Chromate(2-), [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulfonato(3-)]-, disodium

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Acid Orange 154 is not mutagenic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1995

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:

no

Qualifier:

according to guideline

Guideline:

other: EEC Directive 87/302, L 133, p. 61 - 63

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

Name: FAT 20'010/C Lanacron Orange S-2R crude dry
Batch No.: 149
Aggregate State at RT: Solid
Colour: orange
Purity: 78.83 % (active ingredient)
Stability: Pure: stable until December, 1999
Storage: room temperature
Expiration Date: December 1999

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Large stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing; Technical University; D-64293 Darmstadt; F.R.G.) are stored in liquid nitrogen in the cell bank of CCR allowing the repeated use of the same cell culture batch in experimen

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiment I:
without S9 mix: 1, 10, 20, 25, 30, and 40 µg/mL
with S9 mix: 3, 10, 30, 50, 70 and 90 µg/ml

Experiment II:
without S9 mix: 1, 3, 10, 12, 15 and 18 µg/ml
with S9 mix: 3, 10, 30, 50, 70 and 90 µg/ml

Experiment III:
without S9 mix: 12 and 15 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:The solvent was chosen according to its solubility properties and its non-toxicity for the cells.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

nutrient medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

With metabolic activation

Evaluation criteria:

A test article is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response for one of the test points. A test article producing neither a concentration- related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system. A significant response is described as follows: The test article is classified as mutagenic if it induces a reproducible mutation frequency that is at least three times higher than the spontaneous mutation frequency in the experiment at one or more of the concentrations.
The test article is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed. However, in a case by case evaluation this decision depends on the level of the correspon-ding negative control data. If there is by chance a low spontaneous mutation rate in the range normally found (0 - 45 mutants per 10E6 cells) a concentration-related increase of the mutations within this range has to be discussed.

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:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

FAT 20010/C did not induce gene mutations at the HPRT locus in V79 cells.

Executive summary:

A study was performed to investigate the potential of FAT 20010/C to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. This test was performed according to OECD Guideline for Testing of Chemicals, Section 4, No. 476, adopted April 4, 1984, "ln vitro Mammalian Cell Gene Mutation Tests" and EEC Directive 87/302, L 133, p. 61 - 63

The study was performed in two independent main experiments, using identical procedures, both with and without liver microsomal activation.

Experiment I: without S9 mix: 1.0; 10.0; 20.0, 25.0; 30,0 and 40.0 µg/ml

with S9 mix: 3.0; 10.0; 30.0; 50.0; 70.0 and 90.0 µg/ml

Experiment II: without S9 mix: 1.0; 3.0; 10.0; 12,0; 15.0 and 18.0 µg/ml

with S9 mix: 3.0; 10.0; 30.0; 50.0; 70.0 and 90.0 µg/ml

Experiment III: without S9 mix: 12.0 and 15.0 µg/ml

Without metabolic activation strong toxic effects occurred already at concentrations as low as 20 μg/ml. With metabolic activation, the concentration of the test article could be increased up to 90 μg/ml. Although the absolute numbers of colonies in experiment I with metabolic activation indicate no relevant toxicity the colonies per se showed distinct toxic effects at concentrations of 50 μg/ml or above and barely exceeded the limit of 50 cells per colony. In the second experiment these colonies remained almost invisible and clearly did not reach the limit of 50 cells per colony. The cloning efficiency of the cells was reduced below 30 % at the highest concentrations tested. In the first experiment the number of mutant colonies was not substancially increased compared to the frequency of spontaneous mutations at any concentration of the test article. The enhancement factor of three was not reached. There was also no indication of a concentration depend increase of mutant colonies. In the second and third experiments the enhancement factor of three is exceeded in the absence of metabolic activation at almost all concentrations of the test article. This effect however, is due to the rather low values of the solvent controls and does not reflect a true mutagenic effect. The absolute numbers of colonies are well within the historical range of negative controls. In all experiments of this study (with and without S9 mix) the range of the negative controls was from 1.4 up to 16.3 mutants per 10⁶ cells; the range of the groups treated with the test article was from 2.0 up to 26.1 mutants per 10⁶ cells. EMS (0.6 mg/ml) and DMBA (3.85 µg/ml) were used as positive controls and showed a distinct increase in induced mutant colonies. In conclusion, it can be stated that in this mutagenicity assay and under the experimental conditions reported the test article did not induce gene mutations at the HPRT locus in V79 cells.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only 4 tester strains used, no tester strain to detect crosslinking mutagens included

GLP compliance:

no

Remarks:

study predates GLP

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Name: FAT 20010/B
Purity: 57 %

Target gene:

histidine auxotrophic mutants

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: histidine-auxo-trophic mutants

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

25, 75, 225, 675, and 2025 µg/0.1 ml.

Vehicle / solvent:

Acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

phosphate buffer

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N--methyl-N'- nitro-N-nitrosoguanidine

Remarks:

for Strain TA 1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 9(5)aminoacridine hydrochloride monohydrate

Remarks:

for Strain TA 1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Phosphate buffer

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: daunorubicin-HCl

Remarks:

for Strain TA 98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

phosphate buffer

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

for Strain TA 100

Details on test system and experimental conditions:

The test was performed with the following concentrations of the trial substance with and without microsomal activation: 25, 75, 225, 675 and 2025 µg/0.1 ml. The substance was dissolved in acetone. Acetone alone was used for the negative controls (the substances and vehicles used for the positive controls are indicated below). Each Petri dish contained:
1) approx. 20 ml of minimum agar (Agar purified, "Difco" certified, or agar noble (repeat experiment), Difco Laboratories, Detroit, Michigan, U.S.A., Art.No.05650 and 0142-01, plus salts (Vogel-Bonner Medium E) and glucose),
2) 0.1 ml of the solution of the test substance or the vehicle and 0.1 ml of a bacterial culture (in nutrient broth: Bacto Nutrient Broth dehydrated, Difco Laboratories, Detroit, Michigan, U.S.A., Art.No.0003 0.8% plus 0.5% NaCl) in 2.0 ml of soft agar. The soft agar was composed of: 100 ml of 0.6% agar solution (Agar purified, "Difco" certified or agar noble) with 0.6% NaCl and 10 ml of a solution of 1-histidine, 0.5 mM (Fluka, Buchs, Switzerland, Art.No.14400) and +biotin 0.5 mM (Fluka, Buchs, Switzerland, Art.No.53320).

In the experiment in which the substance was metabolically activated, 0.5 ml of an activation mixture was added also. 1 ml activation mixture contains: 0.3 ml S9 fraction of liver from rats induced with Aroclor 1254 (Analabs, Inc., North Haven, Connecticut, U.S.A., No.RCS-088), 8 µmoles MgC12, 33 µmoles KC1, 5 µmoles glucose-6-phosphate, 4 µmoles NADP and 100 µmoles phosphate buffer, pH 7.4.

Evaluation criteria:

When the colonies had been counted, the arithmetic mean was calculated.

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

No bacteriotoxicity is reported up to mthe maximum concentration tested.

Conclusions:

In the experiments performed without and with microsomal activation, treatment with FAT 20010/B led to an increase in the number of backmutant colonies of strains TA 98 and TA 1537. A mutagenic effect was observed at the concentrations of 675 and 2025 µg/ 0.1 ml.

Executive summary:

FAT 20010/B was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium. This test was performed according to guideline similar to OECD TG 471. The investigations were performed with the following concentrations of the trial substance with and without microsomal activation: 25, 75, 225, 675, and 2025 µg/0.1 ml. In order to confirm the results, the experiments were repeated. In the experiment performed without and with microsomal activation on Strains TA 1537 and TA 98, treatment with FAT 20010/B led to an increase in the number of back-mutant colonies at the concentra­tions of 625 and 2025 µg/0.1 mL. Test substance FAT 20010/B exerted a mutagenic action in this test system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In a study performed in 1979, the test substance FAT 20010/B was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium. In the experiment performed without and with microsomal activation on strains TA 1537 and TA 98, treatment with FAT 20010/B led to an increase in the number of back-mutant colonies at the concentrations of 625 and 2025 µg/0.1 ml. Thus the compound FAT 20010/B thus exerted a mutagenic action in this test system. A possible explanation for such positive results in this Ames test might be the presence of aromatic Nitro-groups in the molecular structure. Such nitro-groups might be substrates to bacterial nitro-reductases a class of enzymes being more prominent in bacteria with no or less relevance for eukaryonitc cells. Due to the formation of nitrenium ions by these bacterial nitroreductases attacks of the unprotected ring-chromosom of bacteria might be the consequences with the result of mutations formed. (see below for further details). To further investigate such (false-positive) effects a G

LP-compliant study was performed according to OECD guideline 476 to investigate the potential of FAT 20010/C to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in three independent experiments, using identical procedures, both with and without presence of an artificial metabolic activation system (rat liver S9 mix). Up to the highest investigated concentration no relevant increase in mutant colony numbers was obtained in two independent experiments. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies. In conclusion it can be stated that during the described mutagenicity test and under the experimental conditions reported the test article did not induce gene mutations at the HPRT locus in V79 cells. Therefore, FAT 20010/C is considered to be non-mutagenic in HPRT assay.

Potential false-positive mutagenic effects with substances containing aromatic nitrogroups obtained in bacterial test systems:

Explanation:

Acid Orange 154 was found to exert mutagenic potential in a Salmonella reverse mutation assay, however it did not exert mutagenic action in mammalian cells in vitro (HPRT). Hence, Acid Orange 154 is considered to be not mutagenic. One reason for this phenomenon could be the presence of a nitro-group in the molecule. 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.

 

Assessment:

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. 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].

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 in Salmonella strains 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 [Suter et 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 MutaTM Mouse study was done in colon and liver with five daily treatments at the MTD, and sampling of 3, 7 and 21 days post-treatment. No evidence of a mutagenic potential was found in colon and liver. Likewise, a comet assay did not detect any genotoxic activity in jejunum and liver of rats, after single treatment with a roughly six times higher dose than the transgenic study, which reflects the higher exposure observed in mice. In addition, a radioactive DNA binding assay in the liver of mice and rats did not find any evidence for DNA binding. Based on these results, it was concluded that AMP397 has no genotoxic potential in vivo. It was hypothesized that the positive Ames test was due to activation by bacterial nitro-reductase, as practically all mammalian assays including four in vivo assays were negative, and no evidence for activation by mammalian nitro-reductase or other enzymes were seen. Furthermore, no evidence for excretion of metabolites mutagenic for intestinal cells by intestinal bacteria was found.

Fexinidazole was in pre-clinical development as a broad-spectrum antiprotozoal drug by the Hoechst AG in the 1970s-1980s, but its clinical development was not pursued. Fexinidazole was rediscovered by the Drugs for Neglected Diseases initiative (DNDi) as drug candidate to cure the parasitic disease human African trypanomiasis (HAT), also known as sleeping sickness. The genotoxicity profile of fexinidazole, a 2-substituted 5-nitroimidazole, and its two active metabolites, the sulfoxide and sulfone derivatives were investigated [Tweats et al. 2012]. All the three compounds are mutagenic in the Salmonella/Ames test; however, mutagenicity is either attenuated or lost in Ames Salmonella strains that lack one or more nitroreductase(s). It is known that these enzymes can nitroreduce compounds with low redox potentials, whereas their mammalian cell counterparts cannot, under normal conditions. Fexinidazole and its metabolites have low redox potentials and all mammalian cell assays to detect genetic toxicity, conducted for this study either in vitro (micronucleus test in human lymphocytes) or in vivo (ex vivo unscheduled DNA synthesis in rats; bone marrow micronucleus test in mice), were negative.

 

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 Acid Orange 154 is a bacteria specific effect and not relevant to mammalians.

 

The above arguments were further supported by the negative outcome in the in vitro mammalian cell gene mutation assay with Acid Orange 154. As a result, it was concluded that Acid Orange 154 is not mutagenic. Therefore, further genotoxicity tests have not been conducted.

Justification for classification or non-classification

Based on the available genotoxicity studies, Acid Orange 154 does not need to be classified for genotoxicity according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.