Glycine, N-[5-(acetylamino)-4-[2-(2-bromo-4,6-dinitrophenyl)diazenyl]-2-methoxyphenyl]-N-(2-methoxy-2-oxoethyl)-, methyl ester

Endpoint summary

Additional information

The test item Disperse Blue ANT 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 Blue ANT 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. Precipitation of the test item was observed at the end of the incubation period at the two highest concentrations. Due to the dark color of plates the background lawn was not analysable at the two highest dose levels. Large increases in revertant numbers were observed both in the absence and presence of S9 metabolic activation with all tester strains with the exception of TA1535 and WP2 uvrA without S9 metabolism and WP2 uvrA with S9 metabolism. Due to a toxic effect, induced mutant colonies were markedly reduced at the highest dose level with TA98 and TA 100 tester strain in the presence of S9 metabolism.

Main Assay I: On the basis of toxicity test results, in Main Assay I, using the plate incorporation method, the test item was assayed by using the following dose levels: 5000, 2500, 1250, 625 and 313 µg/plate. Precipitation of the test item was observed at the end of the incubation period at the two highest concentrations. At these dose levels the background lawn was not analysable due to the dark color of plates. The test item induced large increases in the number of revertant colonies using the plate incorporation method in all tester strains in the absence and presence of S9 metabolism with the exception of TA1535 and WP2 uvrA. Since a clear positive response was observed, no further experiment was undertaken.

Conclusion: It is concluded that the test item, Disperse Blue ANT, induces reverse mutation by base substitutions or frameshifts in bacteria in the absence and presence of S9 metabolism in the Salmonella strains TA98, TA100 and TA1537, under the reported experimental conditions.

The test item Disperse Blue ANT (Br) 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 were prepared using dimethylsulfoxide (DMSO). A preliminary cytotoxicity assay was performed, in the absence and presence of S9 metabolic activation. Based on solubility features, the test item was assayed at a maximum dose level of 1250 µg/mL and at a wide range of lower dose levels: 625, 313, 156, 78.1, 39.1, 19.5, 9.77 and 4.88 µg/mL. In the absence of S9 metabolism, no cells survived at almost all the concentrations tested; survival was reduced to 50% of the concurrent negative control value at the lowest dose level (4.88 µg/mL). In the presence of S9 metabolism, severe toxicity was noted at the two highest concentrations, while survival was reduced to 44% of the concurrent negative control value at 313 µg/mL. No relevant toxicity was noted over the remaining concentrations tested. By the end of treatment time, precipitation of test item and/or a coloured film, adhering to the flask surface, was observed at all concentrations tested in the absence of S9 metabolism and at the three highest dose levels in its presence. In order to select the appropriate range of concentrations for the mutation assay, an additional toxicity test was performed in the absence of S9 metabolism using the following lower and closer concentrations: 10.0, 7.14, 5.10, 3.64 and 2.60 µg/mL. Since no adequate toxicity for dose selection was obtained, a third toxicity test was performed in the absence of S9 metabolism, using the following concentrations: 40.0, 20.0, 10.0, 5.00 µg/mL. Severe toxicity was noted at the highest concentration; survival was reduced to 12% and 37% of the concurrent negative control value at 20.0 and 10.0 µg/mL, respectively; while no relevant toxicity was observed at 5.00 µg/mL. Two independent assays for mutation to 6-thioguanine resistance were performed using the following dose levels:

Main Assay I (+S9): 600, 400, 267, 178, 119 and 79.0 µg/mL

Main Assay I (-S9): 40.0, 20.0, 10.0, 5.00, 2.50, 1.25 and 0.625 µg/mL

Main Assay II (+S9): 400, 308, 237, 182, 140 and 108 µg/mL

Main Assay II (+S9): 20.0, 15.4, 11.8, 9.10, 7.00 and 5.39 µg/mL

In the second experiment the concentration range was slightly modified on the basis of the toxicity results obtained in Main Assay I. 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 Blue ANT (Br) 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.

The ability of the test item 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. The test was only performed in males, as no toxicological relevant difference in target organ toxicity was noted.

Samples of bone marrow were collected approximately 24 hours following the final treatment and approximately 48 hours following the second last treatment from 5 males of the main groups randomly selected and from all animals of the positive control group consisting of 5 males. One femur of each animal was removed and bone marrow cells obtained by flushing with foetal calf serum. The cells were centrifuged and a concentrated suspension prepared to make smears on slides. These slides were air-dried, fixed with methanol and then stained with haematoxylin and eosin solutions and mounted with Eukitt. Three slides were made from each animal.

The slides were randomly coded by a person not involved in the subsequent microscope scoring and examined under low power to select one or more slides from each animal according to staining and quality of smears. Four thousand polichromatic erythrocytes (PCEs) per animal were examined for the presence of micronuclei at high power (x 100 objective, oil immersion). At the same time, the numbers of normal and micronucleated normochromatic erythrocytes (NCEs) were also recorded

Findings noted during treatment (discoloured urine/bedding) were considered a proof of absorption.

The ratio of mature to immature erythrocytes and the proportion of immature erythrocytes among total erythrocytes were analysed to evaluate the bone marrow cell toxicity. Based on these results, no relevant inhibitory effect on erythropoietic cell division was observed at any dose level.

Following treatment with the test item, no relevant increase in the number of micronucleated PCEs was observed at any dose level.

A marked increase in the frequency of micronucleated PCEs was observed in the positive control group.

A summary of the results obtained is presented in the following table:

Dose level (mg/kg/day)	Incidence in micronucleated PCEs			PCE/s(PCEs+NCEs) % over the mean control value
	Mean	SE	Range
0.00	0.4	0.2	0.0-1.0	100
62.5	1.2	0.3	0.5-2.3	93
250	1.0	0.4	0.0-2.3	92
1000	1.2	0.4	0.5-2.5	100
Mitomycin-C 2.00 mg/kg	11.3	1.7	7.0 - 17.3	89

The incidence of micronucleated PCEs of the negative control group fell within the historical control range (95% confidence limit). Statistically significant increases in the incidence of micronucleated PCEs over the negative control values were seen in the positive control group. The induced response was compatible with the historical control range, demonstrating the laboratory proficiency in the conduct of the test. Five animals per groups were available for micronucleus slide analysis. Based on the stated criteria, the assay was therefore accepted as valid.

On the basis of the results obtained, it is concluded that Disperse Blue ANT does not induce micronuclei in the polychromatic erythrocytes of treated rats, under the reported experimental conditions.

Justification for selection of genetic toxicity endpoint
The mutagenic effects observed in the Ames test is a bacteria specific effect and not relevant to mammalians.

Short description of key information:
Only bacteria-specific effects were noted in the bacteria reverse mutatiion assay, whereas the mutagenicity study in mammalian cells was negative. In addition, the test substance did not show clastogenic or aneugenic effects.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Mutagenicity Assessment Disperse Blue ANT

The test item Disperse Blue ANT 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 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.

 

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 Blue ANT is a bacteria specific effect and not relevant to mammalians.This conclusion is affirmed by the results of the mutagenicity test in mammalian cells, which was unequivocally negative.

 

References

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.

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.