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: 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

Reference
Reference Type:
study report
Title:
Unnamed
Report Date:
2006

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
(1997)
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
The stability of the test substance at room temperature in the vehicle corn oil over a period of 4 hours was verified analytically.

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Germany GmbH
- Age at study initiation: 5 – 8 weeks
- Weight at study initiation: About 30 g
- Assigned to test groups randomly: The animals were assigned to the test groups according to a randomization plan prepared with an appropriate computer program.
- Housing: individual housing in Makrolon cages, type MI. Fully air-conditioned rooms which central air conditioning
- Diet: Standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water: Drinking water from bottles were available ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- 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
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, corn oil was selected as the vehicle, which had been demonstrated to be suitable in the in vivo micronucleus test and for which historical data are available.
- Concentration of test material in vehicle: 5 g/100 mL, 10 g/100 mL, 20 g/100 mL.
Details on exposure:
- Preparation of dosing solutions: To achieve a solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly. All test substance formulations were prepared immediately before administration.
- Test substance concentrations: The low dose group was given 500 mg test substance/kg body weight or 10 mL/kg body weight of a solution with a concentration of 5 g/100 mL. The intermediate dose group was given 1000 mg test substance/kg body weight or 10 mL/kg body weight of a solution with a concentration of 10 g/100 mL. The top dose group was given 2000 mg test substance/kg body weight or 10 mL/kg body weight of a solution with a concentration of 20 g/100 mL.
- Test procedure and administration: At the beginning of the study, the animals were weighed and the substance to be administered or the amount of volume was related to the specific weight of the individual animals. Male animals per test group were given test substance dissolved in corn oil at dose levels of 500 mg/kg, 1000 mg/kg and 2000 mg/kg body weight. Treatment consisted of two oral administrations. The volume administered was 10 mL/kg body weight.
- Analysis of test substance preparation: The stability of the test substance at room temperature in the vehicle corn oil was determined analytically. For the determination of the test substance concentrations in the vehicle, 6 samples of each dose were taken from the test substance preparations, kept at room temperature until the treatment of the last animal (approximately 1 hour) and then deep-frozen until they were determined analytically. The determination of the concentrations in the vehicle was carried out by means of potentiograph.
Duration of treatment / exposure:
The animals of the vehicle control and the dose groups were treated twice at a 24-hour interval and samples of bone marrow were taken 24 hours after the last treatment.
Frequency of treatment:
twice
Post exposure period:
24 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 1000, 2000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5 males/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Animals of the positive control groups were treated only once and samples of bone marrow were taken after 24 hours. The following positive controls, both dissolved in purified water were administered to male animals: cyclophosphamide (CPP; orally) or vincristine (VCR; intraperitoneally), each in a volume of 10 mL/kg body weight.

Examinations

Tissues and cell types examined:
Bone marrow of the two femora was prepared 24 hours after the second administration. After staining of the preparations, 2000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2000 polychromatic erythrocytes were also recorded.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Dose selection was based on a pretest for the determination of the acute oral toxicity.
DETAILS OF SLIDE PREPARATION:
- Preparation of the bone marrow: The bone marrow was prepared according to the method described by Schmid (The micronucleus test for cytogenetic analysis, Hollaender, A. (ed), Chemical Mutagens, Principles and Methods for their Detection, Volume 4, Plenum Press, New York, 1976; The micronucleus test, Kilbey et al. (eds), Handbook of Mutagenicity Test Procedures, Elsevier Scientific Publishing Company, Amsterdam - New York - Oxford , 1977) and Salamone (Towards an improved micronucleus test. Studies on 3 model agents, mitomycin C, cyclophosphamide and dimethylbenzanthracene, Mut. Res., 74, 347 - 356, 1980). The two femora of the animals sacrificed by cervical dislocation were prepared by dissection and removing all soft tissues. After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with fetal calf serum (FCS) which was at 37°C (about 2 mL/femur). The suspension was mixed thoroughly with a pipette, centrifuged at 300 x g for 5 minutes, the supernatant was removed and the precipitate was resuspended in about 50 μL fresh FCS. One drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette. Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained.
- Staining of the slides: The slides were stained in eosin and methylene blue (modified May-Grünwald solution or Wrights solution) for about 5 minutes. After having briefly been rinsed in purified water, the preparations were soaked in purified water for about 2 - 3 minutes. Subsequently, the slides were stained in Giemsa solution (15 mL Giemsa, 185 mL purified water) for about 15 minutes. After having been rinsed twice in purified water and clarified in xylene, the preparations were mounted in Corbit-Balsam.
MICROSCOPIC EVALUATION: In general, 2000 polychromatic erythrocytes (PCEs) from each animal of every test group are evaluated and investigated for micronuclei (MN). The normochromatic erythrocytes (NCEs) which occur are also scored. The following parameters are recorded:
- Number of polychromatic erythrocytes
- Number of polychromatic erythrocytes containing micronuclei (The increase in the number of micronuclei in polychromatic erythrocytes of treated animals as compared with the solvent control group provides an index of a chromosome-breaking (clastogenic) effect or damage of the mitotic apparatus (aneugenic activity) of the substance tested.)
- Number of normochromatic erythrocytes
- Number of normochromatic erythrocytes containing micronuclei
- Ratio of polychromatic to normochromatic erythrocytes (An alteration of this ratio indicates that the test substance actually reached the target. Individual animals with pathological bone marrow depression may be identified and excluded from the evaluation.)
- Number of small micronuclei (d < D/4) and of large micronuclei (d ≥ D/4) (d = diameter of micronucleus, D = cell diameter). (The size of micronuclei may indicate the possible mode of action of the test substance, i.e. a clastogenic or a spindle poison effect.
Evaluation criteria:
- The mouse micronucleus test is considered valid if the following criteria are met: (1) The quality of the slides must allow the identification and evaluation of a sufficient number of analyzable cells, i.e. ≥ 2000 PCEs and a clear differentiation between PCEs and NCEs. (2) The ratio of PCEs/NCEs in the untreated animals (negative control) has to be within the normal range for the animal strain selected. (3) The number of cells containing micronuclei in negative control animals has to be within the range of the historical control data both for PCEs and for NCEs. (4) The two positive control substances have to induce a significant increase in the number of PCEs containing small and large micronuclei within the range of the historical control data or above.
- A finding is considered positive if the following criteria are met: (1) Significant and dose-related increase in the number of PCEs containing micronuclei. (2) The number of PCEs containing micronuclei has to exceed both the concurrent negative control and the highest value of the historical control range.
- A test substance is considered negative if the following criteria are met: The number of cells containing micronuclei in the dose groups is not significantly above the negative control and is within the historical control data.
Statistics:
- The statistical evaluation of the data was carried out using the program system MUKERN (BASF Aktiengesellschaft).
- The asymptotic U test according to MANN-WHITNEY (modified rank test according to WILCOXON) was carried out to clarify the question whether there were significant differences between the control group and dose groups with regard to the micronucleus rate in polychromatic erythrocytes. The relative frequencies of cells containing micronuclei of each animal were used as a criterion for the rank determination for the U test.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
In a pretest for the determination of the acute oral toxicity, all animals (male and female) tolerated the treatment with 2000 mg/kg body weight, twice recommended as the highest dose according to the OECD Guideline, without clinical signs. Thus, only male animals were used for the cytogenetic investigations. Therefore, a dose of 2000 mg/kg body weight was selected as the highest dose in the present cytogenetic study. 1000 mg/kg and 500 mg/kg body weight were administered as further doses.

RESULTS OF DEFINITIVE STUDY
- Microscopic evaluation: The two oral administrations of corn oil in a volume of 10 mL/kg body weight led to 1.0‰ polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval. After two administrations of the highest dose of 2000 mg/kg body weight, 1.6‰ polychromatic erythrocytes containing micronuclei were found after 24 hours. In the two lower dose groups, rates of micronuclei of about 0.5‰ (1000 mg/kg group) and 0.8‰ (500 mg/kg group) were detected. With 16.8‰ the positive control substance cyclophosphamide for clastogenicity, led to the expected clear increase in the number of polychromatic erythrocytes containing exclusively small micronuclei. With 51.9‰, the positive control substance vincristine for induction of spindle poison effects also led to a clearly enhanced number of polychromatic erythrocytes containing micronuclei with the expected amount of large micronuclei, i.e. 14.9‰. The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the negative control or in the various dose groups. A slight inhibition of erythropoiesis, induced by the treatment of mice with the test substance was detected at all doses administered.
- Clinical examinations: The two oral administrations of the vehicle in a volume of 10 mL/kg body weight were tolerated by all animals without any signs or symptoms. The administration of the test substance did not led to clinical signs of toxicity. Neither the single administration of the positive control substance, cyclophosphamide, in a dose of 20 mg/kg body weight nor that of vincristine in a dose of 0.15 mg/kg body weight caused any evident signs of toxicity.
- Analytical investigations: The stability of the test substance at room temperature in the vehicle corn oil over a period of 4 hours was verified analytically. Since a solution was obtained with the vehicle, it was not necessary to verify the homogeneity analytically. The concentration control analyses of all concentrations revealed that the values were in the expected range of the target concentrations, i.e. were always in a range of 90% - 110% of the nominal concentration.

Any other information on results incl. tables

For assessing skin irritation in vivo a read across to a substance with a very similar chemical composition (EC No. 900 -066 -9; comparable allophanate-type HDI oligomerisation product) is applied.The read across is based on physicochemical and toxicological similarity of the two substances. Especially a 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), as the key study for justification of read-across, confirmed for both substances the same toxicological mode of action and nearly identical potency. 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:

The substance was tested in a Mammalian Erythrocyte Micronucleus Test (OECD 474) for chromosomal damage (clastogenicity) and for its ability to induce spindle poison effects (aneugenic activity) in NMRI mice. For this purpose, the test substance, dissolved in corn oil, was administered twice orally, with a 24-hour interval between administrations, to groups of 5 male animals at dose levels of 500 mg/kg, 1 000 mg/kg and 2 000 mg/kg body weight in a volume of 10 mL/kg body weight in each case. The animals were sacrificed and the bone marrow of the two femora was prepared 24 hours after the second administration. After staining of the preparations, 2 000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2 000 polychromatic erythrocytes were also recorded.

According to the results of the present study, the two oral administrations of the substance did not lead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was always close to the same range

as that of the concurrent negative control in all dose groups and within the range of the historical control data. A slight inhibition of erythropoiesis, determined from the ratio of polychromatic to normochromatic erythrocytes, was detected at all doses administered.

Thus, under the experimental conditions chosen here, the test substance shows no indications for a genotoxic potential in vivo.