Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions: no analysis on stability of test substance in the vehicle reported, instead of at least 2000 immature erythrocytes per animal 1000 were scored for the incidence of micronuclei

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1986
Report Date:
1986

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
References in report: Matter & Schmid, Mutation Res., 1971, 417-425 and on the modification of Heddle, Salomone and others, e.g. Salamone et.al. in Proceedings of the International Program for the evaluation of short-term tests for carcinogenicity (Ashby and Serres, Eds.), Elsevier, Amsterdam, 1980, 686-697.
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (U.K.) Limited, Manston, Kent, UK
- Age at study initiation: 6-8 weeks
- Weight at study initiation: males 27-35 g, females 21-28 g
- Housing: individual in polypropylene and stainless steel cages; non-SPF conditions
- Diet and water: ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 (18-23)
- Humidity (%): 37 (31-46)
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: unspecified
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Immediately prior to dosing, the test compound formulation was prepared. The dose volume used for both vehicle control and test compound treated animals was 10mL/kg bw.

DETAILS ON STUDY DESIGN:
Dose-finding: Prior to micronucleus test a dose finding (1 animal/sex and dose group) and a main toxicity test (5 animals/sex at 5 g/kg) was undertaken. As no mortalities of clinical signs were observed, especially in the 9-day observation period of the main toxicity test, the standard maximum dose of 5 g/kg was selected for the micronucleus study.
In the micronucleus test, 3 groups of male and female mice were dosed once orally with test or control agents, then marrow samples were taken at the following time intervals: 24, 48, and 72 hours (each 5 animals/sex)
Duration of treatment / exposure:
Marrow samples were taken at 24, 48, and 72 hours.
Frequency of treatment:
once
Post exposure period:
24, 48, or 72 hours; 9 days for the previously conducted toxicity test
Doses / concentrations
Remarks:
Doses / Concentrations:
5000 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
15 test animals/15 controls (at each sampling time 24, 48 and 72 hours 5 animals/sex were used)
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide; it was prepared fresh as a 8 mg/mL solution in water and administered to the positive control animals in standard dose volumes of 10 mL/kg.

Examinations

Tissues and cell types examined:
Bone marrow of the femora was prepared 24, 48, and 72 hours after administration. After staining of the preparations, 1000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes occurring per 1000 polychromatic erythrocytes were also recorded.
Details of tissue and slide preparation:
The mice were killed by cervical dislocation. The femora were quickly dissected out and freed of adherent tissue. A small hole was made in the neck of one femur and the marrow flushed, using a 1 ml syringe fitted with a gauge 25 needle, into a heparinised centrifuge tube containing 3 ml of a 1:1 mixture of foetal calf serum and 0.8 % tri-sodium citrate in Sorensen's buffer, pH 6.8. (This mildly hypotonic treatment served to make the micronuclei clearly visible and to distinguish them from surrounding artefacts.) The contents of the tubes were briefly agitated on a vortex mixer to allow separation of the cells.
The centrifuge tubes had previously been given computer generated random numbers unrelated to the original animal numbers, i.e. from this stage onwards the operators were unaware of the dose group from which the samples were taken.
The contents of the tube were centrifuged for 5 min at 1000 r.p.m. and the supernatant fluid discarded, leaving a few drops in the tube. The cells were then resuspended on a vortex mixer in this remaining amount of serum.
Clean slides were assigned numbers corresponding to the tube numbers. A drop of the suspension was placed at one end of the slide and a smear made by drawing the tip of a Pasteur pipette horizontally along the slide. Two slides were prepared from each tube and animal. The smear was left to air dry, fixed in methanol for 15 min and stained 3-5 days later as follows: 1 % May Grunwald in methanol - 5 min., 10 % Giemsa in distilled water - 10 min.
The stained smears were rinsed in 3 changes of distilled water for approximately 1 min. Slides were then briefly dipped in methanol, air dried and cleared in xylene. Permanent slide preparations were obtained by sealing glass coverslips onto the microscopic slides.
The better of the 2 prepared slides was selected for examination and the coded slides read "blind" in a random manner by the same operator. One thousand (1000) polychromatic erythrocytes (PCE) per animal were scored for micronuclei. As a control against inclusion of artefacts, or action of a mutagen on the G2 and/or mitotic phase of the cell cycle, the number of micronucleated normochromatic erythrocyte" (NCE) was also recorded. The PCE/NCE ratio was determined for each animal by counting the number of immature (PCE) per mature (NCE) erythrocytes in a minimum total of 333 erythroblasts (PCE + NCE).
Evaluation criteria:
According to Bruce & Heddle, Can. J. Genet: Cytol., 21, 1979, 319-334; Salamone et.al., in Ashby and de Serres (Eds.), Proceedings of the International Program for the Evaluation of Short-Term Tests for Carcinogenicity, Elsevier, Amsterdam, 1980, 686-697.)
Statistics:
no data

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The numbers of micronuclei in animals dosed with the vehicle were within the historical control range throughout the study.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): There was no increase in the frequency of micronuclei.
- Ratio of PCE/NCE (for Micronucleus assay): There was also no appreciable suppression of marrow proliferation as indicated by shifts in the PCE/NCE ratios. The slightly reduced number of PCE recorded in the 48 h samples was taken to indicate that the test compound had reached the target organ.

Any other information on results incl. tables

For genetic toxicity/chromosome aberration in vivo 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 results of chromosome aberration in vivo 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).

For the study with HDI isocyanurate the only clinical sign was some subdued animals and one mouse with a severed tail, but otherwise no abnormalities were apparent.

Applicant's summary and conclusion

Executive summary:

The substance was tested in a Mammalian Bone Marrow Micronucleus Test similar to OECD TG 474. Based on dose finding and prior toxicity study (observation period 9 days) 5 mice/sex were treated orally with a single limit dose of 5 g/kg of the formulated substance (vehicle: corn oil; no stability testing in the vehicle was reported). Bone marrow of the femora was sampled after 24, 48 and 72 hours. After staining of the preparations, 1000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes occurring per 1000 polychromatic erythrocytes were also recorded.

No increase in the frequencies of micronuclei were observed after treatment, therefore it was concluded that the test substance was devoid of mutagenic activity.

No substance-related toxic symptoms were observed in the study or in the previously conducted toxicity study. There was also no appreciable suppression of marrow proliferation as indicated by shifts in the PCE/NCE ratios. The slightly reduced number of PCE recorded in the 48 h samples was taken to indicate that the test compound had reached the target organ. Under the experimental conditions chosen here, no indications were found for a genotoxic potential in vivo.