Methyl N-[3-(acetylamino)-4-[(2,4-dinitrophenyl)azo]phenyl]-N-(3-methoxy-3-oxopropyl)-β-alaninate

Endpoint summary

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. The test substance or its structural analogue was negative in all other genotoxicity studies.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

The test item Disperse Red 311 was tested positivein a screening Bacteria Reverse Mutation Assay (Ames test) in Salmonella strain TA98, but negative in an in-vivo micronucleus test in mice and an in-vivo Unscheduled DNA Synthesis (UDS) assay in rat hepatocytes.

This positive effect in the bacterial mutation assay is a bacteria-specific effect due to bacterial nitro-reductases, which are highly effective in these bacterial strains, but not in mammalian cells.

This effect was also investigated in-depths in two other nitro-dyes.

The first nitro dye was tested positive in an Ames test, but negative in the following tests:

Study Type	Metabolic activation	Result
Mitotic recombination assay withSaccharomyces cerevisae	Rat liver S9-mix	Negative
Point mutation assay withSaccharomyces cerevisae	Rat liver S9-mix	Negative
HPRT assay with V79 hamster cells	Rat liver S9-mix	Negative
UDS assay with rat hepatocytes	Metabolic competent hepatocytes were used	Negative
Micronucleus assay in vivo (mouse)	In vivo assay	Negative

The seconnd nitro-dye, Disperse Blue 291, was tested positive in an Ames test with nitroreductase and O‑acetyltransferase positive Salmonella typhimurium strains, but negative with nitroreductase and O-acetyltransferase negative strains and in a test for unscheduled DNA synthesis.

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 nitro-compounds. Type I nitroreductases can transfer two electrons from NAD(P)H to form the nitroso and hydroxylamino intermediates and finally the amino group. Type II nitroreductases transfer a single electron to the nitro group, forming a nitro anion radical, which in the presence of oxygen generates the superoxide anion in a futile redox cycle, regenerating the nitro group [de Oliveira et al. 2010].

The positive effect in the bacterial reverse mutation test (Ames) was clearly related to a bacteria-specific metabolism of the test substance, as 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]. This could be also be proved to be true in studies with Disperse Blue 291, which was tested for mutagenic activity in the Salmonella assay with strains with different levels of nitroreductase and O-acetyltransferase[Umbuzeiro et al. 2005]. In this study,Disperse Blue 291 showed mutagenic activity with all standard strains of Salmonella typhimurium tested (TA1537, TA1538, TA98 and TA100), except for TA1535.In nitroreductase and O-acetyltransferasenegative strains (TA98NR, TA98DNP6) not mutagenic activity was observed in the absence of S9, whereas themutagenic activity was increased with the nitroreductaseand/or O‑acetyltransferaseoverproducing strains, (YG1021, YG1024 and YG1041) This shows the importance of the bacterial acetyltransferase enzyme in the activation of Disperse Blue 291. Because of the remarkable increase in the response with the nitroreductase and O‑acetyltransferase overproducing strain (YG1041), it is assumed that the product of the nitroreductaseis a substrate for the O-acetyltransferase. As there was a very slight increase in mutagenicity with TA98NR, TA98, YG1021, TA98DNP6, and YG1024 in the presence of S9, it was assumed that P450 enzymes have also a role in the activation of Disperse Blue 291, besides the bacterial enzymes. This could however not proven true in in-vivo studies with Disperse Blue 291.

It has also been demonstrated in various other publications that this mutagenic activity is a bacteria-specific effect and that these Ames positive nitro-substances are not mutagenic in mammalian assays.

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 Salmonellastrains 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 metaboliteis released by intestinal bacteria, a Muta^TMMouse 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.
• Fexinidazolewas 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 [Tweats et 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 Disperse Red 311 is a bacteria specific effect and not relevant to mammalians.

Disperse Red 311 was not genotoxic in the in-vivo UDS and MNT test. Therefore, a direct genotoxic effect as well as a metabolisation towards genotoxic structures by mammalian species can be excluded.

References

De Oliveira IM, Bonatto D, Pega Henriques JA. Nitroreductases: Enzymes with Environmental Biotechnological and Clinical Importance. InCurrent Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology; Mendez-Vilas, A., Ed.; Formatex: Badajoz, Spain, 2010:1008–1019.

Suter W, Hartmann A, Poetter F, Sagelsdorff P, Hoffmann P, Martus HJ. Genotoxicity assessment of the antiepileptic drug AMP397, an Ames-positive aromatic nitro compound. Mutat Res. 2002 Jul 25;518(2):181-94.

Umbuzeiro GA, Freeman H, Warren SH, Kummrow F, Claxton LD. Mutagenicity evaluation of the commercial product CI Disperse Blue 291 using different protocols of the Salmonella assay. Food and Chemical Toxicology 2005;43:49–56.

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

Additional information

Bacteria reverse mutation

The mutagenic activity of Disperse Red 311 (86.7%purity), was investigated in a screening assay in the plate incorporation test using the Salmonella typhimurium strain TA 98. The test substance was dissolved in DMSO, without correction for purity. For screening purposes, the test substance was tested in strain TA98 only (both in the presence and absence of S9), at dose levels of 5000, 2500, 1000, 500, 200 and 100 µg/plate. As the first experiment gave positive results in both the presence and absence of S9, the test compound was be re-tested at dose levels of 500, 200, 100, 50, 20 and 10 µg/plate in strain TA98 (±S9) only, again using a Plate-Incorporation assay. Reproducible, dose-dependent increases in revertant colony numbers were obtained with and without S9 mix. 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 frameshifts in the genome in the tested strain. As the screening test was positive, no further experiment was conducted.

MNT in-vivo

Disperse Red 311 (86.7%purity), was tested via the oral route of administration in male CD-1 mice at a dose level of 5000mg/kg,thisdose level being the limit dose for this assay. Bone marrow samples were taken 24 and 48 hours after dosing.No statistically or biologically significant increases in the incidence of micronucleated polychromatic erythrocytes, over the vehicle control values, were seen in either sex at either of the sampling times investigated.Comparison of the percentage of polychromatic erythrocytes showed no significant differences between the vehicle control andDisperse Red 311treated animals.The test system positive control, cyclophosphamide, induced statistically significant and biologically meaningful increases in micronucleated polychromatic erythrocytes, compared to the vehicle control values, thus demonstrating the sensitivity of the test system to a known clastogen.It is therefore concluded thatDisperse Red 311, under the conditions of test, is not clastogenic in the mouse bone marrow micronucleus test.

Unscheduled DNA Synthesis

Disperse Red 311 (86.7%purity), was tested for the ability to induce unscheduled DNA synthesis (UDS) in an in vivo rat hepatocyte assay. Male Alpk:APfSD rats were treated with a single oral dose of Disperse Red 311 by gavage at dose levels of 1250 or 2000 mg/kg bodyweight. The latter dose level is the limit dose level for this assay. Animals were killed and hepatocytes isolated and prepared two and sixteen hours after dosing. 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 0.5% (w/v) hydroxy propyl methyl cellulose in 0.1% aqueous polysorbate 80, the vehicle for Disperse Red 311 and with the carcinogen dimethylhydrazine dihydrochloride [DMH.2HC1]. Vehicle-treated rats gave rise to mean net nuclear grain counts of less than zero, whilst hepatocytes from DMH.2HC1-treated animals had mean net nuclear grain counts of greater than +5. These data show that the Background levels of UDS in this study were normal and that the test animals were responsive to a known carcinogen requiring metabolic activation for the demonstration of genotoxic activity. Hepatocytes from Disperse Red 311 treated animals were assessed for UDS at both dose levels tested. Treatments with Disperse Red 311 in no case resulted in a mean net nuclear grain count greater than zero, at either time point investigated. Colouration of the urine was observed in animals dosed with Disperse Red 311, which clearly indicates that the test material was absorbed following administration via the oral route. It is concluded that, when tested up to a limit dose level of 2000 mg/kg, the test sample of Disperse Red 311 did not induce DNA repair (as measured by unscheduled DNA synthesis) in rat liver.

Justification for classification or non-classification

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