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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Only bacteria-specific effects were noted in the bacteria reverse mutation assay, whereas the mutagenicity study in mammalian cells with the structural analogue was negative.

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

Genetic toxicity in vivo

Description of key information

The test substance did not induce DNA repair (as measured by unscheduled DNA synthesis) in rat liver nor did the structural analogue induce micronuclei in the polychromatic erythrocytes of treated rats.

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

Mode of Action Analysis / Human Relevance Framework

The test substance was positive in the Ames assay, but negative in a test for unscheduled DNA synthesis; furthermore, a structural analogue was negative in a mutation assay in mammalian cells and in an in vivo mouse micronucleus assay. The positive effect in the Ames test is a bacteria-specific effect due to bacterial nitro-reductases, which are highly effective in these bacterial strains, but not in mammalian cells.

It is well-known for aromatic nitro compounds to be positive in the Ames assay resulting from metabolism by the bacteria-specific enzyme nitro-reductase (Tweats et al., 2012). However, it has been demonstrated in various publications that this is a bacteria-specific effect and that these Ames positive substances are not mutagenic in mammalian assays.

The nitroreductase family comprises a group of flavin mononucleotide (FMN)- or flavin adenine dinucleotide (FAD)-dependent enzymes that are able to metabolize nitroaromatic and nitroheterocyclic derivatives (nitrosubstituted compounds) using the reducing power of nicotinamide adenine dinucleotide (NAD(P)H). These enzymes can be found in bacterial species and, to a lesser extent, in eukaryotes. The nitroreductase proteins play a central role in the activation of nitrocompounds (de Oliveira et al., 2010).

That the reduction of these nitro-compounds to mutagenic metabolites is a bacteria-specific effect is demonstrated in the following by means of the two compounds AMP397 and fexinidazole.

AMP397 is a drug candidate developed for the oral treatment of epilepsy. The molecule contains an aromatic nitro group, which obviously is a structural alert for mutagenicity. The chemical was mutagenic in Salmonella strains TA97a, TA98 and TA100, all without S9, but negative in the nitroreductase-deficient strains TA98NR and TA100NR. Accordingly, the ICH standard battery mouse lymphomatkand mouse bone marrow micronucleus tests were negative, although a weak high toxicity-associated genotoxic activity was seen in a micronucleus test inV79 cells (Suter et al., 2002). The amino derivative of AMP397 was not mutagenic in wild type TA98 and TA100. To exclude that a potentially mutagenic metabolite is released by intestinal bacteria, a MutaMouse study was done in colon and liver with five daily treatments at the MTD, and sampling of 3, 7 and 21 days post-treatment. No evidence of a mutagenic potential was found in colon and liver. Likewise, a comet assay did not detect any genotoxic activity in jejunum and liver of rats, after single treatment with a roughly six times higher dose than the transgenic study, which reflects the higher exposure observed in mice. In addition, a radioactive DNA binding assay in the liver of mice and rats did not find any evidence for DNA binding. Based on these results, it was concluded that AMP397 has no genotoxic potential in vivo. It was hypothesized that the positive Ames test was due to activation by bacterial nitro-reductase, as practically all mammalian assays including fourin vivoassays were negative, and no evidence for activation by mammalian nitro-reductase or other enzymes were seen. Furthermore, no evidence for excretion of metabolites mutagenic for intestinal cells by intestinal bacteria was found.

Fexinidazole was in pre-clinical development as a broad-spectrum antiprotozoal drug by the Hoechst AG in the 1970s-1980s, but its clinical development was not pursued. Fexinidazole was rediscovered by the Drugs for Neglected Diseases initiative (DNDi) as drug candidate to cure the parasitic disease human African trypanomiasis (HAT), also known as sleeping sickness. The genotoxicity profile of fexinidazole, a 2-substituted 5-nitroimidazole, and its two active metabolites, the sulfoxide and sulfone derivatives were investigated (Tweatset al., 2012). All the three compounds are mutagenic in the Salmonella/Ames test; however, mutagenicity is either attenuated or lost in Ames Salmonella strains that lack one or more nitroreductase(s). It is known that these enzymes can nitroreduce compounds with low redox potentials, whereas their mammalian cell counterparts cannot, under normal conditions. Fexinidazole and its metabolites have low redox potentials and all mammalian cell assays to detect genetic toxicity, conducted for this study either in vitro (micronucleus test in human lymphocytes) or in vivo (ex vivo unscheduled DNA synthesis in rats; bone marrow micronucleus test in mice), were negative.


Based on these data and the common mechanism between the reduction of these nitro-compounds, which is widely explored in literature (de Oliveira et al., 2010), it is concluded, that the mutagenic effects observed in the Ames test with the test and read-across substances is a bacteria specific effect and not relevant to mammalians.


De Oliveira IM, Bonatto D, Pega Henriques JA (2010). Nitroreductases: Enzymes with environmental biotechnological and clinical importance. In: Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology. Mendez-Vilas, A., Ed.; Formatex: Badajoz, Spain. 1008–1019.

Suter W, Hartmann A, Poetter F, Sagelsdorff P, Hoffmann P, Martus HJ (2002). Genotoxicity assessment of the antiepileptic drug AMP397, an Ames-positive aromatic nitro compound. Mutat. Res. 518(2):181-94.

Tweats D, Bourdin Trunz B, Torreele E (2012). Genotoxicity profile of fexinidazole--a drug candidate in clinical development for human African trypanomiasis (sleeping sickness). Mutagenesis. 27(5):523-32.

Additional information

In vitro

In a reverse gene mutation assay in bacteria, strains TA 1535, TA 100, TA 1537, TA 98 of S. typhimurium were exposed to the test item (60% purity) at concentrations of 33, 100, 333, 1000, 2500 and 5000 µg/plate in the presence and absence of mammalian metabolic activation. The test item was tested up to the limit concentration (5000 µg/plate). Toxic effects, evident as a reduction in the number of revenants, were observed with and without S9 mix in strains TA 1535 and TA 1537 at 2500 and 5000 µg/plate, and in strain TA 100 with S9 mix in experiment II. The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. In both experiments, a substantial and dose dependent increase in revertant colony numbers was observed following treatment with the test substance in strains TA 1535, TA 1537, TA 98, and TA 100. However, overlapping toxic effects reduced the number of revertant colonies in strains TA 1535, TA 1537 (with and without metabolic activation), and TA 100 from 1000 up to 5000 µg/plate. Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article induced gene mutations by base pair changes and frameshifts in the genome of the strains TA 1535, TA 1537, TA 98, and TA 100.

A study was conducted to determine the genotoxicity of the structural analogue 01 (93% purity) according to OECD Guideline 476. Chinese hamster lung fibroblasts (V79) were exposed at concentrations of 0, 0.05, 0.1, 0.25, 0.5 and 1.0 µg/mL with or without S-9 metabolic activation. Cell survival and mutations were assessed. In the experiment for direct mutagenicity, N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) served as a positive control. In the S9-mix mediated assay, 7,12-dimethylbenz(a)anthracene (DMBA) was used. Negative and positive control experiments were valid. The substance showed acute toxicity to the V79 cells in the direct assay. However, there were no signs of mutagenicity in the presence or absence of S9 mix at various concentrations up to the limits of toxicity and solubility. Under the study conditions, the test substance was not considered to be mutagenic in Chinese hamster lung fibroblasts (V79).

In vivo

Disperse Violet 93.1 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 dose of Disperse Violet 93.1 by gavage at 1250 and 2000mg/kg bodyweight. The highest test dose, 2000 mg/kg bw 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 and an equal area of cytoplasm 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 this laboratory no negative control animal has shown a mean net nuclear grain count of greater than zero. An [N-C] value of greater than zero is therefore considered indicative of a UDS response. Each experiment was validated by concurrent control treatments of rats with corn oil, the solvent for Disperse Violet 93.1 and with the carcinogens 2-acetylaminofluorene [2AAF] at twelve hours or Nnitrosodimethylamine [NDMA] at four hours. Solvent treated rats gave rise to mean net nuclear grain counts of less than zero, whilst hepatocytes from 2AAF 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 Disperse Violet 93.1 treated animals were assessed for UDS at both dose levels. Treatments with Disperse Violet 93.1 in no case resulted in a mean net nuclear grain count greater than zero, at either time point. It is concluded that, when tested up to 2000 mg/kg body weight, the test sample of Disperse Violet 93.1 did not induce DNA repair (as measured by unscheduled DNA synthesis) in rat liver.

A study was conducted to determine the in vivo genetic toxicity of structural analogue 01 (93% purity) according to OECD Guideline 474. The ability of the test substance to induce cytogenetic damage and/or disruption of the mitotic apparatus in rat bone marrow was investigated measuring the induction of micronuclei in polychromatic erythrocytes. Male and female rats (15/sex/group) were exposed to the test substance at concentration of 0 or 5000 mg/kg bw by gavage in a single application of a 2% carboxymethylcellulose (CMC) distilled water suspension. A positive control group (mitomycin-C, 2.0 mg/kg) was also tested. The examinations were performed at 24, 48 and 72 h by sacrifice of 6 animals per sex. The substance did not show an increase of micronuclei from bone marrow compared to the vehicle control. The values for the positive and negative controls were within the expectation ranges. The experiment was therefore considered valid. Under the study conditions, the test substance was not found to be genotoxic.

Justification for classification or non-classification

Based on the results of in vitro and in vivo testing, no classification for genotoxicity is required for the test substance according to CLP (EC 1272/2008) criteria.