Registration Dossier

Toxicological information

Repeated dose toxicity: inhalation

Currently viewing:

Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
migrated information: read-across based on grouping of substances (category approach)
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 IUCLID pick-list as this should be the maximum score for read-across.

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 (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent

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: about 8 weeks old
- Weight at study initiation: about 125 g (females) and 150 g (males)
- Housing: in groups of five in conventional Makrolon Type III cages (according to Spiegel & Gönnert, Zschr. Versuchstierkunde 1, 38, 1961; 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; 10%
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: During the exposure, the animals were exposed to the test substance aerosol in Plexiglas exposure tubes. The size of the exposure tubes was adjusted to the size of the rat. The exposure tubes were designed in such a manner that the tail of the rat was outside the exposure tube.
- Inhalation chamber: stainless steel, diameter 30 cm, height 28 cm, volume approx. 20 L.
- Method of conditioning air: Usage of two Boge compressors, connected in parallel. Air was conditioned with a VIA compressed air dryer for removal of i. e. water, dust and oil.
- Treatment of exhaust air: 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.
- Method of particle size determination: Aerodynamic Particle Sizer with Laser Velocimeter and for the 25 mg/m³ group a Berner cascade impactor.
The test substance/acetone solution was nebulized as a readily inhalable aerosol using a binary nozzle (Rhema) and a Braun infusion pump with ground glass syringe under dynamic conditions in a 3 L three-necked flask. A preseparator was used to separate larger particles.
All concentrations were generated using an aerosol generator and subsequent secondary dilutions.

TEST ATMOSPHERE
- Brief description of analytical method used: Test substance was coupled to N-4-nitrobenzyl-N-n-propylamin, thereafter the coupling product was determined per HPLC. For 25 mg/m³ group also a gravimetric analysis of the test substance concentration in the exposure atmosphere was performed.
- Samples taken from breathing zone: yes

VEHICLE (if applicable)
- Justification for use and choice of vehicle: Acetone makes it possible to generate an aerosol of very fine particles. Acetone itself exhibits a very low toxicity (Maximum workplace concentration 2400 mg/m³, MAK List, 1983). Accumulated data on the toxic and physical/chemical properties of this solvent are in house available.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
analysis by HPLC after coupling of the test substance to a chromophor
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
Control animals:
yes, concurrent vehicle
yes, sham-exposed
Details on study design:
- Dose selection rationale:
The concentrations tested are based on the following range-finding tests (head/nose exposure):
1. Acute inhalation toxicity study according OECD TG 403 (Bayer AG, 1984, report no. 12785)
2. Subacute inhalation toxicity study according OECD TG 412 (Bayer AG, 1985, report no. 13504)

- Vehicle control exposed to 100 mg/m³ aerosolized acetone

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
The appearance and behavior were evaluated several times on the exposure days, but not during exposure (tube exposure).

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights determined prior to first exposure and once a week thereafter.

OPHTHALMOSCOPIC EXAMINATION: Not performed

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were obtained at the time of necropsy. No preliminary and interim examinations performed, since the concentration range-finding tests showed no indications of specific biochemical changes.
- Anaesthetic used for blood collection: Yes (Diethylether)
- How many animals: Blood samples were obtained from 8 rats per group per sex.
- Parameters checked: Hämatocrit, Hämoglobin, Leukocyte count, Erythocyte count, MCV, MCH, MCHC, Thrombocyte count, Leukocyte differential count, 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 at the time of necropsy. No preliminary and interim examinations performed, since the concentration range-finding tests showed no indications of specific biochemical changes.
- 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, 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, keton bodies.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes

Organ weights: Adrenals, heart, kidneys, liver, lungs, ovaries, spleen, testes, thyroid.

Fixed organs: 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 (mediastinale, 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 (all dose groups and controls)
- microscopic: 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: In each case 2 rats per group per sex were sacrificed at the end of the experiment and perfused with Karnowsky fixative. This procedure permits scanning electon microscopic examinations of the respiratory tract and the olfactory region.
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 the testing a flow/whole body plethysmograph was used.
Statistics:
Comparison of air control group to other groups:rank-test of MANN and WHITNEY (1947), taking into account the modification of WALTER (1951).
Histopathological findings were evaluated using the "pairwise Fisher's test" with preferred R x C Chi-Sqare Test; method of GAD and WEIL (1982).

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 (hyperpnoea, nonspecific signs towards end of study); no substance induced mortality observed.

BODY WEIGHT AND WEIGHT GAIN: Significant effects on body weight only observed for male rats of dose group 26.4 mg/m³. All other groups without substance-related effects on body weight or body weight gain.

HAEMATOLOGY: Rats of the 26.4 mg/m³ group had an increase in leukocytes.

CLINICAL CHEMISTRY/URINALYSIS: No indications of specific damage.

ORGAN WEIGHTS: Lung weights of the 26.4 mg/m³ group absolute and relative significant increased.

PATHOLOGY/HISTOPATHOLOGY: In the 26.4 mg/m³ group indications of inflammatory changes in the lower respiratory tract (increase in macrophages, focal interstitial fibrosis, connective tissue proliferation). No indications for organ damage except for the respiratory organs.

LUNG FUNCTION MEASUREMENTS: Slight reduction in the vital capacity, increase in the functional residual capacity and residual volume in rats of the 26.4 mg/m³ group.

Effect levels

Dose descriptor:
NOAEL
Effect level:
3.3 mg/m³ air (analytical)
Sex:
male/female
Basis for effect level:
other: increased lung weights, inflammatory changes in the lower respiratory tract and effects on lung function 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 dose toxicity a read across to a substance with a similar composition (HDI oligomerisation product, isocyanurate type, EC No. 931 -274 -8, CAS No 28182 -81 -2) is applied. The read across is based on physicochemical and toxicological similarity of the two substances. Especially a recent comparative pulmonary irritant potency study according to TRGS 430 (Technical Rule for Hazardous Substances 430; published by the German Federal Ministry of Labour and Social Affairs, 2009) confirmed for HDI oligomerisation product, allophanate-type the same toxicological mode of action (irritant portal of entry toxicity) with a similar potency (NOAECs for pulmonary irritation: 3.4 mg/m³ for HDI oligomerisation product, allophanate-type versus 3.3 mg/m³ for HDI oligomerisation product, isocyanurate type). For further justification of the grouping and read-across according to regulation (EC) No 1907/2006, Annex XI, 1.5 see document attached to chapter "Assessment Reports".

Applicant's summary and conclusion

Executive summary:

A subchronic toxicity study according to OECD 413 was conducted with 10 male and 10 female rats per dose group. The animals were head/nose only aerosol exposed for 13 weeks (5 days a week, 6h per day) at mean analytical concentrations of 0.5, 3.3, and 26.4 mg/m³ air. The aerosol was of good respirability (MMAD approx. 1.5 µm, GSD approx. 1.4). Rats exposed to air or to the vehicle acetone served as control.
No substance induced mortality was observed. The animals tolerated the treatment virtually without symptoms, except one male of the 26.4 mg/m³ group that exhibited difficult breathing towards the end of the study. Also towards the end of the study a slightly reduced body weight gain was observed for males of the 26.4 mg/m³ group, but not for females.
Lung function tests performed at the end of the study provided indications for a chronic obstructive lung disorder in rats at 26.4 mg/m³.

The absolute and relative lung weights were significantly increased in male and female rats at 26.4 mg/m³. Histopathological examinations in this dose group revealed inflammatory changes in the lower respiratory tract (focal fibrosis, connective tissue proliferation, increase in macrophages). Hematological investigations revealed an increase in leukocytes in these rats. Other hematological, clinical chemistry and urine analysis parameters remained unchanged.

All tests and examinations revealed changes only for rats exposed to 26.4 mg/m³ air. The location of the damage was essentially limited to the lung periphery. All changes were nonspecific and were thus attributed to the primary irritant potential of the substance. There was no evidence of damage to any organs except for the respiratory organs.
The NOAEL was thus 3.3 mg/m³ air.