Registration Dossier

Administrative data

Description of key information

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Klimisch reliability of study is 1 (GLP guideline study); according to ECHA Practical Guide 6 rel. 2 is selected from the pick-list as this should be the maximum score for read-across.
Reason / purpose:
reference to other study
Reference:
Composition 0
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Version / remarks:
(1983)
Deviations:
yes
Remarks:
, no ophtalmological examination
GLP compliance:
yes
Test material information:
Composition 1
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Bor: WISW (SPF-Cpb)
- Source: Winkelmann, Borchen, Germany
- Age at study initiation: approx. 8 weeks
- Weight at study initiation: corresponding to age about 125 g (females) and 150 g (males)
- Housing: in groups of 5 in conventional Makrolon Type III cages (based on A. Spiegel and R. Goennert, Zschr. Versuchstierkunde, 1, 38, 1961 and G. Meister, Zschr. Versuchstierkunde, 7, 144-153, 1965)
- Diet and water: ad libitum
- Acclimation period: at least 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): approx. 50
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose/head only
Vehicle:
other: acetone
Remarks on MMAD:
MMAD / GSD: MMAD approx. 1.5 µm, GSD approx. 1.4.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Method of holding animals in test chamber: Animals were head/nose-only exposed to the aerosolised test article in restrainers made of plexiglas. Restrainer tubes were chosen that accommodated the animal's size. Contamination of the haircoat could largely be avoided by this exposure design.
- Generation of atmospheres: Atmospheres were generated under dynamic conditions using a Braun infusion pump and a binary nozzle (Rhema Labortechnik, Germany).
- Generation of aerosol: The test substance was nebulized using conditioned (dry, oil-free) compressed air (p=600 kPa). 10 % Acetone was added to decrease the viscosity of the test substance. In order to increase the efficiency of the generation of respirable particles and to prevent larger particles from entering the chamber a preseparator/ baffle system was used (Tillery et. al., Environmental Health Perspectives, 16, 1976, 25). The stainless steel inhalation chamber had the following dimensions: diameter = 30 cm, height = 28 cm (volume about 20 L). Details of this modular chamber and its validation have been published previously (Pauluhn, J. of applied Toxicology, 13, 1994, 55).
- Conditioning of compressed air: Compressed air was supplied by Boge compressors and was conditioned (i.e. freed from water, dust, and oil) automatically by a VIA compressed air dryer. Adequate control devices were employed to control supply pressure.
- Inhalation chamber steady-state concentration: The test atmosphere generation conditions provide an adequate number of air exchanges per hour (ca. 39 x, continuous generation of test atmospheres). Under such test conditions steady state is attained within 5 minutes (t95% = 3 x chamber volume/flow rate). 60 - 80% of the inhalation chamber supply air was exhausted via an aerosol filter (cylinder containing cotton wool). The inhalation chambers were operated under negative pressure in hoods. At each exposure port a minimal air flow rate of 0.75 L/min. was provided.
- The temperature was continuously measured by an Arucomb sensor. Representative temperature and humidity values were 25 °C and 34 %, respectively (inhalation chamber with rats).

TEST ATMOSPHERE
- The integrity and stability of the aerosol generation and exposure system was measured continuously by a Ratfisch RS 55 total carbon-analysator.
- Samples taken from breathing zone: yes
- Brief description of analytical method used: HPLC and additionally gravimetric analysis of filter samples (filter: Glass-Fibre-Filter, Sartorius, Goettingen, Germany; digital balance).
HPLC-method: Nitro-reagent-treated glass fibres were exposed to the test atmosphere. The content of isocyanate was analytically detected via HPLC. Gravimetrically determined concentrations were essentially similar to concentrations determined by nitro-reagent derivatisation technique.
- Particle size distribution: The particle-size distribution was analysed using Laser Velocimeter (TSI_APS 3300) and, for the 25 mg/m³ concentration, a Berner cascade impactor. (Hauke, Gmunden, Austria). Aerosol mass < 5 µm was nearly quantitative for all three test substance concentration groups.
- MMAD (Mass median aerodynamic diameter): The respirability of the aerosol was adequate, i.e. the mass median aerodynamic diameter (MMAD) was 1.45 µm at 1.1 mg/m³, 1.36 µm at 4.2 mg/m³, and 1.96 µm at 11.2 mg/m³/ GSD (Geometric st. dev.): 1.32-1.57.

VEHICLE (if applicable)
Acetone; The test substance was nebulised with 10 % acetone (g/v). A vehicle control group was exposed to 100 mg/m³ aerosolised acetone.
- Justification for use and choice of vehicle: Acetone has a very low toxicity (Maximum workplace concentration 2400 mg/m³, MAK List, 1983). Additionally in house-data on the toxicological properties of this solvent are available.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 h/day, 5 days/week
Remarks:
Doses / Concentrations:
0.4, 3.0 and 25 mg/m³
Basis:
other: target conc.
Remarks:
Doses / Concentrations:
0.5, 3.3 and 26.4 mg/m³
Basis:
analytical conc.
No. of animals per sex per dose:
10 test animals/10 controls
Control animals:
yes, concurrent vehicle
yes, sham-exposed
Details on study design:
- Dose selection rationale:
The concentrations tested are based on the following studies (head/nose exposure): Acute Inhalation Toxicity study (OECD TG 403; report no. 12785, Bayer AG, 1984), Subacute Inhalation Toxicity study (OECD TG 412; report no. 13504, Bayer AG, 1985).
The following doses were chosen in this study: 0.5 mg/m³ as the expected NOAEL, 25 mg/m³ as the dose causing clear signs of toxicity, 3.5 mg/m³ as the approximate geometric mean between the other two doses.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
The appearance and behaviour of the animals was evaluated several times on the exposure days, but not during exposure (tube exposure).

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of the animals were determined prior to 1st exposure and once weekly thereafter.

OPHTHALMOSCOPIC EXAMINATION: Not performed

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were obtained prior to necropsy. No preliminary and interim examinations were performed, since no indications of specific biochemical changes were seen in the range-finding study.
- How many animals: Blood samples were obtained from 8 rats per group and sex.
- Parameters checked: Haematocrit, Haemoglobin, Leukocyte count, Erythocyte count, MCV, MCH, MCHC, Thrombocyte count, Leukocyte differential count, and Thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood for glucose determination obtained at week 12. All other blood samples were obtained prior to necropsy. No preliminary and interim examinations performed, since no indications of specific biochemical changes were seen in the range-finding study.
- How many animals: Blood samples were obtained from 8 rats per group per sex.
- Parameters checked: Alanine aminotransferase, Alkaline phosphatase, total protein, Triglycerides, Cholesterol, Bilirubin, Urea, Creatinine, Lactat dehydrogenase, Glutamate dehydrogenase, Cholinesterases, Blood glucose, Serum protein electrophoresis, Triglycerides in the liver tissue, and determination of inorganic ions.

URINALYSIS: Yes
- Time schedule for collection of urine: individually collected overnight during the next to last week of the experiment.
- Metabolism cages used for collection of urine: Yes
- Parameters checked: Blood, protein, glucose, pH, and keton bodies.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals were killed and subjected to a gross pathological examination at the end of the treatment period. 2 rats per group and sex were perfused with Karnowsky fixative at the end of the study. This procedure allows investigations by transmission electron or scanning electron microscopic of the respiratory tract and the olfactory region.
Organ weights of the following organs were determined: Adrenals, heart, kidneys, liver, lungs, ovaries, spleen, testes, thyroid.
The following organs were fixed: Adrenals, aorta, brain, esophagus, eyes, gastrointestinal tract (stomach, duodenum, jejunum, colon), head (nasopharynx, oropharynx, sinus nasales and paranasales), heart, kidneys with pelvis, larynx, liver, lungs (with main stem bronchi), lymph nodes (mediastinal, hilar), muscle (quadriceps, femoralis), ovaries, parathyroids, pituitary, salivary glands (head) with mandibular lymph nodes, skin (rhinarium/nose region), spleen, testes, thymus, thyroid, trachea.

HISTOPATHOLOGY: Yes
The following organs were microscopically examined: Liver, kidneys, adrenals, heart, lungs, thyroid, parathyroids, spleen, testes/ovaries, head (nasopharynx), Larynx, Trachea, esophagus, forestomach and glandula stomach, duodenum, jejunum, colon, hilar lymph nodes, eyes, salivary glands, cervical lymph nodes, aorta, skeletal muscle, thymus, cerebrum, cerebellum, nose, pituitary.
Other examinations:
At the end of the 13-week exposure period, an examination of lung function (forced expiratory measurements, functional residual capacity) using an acetylcholine provocation test on two rats per sex per group was performed. For this examination a flow/whole body plethysmograph was used. The measurement and the interpretation was based on Diamond & O'Donnell, J. Appl. Physiol.: Respirat. Environ. Exercise Physiol. 43, 1977, 942-948; Drazen, Environmental Health Perspectives 56, 1984, 3-9; O'Neil & Raub, Environmental Health Perspectives 36, 1984, 11-22; Nunn, Applied Respiratory Physiology (2nd Edition), Butterworths, London-Boston, 1981; Likens & Mauderly, Respiratory measurements in small laboratory animals: a literature review, Lovelace Biomedical & Environmental Research Institute, 1979; Gross, Journal of Applied Toxicology 1, 1981, 116-123; Palecek, J. Applied Physiology, 27, 1969, 149-156.
Statistics:
Comparison of control groups to other groups: rank-test of Mann & Whitney, Ann. Math. Statist. 18, 1947, 50-60, taking into account the modification of Walter, Mitteilungsblatt Math. Statist. 3, 31-44, 1951, 73-92.
Histopathological findings were evaluated using the "pairwise Fisher's test" with preferred R x C Chi-Sqare Test; method of Gad & Weil, Statistics for Toxicologists, Principles and Methods of Toxicology, ed. A.W. Hayes, Raven Press, New York, 1982, 280.
Details on results:
CLINICAL SIGNS AND MORTALITY: All animals tolerated the treatment without clinical signs, except one male of dose group 26.4 mg/m³ that exhibited hyperpnoea, piloerection and ungroomed fur towards the end of study. No substance induced mortality was observed.

BODY WEIGHT AND WEIGHT GAIN: A test substance-related effect on body weight was observed only for male rats of dose group 26.4 mg/m³.

HAEMATOLOGY: Rats of 26.4 mg/m³ group showed increased leukocyte counts.

CLINICAL CHEMISTRY: No indications for specific toxicological effects.

URINALYSIS: No treatment-related findings.

LUNG FUNCTION MEASUREMENTS: acetyl choline challenge revealed no dose-related effects. No indication of a toxicological significant bronchial hyperreactivity was seen. Animals of the 26.4 mg/m³ group showed a slight reduction of vital capacity, an increase in the functional residual capacity and the residual volume. The total lung capacity was not toxicologically changed. No effects on lung function were detected for animals at or below 3.3 mg/m³.

ORGAN WEIGHTS: Lung weights of animals of the 26.4 mg/m³ group were significantly increased.

PATHOLOGY/HISTOPATHOLOGY: No indications for treatment-related effects were seen at necropsy. Histopathological examinations revealed for these animals indications of inflammatory changes of the lower respiratory tract (increase in macrophages with slight to moderate proliferation of connective tissue; focal interstitial fibrosis). No indications for organ damage except for the lower respiratory tract.
Dose descriptor:
NOAEL
Effect level:
3.3 mg/m³ air
Sex:
male/female
Basis for effect level:
other: Inflammatory changes in the lower respiratory tract at next higher dose level (26.4 mg/m³)
Critical effects observed:
not specified

For repeated inhalation toxicity a read across to HDI oligomers, isocyanurate type (EC 931 -274 -8) is applied. This substance is a close structural analogue to HDI oligomers, iminooxadiazindione type, also derived from catalytic oligomerisation of 1,6 -hexamethylene diisocyanate (HDI; CAS 822 -06 -0) and also belonging to the CAS number 28182-81-2 (Hexane, 1,6 - diisocyanato-, homopolymer).The read across is based on physicochemical and toxicological similarity. In fact, comparison of the toxicological endpoints, that are available for both of the two substances (Acute oral toxicity, Acute inhalation toxicity, Skin and Eye Irritation/Corrosion, Skin Sensitisation, Bacterial mutagenicity (Ames)) reveal good correlation. With respect to Inhalation Toxicity an expert statement is available justifying the read across (Pauluhn, Comparison of pulmonary irritation potency..., Bayer HealthCare AG, 2008).

Therefore, test results obtained for HDI oligomers, isocyanurate type can be transferred to HDI oligomers, iminooxadiazindione type and the data on repeated inhalation toxicity of HDI oligomers, isocyanurate type are also valid for HDI oligomers, iminooxadiazindione type. This approach is in accordance with Annex XI, section 1.5 of the REACH Regulation (Regulation (EC) No 1907/2006).

Executive summary:

A subchronic inhalation toxicity study (90 days) according to OECD TG 413 was conducted on 10 test animals per sex and dose group. In this study animals were head/nose exposed to the aerosolised substance (5 days/week, 6h/day) at concentrations of 0 (vehicle control), 0.5, 3.3, and 26.4 mg/m³. The aerosol was of adequate respirability for the rats (MMAD approx. 1.5 µm, GSD approx. 1.4.). Investigations also included lung function measurements after acetylcholine challenge towards the end of the study.

No substance induced mortality was observed in the course of the study. All animals tolerated the treatment virtually without symptoms, except one male of the high dose group (26.4 mg/m³) that exhibited laboured breathing. A slightly reduced body weight gain was observed for the male rats of the 26.4 mg/m³ group towards the end of the study. Body weight gain was not affected for female animals. Lung function tests provided indications of a chronic obstructive lung disorder in rats of the 26.4 mg/m³ group.
The absolute and relative lung weights were significantly increased in male and female rats of the 26.4 mg/m³ dose group. Histopathologically, inflammatory changes in the respiratory tract (focal fibrosis, proliferation of connective tissue, and increase in macrophages) were seen in the 26.4 mg/m³ dose group and haematological investigations revealed increased leukocyte counts in the same dose group. Other haematological, clinical chemistry and urine analysis parameters remained unchanged.

Conclusively, all tests and examinations provided evidence that changes were confined to the respiratory tract of rats repeatedly exposed to 26.4 mg/m³. The location of the damage was essentially limited to the lung periphery. All changes were nonspecific and are thus attributed to the primary irritant potential of the test substance. There were no indications for damages to organs except for the respiratory tract.
The NOAEL of this study was 3.3 mg/m³.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Study duration:
subchronic
Species:
rat
Quality of whole database:
No signs of systemic toxicity was observed in any of the available repeated inhalation toxicity studies.

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Klimisch reliability of study is 1 (GLP guideline study); according to ECHA Practical Guide 6 rel. 2 is selected from the pick-list as this should be the maximum score for read-across.
Reason / purpose:
reference to other study
Reference:
Composition 0
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Version / remarks:
(1983)
Deviations:
yes
Remarks:
, no ophtalmological examination
GLP compliance:
yes
Test material information:
Composition 1
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Bor: WISW (SPF-Cpb)
- Source: Winkelmann, Borchen, Germany
- Age at study initiation: approx. 8 weeks
- Weight at study initiation: corresponding to age about 125 g (females) and 150 g (males)
- Housing: in groups of 5 in conventional Makrolon Type III cages (based on A. Spiegel and R. Goennert, Zschr. Versuchstierkunde, 1, 38, 1961 and G. Meister, Zschr. Versuchstierkunde, 7, 144-153, 1965)
- Diet and water: ad libitum
- Acclimation period: at least 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): approx. 50
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose/head only
Vehicle:
other: acetone
Remarks on MMAD:
MMAD / GSD: MMAD approx. 1.5 µm, GSD approx. 1.4.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Method of holding animals in test chamber: Animals were head/nose-only exposed to the aerosolised test article in restrainers made of plexiglas. Restrainer tubes were chosen that accommodated the animal's size. Contamination of the haircoat could largely be avoided by this exposure design.
- Generation of atmospheres: Atmospheres were generated under dynamic conditions using a Braun infusion pump and a binary nozzle (Rhema Labortechnik, Germany).
- Generation of aerosol: The test substance was nebulized using conditioned (dry, oil-free) compressed air (p=600 kPa). 10 % Acetone was added to decrease the viscosity of the test substance. In order to increase the efficiency of the generation of respirable particles and to prevent larger particles from entering the chamber a preseparator/ baffle system was used (Tillery et. al., Environmental Health Perspectives, 16, 1976, 25). The stainless steel inhalation chamber had the following dimensions: diameter = 30 cm, height = 28 cm (volume about 20 L). Details of this modular chamber and its validation have been published previously (Pauluhn, J. of applied Toxicology, 13, 1994, 55).
- Conditioning of compressed air: Compressed air was supplied by Boge compressors and was conditioned (i.e. freed from water, dust, and oil) automatically by a VIA compressed air dryer. Adequate control devices were employed to control supply pressure.
- Inhalation chamber steady-state concentration: The test atmosphere generation conditions provide an adequate number of air exchanges per hour (ca. 39 x, continuous generation of test atmospheres). Under such test conditions steady state is attained within 5 minutes (t95% = 3 x chamber volume/flow rate). 60 - 80% of the inhalation chamber supply air was exhausted via an aerosol filter (cylinder containing cotton wool). The inhalation chambers were operated under negative pressure in hoods. At each exposure port a minimal air flow rate of 0.75 L/min. was provided.
- The temperature was continuously measured by an Arucomb sensor. Representative temperature and humidity values were 25 °C and 34 %, respectively (inhalation chamber with rats).

TEST ATMOSPHERE
- The integrity and stability of the aerosol generation and exposure system was measured continuously by a Ratfisch RS 55 total carbon-analysator.
- Samples taken from breathing zone: yes
- Brief description of analytical method used: HPLC and additionally gravimetric analysis of filter samples (filter: Glass-Fibre-Filter, Sartorius, Goettingen, Germany; digital balance).
HPLC-method: Nitro-reagent-treated glass fibres were exposed to the test atmosphere. The content of isocyanate was analytically detected via HPLC. Gravimetrically determined concentrations were essentially similar to concentrations determined by nitro-reagent derivatisation technique.
- Particle size distribution: The particle-size distribution was analysed using Laser Velocimeter (TSI_APS 3300) and, for the 25 mg/m³ concentration, a Berner cascade impactor. (Hauke, Gmunden, Austria). Aerosol mass < 5 µm was nearly quantitative for all three test substance concentration groups.
- MMAD (Mass median aerodynamic diameter): The respirability of the aerosol was adequate, i.e. the mass median aerodynamic diameter (MMAD) was 1.45 µm at 1.1 mg/m³, 1.36 µm at 4.2 mg/m³, and 1.96 µm at 11.2 mg/m³/ GSD (Geometric st. dev.): 1.32-1.57.

VEHICLE (if applicable)
Acetone; The test substance was nebulised with 10 % acetone (g/v). A vehicle control group was exposed to 100 mg/m³ aerosolised acetone.
- Justification for use and choice of vehicle: Acetone has a very low toxicity (Maximum workplace concentration 2400 mg/m³, MAK List, 1983). Additionally in house-data on the toxicological properties of this solvent are available.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 h/day, 5 days/week
Remarks:
Doses / Concentrations:
0.4, 3.0 and 25 mg/m³
Basis:
other: target conc.
Remarks:
Doses / Concentrations:
0.5, 3.3 and 26.4 mg/m³
Basis:
analytical conc.
No. of animals per sex per dose:
10 test animals/10 controls
Control animals:
yes, concurrent vehicle
yes, sham-exposed
Details on study design:
- Dose selection rationale:
The concentrations tested are based on the following studies (head/nose exposure): Acute Inhalation Toxicity study (OECD TG 403; report no. 12785, Bayer AG, 1984), Subacute Inhalation Toxicity study (OECD TG 412; report no. 13504, Bayer AG, 1985).
The following doses were chosen in this study: 0.5 mg/m³ as the expected NOAEL, 25 mg/m³ as the dose causing clear signs of toxicity, 3.5 mg/m³ as the approximate geometric mean between the other two doses.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
The appearance and behaviour of the animals was evaluated several times on the exposure days, but not during exposure (tube exposure).

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of the animals were determined prior to 1st exposure and once weekly thereafter.

OPHTHALMOSCOPIC EXAMINATION: Not performed

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were obtained prior to necropsy. No preliminary and interim examinations were performed, since no indications of specific biochemical changes were seen in the range-finding study.
- How many animals: Blood samples were obtained from 8 rats per group and sex.
- Parameters checked: Haematocrit, Haemoglobin, Leukocyte count, Erythocyte count, MCV, MCH, MCHC, Thrombocyte count, Leukocyte differential count, and Thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood for glucose determination obtained at week 12. All other blood samples were obtained prior to necropsy. No preliminary and interim examinations performed, since no indications of specific biochemical changes were seen in the range-finding study.
- How many animals: Blood samples were obtained from 8 rats per group per sex.
- Parameters checked: Alanine aminotransferase, Alkaline phosphatase, total protein, Triglycerides, Cholesterol, Bilirubin, Urea, Creatinine, Lactat dehydrogenase, Glutamate dehydrogenase, Cholinesterases, Blood glucose, Serum protein electrophoresis, Triglycerides in the liver tissue, and determination of inorganic ions.

URINALYSIS: Yes
- Time schedule for collection of urine: individually collected overnight during the next to last week of the experiment.
- Metabolism cages used for collection of urine: Yes
- Parameters checked: Blood, protein, glucose, pH, and keton bodies.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals were killed and subjected to a gross pathological examination at the end of the treatment period. 2 rats per group and sex were perfused with Karnowsky fixative at the end of the study. This procedure allows investigations by transmission electron or scanning electron microscopic of the respiratory tract and the olfactory region.
Organ weights of the following organs were determined: Adrenals, heart, kidneys, liver, lungs, ovaries, spleen, testes, thyroid.
The following organs were fixed: Adrenals, aorta, brain, esophagus, eyes, gastrointestinal tract (stomach, duodenum, jejunum, colon), head (nasopharynx, oropharynx, sinus nasales and paranasales), heart, kidneys with pelvis, larynx, liver, lungs (with main stem bronchi), lymph nodes (mediastinal, hilar), muscle (quadriceps, femoralis), ovaries, parathyroids, pituitary, salivary glands (head) with mandibular lymph nodes, skin (rhinarium/nose region), spleen, testes, thymus, thyroid, trachea.

HISTOPATHOLOGY: Yes
The following organs were microscopically examined: Liver, kidneys, adrenals, heart, lungs, thyroid, parathyroids, spleen, testes/ovaries, head (nasopharynx), Larynx, Trachea, esophagus, forestomach and glandula stomach, duodenum, jejunum, colon, hilar lymph nodes, eyes, salivary glands, cervical lymph nodes, aorta, skeletal muscle, thymus, cerebrum, cerebellum, nose, pituitary.
Other examinations:
At the end of the 13-week exposure period, an examination of lung function (forced expiratory measurements, functional residual capacity) using an acetylcholine provocation test on two rats per sex per group was performed. For this examination a flow/whole body plethysmograph was used. The measurement and the interpretation was based on Diamond & O'Donnell, J. Appl. Physiol.: Respirat. Environ. Exercise Physiol. 43, 1977, 942-948; Drazen, Environmental Health Perspectives 56, 1984, 3-9; O'Neil & Raub, Environmental Health Perspectives 36, 1984, 11-22; Nunn, Applied Respiratory Physiology (2nd Edition), Butterworths, London-Boston, 1981; Likens & Mauderly, Respiratory measurements in small laboratory animals: a literature review, Lovelace Biomedical & Environmental Research Institute, 1979; Gross, Journal of Applied Toxicology 1, 1981, 116-123; Palecek, J. Applied Physiology, 27, 1969, 149-156.
Statistics:
Comparison of control groups to other groups: rank-test of Mann & Whitney, Ann. Math. Statist. 18, 1947, 50-60, taking into account the modification of Walter, Mitteilungsblatt Math. Statist. 3, 31-44, 1951, 73-92.
Histopathological findings were evaluated using the "pairwise Fisher's test" with preferred R x C Chi-Sqare Test; method of Gad & Weil, Statistics for Toxicologists, Principles and Methods of Toxicology, ed. A.W. Hayes, Raven Press, New York, 1982, 280.
Details on results:
CLINICAL SIGNS AND MORTALITY: All animals tolerated the treatment without clinical signs, except one male of dose group 26.4 mg/m³ that exhibited hyperpnoea, piloerection and ungroomed fur towards the end of study. No substance induced mortality was observed.

BODY WEIGHT AND WEIGHT GAIN: A test substance-related effect on body weight was observed only for male rats of dose group 26.4 mg/m³.

HAEMATOLOGY: Rats of 26.4 mg/m³ group showed increased leukocyte counts.

CLINICAL CHEMISTRY: No indications for specific toxicological effects.

URINALYSIS: No treatment-related findings.

LUNG FUNCTION MEASUREMENTS: acetyl choline challenge revealed no dose-related effects. No indication of a toxicological significant bronchial hyperreactivity was seen. Animals of the 26.4 mg/m³ group showed a slight reduction of vital capacity, an increase in the functional residual capacity and the residual volume. The total lung capacity was not toxicologically changed. No effects on lung function were detected for animals at or below 3.3 mg/m³.

ORGAN WEIGHTS: Lung weights of animals of the 26.4 mg/m³ group were significantly increased.

PATHOLOGY/HISTOPATHOLOGY: No indications for treatment-related effects were seen at necropsy. Histopathological examinations revealed for these animals indications of inflammatory changes of the lower respiratory tract (increase in macrophages with slight to moderate proliferation of connective tissue; focal interstitial fibrosis). No indications for organ damage except for the lower respiratory tract.
Dose descriptor:
NOAEL
Effect level:
3.3 mg/m³ air
Sex:
male/female
Basis for effect level:
other: Inflammatory changes in the lower respiratory tract at next higher dose level (26.4 mg/m³)
Critical effects observed:
not specified

For repeated inhalation toxicity a read across to HDI oligomers, isocyanurate type (EC 931 -274 -8) is applied. This substance is a close structural analogue to HDI oligomers, iminooxadiazindione type, also derived from catalytic oligomerisation of 1,6 -hexamethylene diisocyanate (HDI; CAS 822 -06 -0) and also belonging to the CAS number 28182-81-2 (Hexane, 1,6 - diisocyanato-, homopolymer).The read across is based on physicochemical and toxicological similarity. In fact, comparison of the toxicological endpoints, that are available for both of the two substances (Acute oral toxicity, Acute inhalation toxicity, Skin and Eye Irritation/Corrosion, Skin Sensitisation, Bacterial mutagenicity (Ames)) reveal good correlation. With respect to Inhalation Toxicity an expert statement is available justifying the read across (Pauluhn, Comparison of pulmonary irritation potency..., Bayer HealthCare AG, 2008).

Therefore, test results obtained for HDI oligomers, isocyanurate type can be transferred to HDI oligomers, iminooxadiazindione type and the data on repeated inhalation toxicity of HDI oligomers, isocyanurate type are also valid for HDI oligomers, iminooxadiazindione type. This approach is in accordance with Annex XI, section 1.5 of the REACH Regulation (Regulation (EC) No 1907/2006).

Executive summary:

A subchronic inhalation toxicity study (90 days) according to OECD TG 413 was conducted on 10 test animals per sex and dose group. In this study animals were head/nose exposed to the aerosolised substance (5 days/week, 6h/day) at concentrations of 0 (vehicle control), 0.5, 3.3, and 26.4 mg/m³. The aerosol was of adequate respirability for the rats (MMAD approx. 1.5 µm, GSD approx. 1.4.). Investigations also included lung function measurements after acetylcholine challenge towards the end of the study.

No substance induced mortality was observed in the course of the study. All animals tolerated the treatment virtually without symptoms, except one male of the high dose group (26.4 mg/m³) that exhibited laboured breathing. A slightly reduced body weight gain was observed for the male rats of the 26.4 mg/m³ group towards the end of the study. Body weight gain was not affected for female animals. Lung function tests provided indications of a chronic obstructive lung disorder in rats of the 26.4 mg/m³ group.
The absolute and relative lung weights were significantly increased in male and female rats of the 26.4 mg/m³ dose group. Histopathologically, inflammatory changes in the respiratory tract (focal fibrosis, proliferation of connective tissue, and increase in macrophages) were seen in the 26.4 mg/m³ dose group and haematological investigations revealed increased leukocyte counts in the same dose group. Other haematological, clinical chemistry and urine analysis parameters remained unchanged.

Conclusively, all tests and examinations provided evidence that changes were confined to the respiratory tract of rats repeatedly exposed to 26.4 mg/m³. The location of the damage was essentially limited to the lung periphery. All changes were nonspecific and are thus attributed to the primary irritant potential of the test substance. There were no indications for damages to organs except for the respiratory tract.
The NOAEL of this study was 3.3 mg/m³.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
3.3 mg/m³
Study duration:
subchronic
Species:
rat

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

Mode of Action Analysis / Human Relevance Framework

Additional information

For assessment of repeated dose toxicity a read across to HDI oligomers, isocyanurate type (EC 931 -274 -8) is applied. This substance is a close structural analogue to HDI oligomers, iminooxadiazindione type, also derived from catalytic oligomerisation of 1,6 -hexamethylene diisocyanate (HDI; CAS 822 -06 -0) and also belonging to the CAS number 28182-81-2 (Hexane, 1,6 - diisocyanato-, homopolymer).The read across is based on physicochemical and toxicological similarity. In fact, comparison of the toxicological endpoints, that are available for both of the two substances reveal good correlation.

 Toxicological endpoint  HDI oligomers, isocyanurate type  HDI oligomers, iminooxadiazindione type
 Acute oral toxicity  > 2000 mg/kg  > 2000 mg/kg
 Acute inhalation toxicity (pulmonary irritant study) NOAEL 3 mg/m³   NOAEL 2.1 mg/m³
 Skin Irritation/Corrosion  slight irritation/no classification required  slight irritation/no classification required
 Eye Irritation/Corrosion  very slight irritation/no classification required  very slight irritation/no classification required
 Skin Sensitisation  classification required  classification required
 Bacterial Mutagenicity (Ames)  negative  negative

With respect to Inhalation Toxicity an expert statement is available justifying the read across (Pauluhn, Comparison of pulmonary irritation potency..., Bayer HealthCare AG, 2008; attached to this endpoint summary).

Therefore, test results obtained for HDI oligomers, isocyanurate type can be transferred to HDI oligomers, iminooxadiazindione type and data on repeated dose toxicity of HDI oligomers, isocyanurate type are also valid for HDI oligomers, iminooxadiazindione type. This approach is in accordance with Annex XI, section 1.5 of the REACH Regulation (Regulation (EC) No 1907/2006).

See below discussion of repeated dose toxicity of read-across substance HDI oligomers, isocyanurate type:

"A subchronic inhalation toxicity study (13 weeks) according to OECD TG 413 was conducted on 10 test animals per sex and dose group. In this study the animals were head/nose exposed to the aerosolised substance (5 days/week, 6 h/day) at concentrations of 0 (vehicle control), 0.5, 3.3, and 26.4 mg/m³. The aerosol was of adequate respirability for the rats (MMAD approx. 1.5 µm, GSD approx. 1.4.). Investigations also included lung function measurements after acetylcholine challenge towards the end of the study.

Specified examinations revealed a slightly reduced body weight gain (only observed for male rats), slight impairment of lung function, significantly increased lung weights and, according to histopathology, inflammatory changes in the respiratory tract, all observed in the 26.4 mg/m³ dose group. Additionally, increased leukocyte counts were seen in haematological investigations at this concentration.

All changes seen in the study were nonspecific and thus attributed to the primary irritant potential of the test substance. There were no indications for damages to organs except the respiratory tract.

Overall, the study provided evidence that repeated inhalation toxicity of rats exposed to the substance aerosol at 26.4 mg/m³ causes changes confined to the respiratory tract.
The NOAEL of this study was 3.3 mg/m³.

A second repeated inhalation studyaccording to OECD TG 412 (3 weeks) confirmed this result qualitatively and quantitatively. The NOAEL here was 4.3 mg/m³. As additional observation only a hyperaemia with detritus of the nose mucosa was reported, also related to the irritant properties of the substance-aerosol.

Further available is a more recent repeated inhalation study similar to OECD TG 412 (2 weeks). Based on prior knowledge of the substance's toxicity, some guideline investigations were omitted in this study (i.e. haematology, clinical chemistry, and histopathology of organs other than the respiratory tract) in favour of additional investigations on the asthmatogenic potential of the substance (lung lavage endpoints, blood gas analysis, lung function tests, and in depth lung histopathology). As result transient findings (clinical signs like laboured breathing and reduced condition, reduced body weight, increased lung weights, increase of macrophages and LDH in lung lavage), mostly seen by trend, were reported for animals of the high dose group only (69.2 mg/m³). A transient marginally impaired lung function reported for the same dose level was seen as causally related to the changes in lung weights. No evidence was seen for an asthmatogenic property of the substance, as no infiltration of granulocytes, especially eosinophilic granulocytes, and no indications for a bronchoalveolitis or an airway hyperreactivity was found.

These findings give further proof for the irritant port of entry toxicity of the substance. Moreover, the quantitative result corresponds well to the results of the other studies, as the NOAEL here was 16.3 mg/m³ air and the NOEL 4.6 mg/m³."


Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
For the read-across substance HDI oligomers, isocyanurate type only one subchronic (13 weeks) study available.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
For the read-across substance HDI oligomers, isocyanurate type only one subchronic (13 weeks) study available.

Justification for classification or non-classification

According to Regulation (EC) No 1272/2008, Annex I, no classification is warranted for repeated dose toxicity. Classification as STOT repeated is not justified, due to lack of cumulative toxicity. As it could be shown, no systemic effects were observed in the repeated dose studies. The local effects observed did not give evidence for a major time-dependent change of the response threshold, as the NOAECs were 3.3 mg/m³ for the subchronic key study (concentrations tested 0, 0.5, 3.3, 26.4 mg/m³) and 4.3 mg/m³ for a subacute (3 weeks) study (concentrations tested 0, 4.3, 14.7, 89.8 mg/m³). Moreover, there is scientific evidence that for non-volatile polyisocyanates that act predominantly as pulmonary irritants even the NOAECs from acute pulmonary irritant potency studies, where bronchoalveolar lavage endpoints as markers for pulmonary irritation were investigated (recommended in the Technical Rule for Hazardous Subszances 430, published by the German Federal Ministry of Labour and Social Affairs), does not essentially differ from NOAECs of long(er)-term repeated inhalation studies (Pauluhn, Journal of Applied Toxicology, 24, 231-247, 2004). In fact, the NOAEC of the pulmonary irritant potency study revealed an almost identical NOAEC of 3 mg/m³.