not specified

no remarks

Conclusions:

The exposure of rats to the test substance caused concentration dependent pulmonary irritation as indicated by biochemical and cytological
parameters in BALF, the increased weights of lung and the histomorphology of the lungs. The lowest tested concentration of 5 mg/m3 is the NOAEC
under the current test conditions.

Executive summary:

In a 14-day range finding study, ten male Wistar rats per test group were head-nose exposed to respirable dusts of IPDI homopolymer on 6 hours per day, on 5 consecutive days per week for 2 weeks (10 exposures). The target concentrations were 5, 25 and 125 mg/m3. A concurrent control group was exposed to conditioned air.

 

On each exposure day clinical examination was performed before, during and after exposure. Body weight and rectal temperature of the animals were determined in suitable intervals. Five animals per group were sacrificed on study day 11 (after the last exposure), the lungs were lavaged, and the bronchoalveolar lavage fluid (BALF) was analyzed for markers indicative for injury of the bronchoalveolar region. On study day 14 (3rd day after exposure) the remaining animals were sacrificed, the respiratory tract was histologically processed and examined by light microscopy.

 

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

of 5th and 10th exposure 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 concentration (125 mg/m³):

•increased protein concentration (p≤0.01) in broncho alveolar fluide (BALF)

•increased enzyme acitivities of lactate dehydrogenase, alkaline phosphatase, N-acetyl-β-D-glucosaminidase andγ-glutamyltransferase in BALF (p≤0.01)

•Increased total cell count in BALF (p≤0.01) driven by increased polymorphonuclear neutrophile granulocytes, monocytes and lymphocytes (p≤0.01)

•Lung weight increase

•Lungs: bronchiolo-alveolar hyperplasia

 

Mid concentration (25 mg/m³):

•increased enzyme acitivities of lactate dehydrogenase, alkaline phosphatase, N-acetyl-β-D-glucosaminidase andγ-glutamyltransferase in BALF (p≤0.05)

•Increased total cell count in BALF (p≤0.05) driven by increased polymorphonuclear neutrophile granulocytes (p≤0.01) and lymphocytes (p≤0.05)

•Lungs: bronchiolo-alveolar hyperplasia

 

Low concentration (5 mg/m³)

•No compound-related adverse findings

Therefore the lowest tested concentration of 5 mg/m3 is the NOAEC under the current test conditions.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

2.9 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

The study is valid without restriction (Klimisch score 1).

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

No results from repeated-dose toxicity tests are available for the oral and dermal route of exposure.

Because inhalation is the most likely route of human exposure, a 90 days repeated dose inhalation study (0,3, 15, 75 mg/m³;

6 hours/day on five days/week for 13 weeks; OECD TG 413, Ma-Hock, BMS, 2009) with the test item was conducted.

The exposure of rats to the test substance caused concentration dependent pulmonary irritation as indicated by biochemical and cytological parameters in BALF, increased weights of lung and corresponding histological findings of the lung and the mediastinal lymph nodes. Overall, at the high concentration of 75 mg/m³ several effects were not reversible within 4 weeks recovery period due to the expected low lung clearance rate for poorly soluble particles. No substance-related clinical signs of toxicity were observed. Furthermore, no signs of systemic toxicity were observed in the study.

The lowest tested concentration of 2.9 mg/m³ is the No Observed Adverse Effect Level (NOAEC) under the current test conditions.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
According to REACH Annex IX (Column 1 of section 8.6.2) repeated oral study is not needed, because inhalation is the relevant route of exposure during use.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
Only one sub-chronic (90-day) inhalation study with 3-isocyanatomethyl-3,5,5 -trimethyl- cyclohexyl-isocyanate homopolymer, isocyanurate type (IPDI homopolymer) is available.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
Only one sub-chronic (90-day) inhalation study with 3-isocyanatomethyl-3,5,5 -trimethyl- cyclohexyl-isocyanate homopolymer, isocyanurate type (IPDI homopolymer) is available.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
According to REACH Annex IX (Column 1 of section 8.6.2) repeated dermal study is not needed, because inhalation is the relevant route of exposure during use.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
According to REACH Annex IX (Column 1 of section 8.6.2) repeated dermal study is not needed, because inhalation is the relevant route of exposure during use.

Justification for classification or non-classification

Based on the data regarding repeated dose toxicity the test item must not be classified according to the criteria of EC Directive 67/548/EEC and EC Regulation 1272/2008.