Disodium [3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-4-hydroxy-5-nitrobenzenesulphonato(3-)][2-[(2-hydroxybenzylidene)amino]-4-nitrophenolato(2-)-N2,O1,O2]chromate(2-)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The in vitro studies were conducted with the structural analogue substance Acid Red 414:

In a reverse gene mutation assay in bacteria, strains TA 100, TA 1537, TA 1538 and TA 98 of S. typhimurium were exposed to two batches of Acid Red 414 at concentrations of up to 10000 µg/plate.

Concentrations of up to and including 10000 µg/plate did not cause any bacteriotoxic effects. The total numbers of bacteria remained unchanged. No inhibition of growth was observed.

Evidence of a mutagenic activity of Acid Red 414 was found for both batches. In all Salmonella typhimurium strains used a biologically relevant increase in the mutant count of more than double of the corresponding negative control was found. The lowest effective concentrations for batch Ök.Nr. 1190 and Ök.Nr. 2027 were 10 and 50 µg/plate for Salmonella typhimurium TA 1538, 1000 and 5000 µg/per plate for TA 100, 20 µg/plate for TA 1537 and 500 µg/plate for TA 98, respectively. Both batches of Acid Red 414 showed hence mutagenic effects in the Salmonella/microsome test with and without a metabolic activation.

The present study was conducted in compliance with OECD Guideline For Testing Of Chemicals, 476 "Genetic Toxicology: In vitro Mammalian Cell Gene Mutation Test" in compliance with the Principles of Good Laboratory Practice (GLP).

The study was performed to investigate the potential of Acid Red 414 to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster in vitro.

Two independent experiments were conducted both with and without an exogenous rat liver microsomal activation system (S9-mix).

The compound was suspended in cell culture medium and tested at the following concentrations:

Main experiment with and without metabolic activation: 100, 250, 500, 750, 1000#, 1375*, 1750* µg/ml

Repeat main experiment with and without metabolic activation: 100, 250, 500, 750, 1000* µg/ml

*= because of high toxicity no mutant selection was performed.

#= in the absence of S9-mix no mutant selection was performed

 

The concentration ranges were based on the results of preliminary tests for solubility and toxicity.

In the absence of S9 metabolic activation in both mutation experiments a dose-related decrease in survival was observed reaching 16.8 and 3.8 % of the solvent control value in the microtiter plates at a dose level of 1000 µg/ml. In the presence of S9-mix survival was reduced to 31.2 and 8.2 % of the solvent control value at the same concentration.

With the concentrations of 250 and 750 µg/ml the test compound induced a statistically significant enhancement of the mutation rate over the range of the solvent controls in the presence of a metabolic activation in the second main experiment. But these results were not reproducible, not three fold higher than the solvent control group and not dose-dependent and therefore of no biological relevance.

The sensitivity of the test system was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control compounds.

In conclusion, Acid Red 414 did not induce gene mutation, i.e. was not mutagenic, in this HPRT-test with V79 Chinese hamster cells, in either the presence or absence of metabolic activation.

Based on the results, the target substance Acid Orange 166 is considered mutagenic in bacteria but not mutagenic in mammalian cells.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

August 1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

No E. coli strain tested

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine locus

Species / strain / cell type:

S. typhimurium, other: TA 1538, TA 1537, TA 100, TA 98

Metabolic activation:

with and without

Metabolic activation system:

rat S9-mix

Test concentrations with justification for top dose:

Concentrations used: 10000, 5000, 1000, 500, 100, 50, 10 and 5 µg/plate
No bacteriotoxic effect up to 10000 µg/plate

Vehicle / solvent:

demineralised water

Untreated negative controls:

no

Remarks:

because the vehicle was water

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Cyclophosphamide, trypaflavine, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation: 0.1 mL TS+0.1 mL bacteria+0.5 mL S9+2 mL soft agar: 30 sec at 45 °C
- Incubation period: 48 hours at 37°C

NUMBER OF REPLICATIONS: 4 plates/strain/concentration

DETERMINATION OF CYTOTOXICITY
- Method: - background growth
- marked and dose-dependent reduction in mutant count compared to negative controls
- titer determination

Acceptance criteria:
a) The negative controls had to be within the expected range, as defined by published data (i.e. Maron and Ames, 1983) and the laboratory's own historical data
b) The positive controls had to show sufficient effects, as defined by the laboratory's experience
c) Titer determinations had to demonstrate sufficient bacterial density in the suspension.

An assay which did not comply with at least one of the above criteria was not used for assessment. Furthermore, the data generated in this assay needed to be confirmed by two additional independent experiments. Even if the criteria for points (a), (b) and (c) were not met, an assay was accepted if it showed mutagenic activity of the test compound.

Evaluation criteria:

A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result; this increase should be about twice the amount of negative controls.

Statistics:

N.A.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

TA 100: 1.6-fold increase at cytotoxic concentrations

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

There was no indication of a bacteriotoxic effect of the test item at up to 10000 µg per plate. The total bacteria counts consistently produced results comparable to the negative controls, or differed only insignificantly. Nor was any inhibition of growth noted.

All four strains concerned showed a dose related and biologically relevant increase in mutant counts over those of the negative controls. This applied both to both batches with and without S9 mix.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Both batches of Acid Red 414 showed mutagenic effects in the Salmonella/microsome test with and without a metabolic activation.

Executive summary:

In a reverse gene mutation assay in bacteria, strains TA 100, TA 1537, TA 1538 and TA 98 of S. typhimurium were exposed to two batches of Acid Red 414 at concentrations of up to 10000 µg/plate.

Concentrations of up to and including 10000 µg/plate did not cause any bacteriotoxic effects. The total numbers of bacteria remained unchanged. No inhibition of growth was observed.

Evidence of a mutagenic activity of Acid Red 414 was found for both batches. In all Salmonella typhimurium strains used a biologically relevant increase in the mutant count of more than double of the corresponding negative control was found. The lowest effective concentrations for batch Ök.Nr. 1190 and Ök.Nr. 2027 were 10 and 50 µg/plate for Salmonella typhimurium TA 1538, 1000 and 5000 µg/per plate for TA 100, 20 µg/plate for TA 1537 and 500 µg/plate for TA 98, respectively. Both batches of Acid Red 414 showed hence mutagenic effects in the Salmonella/microsome test with and without a metabolic activation.

 

The positive controls Endoxan, trypaflavine and 2-amino-anthracene acted markedly mutagenic, as can be seen from the biologically relevant increase of mutant colonies compared with the corresponding negative control.

 

This study is classified as acceptable. The study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The in vivo study was conducted with the structural analogue substance Acid Red 414:

The Mammalian Erythrocyte Micronucleus Test according to OECD Test Guideline No 474 was carried out with Acid Red 414 in male and female NMRI mice. The test compound was suspended in deionized water and was given twice at an interval of 24 hours as an orally dose of 2000 mg per kg body weight to male and female mice, based on the results of a previous dose range finding assay. According to the test procedure the animals were killed 24 hours after test substance administration.

Cyclophosphamide was used as positive control substance and was administered once orally at a dose of 50 mg per kg body weight.

The number of polychromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained unaffected by the treatment with Acid Red 414 and was not less than 20% of the control value.

Cyclophosphamide induced a marked statistically significant increase in the number of poly-chromatic cells with micronuclei, indicating the sensitivity of the test system. The ratio of polychromatic erythrocytes to total erythrocytes was not changed to a significant extent.

Under the conditions of the present study the results indicate that Acid Red 414 is not mutagenic in the micronucleus test.

Based on the results, the target substance Acid Orange 166 is considered not clastogenic in mammalians.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

The test item Acid Red 414 was tested positive in the Bacteria Reverse Mutation Assay (Ames test), but was negative in the mutation assay in mammalian cells (HPRT) testing and the in-vivo Micronucleus Test in mice, testing for clastogenicity and aneugenicity. 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.

The positive effect in the bacterial reverse mutation test (Ames) was clearly related to a bacteria-specific metabolism of the test substance, 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]. 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].

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.

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 lymphomatkand mouse bone marrow micronucleus tests were negative, although a weak high toxicity-associated genotoxic activity was seen in a micronucleus test inV79 cells [Suter et al. 2002].The amino derivative of AMP397 was not mutagenic in wild type TA98 and TA100. To exclude that a potentially mutagenic metaboliteis released by intestinal bacteria, a Muta^TMMouse study was done in colon and liver with five daily treatments at the MTD, and sampling of 3, 7 and 21 days post-treatment. No evidence of a mutagenic potential was found in colon and liver. Likewise, a comet assay did not detect any genotoxic activity in jejunum and liver of rats, after single treatment with a roughly six times higher dose than the transgenic study, which reflects the higher exposure observed in mice. In addition, a radioactive DNA binding assay in the liver of mice and rats did not find any evidence for DNA binding. Based on these results, it was concluded that AMP397 has no genotoxic potential in vivo. It was hypothesized that the positive Ames test was due to activation by bacterial nitro-reductase, as practically all mammalian assays including fourin vivoassays were negative, and no evidence for activation by mammalian nitro-reductase or other enzymes were seen. Furthermore, no evidence for excretion of metabolites mutagenic for intestinal cells by intestinal bacteria was found.

 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 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.

As a conclusion, it could be said that 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 Acid Red 414 is a bacteria specific effect and not relevant to mammalians.

Acid Red 414 was not genotoxic in the mammalian in-vitro cell mutagenicity test (HPRT assay) and the in-vivo MNT test. Therefore, a direct genotoxic effect as well as a metabolisation towards genotoxic structures by mammalian species can be excluded.

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.

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.

Justification for classification or non-classification

The test item is not considered genotoxic based on a weight of evidence assessment of the results of an appropriate testing battery with the structural analogue substance Acid Red 414.