N-[2-[(2-chloro-4,6-dinitrophenyl)azo]-5-(diethylamino)-4-methoxyphenyl]acetamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

CA: Not mutagenic

UDS: Not mutagenic

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

No studies on "genetic toxicity" are available for the substance in itself. Nevertheless a study has been conducted with a analogue molecules (Similar Substance 01 and 05). Further information are reported in the Read Across justification attached to section 13.

In vitro Genetic Toxicity

An Ames test is on going and is expected to be negative.

In vivo Genetic Toxicity

In the first in vivo study the substance (Similar Substance 05) was tested for the ability to induce unscheduled DNA synthesis (UDS) in an in vivo rat hepatocyte assay. Male Fischer 344 rats were treated with a single oral doseby gavage at 1250, or 2000 mg/kg body weight. The highest test dose, 2000 mg/kg was the limit test dose for a non-toxic test agent in this assay. Animals were killed and hepatocytes prepared four hours and twelve hours following administration of the chemical. Two independent experiments were carried out for each time point.

Hepatocytes from treated rats were exposed to [³H]-thymidine and the amount of radioactivity incorporated into the nucleus [N] and an equal area of cytoplasm [C] determined by autoradiography. The cytoplasmic grain count was subtracted from that of the nucleus. The value obtained, the mean net nuclear grain count [N-C], is an index of UDS activity. In the respective testing laboratory, no negative control animal has shown a mean net nuclear grain count greater than zero. An [N-C] of more than zero in a treated animal is therefore considered indicative of a UDS response.

Each experiment was validated by concurrent control treatments of rats with corn oil, the solvent for the test substance and with the carcinogens 2-acetylaminofluorene [2AAF] at twelve hours and N-nitrosodimethylamine [NDMA] or 6-p-dimethylaminophenylazobenzthiazole [6BT] at four hours. Solvent treated rats gave rise to mean net grain counts of less than zero, whilst hepatocytes from 2AAF, 6BT or NDMA treated animals had mean net nuclear grain counts of greater than +5. These data showed that background levels of UDS were normal and that the tester animals were responsive to known carcinogens requiring metabolic activation for genotoxic activity.

Hepatocytes from treated animals were assessed for UDS at two dose levels of 1250 and 2000 mg/kg body weight. Treatments with the test substance in no case resulted in a mean net grain count greater than zero, at either time point.

It is concluded that, when tested up to a limit dose of 2000 mg/kg body weight, the test sample did not induce DNA repair (as measured by unscheduled DNA Synthesis) in hepatocytes from rats treated in vivo.In a second in vivo study, carried out on Similar Substance 01, the test item was tested for its genetic toxicity effects according to OECD Guideline 474, in male and female Sprague Dawley Rats. The test substance was suspended in Tylose H4000 G4 PHA 0.5% and was given twice at an interval of 24 hours as an oral dose of 2000 mg per kg body weight. At study start the animals were 6 weeks of age and had mean body weights of 181.2 g (M) and 142.5 g (F). According to the test procedure the animals were killed 24 hours after the last administration
Cyclophosphamide 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 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.
Cyclophosphamide 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.

The test substance is Not Classified for Mutagenic Toxicity.