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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation, OECD 476, negative

in vitro bacteria test, OECD 471 with nitro-reductasi deficient strains: marked reduction in mutagenicity activity

in vitro bacteria test, OECD 471: positive

in vitro chromosomal aberrtion, OECD 473: negative

Genetic toxicity in vivo

Description of key information

in vivo MNT, OECD 474: negative

in vivo UDS, OECD 486: negative

Link to relevant study records
Reference
Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Type of information:
other: read across from analogue substance
Adequacy of study:
key study
Study period:
Since april 21, 1988 to june 01, 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP compliant with international guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: SAVO med. Versuchstierzuchten GmbH
- Weight at study initiation: ~ 120 g
- Assigned to test groups randomly: yes
- Fasting period before study:Approximately 18 hours before treatment with the test article the animals received no food but water ad libitum.
- Housing: single housed in Makrolon Type II, with wire mesh top , granulated soft wood bedding
- Diet: pelleted standard diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):21 ± 3°C
- Humidity(%): 30-70%
- Photoperiod : 12 hours cycle dark/light from artificial light at 6.00 a.m. to 6.00 p.m.

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: deionized water
Duration of treatment / exposure:
28 days
Frequency of treatment:
24 +/- 2 hours for 28 days
Post exposure period:
24 hours after the last application the bone marrow cells were collected for micronuclei analysis.
Remarks:
Doses / Concentrations:
1000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
6 animals x sex x dose (test item)
6 animals x sex x dose (positive control)
6 animals x sex x dose (negative control)
Control animals:
no
Positive control(s):
- Name: Cyclophosphamide
- Route of administration: oral
- Doses / concentrations: 10 ml/kg bw
Tissues and cell types examined:
bone marrow cells were collected for micronuclei analysis.
Details of tissue and slide preparation:
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a 5 ml syringe. The cell suspension was centrifuged at 1,500 rpm 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 (MERCK, D-6100 Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-7800 Freiburg). At least one slide was made from each bone marrow sample.
Evaluation criteria:
A test article is classified as mutagenic if it induces either a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes or a reproducible statistically significant positive response for at least one of the test points.
A test article producing neither a statistically significant dose-relat-ed increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.
This can be confirmed by means of the nonparametric Mann-Whitney test.
However, both biological and statistical significance should be considered together.
Statistics:
Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

A dose of 1000 mg/kg b.w. was administered. 1000 mg/kg b.w. of the test article was close to the maximum tolerated dose. 3 out of 12 treated animals died during the 28 days period (1 male, 2 females).

Conclusions:
The analogue substance was tested for in vivo micronucleus following OECD 474. Under the experiemtnal conditions test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the rat.
Executive summary:

The analogue substance was tested for in vivo micronucleus following OECD 474. This study was performed to investigate the potential of the substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the rat.

The test article was dissolved in deionized water. This solvent was used as negative control. The volume administered orally was 10 ml/kg b.w. The animals received the test article or negative control once per 24 +/- 2 h for 28 days. 24 hours after the last application the bone marrow cells were collected for micronuclei analysis.

Ten animals (5 males, 5 females) per test group with the exception of the test article group in which only 9 animals survived (5 males and 4 females) 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.

A dose of 1000 mg/kg b.w. was administered. 1000 mg/kg b.w. of the test article was close to the maximum tolerated dose. 3 out of 12 treated animals died during the 28 days period (1 male, 2 females).

After treatment with the test article the ratio between PCEs and NCEs was affected as compared to the corresponding negative control thus indicating a test article-dependent effect on the target cells.

In comparison to the corresponding negative controls there was neither for the females nor for the total collective of FAT 40'317/B treated animals a statistically significant enhancement in the frequency of the detected micronuclei.

An appropriate reference mutagen was administered once and used as positive control which showed a distinct increase of induced micronucleus frequency.

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

Additional information

Justification for classification or non-classification

Classification for mutagenicity under Regulation 1272/2008 is warranted for substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans. The classification in Category 2 is based on:

— positive evidence obtained from experiments in mammals and/or in some cases from in vitro experiments, obtained from:

— somatic cell mutagenicity tests in vivo, in mammals; or

— other in vivo somatic cell genotoxicity tests which are supported by positive results from in vitro mutagenicity assays.

The test item is positive to the traditional in vitro gene mutation study in bacteria which triggers first some potential elucidation of bacteria specific mechanisms. An OECD 471 with nitro-reducatse deficient strains was performed which shows a marked reduction of the mutagenic potential. However the trigger for some tests at Annex IX level is still valid. A reliabale and adequate in vitro gene mutation OECD 476 and in vivo genetic toxicity test, OECD 486 UDS Assay, are available showing negative results. Further in vivo Comet Assay OECD 489 is proposed to completely assess and confirm the gene mutation toxicity behaviour of the substance.

Also an in vitro chromosomal aberration OECD 473 and an in-vivo micronucleus OECD 474 are available showing negative results.

Based on the current available information, the substance is not considered mutagenic and a review of the classification will be performed once the OECD 489 results are available.