Butanamide, 2-[2-(2-methoxy-5-nitrophenyl)diazenyl]-N-(2-methoxyphenyl)-3-oxo-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In an bacterial reverse mutation assay (Ames test) with or without metabolic activation (S9 mix) according to OECD guideline 471 (BASF SE, 2008), no significant increase in the numbers of revertant colonies was recorded

In a chromosome aberration test (CA) with or without metabolic activation (S9 mix) according to OECD guideline 473 (BASF SE, 2008), a relevant increase in the number of cells containing numerical chromosomal aberrations was observed.

In a gene mutation study in mammalian cells (HPRT test) with or without metabolic activation (S9 mix) according to OECD Guideline 476 (BASF SE, 2011), no biologically relevant increase of mutants was found.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In a mammalian micro nucleus test according to OECD Guideline 474 in mice (BASF SE, 2008), no statistical significances or biologically relevant increase in the frequency of erythrocytes containing micronuclei was observed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro studies

Bacterial reverse mutation assay

In a bacterial reverse mutation assay conducted according to the OECD Guideline 471 (Bacterial Reverse Mutation Assay), the test substance was evaluated for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium (TA 98, TA 100, TA1535, and TA 1537) and Escherichia coli (E.coli P2 uvrA) (BASF SE, 2009). The following dose levels were evaluated at indicated conditions:

- 1st Experiment: 0, 21.2, 106, 530, 2650, and 5300 μg/plate (standard plate test with and without S9 mix, all strains, in triplicates);

- 2nd Experiment: 0, 331.3, 662.5, 1325, 2650 and 5300 μg/plate (standard plate test with and without S9 mix, only TA 98, in triplicates);

- 3rd Experiment: 0, 21.2, 106, 530, 2650, and 5300 μg/plate (preincubation test with and without S9 mix, all strains, in triplicates).

5300 µg/plate was used as top dose in all experiments due to the purity of the test substance. Acetone was used as vehicle control and appropriate positive control substances were also evaluated. Precipitation of the test substance was found from about 21.2 μg/plate onward with and without S9 mix and a weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions from about 2650 μg/plate onward.

A dose-related biologically relevant increase in the number of his+ revertants was observed in the standard plate test and in the preincubation with the strain TA 98 after the addition of a metabolising system (first experiment: increase at 5300 μg/plate by a factor of 2.5; second experiment: increase at 2650 and 5300 μg/plate by factors of 2.1 and 2.2 respectively; third experiment: increase at 2650 μg/plate by a factor of 2.3). According to the results of the present study, the test substance is weakly mutagenic in the Salmonella typhimurium mutation assay under the experimental conditions chosen. However, based on the occurrence of precipitation in all doses showing increased revertant rates it cannot be ruled out that the impurities are the cause of these findings.

 

Chromosome aberration assay

In a chromosome aberration assay conducted according to the OECD Guideline 473 (In vitro Mammalian Chromosome Aberration Test), the substance was assessed for its potential to induce structural chromosomal aberrations (clastogenic activity) and/or changes in the number of chromosomes (aneugenic activity) in V79 cells in vitro both in the absence and the presence of a metabolising system (BASF SE, 2008). Based on the results of an initial range-finding cytotoxicity test, where the test substance showed weak toxicity of about 50 % of control at high concentrations (and at which distinct test substance precipitation was observed that interfered with the determination of cytotoxic parameters), the following doses were tested under the indicated conditions:

- 1st Experiment: 4-hour exposure; 18-hour sampling time; with and without S9 mix; 0, 0.5, 1.0, 2.0, 4.0, 8.0, 16.0, and 32.0 μg/mL treatment groups (only 0, 2.0 and 4.0 µg/mL test groups were evaluated for chromosomal aberrations);

- 2nd Experiment: 4-hour exposure; 18-hour sampling time; with and without S9 mix; 0.5, 1.0, 2.0, 3.9, 7.8, 15.8, 31.3, 62.5, 125.0, 500.0, and 1000.0 µg/mL treatment groups (only 0, 1.0, 2.0 and 3.9 µg/mL test groups were evaluated for chromosomal aberrations). A sample of at least 100 metaphases for each culture was analyzed for chromosomal aberrations (except for the positive control cultures, there were only 50 metaphases scored due to clearly increased aberration rates).

According to the results of the present in vitro cytogenetic study, the test substance led to a dose-dependent and biologically relevant increase in the number of structural chromosomal aberrations incl. and excl. gaps with metabolising system after a treatment time of 4 hours and a sampling time of 18 hours. At the same time, the structural chromosome aberration rates of the vehicle control groups and the increase in the frequencies of structural chromosome aberrations induced by the positive control substances (clearly demonstrated the sensitivity of the test system and of the metabolic activity of the S9 mix employed) were within the range of the historical negative and positive control data, respectively and, thus, fulfilled the acceptance criteria of this study. Therefore, the conclusion is drawn that the test substance is a chromosome-damaging (clastogenic) substance using V79 cells in the presence of metabolic activation under the experimental conditions chosen.

Mammalian Cell Gene Mutation assay

In a mammalian Cell Gene mutation Assay conducted according to OECD Guideline 476 (mammalian cell gene mutation test), the substance was evaluated for its mutagenic potential based on the ability to induce basepair and frameshift mutations, as well as deletions in the HPRT locus in chinese hamster ovary cells (CHO) (BASF SE, 2011). Two independent experiments were carried out with and without metabolic activation (S9 mix from induced rats). Regarding the solubility of the substance and according to an initial range finding cytotoxicity test for the determination of the experimental doses, the following doses were tested and evaluated in the study:

- 1st Experiment without S9 mix (4-hour exposure period) 0, 1.5, 3.0, 6.0, 50.0, and 100.0 μg/mL

with S9 mix (4-hour exposure period) 0, 1.5, 3.0, 6.0, 187.5, 375.0, and 750.0 μg/mL

- 2nd Experiment without S9 mix (24-hour exposure period) 0, 1.3, 2.5, 5.0, 10.0, 50.0, 100.0, and 200.0 μg/mL

with S9 mix (4-hour exposure period) 0, 1.3, 2.5, 5.0, 10.0, 200.0, 400.0, and 750.0 μg/mL

After an attachment period of 20 - 24 hours and a treatment period of 4 hours both with and without metabolic activation and 24 hours without metabolic activation, an expression phase of about 6 - 8 days and a selection period of about 1 week followed. The colonies of each test group were fixed with methanol, stained with Giemsa and counted. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, EMS (ethyl methanesulfonate) and MCA (methylcholanthrene), led to the expected increase in the frequencies of forward mutations. In this study, in the 1st and 2nd experiment in the absence of metabolic activation the highest concentrations tested for gene mutations were clearly cytotoxic. Besides, in both experiments in the presence of metabolic activation no cytotoxicity was observed up to the highest concentration that could be prepared in the most suitable vehicle DMSO. On the basis from the results of the present study, the test substance did not cause any relevant increase in the mutant frequencies either without S9 mix or after adding a metabolizing system in two experiments performed independently of each other.

Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

 

In vivo studies

Mouse micronucleus assay

The ability of the test substance to cause chromosomal damage (clastogenicity) or to induce spindle poison effects (aneugenic activity) was tested in NMRI mice according to the OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test; BASF SE, 2008). For this purpose, the test substance (suspended in corn oil) or vehicle alone, was administered once intraperitoneally to male animals at dose levels of 0, 375 mg/kg, 750 mg/kg and 1500 mg/kg body weight in a volume of 15 mL/kg body weight per mice. Both cyclophosphamide (for clastogenicity) and vincristine sulfate (for spindle poison effects) were used as positive control substances.

The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the highest dose group of 1500 mg/kg bw and in the vehicle controls. In the test groups of 750 mg/kg and 375 mg/kg bw and in the positive control groups, only the 24-hour sacrifice interval was investigated. After staining of bone marrow cell preparations, 2000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei, as well as for normocytes with and without micronuclei occurring per 2000 polychromatic erythrocytes.

According to the results of the present study, the single intraperitoneal administration of the test substance did not lead to any relevant increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was always close to the same range as the concurrent vehicle control in all dose groups and at all sacrifice intervals (both test substance and vehicle control rates were within the range of the historical vehicle control data). A clear inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected in the 48-hour sacrifice interval. The positive control substance clearly led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei.

Therefore, under the experimental conditions chosen here, the test substance does not have any chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.

Conclusion:

An indication that the test substance may cause chromosome-damaging (clastogenic) effect or impairment of chromosome distribution in the course of mitosis (aneugenic activity) was obtained from the in vitro study in V79 mammalian cells (OECD Guideline 473). This was however not confirmed in vivo after single IP treatment of male NMRI mice (OECD Guideline 474), where bone marrow cells were analysed for clastogenic and aneugenic effects (in the presence of a clear inhibition of erythropoiesis as sign that the bone marrow is a target organ). According to the results observed in the Ames test (+S9 mix) (OECD Guideline 473) the test substance is weakly mutagenic. However, based on the occurrence of precipitation in all doses showing increased revertant rates it cannot be ruled out that the impurities are the cause of these findings. This argumentation is supported by the negative results of the HPRT mammalian cell gene mutation assay (OECD guideline 476) with and without metabolic activation (S9 mix) in CHO cells.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008.

As consequence the test substance should not be considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.