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Administrative data

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, whereas the mutagenicity study in mammalian cells was negative. In addition, the test substance did not show clastogenic or aneugenic effects.

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:
09 December 2014 to 03 March 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
The test item 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 HPRT-competent 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 stock of V79 cells are stored in liquid nitrogen and subcoltures 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 and 3263
Test concentrations with justification for top dose:
A preliminary cytotoxicity assay was performed at the following dose levels: 625, 313, 156, 78.1, 39.1, 19.5, 9.77, 4.88 and 2.44 µg/mL.
Two independent assays for mutation to 6-thioguanine resistance were performed using the following concentrations:
Main Assay I (+S9): 625, 313, 156, 78.1, and 39.1 µg/mL
Main Assay I (-S9): 156, 78.1, 39.1, 19.5, and 9.77 µg/mL
Main Assay II (+S9): 156, 78.0, 39.0, 19.5 and 9.75 µg/mL
Main Assay II (-S9): 39.0, 19.5, 9.75, 4.88 and 2.44 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. 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.
Determination of mutant frequency: A single expression time was used for each experiment: Day 6 in Main Assay I and Day 8 in Main Assay II. At the expression time, each culture was trypsinised, resuspended in complete medium and counted by microscopy. After dilution, an estimated 1 x 105 cells 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 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.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Survival after treatment: No relevant toxicity was observed at any concentration tested, in any experiment in the absence or presence of S9 metabolis.
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 replicate culture was not a significant factor in explaining the observed variation in the data, in the absence and presence of S9 metabolism, in Main Assay I and II. Dose level was a significant factor (p < 0.05%) in explaining the observed variation in the data, in Main Assay I in the absence of S9 metabolism.
Conclusions:
It is concluded that the test item 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 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. Based on solubility features, the test item was assayed at a maximum dose level of 625 µg/mL and at a wide range of lower dose levels: 313, 156, 78.1, 39.1, 19.5, 9.77, 4.88 and 2.44 µg/mL. No relevant toxicity was observed at any concentration tested, in the absence or presence of S9 metabolism. A coloured film, adhering to the flask surface, was noted starting from 39.1 µg/mL, in the absence of S9 metabolism and at the three highest dose levels in the presence of S9 metabolism.

Two independent assays for mutation to 6-thioguanine resistance were performed using the following concentrations:

Main Assay I (+S9): 625, 313, 156, 78.1, and 39.1 µg/mL

Main Assay I (-S9): 156, 78.1, 39.1, 19.5, and 9.77 µg/mL

Main Assay II (+S9): 156, 78.0, 39.0, 19.5 and 9.75 µg/mL

Main Assay II (-S9): 39.0, 19.5, 9.75, 4.88 and 2.44 µg/mL

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 the substance 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

The test item 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 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 highest concentration. 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 WP2 uvrA with S9 metabolism. Due to a toxic effect, induced mutant colonies were markedly reduced at the highest dose level with TA1537 tester strain both in the absence and 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. Since a clear positive response was observed, no further experiment was undertaken.

Conclusion: It is concluded that the test item 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.

The test item 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. Based on solubility features, the test item was assayed at a maximum dose level of 625 µg/mL and at a wide range of lower dose levels: 313, 156, 78.1, 39.1, 19.5, 9.77, 4.88 and 2.44 µg/mL. No relevant toxicity was observed at any concentration tested, in the absence or presence of S9 metabolism. A coloured film, adhering to the flask surface, was noted starting from 39.1 µg/mL, in the absence of S9 metabolism and at the three highest dose levels in the presence of S9 metabolism.

Two independent assays for mutation to 6-thioguanine resistance were performed using the following concentrations:

Main Assay I (+S9): 625, 313, 156, 78.1, and 39.1 µg/mL

Main Assay I (-S9): 156, 78.1, 39.1, 19.5, and 9.77 µg/mL

Main Assay II (+S9): 156, 78.0, 39.0, 19.5 and 9.75 µg/mL

Main Assay II (-S9): 39.0, 19.5, 9.75, 4.88 and 2.44 µg/mL

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 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

1.1

0.5

0.3-3.0

100

62.5

1.1

0.2

0.8-1.8

95

250

1.5

0.1

1.0-1.8

101

1000

1.4

0.3

0.0-1.8

92

Mitomycin-C

2.00 mg/kg

 

 

11.3

 

1.4

 

7.8-16.0

 

88

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 does not induce micronuclei in the polychromatic erythrocytes of treated rats, under the reported experimental conditions.

Justification for classification or non-classification

Mutagenicity Assessment Disperse Green GNA

The test item 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 MutaTMMouse 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 the test item 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. InCurrent Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology; Mendez-Vilas, A., Ed.; Formatex: Badajoz, Spain, 2010:1008–1019.