2-substitutedalkyl N-{3-acetamido-4-[(2-cyano-4,6-disubstitutedphenyl)diazenyl]phenyl}-N-benzyl-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 mutatiion assay, whereas the mutagenicity study in mammalian cells was negative.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 January 2015 to 30 April 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Compliant with GLP and testing guidelines; coherence among data, results and conclusion.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

The test item Disperse Blue 1092 was examined for mutagenic activity by assaying for the induction of 6-thioguanine resistant mutants in Chinese
hamster V79 cells after in vitro treatment. 6-thioguanine can be metabolised by the enzyme hypoxanthine-guaninphosphoribosyl-transferase (HPRT) into nucleotides, which are used in nucleic acid synthesis resulting in the death of HPRTcompetent cells. HPRT-deficient cells, which are presumed to arise through
mutations in the HPRT gene, cannot metabolise 6-thioguanine and thus survive and grow in its presence.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: EMEM medium supplemented with 10% Foetal Calf Serum (EMEM complete)
- Properly maintained: yes; Permanent stocks of the V79 cells are stored in liquid nitrogen. Subcultures are prepared from the frozen stocks for experimental
use.
- Periodically checked for Mycoplasma contamination: yes
- The karyotype, generation time, plating efficiency and mutation rates (spontaneous and induced) have been checked in this laboratory.
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 tissue fraction: Species: Rat; Strain: Sprague Dawley; Tissue: Liver Inducing Agents: Phenobarbital – 5,6-Benzoflavone Producer: MOLTOX, Molecular Toxicology, Inc. Batch Numbers: 3332, 3350 and 3417

Test concentrations with justification for top dose:

A preliminary cytotoxicity assay was performed. Based on solubility features, the test item was assayed at a maximum dose level of 39.1 μg/mL and at a wide range of lower dose levels: 19.6, 9.78, 4.89, 2.44, 1.22, 0.611, .0305 and 0.153 μg/mL.
Two independent assays for mutation to 6-thioguanine resistance were performed using the following dose levels:
Main Assay I (+S9): 78.1, 39.1, 19.5, 9.77, 4.88, and 2.44 μg/mL.
Main Assay I (-S9): 39.1, 19.5, 9.77, 4.88, and 2.44 μg/mL.
Main Assay II (+S9): 80.0, 44.4, 24.7, 13.7, 7.62 and 4.23 μg/mL.
Main Assay II (-S9): 40.0, 25.0, 15.6, 9.77, 6.10 and 3.81 μg/mL.

Vehicle / solvent:

Test item solutions were prepared using dimethylsulfoxide (DMSO).

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

A preliminary cytotoxicity test was undertaken in order to select appropriate dose levels for the mutation assays. Treatments were performed both in the absence and presence of S9 metabolism; a single culture was used at each test point and positive controls were not included.
Two mutation assays were performed including negative and positive controls, in the absence and presence of S9 metabolising system. Duplicate cultures were prepared at each test point, with the exception of the positive controls which were prepared in a single culture. On the day before the experiment, sufficient numbers of 75 cm2 flasks were inoculated with 2 million freshly trypsinised V79 cells from a common pool. The cells were allowed to attach overnight prior to treatment. Following treatment, the cultures were incubated at 37°C for three hours. At the end of the incubation period, the treatment medium was removed and the cell monolayers were washed with PBS. Fresh complete medium was added to the flasks which were then returned to the incubator at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) to allow for expression of the mutant phenotype.
Determination of survival: The following day, the cultures were trypsinised and an aliquot was diluted and plated to estimate the viability of the cells.
Subculturing:On Day 3, the cell populations were subcultured in order to maintain them
in exponential growth. When Day 8 is used as expression time, subculturing was performed on Day 4 and Day 6.
Determination of mutant frequency: A single expression time was used for each experiment. Day 8 (Main I) and Day 6 (Main II) were used alternatively in the experiments. At the expression time, each culture was trypsinised, resuspended in complete medium and counted
by microscopy. After dilution, an estimated 1 x 10^5cells were plated in each of five 100 mm tissue culture petri dishes containing medium supplemented
with 6-thioguanine (at 7.5 μg/mL). These plates were subsequently stained with Giemsa solutions and scored for the presence of mutants. After dilution,
an estimated 200 cells were plated in each of three 60 mm tissue culture petri dishes. These plates were used to estimate Plating Efficiency (P.E.).

Evaluation criteria:

For a test item to be considered mutagenic in this assay, it is required that:
– There is a five-fold (or more) increase in mutation frequency compared with the solvent controls, over two consecutive doses of the test item. If only the highest practicable dose level (or the highest dose level not to cause unacceptable toxicity) gives such an increase, then a single treatment-level will suffice.
– There must be evidence for a dose-relation (i.e. statistically significant effect in the ANOVA analysis).

Statistics:

The results of these experiments were subjected to an Analysis of Variance in which the effect of replicate culture, expression time and dose level in
explaining the observed variation was examined. For each experiment, the individual mutation frequency values at each test point were transformed
to induce homogeneous variance and normal distribution. The appropriate transformation was estimated using the procedure of Snee and Irr (1981), and
was found to be y = (x + a)b where a = 0 and b = 0.275. A two way analysis of variance was performed (without interaction) fitting to two factors:
– Replicate culture: to identify differences between the replicate cultures treated.
– Dose level: to identify dose-related increases (or decreases) in response, after allowing for the effects of replicate cultures and expression time.
The analysis was performed separately with the sets of data obtained in the absence and presence of S9 metabolism.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Survival after treatment:
In Main Assay I, no relevant toxicity was observed at any concentration tested, in the absence or presence if S9 metabolic activation.
In Main Assay II, in the absence of S9 metabolism, mild toxicity was observed at the two highest dose levels of 25.0 and 40.0 μg/mL reducing survival to 61% and 55%, respectively; while no relevant toxicity was noted over the remaining concentrations tested. In the presence of S9 metabolism, no relevant toxicity was observed at any concentration tested.

Mutation results:
No relevant increases over the spontaneous mutation frequency were observed in any experiment, at any treatment level either in the absence or presence of S9 metabolic activation. Analysis of variance indicated that dose levels and replicate culture were not significant factors in explaining the observed variation in the data, in the absence and presence of S9 metabolism, in Main Assay I and II.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

It is concluded that Disperse Blue 1092 does not induce mutation in Chinese hamster V79 cells after in vitro treatment, in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Executive summary:

The test item Disperse Blue 1092 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 beta-naphthoflavone. Test item solutions 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 39.1 μg/mL and at a wide range of lower dose levels: 19.6, 9.78, 4.89, 2.44, 1.22, 0.611, .0305 and 0.153 μg/mL. A reduction of cell viability was observed at the two highest dose levels (39.1 and 19.6 μg/mL), in the absence of S9 metabolism while no relevant toxicity was observed at any dose levels, in the presence of S9 metabolism. Precipitation of the test item was noted at the highest concentration tested (39.1 μg/mL), in the absence and presence of S9 metabolism; a coloured film, adhering to the flask surface, was noted starting from 2.44 μg/mL, in the absence of S9 metabolism. Two independent assays for mutation to 6-thioguanine resistance were performed using the following concentrations:

Main Assay I (+S9): 78.1, 39.1, 19.5, 9.77, 4.88, and 2.44 μg/mL.

Main Assay I (-S9): 39.1, 19.5, 9.77, 4.88, and 2.44 μg/mL.

Main Assay II (+S9): 80.0, 44.4, 24.7, 13.7, 7.62 and 4.23 μg/mL.

Main Assay II (-S9): 40.0, 25.0, 15.6, 9.77, 6.10 and 3.81 μg/mL.

Since, in the preliminary toxicity test, no precipitation of the test item was noted in the presence of S9 metabolic activation, in the first experiment 78.1 μg/mL was selected as the maximum dose level to be tested in the presence of S9 metabolism. The concentration range of the second experiment was slightly modified on the basis of the results obtained in the first experiment. 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 1092 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

The test item Disperse Blue 1092 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 Blue 1092 was assayed in the toxicity test at a maximum concentration of 781 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 247, 78.1, 24.7, and 7.81 µg/plate. Precipitation of the test item was observed at the end of the incubation period at the highest concentration. In addition, for the tester strains WP2 uvrA and TA98, precipitation was also observed at the next lower concentration of 247 µg/plate in the presence of S9 metabolism. No toxicity was observed with any tester strain in the absence or presence of S9 metabolism. Dose related increases in revertant numbers were observed both in the absence and presence of S9 metabolism with all tester stains with the exception of WP2 uvrA.

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: 781, 391, 195, 97.6 and 48.8 µg/plate. Precipitation of the test item was observed at the end of the incubation period at the highest concentration 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 statistically significant and biologically relevant increases in the number of revertant colonies in the absence and presence of S9 metabolism with all tester strains with the exception of WP2 uvrA. Since a clear positive response was observed, no further experiment was undertaken.

Conclusion: It is concluded that the test item Disperse Blue 1092 induces reverse mutation in all Salmonella strains in the absence and presence of S9 metabolism, under the reported experimental conditions.

The test item Disperse Blue 1092 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 beta-naphthoflavone. Test item solutions 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 39.1 μg/mL and at a wide range of lower dose levels: 19.6, 9.78, 4.89, 2.44, 1.22, 0.611, .0305 and 0.153 μg/mL. A reduction of cell viability was observed at the two highest dose levels (39.1 and 19.6 μg/mL), in the absence of S9 metabolism while no relevant toxicity was observed at any dose levels, in the presence of S9 metabolism. Precipitation of the test item was noted at the highest concentration tested (39.1 μg/mL), in the absence and presence of S9 metabolism; a coloured film, adhering to the flask surface, was noted starting from 2.44 μg/mL, in the absence of S9 metabolism. Two independent assays for mutation to 6-thioguanine resistance were performed using the following concentrations:

Main Assay I (+S9): 78.1, 39.1, 19.5, 9.77, 4.88, and 2.44 μg/mL.

Main Assay I (-S9): 39.1, 19.5, 9.77, 4.88, and 2.44 μg/mL.

Main Assay II (+S9): 80.0, 44.4, 24.7, 13.7, 7.62 and 4.23 μg/mL.

Main Assay II (-S9): 40.0, 25.0, 15.6, 9.77, 6.10 and 3.81 μg/mL.

Since, in the preliminary toxicity test, no precipitation of the test item was noted in the presence of S9 metabolic activation, in the first experiment 78.1 μg/mL was selected as the maximum dose level to be tested in the presence of S9 metabolism. The concentration range of the second experiment was slightly modified on the basis of the results obtained in the first experiment. 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 1092 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.

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.

Justification for classification or non-classification

Mutagenicity Assessment Disperse Blue 1092

The test item Disperse Blue 1092 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 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 metaboliteis released by intestinal bacteria, a Muta^TM 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.

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 1092 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.