4-[[2-methoxy-4-[(4-nitrophenyl)azo]phenyl]azo]phenol

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 mutation assay, whereas the mutagenicity study in mammalian cells was negative.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Ames test

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

S-9 fraction (rat liver homogenate)

Test concentrations with justification for top dose:

0, 4, 20, 100, 500, 2500 and 5000 µg/plate
(3 test plates per dose or per control)

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

other: 2-aminoanthracene, N-methyl-N'-nitro-N-nitroso-guanidine, 4-nitro-o-phenylenediamine

Details on test system and experimental conditions:

To 2 mL of molten top agar in a sterile test-tube, were added 0.1 mL of the tester strain culture, graded quantities of the test substance in 0.1 mL solution and, for the S-9 series, 0.5 mL of S-9 Mix. The contents of the test-tube were rapidly mixed and poured onto the surface of previously prepared minimal agar plates with Vogel-Bonner E mixture. The plate were incubated upside down at 37°C for 2 days, after which the number of revertants colonies appearing was counted.

Evaluation criteria:

Doubling of the spontaneous mutation rate (control)

Key result

Species / strain:

other: Salmonella typhimurium TA98, TA100, TA1535, and TA1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Positive and negative controls were valid

Without metabolic activation:

TA1535: Weakly positive reaction at 5000 µg/plate (factor 3.0). TA100: Mutagenicity was observed over a dose range of 500 µg - 5000 µg/plate (factor 2.4 - 5.3). TA1537: Positive reaction from about 20 µg/plate (factor 2.11 onward with an increase in the number of his revertants by a factor of 21.3 at 5000 µg/plate. TA98: A positive reaction was detected from about 20 µg/plate (factor 2.9 3.21 onward; increase in the number of mutant colonies by a factor of 31.2 at 5000 µg/plate).

With metabolic activation:

TA1535: Slight increase in the number of mutant colonies at 500 µg - 5000 µg/plate (factor 2.4 - 3.7). TA100: Weakly positive reaction from about 100 µg/plate (factor 1 .6) onward up to 5000 µg/plate (factor 5.8). TA1537: Increase in the number of mutant colonies from about 100 µg/plate (factor 4.3) onward up to 2500 µg - 5000 µg/plate (factor 17.5 - 18 .0). TA98: Mutagenicity was observed from about 20 µg/plate (factor 3.0 - 3.3) onward with a maximum increase in the number of his revertants by a factor of 39.4 at 2500 µg/plate in the 1st experiment.

Conclusions:

Under the study conditions, the test substance was considered to be mutagenic in Salmonella typhimurium TA98, TA100, TA1535, and TA1537.

Executive summary:

A study was conducted to determine the mutagenic potential of the test substance (in the form of a powder of 94.5% purity) according to OECD Guideline 471. Four strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) were exposed to the test substance at concentrations of 4.0 to 5000.0 µg/plate with or without metabolic activation (S-9 Mix) for 48 h. Negative and positive controls were valid. The test substance with and without metabolic activation induced mutagenic activity in bacteria; the number of revertants was greater than twice the number of spontaneous mutations. Under the study conditions, the test substance was considered to be mutagenic (Engelbart, 1991).

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From September 22, 2015 to December 01, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

In Main Assay I the mutant frequency was evaluated at Day 9, instead of Day 8 as indicated in the Study Protocol. This deviation was not considered to have affected the integrity of the study.

GLP compliance:

yes

Type of assay:

other: HPRT mutation system in Chinese hamster V79 cells

Target gene:

enzyme hypoxanthine-guaninphosphoribosyl-transferase (HPRT)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S-9 (from Sprague-Dawley rat liver)

Test concentrations with justification for top dose:

0.977 to 250 µg/mL (to avoid recipitation of the test substance and DMSO toxicity) for 3h.
(Further reduced to 200 µg/mL because of opacity of treatment mixture, slight precipitation, and toxicity).

Vehicle / solvent:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

Mutation assay:
-Treatment of cell cultures. Two experiments 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. A number of cells was then replated in order to maintain the treated cell populations; the number of cells taken forward was adjusted according to the expected viability of the cultures, to give one million viable cells.
- Subculturing. On Day 3 or Day 4, the cell populations were subcultured in order to maintain them in exponential growth. When Day 9 is used as expression time, subculturing was performed also on Day 6. The number of cells taken forward was adjusted according to the expected viability, to give at least one million viable cells seeded in cell culture flasks.
- Determination of mutant frequency. A single expression time was used for each experiment: Day 9 in Main AssayI and Day 6 in Main Assay II. At the expression time, each culture was trypsinised, resuspended in complete medium and counted by microscope. After dilution, an estimated 1 x 10E05 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.).

Rationale for test conditions:

Preliminary studies

Evaluation criteria:

The assay was considered valid if the following criteria were met:
– The absolute plating efficiencies in the negative control cultures fell within the range of 50-130%.
– The mutant frequencies in the negative control cultures fell within the range of 1-100 x 106 viable cells.
– The total number of cells at Day 1 and Day 3 in the negative control cultures was higher than 5 x 106.
– The total number of cells at Day 4 in the negative control cultures was higher than 10 x 106.
– The absolute plating efficiencies in the positive control cultures fell within the range of 10-130%.
– The mutant frequencies in the positive control cultures fell within the distribution range of the historical control data (calculated as the 1° and 99° percentiles).
– At least four dose levels had to be analyzable (survival higher than 10%).

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:

cytotoxicity

Remarks:

in absence of metabolic activation at the two highest dose tested

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The mean total number and the morphology of the cells were normal. The mutant frequencies in the negative control cultures fell within the normal range. Treatment with the positive controls gave marked responses in all experiments indicating the correct functioning of the test system. The study was accepted as valid.

For a test substance to be considered mutagenic in this assay, it was required that:

– There was a five-fold (or more) increase in mutation frequency compared with the solvent controls, over two consecutive doses of the test substance. 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).

Survival after treatment:

- In the absence of S-9 metabolism, treatment at the two highest dose levels yielded a reduction of relative survival to approximately 60% of the negative control, while no relevant toxicity was observed over the remaining concentrations tested.

- In the presence of S-9 metabolism, no relevant toxicity was observed at any concentration tested.

(Opacity of the treatment medium was noted starting from 50.0 µg/mL, in the absence of S9 metabolism. Opacity and slight precipitation were observed at the highest and two highest dose levels, in the absence and presence of S9 metabolism, respectively).

Mutation results:

In absence of S-9 metabolic activation, no five-fold increase in mutation frequency over the concurrent solvent control was noted; analysis of variance indicated that dose level and replicate culture were not significant factors in explaining the observed variation in the data.

In the presence of S-9 metabolic activation, a five-fold increase in mutation frequency was observed at the three highest concentrations; dose level was a significant factor (p< 0.01%) since mutation frequencies were greater at higher concentrations. However, all mutation frequencies fell within the historical control range, with the only exception of one replicate culture at the highest concentration tested. Moreover, the mutation frequency of the negative control in the presence of S-9 was very low if compared with the spontaneous mutation frequency observed in the concurrent non-activated series and fell in the lower part of the distribution range of historical control data. Based on these remarks, the observed increase was considered a chance event not related to the action of the test item and of no biological significance.

Conclusions:

Under the study conditions, the test substance was not considered to be mutagenic in Chinese hamster lung fibroblasts (V79).

Executive summary:

A study was conducted to determine the genotoxicity of the test substance (in the form of a brown solid of 94.8% purity) according to OECD Guideline 476 and EU Method B.17. Chinese hamster lung fibroblasts (V79) were exposed at concentrations of 6.25 to 200 µg/mL with or without S-9 metabolic activation. Cell survival and mutations were assessed. In the absence of S-9 metabolism, treatment at the two highest dose levels yielded a reduction of relative survival to approximately 60% of the negative control, while no relevant toxicity was observed over the remaining concentrations tested. In the presence of S-9 metabolism, no relevant toxicity was observed at any tested concentration. No reproducible and biologically relevant increase in mutant frequency was observed at any dose level, in the absence or presence of S-9 metabolic activation. No reproducible evidence of a dose effect relationship was noticed. Under the study conditions, the test substance was not considered to be mutagenic in Chinese hamster lung fibroblasts (V79) (Venturella, 2015).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The test substance did not induce micronuclei in the polychromatic erythrocytes of treated rats.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From September 09, 2015 to December 02, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

but considered to have not affected the integrity of the study

Principles of method if other than guideline:

This study is included in a combined repeated dose toxicity study with the reproduction/developmental toxicity screning test in rats. Here, only the assessment of genotoxicity will be adressed. The ability of the test substance 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.

GLP compliance:

yes

Type of assay:

other: Mammalian Erythrocyte Micronucleus test

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

- A total of 130 Hsd: Sprague Dawley SD rats (65 males and 65 virgin females), 6 to 7 weeks old and weighing 176 to 200 g for males and 151 to 175 g for females, were ordered from Harlan Italy s.r.l., San Pietro al Natisone (UD), Italy. 5 animals per cage.
- Acclimatisation period of approximately 3 to 9 weeks, depending on the type of treatment.
- Temperature and relative humidity: 22+/- 2°C and 55 +/- 15%, respectively.
- Artificial light for 12 hours.
- Feed (commercially available laboratory rodent diet (4 RF 21) and water: ad libitum.

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

The test substance was administered orally by gavage to animals at 5 mL/kg bw. The oral route was selected as it is a possible route of exposure of the test substance in man.

Duration of treatment / exposure:

Main groups:
- Males animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing, through the mating period and thereafter at least until the minimum total dosing period of 28 days has been completed including the day before necropsy. Dose volumes will be adjusted once per week for each animal according to the last recorded body weight.
- Females animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until at least up to, and including, Day 3 post partum or the day before sacrifice. Dose volumes will be adjusted once per week for each animal according to the last recorded body weight. During the gestation period, dose volumes will be calculated according to individual body weight on Days 0, 7, 14 and 20 post coitum and on Day 1 post partum. Thereafter individual dose volumes will remain constant.
Positive control group:
- Animals will receive a single dose approximately 24 hours before sacrifice.

Frequency of treatment:

once daily

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Dose / conc.:

600 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Mitomycin-C (2.00 mg/kg)

Tissues and cell types examined:

- Bone marrow
- Erythrocytes

Details of tissue and slide preparation:

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 and 5 females of the main groups randomly selected and from all animals of the positive control group. 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. At least three slides were made from each animal.
At first, only slides from male animals were examined. Subsequently, in order to obtain bone marrow toxicity information, slides from two female animals treated at the high dose level and two females from the vehicle control group, were scored.

Evaluation criteria:

- 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.
- The test substance was considered to induce micronuclei if a statistically significant increase in the micronucleus incidence of polychromatic erythrocytes (at P<0.05) was observed in any treatment group and a dose-effect relationship was demonstrated. Where statistically significant increases in the incidence of micronucleated PCEs were observed, but all results were inside the distribution of negative control values within this laboratory, then historical control data were used to demonstrate that these increases did not have any biological significance.

Statistics:

Only counts obtained from polychromatic cells were subjected to statistical analysis and the original observations (and not micronucleus frequencies per 1000 cells) were used. The variation between individual animals within each treatment group was assessed by χ2 calculation. In case of no significant heterogeneity within either group, the χ2 test was employed to compare treated groups with the vehicle control. If at least one of the groups was not homogeneous, the variance ratio (F) value was calculated from the betweengroup and within-group χ2 values to show the significance of any difference between treated and vehicle control groups. In addition, a test for a linear trend (Snedecor and Cochran) was performed in order to evaluate dose-effect relationship.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- Following treatment with the test substance, no relevant increase in the number of micronucleated PCEs over the concurrent negative control (where values were within the historical control range) was observed at any dose level. A marked increase in the frequency of micronucleated PCEs was noticed in the positive control group.
- Bone marrow cell toxicity. 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.

No relevant differences in clinical signs were observed between male and female animals from the main and recovery groups. The only difference was seen in the post-partum phase, probably due to the stress of delivery and weaning. Also the preliminary scoring of slides from female animals did not show substantial differences between sexes, in terms of bone marrow toxicity and incidence of micronucleated cells. Based on these results, the genotoxicity assessment was performed including male animals only.

Conclusions:

Under the study conditions, the test substance did not induce micronuclei in the polychromatic erythrocytes of treated rats.

Executive summary:

A combined study was conducted to determine the repeated dose toxicity, the reproductive and the in vivo genetic toxicity of the test substance, according to OECD Guidelines 422 and 474. The ability of the test substance 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. Male rats (5 in each groups) were exposed to the test substance at concentrations of 0, 100, 250 and 600 mg/kg bw/day for periods ranging from 29 to 42 days (depending on the dosing, the symptoms, and the timing of sacrifice). A positive control group (mitomycin-C, 2.0 mg/kg) was also tested. Following treatment with the test substance, no relevant increase in the number of micronucleated PCEs over the concurrent negative control (where values were within the historical control range) was observed at any dose level. A marked increase in the frequency of micronucleated PCEs was noticed in the positive control group. 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. No relevant inhibitory effect (no toxicity) on erythropoietic cell division was observed at any dose level. The values for the positive and negative controls were within the expectation ranges. The experiment was therefore considered valid. Under the study conditions, the test substance did not induce micronuclei in the polychromatic erythrocytes of treated rats (Rossiello, 2016).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro

A study was conducted to determine the mutagenic potential of the test substance (in the form of a powder of 94.5% purity) according to OECD Guideline 471. Four strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) were exposed to the test substance at concentrations of 4.0 to 5000.0 µg/plate with or without metabolic activation (S-9 mix) for 48 h. Negative and positive controls were valid. The test substance with and without metabolic activation induced mutagenic activity in bacteria; the number of revertants was greater than twice the number of spontaneous mutations. Under the study conditions, the test substance was considered to be mutagenic (Engelbart, 1991).

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 this is a bacteria-specific effect and that these Ames positive substances are not mutagenic in mammalian assays (de Oliveira et al., 2010), as was also shown in the following HPRT assay in mammalian cells:

A study was conducted to determine the genotoxicity of the test substance (in the form of a brown solid of 94.8% purity) according to OECD Guideline 476 and EU Method B.17. Chinese hamster lung fibroblasts (V79) were exposed at concentrations of 6.25 to 200 µg/mL with or without S-9 metabolic activation. Cell survival and mutations were assessed. In the absence of S-9 metabolism, treatment at the two highest dose levels yielded a reduction of relative survival to approximately 60% of the negative control, while no relevant toxicity was observed over the remaining concentrations tested. In the presence of S-9 metabolism, no relevant toxicity was observed at any tested concentration. No reproducible and biologically relevant increase in mutant frequency was observed at any dose level, in the absence or presence of S-9 metabolic activation. No reproducible evidence of a dose effect relationship was noticed. Under the study conditions, the test substance was not considered to be mutagenic in Chinese hamster lung fibroblasts (V79) (Venturella, 2015).

In vivo

A combined study was conducted to determine the repeated dose toxicity, the reproductive and the in vivo genetic toxicity of the test substance, according to OECD Guidelines 422 and 474. The ability of the test substance 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. Male rats (5 in each groups) were exposed to the test substance at concentrations of 0, 100, 250 and 600 mg/kg bw/day for periods ranging from 29 to 42 days (depending on the dosing, the symptoms, and the timing of sacrifice). A positive control group (mitomycin-C, 2.0 mg/kg) was also tested. Following treatment with the test substance, no relevant increase in the number of micronucleated PCEs over the concurrent negative control (where values were within the historical control range) was observed at any dose level. A marked increase in the frequency of micronucleated PCEs was noticed in the positive control group. 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. No relevant inhibitory effect (no toxicity) on erythropoietic cell division was observed at any dose level. The values for the positive and negative controls were within the expectation ranges. The experiment was therefore considered valid. Under the study conditions, the test substance did not induce micronuclei in the polychromatic erythrocytes of treated rats (Rossiello, 2016).

Justification for classification or non-classification

Based on the results of in vitro and in vivo testing, no classification for genotoxicity is required for the test substance according to CLP (EC 1272/2008) criteria.

Mutagenicity assessment

The substance was positive in the Ames test, but 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 (de Oliveira et al., 2010).

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). The fact that the reduction of these nitro-compounds to mutagenic metabolites is a bacteria-specific effect is demonstrated with the example 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 in V79 cells (Suteret 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 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 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.

Fexinidazolewas 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 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.

References

De Oliveira IM, Bonatto D, Pega Henriques JA (2010). 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:1008–1019.

Suter W, Hartmann A, Poetter F, Sagelsdorff P, Hoffmann P, Martus HJ (2002). Genotoxicity assessment of the antiepileptic drug AMP397, an Ames-positive aromatic nitro compound. Mutat. Res. 518(2):181-94.

Tweats D, Bourdin Trunz B, Torreele E (2012). Genotoxicity profile of fexinidazole--a drug candidate in clinical development for human African trypanomiasis (sleeping sickness). Mutagenesis 27(5):523-32.

  

Conclusion

Based on these data and the common mechanism between the reduction of these nitro-compounds, which is widely explored in literature, it is concluded, that the mutagenic effects observed in the Ames test is a bacteria specific effect and not relevant to mammals.