[No public or meaningful name is available]

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

GLP compliance:

yes (incl. QA statement)

Remarks:

Experimental Toxicology and Ecology, BASF Aktiengesellschaft, 67056 Ludwigshafen, Germany

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

900-066-9

EC Number:

900-066-9

IUPAC Name:

900-066-9

Constituent 2

Constituent 3

Reference substance name:

2-ethylhexyl N-(6-isocyanatohexyl)-N-(6-isocyanatohexylcarbamoyl)carbamate

Cas Number:

197393-84-3

Molecular formula:

C24 H42 N4 O5

IUPAC Name:

2-ethylhexyl N-(6-isocyanatohexyl)-N-(6-isocyanatohexylcarbamoyl)carbamate

Test material form:

other: Liquid

Details on test material:

- Homogeneity: The homogeneity of the test substance was guaranteed on account of the high purity.
- Storage stability: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor.
- Physical state, appearance: Liquid, colorless - yellowish
- Storage conditions: Room temperature (N2 conditions)

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system 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
- 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.

Frequency of treatment:

The animals of the vehicle control and the dose groups were treated twice at a 24-hour interval, animals of the positive control groups were treated only once

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):

The following positive controls, both dissolved in purified water were administered to male animals once orally: cyclophosphamide (CPP) or intraperitoneally vincristine (VCR) each in a volume of 10 mL/kg body weight.

Examinations

Tissues and cell types examined:

Bone marrow of the two femora was prepared
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, W. and SALAMONE, M. et al. 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 examined

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.