Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
Four Klimisch-1-rated studies have been conducted, one in-vivo mammalian micronucleus mutagenicity test, one in-vitro mammalian cells mutagenicity test, one in-vitro mammalian chromosome aberration test, and one bacterial Ames Test. With the exception of the chromosome aberration test, all study results were negative. As outcome of the weight of evidence approach, the registration substance is considered not genotoxic.
Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1996
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: BRL, CH-4414 Füllingsdorf
- Age at study initiation: 9-13 weeks
- Weight at study initiation: males mean value 31.3 ± 3.4 g, females mean value 24.3 ± 1.7 g
- Assigned to test groups randomly: yes
- Housing: individually in Makrolon Type I cages with wire mesh top and granulated soft wood bedding
- Fasting period before study: approximately 18 hours
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 7 °C
- Humidity (%): 15-70 %
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: unspecified
Vehicle:
- Vehicle used: Deionised water
- Justification for choice of solvent/vehicle: The vehicle was chosen to its relative non-toxicity for the animals.
- Amount of vehicle (if gavage or dermal): 20 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment, the test article was dissolved in deionised water.
Duration of treatment / exposure:
Single administration.
Frequency of treatment:
Single oral administration of test item and controls, respectively.
Post exposure period:
Sampling of the bone marrow was done 24 (all dose groups) and 48 hours (only high dose group) after treatment, respectively.
Remarks:
Doses / Concentrations:
200, 670, and 2000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
6 males and 6 females per dose/control group
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive control substance: cyclophosphamide
- Route of administration: orally
- Doses / concentrations: 40 mg/kg bw at 10 mL/kg bw in deionised water
Tissues and cell types examined:
femural bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
In a pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 2000 mg/kg b.w., the substance dissolved in deionised water. As no severe toxic reactions were observed within 48 after exposure, 2000 mg/kg bw was estimated to be suitable as high dose concentration.

DETAILS OF SLIDE PREPARATION:
The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald /Giemsa. Cover slips were mounted with Eukitt. At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 1000 PCEs. The analysis was performed with coded slides.
Five animals per sex and group were evaluated as described. The remaining 6th animal of each test group was evaluated in case an animal had died in its test group.
Evaluation criteria:
A test article is classified as mutagenic if it induces either a dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistically significant positive response for at least one of the test points.
A test article producing neither a dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant positive response at any of the test points is considered non-mutagenic in this system.
Statistics:
Significant increase can be confirmed by means of the nonparametric Mann-Whitney test. However, both biological and statistical significance should be considered together.
Sex:
male/female
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
- Dose range: limit test with 2000 mg/kg bw
- Solubility: well soluble at the tested concentration
- Clinical signs of toxicity in test animals: reduction of spontaneous activity in 4 of 4 animals (fully reversible within 48h), eyelid closure in 2 of 4 animals (fully reversible within 24h), apathy in 2 of 4 animals (fully reversible within 24h),

RESULTS OF DEFINITIVE STUDY
- Ratio of PCE/NCE (for Micronucleus assay): The mean number of normochromatic erythrocytes was increased after treatment with the test article at preparation interval 48 as compared to the mean value of NCEs of the vehicle control. However, the enhancement of the mean value was caused mainly by the results of two males (62 = 2160 NCEs per 1000 PCEs; 63 = 1377 NCEs per 1000 PCEs). Therefore, it cannot be concluded that the test article induced cytotoxic effects. Mean data are given in Table 1.
- Statistical evaluation: In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test article. The mean values of micronuclei observed after treatment with the substance were in the range of the vehicle control group. Cyclophosphamide administered was used as positive control which showed a statistically significant increase of induced micronucleus frequency.
Conclusions:
Interpretation of results (migrated information): negative
The substance is considered to be non-mutagenic in this micronucleus assay.
Executive summary:

This study was performed according to the First Addendum to the OECD Guideline for Testing of Chemicals, Section 4, No. 474, adopted May 26, 1983, „Micronucleus Test" and the method laid down in EEC Directive 92/69, L 383, Annexe V, B 12, dated December 29, 1992 to investigate the potential of the substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

The test article was dissolved in deionised water. Deionised water was used as vehicle control. The volume administered orally was 20 ml/kg bw 24 h and 48 h after a single administration of the test article the bone marrow cells were collected for micronuclei analysis.

Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei.

To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE.

The following dose levels of the test article were investigated:

24 h preparation interval: 200, 670 and 2000 mg/kg bw

48 h preparation interval: 2000 mg/kg bw

The highest guideline-recommended dose (2000 mg/kg) was estimated by a pre-experiment to be suitable. The animals expressed slight toxic reactions.

After treatment with the test article at preparation interval 48 h the mean number of normochromatic erythrocytes was increased as compared to the mean value of NCEs of the vehicle control. However, the enhancement of the mean value was caused mainly by the results of two males (62 = 2160 NCEs per 1000 PCEs; 63 = 1377 NCEs per 1000 PCEs). Therefore, it cannot be concluded that the test article induced cytotoxic effects.

In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test article and with any dose level used. 40 mg/kg bw cyclophosphamide administered per os was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

Therefore, the substance is considered to be non-mutagenic in this micronucleus assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

In-vivo micronucleus study:

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

In-vitro mammalian cells mutagenicity test:

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the registration substance is considered to be non-mutagenic in this HPRT assay.

In-vitro mammalian chromosome aberration test:

In conclusion, as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro, it can be stated that in the study described and under the experimental conditions reported, the registration substance induced reproducibly structural chromosome aberrations in the presence of S9 mix within a concentration range where precipitation of the test article was observed. Therefore, the registration substance is considered to be genotoxic in this chromosome aberration test.

Bacterial reverse mutation study (Ames Test):

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the registration substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Weight of evidence approach:

Four Klimisch-1-rated studies have been conducted on genotoxicity. The mammalian chromosome aberration test gave a positive result in the presence of metabolic activation, while all of the three other studies revealed a negative outcome. As the majority of study results are not indicating genotoxic properties of the test item, and in particular because the in-vivo micronucleus study gave a negative result, the registration substance is considered as not genotoxic.

Justification for classification or non-classification

The registration substance shall be considered as not genotoxic, thus no classification applies.