Methyl N-[4-[(2-bromo-6-chloro-4-nitrophenyl)azo]phenyl]-N-(3-methoxy-3-oxopropyl)-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 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

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From September 12, 1997 to October 13, 1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Test Item purity < 50%

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: "Chemikaliengesetz" (Chemicals Act) of the Federal Republic of Germany, „Anhang 1" (Annexe 1) dated July 25, 1994 („BGBl. I 1994", pp. 1703)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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:

Mammalian Microsomal Fraction S9 mix (liver)

Test concentrations with justification for top dose:

Triplicates: 0, 33, 100, 333, 1000, 2500 and 5000 ug/plate (active ingredient) (based upon the results of the pre-experiment)
(The test substance precipitated weakly at 2500 and 5000 ug/plate in the overlay agar. The undissolved particles of the test substance had no influence on the data recording.)

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene

Details on test system and experimental conditions:

Two independent Salmonella typhimurium reverse mutation assays:
Experiment I was performed as a plate incorporation assay. Since a positive result was obtained in this experiment, experiment II was performed as a plate incorporation assay as well.

Rationale for test conditions:

The Salmonella typhimurium histidine (his) reversion system measures his" -> his+ reversions. The S. typhimurium strains are constructed to differentiate between base pair (TA 1535, TA 100) and frameshift (TA 1537, TA 98) mutations.

Evaluation criteria:

The generally accepted conditions for the evaluation of the results are:
- corresponding background growth on both negative control and test plates,
- normal range of spontaneous reversion rates.

- A test substance is considered positive if either a biologically relevant and reproducible dose related increase in the number of revertants or a biologically relevant and reproducible increase for at least one test concentration is induced. A test substance producing neither a biologically relevant and reproducible dose related increase in the number of revertants nor a biologically relevant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.

-A biologically relevant response is described as follows:
A test asubstance is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate in strains TA 98 and TA 100 or thrice on TA 1535 and TA 1537. Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test substance regardless whether the highest dose induced the criteria described above or not.

Statistics:

Toxicity of the test substance was evidenced by a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn. The colonies were counted. The individual and mean values of the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates, were measured.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

with metabolic activation

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate (a.i.)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

without metabolic activation

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate (a.i.)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

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

Key result

Species / strain:

S. typhimurium TA 100

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:

- Cytotoxicity: In experiment I, toxic effects evident as a reduction in the number of revertants occurred at the highest concentration in strain TA 1535 with S9 mix and in strain TA 1537 without S9 mix. In experiment II, toxic effects occurred at the highest concentration in strains TA 1535 and TA 1537 without S9 mix.
The plates incubated with the test substance showed normal background growth up to 5000 ug/plate with and without S9 mix in all strains used.

- Genotoxicity: In both experiments, substantial and dose dependent increases in revertant colony numbers were observed following treatment with the test substance with and without metabolic activation in strains TA 1537, TA 98 and TA 100. The number of colonies reached or exceeded the threshold of twice (strains TA 98 and TA 100) and thrice (strain TA 1537) the number of the corresponding solvent control at concentrations as low as 33 ug/plate and above. In experiment I a dose dependent increase in revertant colony numbers was observed in strain TA 1535 with and without S9 mix. In the absence of metabolic activation the threshold of thrice the number of the corresponding solvent control was not quite reached. In the presence of metabolic activation the threshold was exceeded at 2500 ug/plate. In experiment II, a dose-dependent increase was observed in strains TA 1535 and TA 1537 in the presence of metabolic activation but the threshold was not reached. In the absence of metabolic activation the threshold was exceeded at 2500 ug/plate in strain TA 1537.
- Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

Conclusions:

Under the study conditions, the test substance was considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.

Executive summary:

A study was conducted to determine the in vitro mutagenic potential of the test substance according to OECD Guideline 471, in compliance with GLP. The assay was performed in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 in two independent experiments both with and without liver microsomal activation (S9 mix). Each concentration and the controls were tested in triplicate. The test substance was tested at the following concentrations: 33, 100, 333, 1000, 2500 and 5000 ug/plate. In Experiment I, cytotoxic effects evident as a reduction in the number of revertants occurred at the highest concentration in strain TA 1535 with S9 mix and in strain TA 1537 without S9 mix. In Experiment II, cytotoxic effects occurred at the highest concentration in strains TA 1535 and TA 1537 without S9 mix. The plates incubated with the test substance showed normal background growth up to 5000 ug/plate with and without S9 mix in all strains used. In both experiments, substantial and dose dependent increases in revertant colony numbers were observed following treatment with the test substance with and without metabolic activation in strains TA 1537, TA 98 and TA 100. Appropriate reference mutagens were used as positive control and showed a distinct increase in induced revertant colonies. Therefore, the test substance induced gene mutations by base pair changes and frameshifts in the genome of the Salmonella typhymurium strains TA98, TA100, TA1535 and TA1537. Under the study conditions, the test substance was considered to be mutagenic in this Salmonella typhimurium reverse mutation assay (Wollny, 1997).

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 17, 2015 to November 10, 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 II, the mutant frequency was evaluated at Day 9, instead of Day 8. This deviation was not considered to have affected the integrity of the study.

Qualifier:

according to guideline

Guideline:

other: Test method B.17 ‘in vitro mammalian cell gene mutation test’ described in Council Regulation (EC) No. 440/2008.

Deviations:

yes

Remarks:

In Main Assay II, the mutant frequency was evaluated at Day 9, instead of Day 8. This deviation was not considered to have affected the integrity of the study.

GLP compliance:

yes

Type of assay:

bacterial forward mutation assay

Target gene:

enzyme hypoxanthine-guanin phosphoribosyl-transferase (HPRT)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (from Sprague-Dawley rat liver)

Test concentrations with justification for top dose:

600, 300, 150, 75.0, 37.5, 18.8, 9.38, 4.69 and 2.34 µg/mL (exposure of 3 h).
(By the end of treatment, precipitation of the test substance was noted starting from 75.0 µg/mL in the absence of S9 metabolism and at the three highest dose levels in its presence. Opacity of the treatment medium was observed starting from 18.8 µg/mL, in the absence of S9 metabolism. Selection of dose levels used in Main Assay I was performed taking into account precipitation and toxicity observed in the preliminary cytotoxicity assay. The dose range used in Main Assay II was modified to focus on the highest concentrations that could be tested. The highest concentrations were finally: 120 or 150 and 300 µg/mL, in the absence and presence of S9 metabolism, respectively).

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

- Cytotoxicity assay (preliminary test - large range of concentrations)
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.
- Mutation assay (determination of survival and determination of mutant frequency)
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.

Evaluation criteria:

For a test substance 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 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).

Statistics:

- 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

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 300 µg/mL (Experiments I and II)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above 37.5 µg/mL (Experiment I) and at and above 60 µg/mL (Experiment II)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- Survival:
In Main Assay I, in the absence of S9 metabolism, moderate reduction in relative survival (RS=18-19%) was noted at the two highest dose levels (150.0 and 75.0 µg/mL), treatment at 37.5 µg/mL yielded a reduction of relative survival to 36% of the negative control, while no relevant toxicity was noted over the remaining concentrations tested. In the presence of S9 metabolism, slight reduction in relative survival (RS=70%) was noted at the highest dose level (300 µg/mL), while no relevant toxicity was observed over the remaining concentrations tested. In Main Assay II, in the absence of S9 metabolism, treatment at the highest dose (120 µg/mL) level yielded a reduction of relative survival to 11% of the negative control, moderate toxicity (RS=55%) was noted at the next lower concentration of 60.0 µg/mL, while no relevant toxicity was observed over the remaining concentrations tested. In the presence of S9 metabolism, moderate toxicity (RS=55%) was noted at the highest dose level (300 µg/mL), while no relevant toxicity was observed over the remaining concentrations tested. Opacity of the treatment medium was noted starting from 30.0 µg/mL, in the absence of S9 metabolism. Opacity and slight precipitation were observed at the highest dose level, both in the absence and presence of S9 metabolism.
- Mutation results:
No five-fold or greater increase in mutant frequency, compared with the negative control, was observed at the highest dose level or at two consecutive doses of the test substance, in the absence or presence of S9 metabolic activation. No reproducible evidence of a dose effect relationship was noticed. Marked increases were obtained with the positive control treatments, indicating the correct functioning of the assay system.
- Osmolality and pH measurements:
The addition of the test substance solution did not have any obvious effect on the osmolality or pH of the treatment medium.

.

Conclusions:

Under the study conditions, the test substance did not induce gene mutation in Chinese hamster V79 cells (HPRT).

Executive summary:

A study was conducted to determine the genetic toxicity in vitro of the test substance according to OECD Guideline 476 and EU Method B.17, in compliance with GLP. In an in vitro mammalian cell gene mutation test (forward gene mutation in HPRT), Chinese hamster V79 cells were exposed to the test substance at concentrations ranging from 2.34 to 300.0 µg/mL with or without metabolic activation (S9 mix). The positive and negative controls were valid. In Main Assay I, in the absence of S9 mix, moderate reduction in relative survival (RS=18-19%) was noted at the two highest dose levels (150.0 and 75.0 µg/mL), treatment at 37.5 µg/mL yielded a reduction of relative survival to 36% of the negative control, while no relevant toxicity was noted over the remaining concentrations tested. In the presence of S9 mix, slight reduction in relative survival (RS=70%) was noted at the highest dose level (300 µg/mL), while no relevant toxicity was observed over the remaining concentrations tested. In Main Assay II, in the absence of S9 mix, treatment at the highest dose (120 µg/mL) level yielded a reduction of relative survival to 11% of the negative control, moderate toxicity (RS=55%) was noted at the next lower concentration of 60.0 µg/mL, while no relevant toxicity was observed over the remaining concentrations tested. In the presence of S9 mix, moderate toxicity (RS=55%) was noted at the highest dose level (300 µg/mL), while no relevant toxicity was observed over the remaining concentrations tested. Opacity of the treatment medium was noted starting from 30.0µg/mL, in the absence of S9 mix. Opacity and slight precipitation were observed at the highest dose level, both in the absence and presence of S9 mix. No five-fold or greater increase in mutant frequency, compared with the negative control, was observed at the highest dose level or at two consecutive doses of the test substance, in the absence or presence of S9 mix. No reproducible evidence of a dose effect relationship was noticed. Under the study conditions, it was concluded that the test substance did not induce gene mutation in Chinese hamster V79 cells after in vitro treatment, in the absence or presence of S9 metabolic activation (Bisini, 2016).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

other: Mammalian erythrocyte micronucleus test

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

oral: gavage

Details on exposure:

Males - Animals were dosed once a day, 7 d/week, for 2 consecutive weeks prior to pairing, through the mating period and thereafter through the day before necropsy (Days 38 and 39 of study). Males were treated for a total of 37 or 38 d.
Females - Animals were dosed once a day, 7 d/week, for 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 3 post partum (for at least 40 d).

Duration of treatment / exposure:

Males - 37-38 d
Females - 40 d

Frequency of treatment:

Daily

Dose / conc.:

62.5 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

reduced from high dose of 1000 mg/kg bw/day due to toxicity

No. of animals per sex per dose:

5 per sex per gourp

Control animals:

yes, concurrent vehicle

Positive control(s):

Mitomycin C

Tissues and cell types examined:

Erythrocytes from bone marrow

Details of tissue and slide preparation:

Extraction of bone marrow
The last two treatments were performed at approximately 24 hour interval. Samples of bone marrow were collected approximately 24 hours following the final treatment and approximately 48 hours following the second last treatment from the same 5 males and 5 females of the main groups randomly selected for clinical pathology investigation. Samples of bone marrow were also collected approximately 24 hours after the single treatment from all animals of Group 7 (Positive Control group). One femur of each animal was rapidly dissected out and cleaned of surrounding tissue. In order to extract the bone marrow, the bone was cut at the proximal end, and irrigated with foetal calf serum using a syringe. The suspension of cells was aspirated, and this procedure was repeated several times.

Preparation of the smears
The suspension thus obtained was centrifuged at 1000 rpm for at least 5 minutes and the supernatant was completely removed. The cells of the sediment were resuspended and transferred onto clean microscope slides as smear preparations. They were air-dried and then fixed with methanol for 10 minutes. Subsequently slides were stained with haematoxylin and eosin solutions. Finally, slides were rinsed in distilled water and allowed to dry.

Scoring of the slides and data analysis
In the first instance, only slides from males were evaluated. In addition, since inter-sex differences of toxicity were observed, also slides from females were evaluated. For each animal, four slides were prepared. These slides were randomised and coded by staff not subsequently involved in the scoring. The adequate quality and a sufficient number of cells were evaluated before scoring. Scoring was performed using a microscope and highpower objective. Immature polychromatic erythrocytes (PCEs) stain a pink-purple colour (since they retain basic ribosomal material for approximately 24 hours after enucleation), and can be distinguished from the pink normochromatic erythrocytes (NCEs). Erythrocytes lack nuclei, making micronuclei obvious when present; the criteria of Schmid (1976) were used to score micronuclei. Four thousand polychromatic erythrocytes(PCEs) per animal were scored for the presence of micronuclei. At the same time the number of normal and normochromatic erythrocytes (NCEs) were also recorded. The proportion of immature
erythrocytes among total erythrocytes gives an indication of the toxicity of the treatment; a reduction in the proportion indicates inhibition of cell division. Finally, the incidence of micronucleated PCEs provides an index of induced genetic damage.

Evaluation criteria:

The assay is considered valid if the following criteria are met:
1. The incidence of micronucleated PCEs of the vehicle control group falls within the historical negative control range.
2. The positive control item results falls within the historical control range and are significantly increased, at statistical analysis, when compared with the concurrent negative control
3. 5 animals per sex and per group are available for slide analysis.

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

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Under the study conditions, the test substance was considered to be non-genotoxic in the rat.

Executive summary:

A study was conducted to determine the genotoxic potential of the test substance according to OECD Guideline 474, in compliance with GLP. The micronucleus test was conducted on 5 male and 5 female rats in order to assess the ability of the test substance to induce cytogenetic damage in rat bone marrow, as measured by the induction of micronuclei in polychromatic erythrocytes. Rats were administered the test substance orally by gavage. Main group animals were exposed to concentrations of 62.5, 250, 500, 1000 and 1000/500 mg/kg bw/day. Males animals were dosed once a day, 7 d/week, for 2 consecutive weeks prior to pairing, through the mating period and thereafter through the day before necropsy (Days 38 and 39 of study). Female animals were dosed once a day, 7 d/week, for 2 consecutive weeks prior to pairing and thereafter during pairing, post-coitum and post-partum periods until Day 3 post-partum (for at least 40 d). Mitomycin C was used as the positive control substance. No relevant inhibitory effect on erythropoietic cell division was observed at any dose level. Based on these observations, the test substance was not considered to be genotoxic in rats (Sisti, 2015).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

The structural analogue was tested positive in the Ames test, but was negative in the mutation assay in mammalian cells as well as in the micronucleus assay in vivo. 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. As the test substance has the same features as the structural analogue, which is the nitro-group in para-position, it is highly likely that the outcome of the Ames test would also be positive.

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. Type I nitroreductases can transfer two electrons from NAD(P)H to form the nitroso and hydroxylamino intermediates and finally the amino group. Type II nitroreductases transfer a single electron to the nitro group, forming a nitro anion radical, which in the presence of oxygen generates the superoxide anion in a futile redox cycle, regenerating the nitro group [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.

AMP397is 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 lymphomatikand mouse bone marrow micronucleus tests were negative, although a weak high toxicity-associated genotoxic activity was seen in a micronucleus test inV79 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 potentialin 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.

 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 (Tweatset 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 eitherin vitro(micronucleus test in human lymphocytes) orin vivo(ex vivounscheduled DNA synthesis in rats; bone marrow micronucleus test in mice), were negative.

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 Orange 30 is a bacteria specific effect and not relevant to mammalians.

References

De Oliveira IM, Bonatto D, Pega Henriques JA (2010). Nitroreductases: Enzymes with environmental biotechnological and clinical importance. In: Current Research, Technology and EducationTopics 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.

Additional information

Genetic toxicity in vitro:

A study was conducted to determine the in vitro mutagenic potential of the test substance according to OECD Guideline 471, in compliance with GLP. The assay was performed in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 in two independent experiments both with and without liver microsomal activation (S9 mix). Each concentration and the controls were tested in triplicate. The test substance was tested at the following concentrations: 33, 100, 333, 1000, 2500 and 5000 ug/plate. In Experiment I, cytotoxic effects evident as a reduction in the number of revertants occurred at the highest concentration in strain TA 1535 with S9 mix and in strain TA 1537 without S9 mix. In Experiment II, cytotoxic effects occurred at the highest concentration in strains TA 1535 and TA 1537 without S9 mix. The plates incubated with the test substance showed normal background growth up to 5000 ug/plate with and without S9 mix in all strains used. In both experiments, substantial and dose dependent increases in revertant colony numbers were observed following treatment with the test substance with and without metabolic activation in strains TA 1537, TA 98 and TA 100. Appropriate reference mutagens were used as positive control and showed a distinct increase in induced revertant colonies. Therefore, the test substance induced gene mutations by base pair changes and frameshifts in the genome of the Salmonella typhymurium strains TA98, TA100, TA1535 and TA1537. Under the study conditions, the test substance was considered to be mutagenic in this Salmonella typhimurium reverse mutation assay (Wollny, 1997).

A study was conducted to determine the genetic toxicity in vitro of the test substance according to OECD Guideline 476 and EU Method B.17, in compliance with GLP. In an in vitro mammalian cell gene mutation test (forward gene mutation in HPRT), Chinese hamster V79 cells were exposed to the test substance at concentrations ranging from 2.34 to 300.0 µg/mL with or without metabolic activation (S9 mix). The positive and negative controls were valid. In Main Assay I, in the absence of S9 mix, moderate reduction in relative survival (RS=18-19%) was noted at the two highest dose levels (150.0 and 75.0 µg/mL), treatment at 37.5 µg/mL yielded a reduction of relative survival to 36% of the negative control, while no relevant toxicity was noted over the remaining concentrations tested. In the presence of S9 mix, slight reduction in relative survival (RS=70%) was noted at the highest dose level (300 µg/mL), while no relevant toxicity was observed over the remaining concentrations tested. In Main Assay II, in the absence of S9 mix, treatment at the highest dose (120 µg/mL) level yielded a reduction of relative survival to 11% of the negative control, moderate toxicity (RS=55%) was noted at the next lower concentration of 60.0µg/mL, while no relevant toxicity was observed over the remaining concentrations tested. In the presence of S9 mix, moderate toxicity (RS=55%) was noted at the highest dose level (300 µg/mL), while no relevant toxicity was observed over the remaining concentrations tested. Opacity of the treatment medium was noted starting from 30.0 µg/mL, in the absence of S9 mix. Opacity and slight precipitation were observed at the highest dose level, both in the absence and presence of S9 mix. No five-fold or greater increase in mutant frequency, compared with the negative control, was observed at the highest dose level or at two consecutive doses of the test substance, in the absence or presence of S9 mix. No reproducible evidence of a dose effect relationship was noticed. Under the study conditions, it was concluded that the test substance did not induce gene mutation in Chinese hamster V79 cells after in vitro treatment, in the absence or presence of S9 metabolic activation (Bisini, 2016).

Genetic toxicity - in vivo:

A study was conducted to determine the genotoxic potential of the test substance according to OECD Guideline 474, in compliance with GLP. The micronucleus test was conducted on 5 male and 5 female rats in order to assess the ability of the test substance to induce cytogenetic damage in rat bone marrow, as measured by the induction of micronuclei in polychromatic erythrocytes. Rats were administered the test substance orally by gavage. Main group animals were exposed to concentrations of 62.5, 250, 500, 1000 and 1000/500 mg/kg bw/day. Males animals were dosed once a day, 7 d/week, for 2 consecutive weeks prior to pairing, through the mating period and thereafter through the day before necropsy (Days 38 and 39 of study). Female animals were dosed once a day, 7 d/week, for 2 consecutive weeks prior to pairing and thereafter during pairing, post-coitum and post-partum periods until Day 3 post-partum (for at least 40 d). Mitomycin C was used as the positive control substance. No relevant inhibitory effect on erythropoietic cell division was observed at any dose level. Based on these observations, the test substance was not considered to be genotoxic in rats (Sisti, 2015).

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.