alkyl substituted aryl diazo heterocyclic aryl ester

Endpoint summary

Additional information

The test item Disperse Red DYGJ 0702 was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone. The test item was used as a solution in dimethylsulfoxide (DMSO).

Toxicity test: The test item Disperse Red DYGJ 0702 was assayed in the toxicity test at a maximum concentration of 5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 µg/plate. Heavy precipitation of the test item was observed at the end of the incubation period at the highest concentration tested which iterfered the evaluation of background lawn and in some cases also with the colony counting. Toxicity, as indicated by thinning of background lawn, reduction in revertant numbers or microcolony formation was observed with all tester strains at the two higher dose levels, in the absence and presence of S9 metabolism. Toxicity was more pronounced with TA100 tester strain in the absence of S9, where thinning of background lawn was observed over the whole dose range tested.

Main Assay I: On the basis of toxicity test results, in Main Assay I, using the plate incorporation method, the test item was assayed at the following dose levels:

TA1535 ( ± S9) : 2500, 1250, 625, 313 and 156

TA1537 ( ± S9): 1250, 625, 313, 156, 78.1 and 39.1

WP2 uvrA (-S9): 5000, 2500, 1250, 625 and 313

(+S9): 2500, 1250, 625, 313 and 156

TA98 ( ± S9): 2500, 1250, 625, 313 and 156

TA100 ( ± S9): 1250, 625, 313, 156 and 78.1

Toxicity as indicated by thinning of background lawn was observed with all tester strains at the highest or two highest dose levels in the absence and presence of S9 metabolism. Toxicity was more pronounced with TA1537 and TA100 in the absence of S9 metabolism where thinning of background lawn was also present at lower dose levels. Precipitation of the test item which did not interfere with scoring was observed at the end of the incubation period at the highest concentration tested in the absence of S9 metabolism for all tester strains and at one or two next lower dose levels for TA1535 and WP2 uvrA respectively. In the presence of S9 metabolism, precipitation of the test item which did not interfere with scoring was observed at the highest concentration tested for the tester strain TA1535 and at the two highest concentrations tested for WP2 uvrA. The test item induced large increases in the number of revertant colonies in all tester strains in the absence and presence of S9 metabolism. Since a clear positive response was observed, no further experiment was undertaken

Conclusion: It is concluded that the test item Disperse Red DYGJ 0702 induces reverse mutation by base substitutions or frameshifts in Salmonella typhimurium and Escherichia coli in the absence and presence of S9 metabolism, under the reported experimental conditions.

However, 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 is considered to be a bacteria specific effect and not relevant to mammalians. It has been demonstrated in various publications that these bacteria-specific Ames positive substances are not mutagenic in mammalian assays as was also shown in the following HPRT assay in mammalian cells.

The test item Disperse Red DYGJ 0702 was examined for mutagenic activity by assaying for the induction of 6-thioguanine resistant mutants in Chinese hamster V79 cells after in vitro treatment. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone. Test item solutions/suspensions were prepared using dimethylsulfoxide (DMSO). A preliminary cytotoxicity assay was performed. Based on solubility features, the test item was assayed, in the absence and presence of S9 metabolism, at a maximum dose level of 125 μg/mL and at a wide range of lower dose levels: 62.5, 31.3, 15.6, 7.81, 3.91, 1.95, 0.987 and 0.488 μg/mL. Since neither relevant toxicity nor precipitation of the test item was observed at any concentration tested, in the absence or presence of S9 metabolism, a higher range of concentrations was selected for the mutation assays. Two independent assays for mutation to 6-thioguanine resistance were performed using the following dose levels:

Main Assay I (- S9): 500, 250, 125, 62.5, 31.3, 15.6 and 7.81 μg/mL.

Main Assay I (+ S9): 500, 250, 125, 62.5, 31.3 and 15.6 μg/mL

Main Assay II (- S9): 278, 154, 85.7, 47.6, 26.5 and 14.7 μg/mL

Main Assay II (+ S9): 500, 278, 154, 85.7, 47.6 and 26.5 μg/mL

The dose range used in Main Assay II was modified taking into account precipitation and toxicity observed by the end of treatment in the previous Main Assay. No reproducible five-fold increases in mutant numbers or mutant frequency were observed following treatment with the test item at any dose level, in the absence or presence of S9 metabolism. Negative and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolism. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system. It is concluded that Disperse Red DYGJ 0702 does not induce gene mutation in Chinese hamster V79 cells after in vitro treatment, in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Short description of key information:
Only bacteria-specific effects were noted in the bacteria reverse mutation assay, whereas the mutagenicity study in mammalian cells was negative.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Mutagenicity Assessment Disperse Red DYGJ 0702

The test item Disperse Red DYGJ 0702 was tested positive in the Ames test, but was negative in the mutation assay in mammalian cells. 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.

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 inSalmonellastrains TA97a, TA98 and TA100, all without S9, but negative in the nitroreductase-deficient strains TA98NR and TA100NR. Accordingly, the ICH standard battery mouse lymphoma tk and 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.

 

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 [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. Thus, fexinidazole does not pose a genotoxic hazard to patients and represents a promising drug candidate for HAT.

 

Conclusion

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 DYGJ 0702 is a bacteria specific effect and not relevant to mammalians.

References

De Oliveira IM, Bonatto D, Pega Henriques JA. 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, 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.

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.