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Additional information

The test item Disperse Yellow DYLA 1306 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: Based on results obtained in a preliminary solubility trial, the test item Disperse Yellow DYLA 1306 was assayed in the toxicity test at a maximum concentration of 248 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 78.4, 24.8, 7.84 and 2.48 µg/plate. Precipitation of the test item was observed at the end of the incubation period at the two highest concentrations. No toxicity was observed with any tester strain in the absence or presence of S9 metabolism. Increases in revertant numbers were observed in the presence of S9 metabolism with TA100 tester strain.

Main Assay: 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: 248, 124, 62.0, 31.0 and 15.5 μg/plate.

Precipitation of the test item was observed at the end of the incubation period at the two highest concentrations both in the absence and presence of S9 metabolic activation.

No toxicity was observed with any tester strain at any dose level in the absence or presence of S9 metabolism. The test item induced increases in the number of revertant colonies in the presence of S9 metabolism with TA100 tester strain reaching a two-fold increase at the highest concentration tested compared to the vehicle control, but not compared to the untreated control (MF 1.8 -fold).

Following the approval of a Protocol Amendment, an additional confirmatory experiment (Main Assay II) was performed in which TA100 tester strain was treated in the presence of S9 metabolism at the dose levels of 248, 165, 110, 73.3, 48.9 and 32.6 μg/plate.

Dose related increases in revertant numbers were observed. These increases were greater than twice the control values at the two highest dose levels, thus confiming the positive response with TA100 tester strain in the presence of S9 metabolism. No further experiment was undertaken.

Conclusion

It is concluded that the test item Disperse Yellow DYLA 1306 induces reverse mutation in Salmonella typhymurium TA100 tester strain in the presence of S9 metabolism, under the reported experimental conditions.

However, this effects was considered to be a bacteria-specific effect, as illustrated below and proven by the mutagenicity test in mammalian cells.

The test item Disperse Yellow DYLA 1306 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 100 μg/mL and at a wide range of lower dose levels: 50.0, 25.0, 12.5, 6.25, 3.13, 1.56, 0.781 and 0.391 μg/mL. No relevant toxicity was observed at any concentration tested, in the absence or presence of S9 metabolism. Precipitation of the test item was noted starting from 12.5 μg/mL, both in the absence and presence of S9 metabolism. Two independent assays for mutation to 6-thioguanine resistance were performed using dose levels described in the following table:

Main Assay I (+/- S9): 25.0, 12.5, 6.25, 3.13, 1.56 and 0.781 μg/mL

Main Assay II (+/- S9): 10.0, 6.25, 3.91, 2.44 and 1.53 μg/mL

The dose range used in Main Assay II was modified taking into account precipitation 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 Yellow DYLA 1306 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.

Hence, Disperse Yellow DYLA 1306 is not mutagenic.


Short description of key information:
The mutagenic effects observed in the Ames test with the test item is a bacteria specific effect which is well investigated in nitro-compounds and is not relevant to mammalians.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Mutagenicity Assessment Disperse Yellow DYLA 1306

The test item Disperse Yellow DYLA 1306 was tested weakly positive in the Ames test with metabolic activation in Samonella typhimurium TA100 but negative in an HPRT 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 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 lymphoma tk and mouse bone marrow micronucleus tests were negative, although a weak high toxicity-associated genotoxic activity was seen in a micronucleus test in V79 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 MutaTM Mouse 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 four in vivo assays 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.

 

 

The fact that this effect was only observed with metabolic activation and only in Samonella typhimurium TA100, is due to the fact that the metabolic activation of the aromatic amine group has been shown to be primarily prevented by steric hindrance [Klein et al 2000; Kazius et al 2005]. Hence, the nitroreductase could only activate the structure after the protective group has been removed by enzymes of the metabolising system. However this is only the case in one Salmonella strain with plasmid pKM101, whose mucAB gene products enhance SOS mutagenesis; which makes strain TA100 more sensitive for mutagen detection [Prival et Zeiger 1998].

 

 

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 Yellow DYLA 1306 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. InCurrent Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology; Mendez-Vilas, A., Ed.; Formatex: Badajoz, Spain, 2010:1008–1019.

Kazius J, McGuire R, Bursi R: Derivation and validation of toxicophores for mutagenicity prediction. J Med Chem 2005;48:312–320.

Klein M, Voigtmann U, Haack T, Erdinger L, Boche G. From mutagenic to nonmutagenic nitroarenes: effect of bulky alkyl substituents on the mutagenic activity of 4-nitrobiphenyl in Salmonella typhimurium. Part I. Substituents ortho to the nitro group and in 2’-position. Mutat. Res. 2000;467:55-68.

Prival MJ, Zeiger E. Chemicals mutagenic in Salmonella typhimurium strain TA1535 but not in TA100, Mutat. Res. 1998;412:251–260.

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.