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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro bacterial reverse mutation assay

Non Mutagenic

in vitro gene mutation study in mammalian cells

Non mutagenic

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

Genetic toxicity in vivo

Description of key information

Non clastogenic

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

Additional information

The assessment of mutagenicity potential of the substance is based on one in vitro studies, carried out on Similar Substance 01, and one in vivo study, carried aout on Similar Substance 02.

Further information are reported in the Read Across justification attached to section 13.

IN VITRO BACTERIAL REVERSE MUTATION TEST

An ames test is in progress and expected negative.

IN VITRO MAMMALIAN CELL GENE MUTATION TEST

The substance was tested on chinese hamster cells V79 according to OECD 476. Test article was a greenblack powder with purity of 54.62%. The test substance was dissolved in DMSO. The cells were treated in the experiments with metabolic activation for 5 hours and in the experiments without metabolic activation for 21 hours.

A preliminary range finding test was run assessing cytotoxicity. The substance was tested at concentrations up to 500.0 µg/ml. Higher concentration could not be applied due to solubility limitations in the vehicle. In the part with metabolic activation, at the highest concentration a 100 % growth inhibiting effect could be seen, while the next lower concentration of 250.0 µg/ml inhibited 90.92 %. Without metabolic activation treatment with substance proved growth inhibiting down to the concentration of 125 µg/ml. The next lower concentration of 62.5 µg/ml revealed an acute inhibition of growth of 63.99 %. Accordingly, 250.0 µg/ml with and 100.0 µg/ml without metabolic activation were chosen as highest concentrations for the first mutagenicity assay.

Mutagenicity test with metabolic activation: the original experiment was performed at the following concentrations: 9.26, 27.78, 83.33 and 250.0 µg/ml. The mean growth inhibiting value found at the highest concentration after treatment was 71.35 %. No toxicity was seen after expression.

In the confirmatory experiment the concentrations applied were 11.11, 33.33, 100.0 and 300.0 µg/ml. The highest concentration revealed a mean acute growth inhibition of 93.76%. The mean growth inhibitory effect after the expression period was 12.28 %.

Mutagenicity test without metabolic activation: the original experiment was performed at the following concentrations: 3.70, 11.11, 33.33 and 100.0 µg/ml. The mean growth inhibition value found at the highest concentration after treatment was 38.33 %. No toxicity was seen after expression.

In the confirmatory experiment the concentrations applied were 5.56, 16.67, 50.0 and 150.0 µg/ml. The highest concentration revealed a mean acute growth inhibitory effect of 71.47 %. No toxicity was seen after expression.

For both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no relevant increase of the mutant frequencies as determined by the screening with 6-TG.

Based on the results of two independently performed experiments and under the given experimental conditions, it is concluded that the substance and its metabolites did not show any mutagenic activity in this forward mutation system.

IN VIVO MAMMALIAN SOMATIC CELL STUDY: CYTOGENICITY/ERYTHROCYTE MICRONUCLEUS

The study was conducted to investigate the potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the rat according to OECD Guideline 474, EPA OPPTS 870.5395 and EU Method B.12, in compliance with GLP.

The test compound was dissolved in deionized water and was given twice at an interval of 24 hours as oral doses of 2000 mg per kg body weight to male and female rats (Hsd:Sprague Dawley). At study start the animals were 6 weeks of age and had mean body weights of 181.4 g (M) and 146.5 g (F). According to the test procedure the animals were killed 24 hours after the last administration.

Endoxan® was used as positive control substance and was administered once orally at a dose of 40 mg per kg body weight.

The number of polychromatic erythrocytes containing micronuclei was not significantly increased compared with the control. The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained unaffected by the treatment with the test substance and differed less than 20 % from the control value.

Endoxan®induced a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the test system. The ratio of polychromatic erythrocytes to total erythrocytes was not changed to a significant extent.

Under the conditions of the present study the results indicate that the test substance is not clastogenic in the micronucleus test in vivo.

Justification for classification or non-classification

GERM CELL MUTAGENICITY

This hazard class is primarily concerned with substances that may cause mutations in the germ cells of humans that can be transmitted to the progeny. However, the results from mutagenicity or genotoxicity tests in vitro and in mammalian somatic and germ cells in vivo are also considered in classifying substances and mixtures within this hazard class.

Category 1: Substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans. Substances known to induce heritable mutations in the germ cells of humans.

Categoty 2: 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.

Note: Substances which are positive in in vitro mammalian mutagenicity assays, and which also show chemical structure activity relationship to known germ cell mutagens, shall be considered for classification as Category 2 mutagens.

Classification for heritable effects in human germ cells is made on the basis of well conducted, sufficiently validated tests as:

- In vivo somatic cell mutagenicicty tests such as these indicated in paragraph 3.5.2.3.5:

— mammalian bone marrow chromosome aberration test;

— mouse spot test;

— mammalian erythrocyte micronucleus test.

- In vitro mutagenicity tests such as these indicated in 3.5.2.3.8:

— in vitro mammalian chromosome aberration test;

— in vitro mammalian cell gene mutation test;

— bacterial reverse mutation tests.

An in vitro mammalian cell gene mutation test is available and the test substance does not show any mutagenic effect.

As per REACH Regulation requirements, a further in vivo study is considered in which the test substance didn’t induce increase the number of polychromatic cells with micronuclei.

Therefore, as reported in the Flow chart R.7.7 -1, no further test are required and it is possible to conclude that the test substance is Not Classified for Mutagenic Toxicity.

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