Registration Dossier

Administrative data

Description of key information

The local NOAEC for IPDI oligomers, allophanate type is 2.9 mg/m³ based on read-across from a subchronic (90-day) inhalation study in rats with the structurally similar IPDI oligomers, isocyanurate type (Ma-Hock, 2009). In this study no systemic toxicity was observed up to the highest test concentration (systemic NOAEC: > 75 mg/m³).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
repeated dose toxicity: oral, other
Remarks:
acc. to OECD TG 422
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Aug to Dec 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Crl: WI(Han)
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Males: 10 weeks; Females: 13 weeks
- Weight at study initiation: Males: 295-322 g; Females: 194-250 g
- Housing:On arrival and following the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm). During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm). During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm). During the lactation phase, females were housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cageenrichment, bedding material, food and water.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): > 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Details on route of administration:
Application volume: 5 mL/kg
Vehicle:
other: dried corn oil
Details on oral exposure:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. Until 6 Oct 2017, the dosing formulations were prepared daily as a solution and dosed within 2 hours after completion of the preparation of the test item. From 7 Oct 2017 (Day 24 of treatment) onwards, dosing formulations were prepared in daily portions as a solution and dosed within 24 hours after completion of the preparation of the test item, as stability was confirmed over a period of 24 hours at room temperature.

Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test item. Any residual volumes were discarded.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Concentration Analysis
Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ±10 % for solutions of target concentration.

- Homogeneity Analysis
Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was <=10 %.

- Stability Analysis
During the course of this study at one occasion during the treatment phase, stability of the prepared formulation was determined at 24 hours at room temperature. Duplicate sets of each sample (approximately 500 mg) were sent to the analytical laboratory. Stability results were considered acceptable if the sample analysis results were within or equal to ±10 % of the concentration determined by the initial analysis of each formulation.
Duration of treatment / exposure:
The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 29 days. Males were treated for 29 days (most males) or 33 days (two males used for re-mating), up to and including the day before
scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females that delivered were treated for 50-64 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to
conception, the duration of pregnancy and 13-15 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver were treated for 42 or 53 days.

Female nos. 45, 48 (Group 1), 51, 55 and 60 (Group 2) were not dosed on one occasion as these females were littering at the moment of dosing. The omission of one day of dosing over a period of several weeks was not considered to affect the toxicological evaluation.
Frequency of treatment:
Once daily at approximately the same time each day.
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale
The dose levels were selected based on the results of a 10-day dose range finder with oral administration of 500 and 1000 mg/kg IPDI oligomers, allophanate type in the rat (Test Facility Study No. 518884) and in an attempt to produce graded responses to the test item.

Based on the results of the dose range finder, selected dose levels for the main study were 100, 300 and 1000 mg/kg. The peak effect of occurrence of clinical signs occurred at 3 hours after dosing. This time point was taken into account to set a time range within which clinical observations and functional observation tests were conducted after dosing in the main study.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were conducted in a standard arena beginning before the first administration of the test item and then once weekly throughout treatment. These observations were conducted 3 hours (±30 min) post-dose.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. In order to monitor the health status, female no. 74 (Group 4) was also weighed on Day 6 of the mating period. Female no. 49 (Group 1) was also weighed on Day 25 post-coitum in order to monitor the pregnancy status. A fasted weight was recorded on the day of necropsy.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FUNCTIONAL TESTS: Yes
- Time schedule for examinations: Functional tests (hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength, locomotor activity) were performed on the selected 5 males during Week 4 of treatment and the selected 5 females during the last week of lactation (i.e. PND 11-13). These tests were performed 3 hours (±30 min) post-dose, after completion of clinical observations.

CLINICAL PATHOLOGY: Yes
Blood of F0-animals (except for animals which were sacrificed in extremis or found dead) was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m., from the retro-orbital sinus under anesthesia using isoflurane in the animal facility. When non-serum samples were clotted, additional blood samples were obtained in the necropsy room (if possible). After collection all samples were transferred to the appropriate laboratory for analysis. F0-females were not fasted prior to blood sampling and necropsy. F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available.
- Hematology parameters: White blood cells (WBC), Red Blood Cell Distribution Width (RDW), Neutrophil (absolute), Haemoglobin, Lymphocyte (absolute), Haematocrit, Monocyte (absolute), Mean corpuscular volume (MCV), Eosinophil (absolute), Mean corpuscular haemoglobin (MCH), Basophil (absolute), Mean corpuscular haemoglobin concentration (MCHC), Red blood cells, Platelet, Reticulocyte (absolute).
- Coagulation parameters: Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT)
- Clinical chemistry parameters: Alanine aminotransferase (ALAT), Creatinine, Aspartate aminotransferase (ASAT), Glucose, Alkaline Phosphatase (ALP), Cholesterol, Total protein, Sodium, Albumin, Potassium, Total Bilirubin, Chloride, Bile Acids, Calcium, Urea, Inorganic Phosphate (Inorg. Phos).
- Thyroid hormone: Blood samples were processed for serum, and serum was analyzed for total Thyroxine (T4). Measurement of T4 was conducted for F0-males. For the F0-generation, assessment of T4 (females) and Thyroid Stimulating Hormone (TSH; both sexes) was considered not relevant because no toxicologically relevant changes were noted in T4 in F0-males or in the weight and morphology of the thyroid of F0-animals of both sexes.
Sacrifice and pathology:
SACRIFICE
- Animals surviving until scheduled euthanasia were weighed, and deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination.
- Scheduled necropsies were conducted on the following days: Males (which sired and failed to sire): Following completion of the mating period (a minimum of 28 days of administration);
Females which delivered: PND 14-16; Females which failed to deliver: With evidence of mating: Post-coitum Day 26-27 (Nos. 49, 65, 69 and 75). Without evidence of mating: 25 days after the last day of the mating period (No. 46).

GROSS NECROPSY
- All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.

HISTOPATHOLOGY / ORGAN WEIGHTS
- Representative samples of the tissues identified in the tables below were collected from all animals and preserved in 10 % neutral buffered formalin (neutral phosphate buffered 4 % formaldehyde solution, Klinipath, Duiven, The Netherlands), unless otherwise indicated.

- Table 1: Tissue Collection and Preservation for all selected animals, and all animals that died spontaneously or were sacrificed in extremis: Animal identification, artery, aorta, body cavity, nasopharynx, bone marrow, bone (femur and sternum), brain (seven levels), cervix, epididymis, esophagus, eye, adrenal gland, coagulation gland, harderian gland, lacrimal gland, mammary gland, parathyroidc gland, pituitary gland, prostate gland, salivary gland, seminal vesicle gland, thyroid gland, gross lesions/masses, gut-associated lymphoid tissue, heart, kidney, large intestine (cecum, colon, rectum), larynx, liver, lung, lymph node (mandibular and mesenteric site), skeletal muscle, optic nerve, sciatic nerve, ovaries, pancreas, skin, small intestine (duodenum, ileum, jejunum), spinal cord,spleen, stomach, testes, thymus, tongue, trachea, urinary bladder, uterus, vagina.

- Table 2: Tissue Collection and Preservation for all remaining animals (incl. males that failed to sire females that failed to deliver pups; non-pregnant, implantation sites only):
Animal identification, cervix, epididymis, coagulation gland, mammary gland, parathyroidc gland, pituitary gland, prostate gland, seminal vesicle gland, thyroid gland, gross lesions/masses, ovaries, testes, uterus, vagina.
Other examinations:
For examinations on reproductive function/performance of parental animals as well as on developmental toxicity of pups see section 7.8.1.
Statistics:
All statistical tests were conducted at the 5 % significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1 % and 5 % levels.

Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.

The following pairwise comparisons were made: Group 2 vs. Group 1, Group 3 vs. Group 1 and Group 4 vs. Group 1.

Parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).

Non-Parametric: Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis.

Incidence: An overall Fisher’s exact test was used to compare all groups at the 5 % significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related clinical signs of toxicity were noted during daily clinical observations or during weekly arena observations. Salivation seen in animals of all groups, most frequently at 300 and 1000 mg/kg, was considered to be a physiological response rather than a sign of systemic toxicity considering its slight severity and the time of occurrence (i.e. after dosing). Any other clinical signs noted incidentally occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No mortality occurred during the study period that was considered to be related to treatment with the test item. There were two premature deaths, both due to gavage related trauma (female nos. 54 and 74 of the 100 and 1000 mg/kg groups, respectively). These deaths were considered not to be test item-related.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related changes in clinical chemistry parameters. The mean concentration of bile acids appeared higher in males at 1000 mg/kg. The difference from controls was not statistically significant and could largely be explained by the high value in male no. 31 (value of 81.3 μmol/L). In the absence of associated adverse anatomic pathology changes, this isolated finding was considered not to represent an adverse effect of the test item. Isolated statistically significant variations noted in clinical chemistry parameters were considered unrelated to treatment due to the lack of a dose-related trend.

Thyroid hormone analyses: Serum levels of T4 in F0 males were not affected by treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional observation parameters were considered not to be affected by treatment. Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was not affected by treatment. The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically relevant alterations in organ weights. Compared to the control group, mean thyroid weights (absolute and relative to body weight) appeared somewhat higher in all groups of treated males, reaching statistical significance at 1000 mg/kg). The relative differences showed no dose-related trend. These changes were considered to have (partly) arisen as a result of slightly low control values, particularly in control male no. 3 for which a reduced size of the thyroid was noted at necropsy. Individual relative thyroid weight of the majority of the treated males was similar to the historical control mean. In the absence of any test item-related macroscopic or microscopic finding in the thyroid gland this increase in thyroid gland weight was considered non-adverse. The statistically significantly higher uterus weights (absolute and relative to body weight) noted in 100 mg/kg females were considered unrelated to treatment due to the lack of a dose-related trend.
Gross pathological findings:
no effects observed
Description (incidence and severity):
All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. These findings were therefore considered to be unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic observations. All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

There were no remarkable findings in the reproductive organs of the 100 mg/kg male (no. 14) which was not used for pairing due to the premature death of the pertaining female (no. 54). There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item, and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no treatment-related effects observed
Critical effects observed:
no
Executive summary:

Ten male and ten female Wistar Han rats per group were treated with IPDI oligomers, allophanate type by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg according to OECD TG 422. The rats of the control group received the vehicle, dried corn oil, alone. Males were treated for 2 weeks prior to mating, during mating, and up to termination (29 to 33 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and 13-15 days of lactation (50 to 64 days). Females that failed to deliver pups were treated for 42 or 53 days.

Formulation analysis showed that the formulations were prepared accurately and homogeneously, and that the formulations were stable for at least 24 hours at room temperature under normal laboratory light conditions.

No parental toxicity was observed up to the highest dose level tested (1000 mg/kg). Treatment with the test item up to 1000 mg/kg was well tolerated as indicated by the absence of adverse changes in the parental parameters and examinations in this study (i.e. clinical appearance, body weight, food consumption, functional tests, haematology and clinical chemistry, macroscopic examination, organ weights, and microscopic examination). The only treatment-related finding in this study consisted of slight salivation after dosing, noted most frequently at 300 and 1000 mg/kg. This was regarded as a physiological response rather than a sign of systemic toxicity.

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the parental NOAEL of IPDI oligomers, allophante type was at least 1000 mg/kg bw.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The study is GLP compliant and is of high quality (Klimisch score=1)

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Referenceopen allclose all

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
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Version / remarks:
(1981)
GLP compliance:
yes (incl. certificate)
Limit test:
no
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
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
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.
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
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.
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)
Critical effects observed:
not specified
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.

 

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
JUSTIFICATION FOR READ-ACROSS FROM SUPPORTING SUBSTANCE (STRUCTURAL ANALOGUE OR SURROGATE)
For 3-Isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate, oligomers, allophanate type (IPDI oligomers, allophanate type) no subchronic (90-day) toxicity study is available. Therefore, subchronic inhalation toxicity data of the structurally similar 3-Isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate homopolymer, isocyanurate type (IPDI oligomers, isocyanurate type) were used via read-across since these data are regarded as representative. IPDI oligomers, isocyanurate type is also an IPDI-based polyisocyanate and is, moreover, contained to approx. 50 % in IPDI oligomers, allophanate type. The subchronic inhalation study with IPDI oligomers, isocyanurate type (Ma-Hock, 2009) confirmed that after 90 days of exposure only local irritant effects were seen, but no systemic toxicity was observed. Taken together, the available data are considered adequate and sufficient for the purpose of classification and labelling and risk assessment (for additional information see justification for the read-across approach to IPDI oligomers, allophanate type in section "Attached justification").
Reason / purpose:
read-across source
Details on inhalation exposure:
-
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.
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)
Critical effects observed:
not specified
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.

 

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Version / remarks:
(2009)
Qualifier:
according to
Guideline:
other: OECD Guidance Document No. 39 (2009)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
- Stability under test conditions: Stability certified for the duration of study.
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Hsd Cpb:WU (SPF)
- Source:Harlan-Nederland, AD Horst, Netherlands
- Age at study initiation: 2 months
- Weight at study initiation (mean): males 254 g, females 184 g (variation of individual weights did essentially not exceed ±10 per cent of the group means)
- Housing: singly in conventional Makrolon® Type IIIh cages (based on A. Spiegel and R. Goennert, Zschr. Versuchstierkunde, 1. 38 (1961) and G. Meister, Zschr. Versuchstierkunde, z. 144~153 (1965)).
- Diet and water: ad libitum
- Acclimation period: approximately 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 40-60
- Air changes (per hr): approximately 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
other: n-butyl acetate
Remarks on MMAD:
MMAD / GSD: Throughout the test groups the average MMAD was aprox. 1.6 (GSD ~2.3).
The percentage of particle <3 µm was in the range of 75.5-77.3 %. Thus, particle-size distribution achieved is adequate to reach all potential target structures of the respiratory tract.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Rats were exposed under dynamic directed-flow nose-only exposure conditions.
- Exposure apparatus: Details of the nose-only exposure system, including its validation, have been published previously (Pauluhn, Journal of Applied Toxicology 13, 1994, 55-62; Pauluhn and Thiel, J. Appl. Toxicol. 27, 2007, 160-167). Each segment of the aluminum inhalation chamber had the following dimensions: inner diameter = 14 cm, outer diameter = 35 cm (two-chamber system), height = 25 cm (internal volume = about 3.8 L). To be able to perform all measurements required to define exposure in a manner that is similar to the exposure of rats, a 'two segment' chamber were used in all groups.
- Method of holding animals in test chamber: Animals were exposed in Plexiglas exposure restrainers. Restrainers were chosen that accommodated the animals' size. These restrainers were designed so that the rat's tail remained outside the restrainer, thus restrained-induced hyperthermia can be avoided.
- Source and rate of air: Compressed air was supplied by Boge compressors.
- Method of conditioning air: Compressed air was conditioned (i.e. freed from water, dust, and oil) automatically by a VIA compressed air dryer.
- Atmosphere generation: Under dynamic conditions the test substance is atomized into the baffle (pre-separator) of the inhalation chamber. For atomization a binary nozzle and conditioned compressed air (15 L/min) was used. The representative dispersion pressure was in the range of 500 to 600 kPa (constant liquid feed at 70 µL/min through the nozzle maintained at room temperature. The test article was fed into the nozzle system using a digitally controlled pump (Harvard PHD 2000 infusion pump). In the vehicle control group a constant liquid feed at 21.5 µL/min butyl acetate (specific density ~0.88 g/cm³), including dilution cascades, were used to attain the targeted concentration, i.e., the same aerosolization principles were used at all exposure levels. In order to increase the efficiency of the generation of fine particles likely to evaporate and to prevent larger particles from entering the chamber a glass-pre-separator/baffle system was used. Overall, this pre-separator systems favors the evaporation of the test substance.
- Temperature, humidity: Temperature and humidity measurements are performed by the computerizedvData Acquisition and Control System using FTF11 sensors (ELKA ELEKTRONIK, Ludenscheid, Germany). The position of the probe was at the exposure location of rats.
- Air flow rate: At each exposure port a minimal air flow rate of 0.75 L/min was provided. The test atmosphere can by no means be diluted by bias-air-flows.
- Air change rate: The test atmosphere generation conditions provide an adequate number of air exchanges per hour [30 L/min x 60 min/(2 x 3.8 L/chamber) = 237, continuous generation of test atmosphere].
- Method of particle size determination: The particle-size distribution was analyzed using a BERNER-TYPE AERAS low-pressure critical orifice cascade impactor (Hauke, Gmunden, Austria).
- Treatment of exhaust air: The exhaust air was purified via filter systems. These filters were disposed of by Bayer Healthcare Pharmaceuticals AG.
The ratio between supply and exhaust air was selected so that 90 % of the supplied air was extracted via the exhaust air location and, if applicable, via sampling ports. Aerosol scrubbing devices were used for exhaust air clean-up. During sampling, the exhaust air was reduced in accordance with the sampling flow rate using a computerized Data Acquisition and Control System so that the total exhaust air flow rate was adjusted on-line and maintained at the specified 90 %. The slight positive balance between the air volume supplied and extracted ensured that no passive influx of air into the exposure chamber occurred (via exposure restrainers or other apertures). The slight positive balance provides also adequate dead-space ventilation of the exposure restrainers.

TEST ATMOSPHERE
- Brief description of analytical method used: The concentration of the non-volatile components of the test-substance was determined by gravimetric analysis (filter: glass-'fiber filter, Sartorius, Gottingen, Germany; digital balance). Filters were pre-conditioned for 60 min @70 °C prior to gravimetric analysis.
The relative proportion of constituents prone to evaporate is determined as follows: aliquots of the test substance were added onto glass fiber filters and the filters were allowed to dry under specified conditions (at 70 °C drying temperature) over a time period of maximal 3 hours. During this time course that time of drying will be defined at which stable conditions are attained. Taking into account that the formulation contained 13.5 % volatile substances, gravimetric analyses could be converted to the formulation by multiplication of concentrations by 100/(100-13.5). Since the substance of the registration is solely 3-Isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate, oligomers, allophanate type no concentration values for the formulation (test substance 80 % in butyl acetate) are reported in this study endpoint record.
To monitor the integrity and stability of the aerosol generation and exposure system either a RAM-1/RAS-2 real-time aerosol photometer (MIE, Bedford, Massachusetts, USA) or Microdust Pro real-time aerosol photometer (Casella, USA) was used.
- Samples taken from breathing zone: yes

VEHICLE (if applicable)
n-butyl acetate (Sigma-Aldrich, LOT: 66096JM)
- Purity of vehicle: urethane grade >/= 99.5 %
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test-substance concentration was determined by gravimetric analysis (filter: glass-'fiber filter, Sartorius, Gottingen, Germany; digital balance). Filters were pre-conditioned for 60 min at 70 °C prior to gravimetric analysis.
All concentrations refer to the nonvolatile fraction of the test article!
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
6 hours/day, 5 days/week
Remarks:
Doses / Concentrations:
5, 25, 125 mg/m³
Basis:
other: target concentrations
Remarks:
Doses / Concentrations:
4.5, 25.8, 122 mg/m³
Basis:
other: gravimetric concentrations (non-volatile fraction of the test article)
No. of animals per sex per dose:
5
Additional 5 animals for recovery groups (High-dose and control)
Additional 6 males for bronchoalveolar lavage groups
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Data on acute inhalation toxicity and on repeated dose toxicity (pilot study 5x6h; both studies included in IUCLID) served as basis for dose selection. Based on the findings of the pilot study, 5 mg/m³ was expected to be the NOAEL and 25 mg/m³ was likely to be in the range where portal-of-entry related local effects would start to occur.
- Satellite groups - bronchoalveolar lavage analysis: Additional 6 male rats per concentration were subjected to bronchoalveolar lavage at the end of the 4-week exposure period. The rationale for setting such a satellite group is that changes in the pulmonary tract can be detected most sensitively in the for branchoalveolar lavage fluid.
- Post-exposure recovery period in satellite groups: Additional 5 rats/sex for control and high dose group were allowed to recover during a 4-week postexposure period.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes, all animals
- Time schedule: The appearance and behavior of each rat was examined carefully at least twice on exposure days (before and after exposure) and once a day during the exposure-free recovery period.
Cage side observations included, but were not limited to, changes in the skin and hair-coat, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous system, and sensori- as well as somatomotor activity and behavior pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhea, lethargy, somnolence and prostration. The time of death was recorded as precisely as possible, if applicable.

DETAILED CLINICAL OBSERVATIONS: Yes, all animals
- Time schedule: During the course of study, additional clinical observations which took into account the pattern of examination consistent with a Functional Observational Battery (FOB). Measurements were made in 5 rats/sex/group. Each rat was first observed in its home cage and then individually examined. The following reflexes were tested, based on recommendations made by Irwin (Psychopharmacologica 13, 1968, 222-257) and Moser et. al. (Fundamental and Applied Toxicology, 11, 1988, 189-206): visual placing response and grip strength on wire mesh (wire-mesh gridgripping resistance of he animal to pull), abdominal muscle tone, corneal and pupillary reflexes, pinnal reflex, righting reflex, tail-pinch response, startle reflex with respect to behavioral changes stimulated by sounds (finger snapping) and touch (back).

RECTAL TEMPERATURES: Yes, all animals
- Time schedule for examinations: The rectal (colonic) temperatures were measured at several time points shortly after cessation of exposure (within 1/2 hour of cessation of exposure) using digital a rectal probe (H. Sachs, March, Germany). Measurements were made in 5 rats/sex/main group following the first exposure day, at end of the exposure period, and third postexposure day.

BODY WEIGHT: Yes, all animals
- Time schedule for examinations: Body weights of all animals were measured on a twice per week basis on Fridays and Mondays (before exposure) during the exposure period and once weekly (Mondays) during the postexposure period.

FOOD AND WATER CONSUMPTION: Yes, all animals
Food and water consumption were determined on a per week basis.

OPHTHALMOSCOPIC EXAMINATION: Yes, all animals
Eye examinations were performed prior to the first exposure and towards the end of the exposure period.

HAEMATOLOGY: Yes, blood was sampled at the end of the 4-week exposure period on five animals/sex/group
The terminal blood samples were obtained by cardiac puncture of the deeply anesthetized, non-fasted rats (Narcoren®; intraperitoneal injection).
- Parameters checked: Hematocrit, Hemoglobin, Leukocytes, Erythrocytes (Mean corpuscular volume, Mean corpuscular hemoglobin concentration, Mean corpuscular, hemoglobin), Thrombocyte count, Reticulocytes, Leukocyte differential count (Lymphocytes, Granulocytes, Segmented neutrophils, Eosinophilic neutrophils, Basophils, Monocytes, Plasma cells, miscellaneous abnormal cell types), Prothrombin time (PT, Quick value, "Hepato Quick").

CLINICAL CHEMISTRY: Yes, blood was sampled at the end of the 4-week exposure period on five animals/sex/group
Blood from fasted animals was not used for the glucose determination. The fasting of animals in connection with inhalation toxicity studies has not proven judicious and successfuL This would have resulted in an unduly long fasting period and, correspondingly, effects on the body weight and the body weight/organ weight ratio cannot be ruled out In view of these very likely artifacts, the glucose was determined on non-fasted rather than fasted animals as a deviation from the test guideline.
- Parameters checked: Aspartate aminotransferase, optimized (ASAT), Alanine aminotransferase, optimized (ALAT), Glutamate dehydrogenase (GLDH), gamma-Giutamylaminotransferase (gamma-GT), Lactate dehydrogenase (LDH), Alkaline phosphatase (APh), Albumin, Bilirubin (total), Blood glucose, Calcium, Chloride, Cholesterol, Creatine Kinase, Creatinine, Magnesium, Phosphate, Potassium, Sodium, Total protein, Triglycerides, Urea.

BRONCHOALVEOLAR LAVAGE: Yes, six male rats/dose group
Samples of bronchoalveolar lavage fluid were collected from the lungs of rats (six male rats/group) at the end of the exposure period (one day after the last exposure). In BAL-fluid (BALF), several indicators of pulmonary damage were assessed: Alkaline phosphatase, Soluble collagen, Lactate dehydrogenase (LDH), N-Acetylglucosaminidase (beta-NAG), Phospholipids, Total protein, gamma-Glutamyltransferase (gamma-GT), Total number of lavaged cells, including the volume and diameter, Cytodifferentiation
Sacrifice and pathology:
All surviving rats were sacrificed at the end of the exposure and postexposure observation period using sodium pentobarbital (Narcoren®; at least 120 mg/kg body weight, intraperitoneal injection) as anesthetic. Complete exsanguination was by severing the aorta abdominalis. Consideration was given to performing a gross necropsy on animals as indicated by the nature of toxic effects, with particular reference to changes related to the respiratory tract. All gross pathological changes were recorded and evaluated.

GROSS PATHOLOGY: Yes, all rats were given a grosspathological examination.
The following organs were weighted at necropsy after exsanguination; paired organs were weighted together; the organ-to-body relationships are specified in both absolute and relative terms, the body weight and well as the brain weight were used as a basis for these calculations as determined prior to necropsy of each animal: Adrenal glands, Brain, Heart, Kidneys, Liver, Lung (incl. trachea), Ovaries, Spleen, Testes, Thymus.
The organ-to-body relationships are specified in both absolute and relative terms. The body weight and well as the brain weight were used as a basis for these
calculations as determined prior to necropsy of each animal.

HISTOPATHOLOGY: Yes
The following organs were evaluated at least for high dose and control groups after being fixed (e.g. in 10 % neutral buffered formalin or Davidson's solution): Adrenal glands, Aorta, Brain, Epididymides, Esophagus, Eyes, Eyelids, Exorbital lacrimal glands, Femur (with knee joint), Harderian gland, Head with nasal cavity (all concentration groups examined including recovery groups), Heart, Intestine (incl. Peyer's Patches; Duodenum, Jejunum, Ileum, Caecum, Colon, Rectum), Kidneys, Larynx (all concentration groups examined including recovery groups), Liver, Lungs (all concentration groups examined including recovery groups), Lymph nodes (lung associated (all concentration groups examined including recovery groups), mandibular, mesenteric, popliteal), Optic nerves, Ovaries with oviducts, Pancreas, Pharynx (all concentration groups examined including recovery groups), Pituitary gland, Prostate, Salivary glands (parotid, submandibular, sublingual), Sciatic nerve, Skeletal muscle (tigh), Skin (mammary region), Skin (muzzle) (all concentration groups examined including recovery groups), Seminal vesicles (incl. coagulating glands), Spinal cord (cervical thoracic, lumbar), Spleen, Sternum, Stomach (forestomach and glandular stomach), Testes, Thymus, Thyroid glands (with parathyroids), Tongue, Trachea (incl. bifurcation)(all concentration groups examined including recovery groups), Ureters, Urinary bladder, Uterus (with cervix), Vagina.
Statistics:
Statistical tests on body weights and weight gain as well as on absolute organ weights or relative log10-transformed organ weights are analyzed using the Dunnett Exact Homogeneous Test. Food/water intake per animal and day are calculated and analyzed by the adjusted Mann-Whitney U-tests. Terminal body weights (TWB) serve as covariate for calculation of the organ-to-body-weight ratio (percentage). Likewise, the Dunnett Exact Homogeneous or Heterogeneous Test, the Dunnett Exact Homogeneous Test after log1 0-transformation or the Bonferroni/Mann-Whitney U-test are used for the statistical analysis of clinical pathology parameters.
Descriptive statistics were provided per sex, dose group and time point for all parameters that were recorded with a specified unit. This included measures of general tendency (mean and median (median not given for food and water intake)) and general variability (standard deviation, minimum and maximum) as appropriate.
For continuous variables, the statistical test procedure was based on prior knowledge of the respective variable derived from previous studies. For normally distributed variables with equal variances across treatment groups Dunnett's tests were performed. Heteroscedastic normally distributed variables were analysed using appropriately adjusted Dunnett's tests, using Satterthwaite adjustments for the degrees of freedom and taking the different variances within the groups into account. For log-normally distributed variables, Dunnett's tests were performed after log transformation of the original values. If experience with historical data indicated that the assumptions for parametric analyses are violated, Bonferroni-adjusted Mann-Whitney U-tests were employed in the analyses. For small sample sizes, the exact version of this test was used.
Details on results:
MORTALITY
All exposures were tolerated without test substance-induced mortality.

CLINICAL SIGNS (INCLUDING BODY WEIGHTS, FOOD AND WATER CONSUMPTION, RECTAL TEMPERATURES AND EXAMINATION OF REFLEXES)
The rats exposed up to 4.5 and 25.8 mg/m³ did not display substance specific clinical signs while at 122 mg/m³ the following clinical signs were recorded: labored and irregular breathing, tachypnea, motility reduced, atony, high-legged gait, piloerection, haircoat ungroomed, hairless areas.
Conclusive changes in body temperature, reflexes, body weights, and feed/water consumption did not occur. Of note is that all findings were most pronounced during the first week and waned during the remaining exposure weeks. During this week the transiently significantly decreased body weights coincided with the significantly decreased food/water consumption. This constellation appears to suggest that nonspecific sensory irritation is the cause for these findings which decreased to normal at continued exposure.

OPHTHALMOSCOPIC EXAMINATION
Ophthalmology was unremarkable.

HAEMATOLOGY
There was no evidence of any adverse hematological effects.

CLINICAL CHEMISTRY
Clinical pathology did not reveal any pathodiagnostically relevant effects considered to be causally related to the exposure to the test article aerosol.

ORGAN WEIGHTS
There were no statistically significant or conclusive changes in absolute or relative organ weights with the exception of increased lung weights at 25.8 and 122 mg/m³. Lung weights showed a clear trend towards reversibility at the end of the 4-week postexposure period. At similar exposure concentrations, the thymus weights were decreased and adrenal weights were increased, especially in female rats.

GROSS PATHOLOGY
Rats exposed at 122 mg/m³ displayed enlarged lung-associated lymph nodes and focal discolorations of lungs.

HISTOPATHOLOGY
Histopathology findings at the end of the exposure period consisted of epithelial changes and increased inflammatory infiltrates of the larynx at 122 mg/m³. Hypercellularity of the epithelial cells occurred at the bronchiolo-alveolar junction. At the alveolar level septal thickening with inflammatory infiltrates and enlarged/foamy alveolar macrophages were observed at 25.8 and 122 mg/m³. Concurrently bronchus associated lymphoid tissue (BALT) was increased. In lung-associated-lymph nodes (LALN), an increased cellularity of the paracortex, including an increased influx of macrophages with foamy appearance, was apparent at 25.8 and 122 mg/m³. While the laryngeal changes regressed during the 4-week postexposure period, the pulmonary and LALN changes remained by and large unchanged. In the adrenals of the female rats exposed at 122 mg/m³ a minimal to slight hypertrophy of the zona fasciculata was apparent. All other findings seen in the organs/tissues evaluated in this subacute inhalation study were equally distributed between controls and substance-exposed groups and/or are known as spontaneous findings from previous studies. Collectively, histopathology did not reveal any adverse effects at 4.5 mg/m³.

BRONCHOALVEOLAR lAVAGE
Determinations in bronchoalveolar lavage fluids resulted in effects suggestive of alveolar irritation at 25.8 and 122 mg/m³ and included an increase neutrophilic granulocyte and total cell count, elevations of protein, collagen, LDH, beta-NAG, and gamma-GT. At 4.5 mg/m³ alveolar macrophages containing cellular debris and, possibly, polymerized reaction products of the test article, were apparent; however, without any increase in total BAL-cell counts.
Dose descriptor:
NOAEC
Effect level:
4.5 other: mg/m³ air (non-volatile fraction of the test article)
Sex:
male/female
Basis for effect level:
other: based on pulmonary irritant effects (histopathology, bronchoalveolar lavage) at next higher concentration (25.8 mg/m³)
Critical effects observed:
not specified

This study revealed evidence of mild-to-moderate laryngeal irritation at 25.8 and 122 mg/m³. The squamous metaplasia of the respiratory epithelium overlaying the ventral aspects of the larynx is a common finding in rat inhalation studies. Of note is, that this appears to be an adaptive response to recurrent irritation and progression of this lesion into neoplasia has never been seen. It is reversible in nature and an indicator of the especially high susceptibility of the rodent larynx to any mild irritant (DeSesso, The relevance to humans of animal models for inhalation studies of cancer in the nose and upper airways. Quality Assurance Good Practice, Regulation, and Law 2, 213-231, 1993; Kaufmann et al., Exp. Toxicol. Pathol. 61, 591 -603, 2009). Evidence of airway irritation proximal or distal the larynx was not found.

Histopathology findings were confined to the alveolar level and were complementary to the changes in lung weights and bronchoalveolar lavage.

The apparent increased involution of thymus at 122 mg/m³, in association with increased adrenal weights, is consistent with the histopathology findings which are typical of an activation of the hypothalamic-pituitary-adrenal axis as a result of acute sensory distress to rats. This response is considered to be a secondary, relatively rat-specific phenomenon and is not considered to be primarily adverse.

Overall, as long as adverse changes occurred they were confined to portal-of-entry related local irritant effects of (larynx and alveoli). Taking all findings into account, 4.5 mg/m³ constitutes the NOAEL for respiratory tract irritation. In regard to extrapulmonary toxicity, no effects were found up to the maximum concentration examined.

Executive summary:

A subacute (28-day) inhalation toxicity study according to OECD TG 412 was conducted with the test substance (80 % in butyl acetate) as liquid aerosol. In this study male and female rats were nose-only exposed to actual concentrations of 0 (butyl acetate control), 4.5, 25.8 and 122 mg/m³ test substance (non-volatile fraction of the test article). The aerosol proved to have an adequate respirability with an average MMAD of approximately 1.6 µm (GSD ~ 2.3). Two satellite groups were investigated in the study: 5 additional rats per sex and concentration (control and high-dose group) were allowed to recover during a 4-weeks postexposure period and 6 additional male rats per concentration were subjected to bronchoalveolar lavage at the end of the 4-week exposure period.

                         

Rats exposed to 4.5 and 25.8 mg/m³ did not display substance specific clinical signs while at 122 mg/m³ unspecific clinical signs and signs of respiratory distress were recorded. These signs were most pronounced during the first week and waned during the remaining exposure weeks. Conclusive changes in body temperature, reflexes, body weights, and feed/water consumption did not occur. There was no evidence of any adverse hematological effects. Likewise, clinical pathology did not reveal any relevant effect considered test-substance related. Mild-to-moderate laryngeal irritation observed at 25.8 and 122 mg/m³ was considered to be an adaptive response. The squamous metaplasia of the respiratory epithelium overlaying the ventral aspects of the larynx is a common finding in rat inhalation studies. It is reversible in nature and an indicator of the especially high susceptibility of the rodent larynx to any mild irritant (DeSesso, The relevance to humans of animal models for inhalation studies of cancer in the nose and upper airways. Quality Assurance Good Practice, Regulation, and Law 2, 213-231, 1993; Kaufmann et al., Exp. Toxicol. Pathol. 61, 591 -603, 2009). Evidence of airway irritation proximal or distal the larynx was not found.

Histopathology findings at 25.8 and 122 mg/m³ were confined to the alveolar level. These findings included hypercellularity of the epithelial cells at the bronchiolo-alveolar junction and septal thickening with inflammatory infiltrates and enlarged/foamy alveolar macrophages at the alveolar level. Concurrently bronchus associated lymphoid tissue (BALT) was increased. In lung-associated-lymph nodes (LALN) increased cellularity of the paracortex, including an increased influx of macrophages with foamy appearance, was apparant at 25.8 and 122 mg/m³. Changes in lung weights (at 25.8 and 122 mg/m³; clear trend towards reversibility at the end of the 4-week post exposure period) and bronchoalveolar lavage were complementary to histopathology. At 4.5 mg/m³ alveolar macrophages containing cellular debris and, possibly, polymerized reaction products of the test article, were apparent; however, without any increase in total BAL-cell counts.

All other adverse findings seen in the organs/tissues evaluated in this study were equally distributed between controls and substance-exposed groups and/or are known as spontaneous findings from previous studies. Collectively, histopathology did not reveal any adverse effects at 4.5 mg/m³.

Overall, as long as adverse changes occurred they were confined to portal-of-entry related local irritant effects of larynx and alveoli. Taking all findings into account, 4.5 mg/m³ (non-volatile fraction of the test article) constitutes the no-observed-adverse-effect-level (NOAEL) for respiratory tract irritation. In regard to extrapulmonary toxicity, no effects were found up to the maximum concentration examined.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
75 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
The study is GLP compliant and is of sufficient quality (Klimisch score = 2)

Repeated dose toxicity: inhalation - local effects

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 GLP compliant and is of sufficient quality (Klimisch score = 2)

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

In a combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test ten male and ten female Wistar rats per group were treated with IPDI oligomers, allophanate type by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg according to OECD TG 422 (van de Ven, 2018). The rats of the control group received the vehicle, dried corn oil, alone. Males were treated for 2 weeks prior to mating, during mating, and up to termination (29 to 33 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and 13-15 days of lactation (50 to 64 days). Females that failed to deliver pups were treated for 42 or 53 days. No parental toxicity was observed up to the highest dose level tested (1000 mg/kg). Treatment with the test item up to 1000 mg/kg was well tolerated as indicated by the absence of adverse changes in the parental parameters and examinations in this study (i.e. clinical appearance, body weight, food consumption, functional tests, haematology and clinical chemistry, macroscopic examination, organ weights, and microscopic examination). The only treatment-related finding in this study consisted of slight salivation after dosing, noted most frequently at 300 and 1000 mg/kg. This was regarded as a physiological response rather than a sign of systemic toxicity. In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the parental NOAEL of IPDI oligomers, allophanate type was at least 1000 mg/kg bw.

A subacute (28-day) inhalation toxicity study according to OECD TG 412 was conducted with IPDI oligomers, allophanate type, 80 % in butyl acetate as liquid aerosol (Pauluhn, 2012). In this study male and female rats were nose-only exposed to actual concentrations of 0 (butyl acetate control), 4.5, 25.8 and 122 mg/m³ test substance (non-volatile fraction of the test article). The aerosol proved to have an adequate respirability for the rat (MMAD ~ 1.6 µm, GSD ~ 2.3). Two satellite groups were investigated in the study: 5 additional rats per sex and concentration (control and high dose group) were allowed to recover during a 4-week post exposure period and 6 additional male rats per concentration were subjected to bronchoalveolar lavage at the end of the 4-week exposure period. Rats exposed to 4.5 and 25.8 mg/m³ did not display substance specific clinical signs while at 122 mg/m³ unspecific clinical signs and signs of respiratory distress were recorded. These signs were most pronounced during the first week and waned during the remaining exposure weeks. Conclusive changes in body temperature, reflexes, body weights, and feed/water consumption did not occur. There was no evidence of any adverse hematological effects. Likewise, clinical pathology did not reveal any relevant effect considered test-substance related. Mild-to-moderate laryngeal irritation observed at 25.8 and 122 mg/m³ was considered to be an adaptive response. The squamous metaplasia of the respiratory epithelium overlaying the ventral aspects of the larynx is a common finding in rat inhalation studies. It is reversible in nature and an indicator of the especially high susceptibility of the rodent larynx to any mild irritant (DeSesso, The relevance to humans of animal models for inhalation studies of cancer in the nose and upper airways. Quality Assurance Good Practice, Regulation, and Law 2, 213-231, 1993; Kaufmann et al., Exp. Toxicol. Pathol. 61, 591 -603, 2009). Evidence of airway irritation proximal or distal the larynx was not found. Histopathology findings at 25.8 and 122 mg/m³ were confined to the alveolar level. These findings included hypercellularity of the epithelial cells at the bronchiolo-alveolar junction and septal thickening with inflammatory infiltrates and enlarged/foamy alveolar macrophages at the alveolar level. Concurrently bronchus associated lymphoid tissue (BALT) was increased. In lung-associated-lymph nodes (LALN) increased cellularity of the paracortex, including an increased influx of macrophages with foamy appearance, was apparent at 25.8 and 122 mg/m³. Changes in lung weights (at 25.8 and 122 mg/m³; clear trend towards reversibility at the end of the 4-week post exposure period) and bronchoalveolar lavage were complementary to histopathology. At 4.5 mg/m³ alveolar macrophages containing cellular debris and, possibly, polymerized reaction products of the test article, were apparent; however, without any increase in total BAL-cell counts. All other adverse findings seen in the organs/tissues evaluated in this study were equally distributed between controls and substance-exposed groups and/or are known as spontaneous findings from previous studies. Collectively, histopathology did not reveal any adverse effects at 4.5 mg/m³. Overall, as long as adverse changes occurred they were confined to portal-of-entry related local irritant effects of larynx and alveoli. Taking all findings into account, 4.5 mg/m³ (non-volatile fraction of the test article) constitutes the NOAEL for respiratory tract irritation. In regard to extrapulmonary toxicity, no effects were found up to the maximum concentration examined. This result is consistent with the outcome of a 5x6 hours pilot study (Pauluhn, 2011), where the NOAEL was 5.5 mg/m³ (concentrations 0, 5.5, 23.4 and 126 mg/m³) due to evidence of respiratory tract irritation at 126 mg/m³ and borderline increased lung weights at 23.4 mg/m³.

Since no subchronic inhalation study is available for IPDI oligomers, allophanate type a corresponding study with IPDI oligomers, isocyanurate type was used as a key study via read-across. In this 90-day inhalation study, 10 male and 10 female Wistar rats per test group were head-nose exposed to respirable dust of IPDI oligomers, isocyanurate type on 6 hours per day and 5 consecutive days per week for 13 weeks (65 exposures) (Ma-Hock, 2009). 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. Summarizing the observed adverse findings, 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 3 mg/m³ (analytical conc. 2.9 mg/m³) is the NOAEC for the test conditions applied.

 

Read across from other aliphatic isocyanates, e.g. the structurally very close IPDI oligomers, isocyanurate type, shows that the mode of action described above is the common mode of action in this group. Repeated exposure lead to local irritant effects at the site of first contact, the port-of-entry. No systemic toxicity is observed for any of the substances. Scientific evidence exist that the NOAECs of aerosolised polyisocyanates are independent from the duration of exposure. Even the NOAECs from acute irritation potency studies, when conducted according to Technical Rule for Hazardous Substances 430 [1] do not essentially differ from NOAECs of long(er)-term repeated inhalation studies [2]. On this basis it is not expected that a longer duration of the study would change the response threshold.

However, based on read-across from a subchronic (90-day) study of the structurally similar IPDI oligomers, isocyanurate type (Ma-Hock, 2009), the NOAEC of 2.9 mg/m³ was used as point of departure for the delineation of DNELs. For the justification of read-across to IPDI oligomers, allophanate type see the document attached to this endpoint summary.


[1] Technical Rule for Hazardous Substances (TRGS) 430, published by the German Federal Ministry of Labour and Social Affairs, last update 2009; exposure 1x6 h, NOAECs based on bronchoalveolar lavage fluid analysis

[2] Pauluhn, J., Journal of Applied Toxicology, 24, 231-247, 2004



Justification for classification or non-classification

According to Regulation (EC) No 1272/2008, Annex I, no classification is warranted for repeated dose toxicity.

This is substantiated for the substance and moreover for other monomeric and homopolymeric isocyanates by the outcome of repeated dose studies, where solely changes resulting from unspecific local effects are revealed. The severity of the effects depends on the concentration and not on exposure duration. This can be shown for IPDI oligomers, allophanate type, by the comparison of two repeated inhalation studies with 5 x 6 and 20 x 6 hours of exposure. The outcome in these studies does not essentially differ (NOAECs 5.5 mg/m³ vs. 4.5 mg/m³, respectively), therefore a non-cumulative mechanism is indicated and no classification on STOT repeated is warranted.