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Repeated dose toxicity: inhalation

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Administrative data

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.
Cross-reference
Reason / purpose:
reference to other study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1987
Report Date:
1987
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
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

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Stability under test conditions: Stability in the vehicle used confirmed

Test animals

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

Administration / exposure

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
Doses / concentrationsopen allclose all
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.

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

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)

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

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).

Applicant's summary and conclusion

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.