Chromate(2-), [2,4-dihydro-4-[(2-hydroxy-4-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

Assessment report on the mutagenicity of FAT 20060

Introduction

Toxic substances can be split in natural substances which are generated because of metabolic activities of organisms and in xenobiotics (chemicals) produced by industrial processes. Many xenobiotic compounds are recalcitrant and persist in the environment for long time because they contain structures or substructures that are not present in nature and are therefore not or limited biodegradable. From an evolutionary point of view such xenobiotics have been released very recently and therefore only a limited number of organisms developed suitable mechanisms to degrade those chemicals in order to detoxify them or to use them as a source of energy (for review see Roldán, 2008). Nitroaromatic compounds are very rare in nature. Only a few products are known such as e.g. chloramphenicol (Ahmed & Vining, 1983) or oxypyrrolnitrine. Some plants specialized to produce nitroglycosides as defence mechanisms like Astragalus sp. (Anderson 1993) but the vast majority of nitroaromatic compounds are man-made and released into the environment because of industrial processes (e.g.polyurethans), explosives (TNT), drugs, pesticides, plastics, and dyes. Mono-nitroaromatic substances can be degraded by several bacterial strains includingSalmonellastrains. Poly-nitrated aromatic compounds are more difficult to degrade and mostly persist as recalcitrant pollutants in the environment.

Bacterial nitro-reductases

The family of nitro-reductases comprises a group of enzymes being dependent of either flavin micronucleotide (FMN) or flavin adenine dinucleotide (FAD) that catalyse the reduction of nitro- groups present in nitroaromatic and nitroheterocyclic derivatives using the reducing power of nicotinamide adenine dinucleotide (NAD(P)H). (De Oliveira, 2007, De Oliveira, 2010). These enzymes can be found in prokaryotes as well as in eukaryotic cells. While these classes of enzymes play a major role in activation of prototoxins and detoxification of toxic substances in bacterial systems, the contribution to such events is low in eukaryotic cells. Nitro-reductases also play a central role in the activation of nitro-compounds and gained recent interest and importance for mediating toxicity of nitro-substituted compounds as well as clinical importance in chemotherapeutic cancer treatment.

Based on their ability to reduce nitro-groups in the presence or absence of molecular oxygen by one or two electron transfers nitro-reductases have been grouped into two categories. Type 1 or oxygen-insensitive nitro-reductases catalyse the sequential transfer of two electrons from NADH or NADPH to the nitro-group of nitro-substituted compounds in the presence or absence of molecular oxygen. The result of this reaction is the formation of instable nitroso and hydroxylamine intermediates which finally are processed to an amino group (Figure 2). The Type 2 or oxygen-sensitive nitro-reductases catalyse one electron reductions of the nitro-group in presence of molecular oxygen. Transfer of one electron to the nitro-group results in formation of a nitro-anion radical that subsequently may react with molecular oxygen forming a superoxide radical while the original nitro-group is regenerated (futile cycle). That means in presence of molecular oxygen large amounts of superoxide radicals can be formed leading to oxidative stress. In absence of molecular oxygen (under anaerobic conditions) instead, nitro-reductases can mediate a second electron transfer forming a nitroso-group. It could be demonstrated that Type 1 nitro-reductases participate in chemical breakdown of a large variety of nitro-compounds including e.g., nitrofurans, nitrobenzenes, nitrophenols and nitrotoluenes.

Polynitroaromatic compounds might be also transformed by two types of reductive pathways. One pathway involves the reduction of the aromatic ring by addition of hydroxide ions through hydride transferases forming a hydride-Meisenheimer complex which will be further metabolized with concomitant release of nitrite (Roldanet al.). The second pathway involves the reduction of nitro-groups to form hydroxylamino or amino-groups catalysed by nitro-reductases. Nitrate or ammonium released by these degenerative pathways can be used as nitrogen source for bacterial growth (figure 4).

In eukaryotic cells and in mammals especially almost no phylogenetic homologues are related to bacterial nitro-reductases. Functionally xanthine dehydrogenase and NAD(P)H-quinone oxidoreductase are related to bacterial Type 1 nitro-reductases but do not exhibit the domain characteristic of this family. Similarly, aldehyde oxidase, cytochrome c oxidase and NADPH cytochrome P450 reductase show functional analogy with bacterial type II nitro-reductases.

Relevance of bacterial nitro-reductases in the Ames-test

TheSalmonella/microsome assay developed by Bruce Ames, et al. in the 1975 has been adopted into an OECD guideline (OECD 471) and is widely used as a starting point for mutagenicity screening. OECD guideline 471 requires fiveSalmonellaandE. colitester strains to be tested in presence and absence of an artificial cocktail of metabolizing enzymes (Aroclor-induced rat liver S9-mix) to simulate mammalian liver metabolism. The assay itself is based on the induction of reverse mutations in thoseSalmonellaandE. colistrains while different strains can be used to detect different types of mutations. Often tester strains TA97, TA98, TA100 and TA1535 are used in combination withE. coliWP2/uvrA as a standard set of tester strains to cover all kind of mutational events that may happen to the primary DNA sequence. Since all these bacterial tester strains described in OECD guideline 471 to be used as a standard set are nitro-reductase positive, substances harvesting one or more nitro-groups within their chemical structure might be metabolized during the incubation time of the assay producing a high amounts of oxygen radicals under aerobe conditions. These radicals may in turn lead to bacterial DNA damage resulting either in mutations or in bacteriotoxicity. In case these mutations would involve the target sequence of the tester strain, false positive results would be observed for the test substance under the conditions used for this assay.

Indeed, such false positive results have been observed during drug development and chemical safety assessment and published in literature.

 

In 2002 Willi Suter described positive mutagenic effects of AMP397 in the standard Ames test [Suter, 2002]. The target substance contains a nitro-group as chemical alert structure within the molecule and was therefore intensively studied for mutagenic effects. Indeed, the test substance demonstrated to be positive in tester strains TA97a, TA98 and TA100 without metabolic activation, while being negative in the nitro-reductase-deficient tester strains TA98NR and TA100NR. In addition, the amino derivative of the test substance was also negative in wild type strains TA97a, TA98 and TA100 without metabolic activation leading to the conclusion that the nitro-group metabolized by bacterial nitro-reductases might be responsible for the positive outcome of the test rather than the unmetabolized mother compound itself. Further investigation of the test article in the Muta^TMMouse assayin vivo(target organs colon and liver), in the Comet assayin vivo(target organs jejunum and liver), in the Micronucleus testin vivo(bone marrow) as well as a DNA binding study investigated in rats and mice could not confirm the positive results obtained with the Ames test on standard tester strainsin vitrofurther supporting the hypothesis of metabolic toxification of the test substance by bacterial nitro-reductases.

 

A second example was published by David Tweats and co-workers in 2012. Fexinidazole a drug candidate for human African trypanomiasis and its two active metabolites have been tested positive in the Ames test for tester strains TA98, TA100 and TA1535 without S9 mix and in TA102 and TA1537 with metabolic activation. Interestingly, when testing nitro-reductase deficient strains TA98NR, TA100NR, TA102NR, TA1535NR and TA1537NR mutagenicity was either lost (e.g. TA98 vs. TA 98NR) or significantly attenuated (TA100 vs. TA100NR). In strains lacking both Type 1 and Type 2 nitro-reductase (TA98NR and TA1535NR) mutagenic effects have been totally abolished while strains lacking only Type 1 nitro-reductase (TA100NR) or strains lacking only Type 2 nitro-reductase remained positive. Mutagenic effects observed with individual tester strains have been further elevated in the presence of Aroclor 1254-induced rat liver S9 mix. These results suggest that the mutagenic effects observed are not resulting because of intrinsic properties of the test items but are a result of bacterial nitro-reductase activity. This finding is further supported by the fact that all three structures the main compound and its two metabolites have been tested negative in the micronucleus testin vitroandin vivoas well as in theex vivoUDS test in rat liver cells. In summary, the authors could demonstrate that the mutagenic effects observed in the bacterial testing system is due to the action of bacterial nitro reductases forming reactive radical species with potential to react with the DNA and thus cause genetic alterations which might finally result in mutations detected by the assay.

 

Also, in 2012 Izet Kapetanovic and co-workers published data on a PPAR-gamma antagonist under clinical investigation. 2-chloro-5-nitro-N-phenylnezamide (GW9662) has been screened for mutagenic effects in the Ames test involving tester strains TA98 and TA100 as well as their nitro-reductase deficient variants TA98NR and TA100NR in presence and absence of an artificial metabolizing agent (Aroclor 1254-induced rat liver S9 mix). As a result, the test substance demonstrated positive (mutagenic) effects in strains TA98 and TA100 while no increased number of revertants could be observed when nitro-reductase deficient variants TA98NR and TA100NR have been incubated with the chemical.

 

But not only drugs and their metabolites are known for false positive effects observed in the bacterial forward mutation assay. Further evidence for industrial chemicals was published by David Josephyet al.in 2015. Azo compounds are found in all functional classes of dyes, including acid, basic, disperse, reactive and solvent dyes [Freeman, 2013]. Among these especially disperse dyes frequently demonstrate positive results in the Ames test [Ferraz,et al. 2011]. The authors tested the mutagenicity of 2-cyano-4-nitroaniline (CNNA) and 2,6-dicyano-4-nitroaniline (CNCNNA), components of azo dyes such as Disperse Blue 165 and Disperse Red 073 in the Ames test strains. Both compounds are extraordinarily potent frameshift mutagens. In strains deficient for the enzyme nitro-reductase (TA 98NR, TA 102NR) mutagenic effects caused by the toxicants could be significantly reduced compared to mutagenic effects induced in their respective wild type strains. Overexpression of the enzyme N-acetyl transferase in tester strain YG1024 enhanced test substance induced mutation rate which is in line with the metabolic activation route of nitroaromatic compounds inSalmonellastrains: enzymatic reduction of the nitro-group to a hydroxylamine, subsequently acetyl coenzyme A dependent O-acetylation catalysed by NAT followed by the generation of an N-acetoxy ester and fission of the N-acetoxy ester which finally generates a DNA-reactive nitrenium ion that is able to form covalent adducts e.g. at the C8 position of guanine [Vance and Levine 1984, Novak 1993, Watanabe 1994, Josephy and Novak 2013]. As a conclusion, the authors were able to demonstrate that the mutagenic effects observed with dyes harvesting one or more nitro-groups within their chemical structure are due to the metabolic activity of nitro-reductase rather than to an intrinsic mutagenic effect by the test chemical itself.

Evaluation and assessment of FAT 20060

FAT 20060 has been tested for mutagenic effects in the Ames test according to OECD 471 involvingSalmonellatester stains TA98, TA100, TA1535 and TA1537 with and without metabolic activation. In addition, it has been investigated for mutagenic effects in eukaryotic cells using the HPRT test according to OECD guideline 476 and it has been investigated for clastogenic effects in Chinese hamster V79 cellsin vitroaccording to OECD guideline 473.

 

The bacterial reverse mutation assay was performed in two independent experiments both with and without liver microsomal activation. Experiment I was performed as a plate incorporation assay and Experiment II as a pre-incubation assay using Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100. Toxic effects, evidenced by a reduction in the number of revertants, occurred in strain TA1535 at 1000, 2500 and 5000 µg/plate with S9 mix in Experiment I, as well as at 2500 (with S9 mix) and 5000 µg/plate (with and without S9 mix) in Experiment II. The plates incubated with the test substance showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. A significant dose-dependent increase in revertant colony numbers was observed in strain TA1537 with S9 mix in Experiments I and II and in strain TA98 without S9 mix in Experiment II. Based on the findings of the study, it was concluded that the test substance induced gene mutations by frameshifts in the genome of strains TA1537 and TA98. Therefore, it is considered to be mutagenic in thisSalmonella typhimuriumreverse mutation assay.

To verify these results in mammalian cells, a study was performed to investigate the potential of the test substance to induce gene mutations at the HPRT locus in Chinese hamster V79 cells according to OECD Guideline 476. The assay was performed in three independent experiments, using identical procedures, two settings with and three settings without liver microsomal activation. Due to unexpectedly strong toxic effects, an additional experiment without metabolic activation was required to complete the data of Experiment I. Without metabolic activation, strong toxic effects occurred already at concentrations as low as 20 µg/mL. With metabolic activation, the concentration of the test substance could be increased up to 200 µg/mL. The cloning efficiency of the cells was reduced below 30 % at the highest concentrations tested. The number of mutant colonies was not increased compared to the frequency of spontaneous mutations at any concentration of the test substance. There was also no indication of a concentration-dependent increase of mutant colonies. Hence, based on the above findings, it was concluded that the test substance did not induce gene mutations at the HPRT locus in V79 cells.

The clastogenic potential of FAT 20060 was evaluated in anin vitrochromosomal aberration assay performed with Chinese hamster V79 cells. The assay included two experiments both with and without metabolic activation. In both experiments, no biologically relevant increase of the aberration rates was noted after treatment with the test item without and with metabolic activation. The aberration rates of all dose groups treated with the test item were within the historical control data of the negative control. In the experiments I and II without and with metabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item as compared to the negative controls. The positive controls induced the appropriate responses. There was no evidence of chromosome aberration induced over background. Further tests to evaluate potential mutagenic effectsin vivoare currently not available.

Summary and conclusion

Based on the assessment conducted above it is most likely that the positive results observed in the Ames test result from mutations induced by reactive oxygen species and other reactive chemical breakdown products formed because of intrinsic bacterial nitro-reductase activity followed by further metabolic chemical breakdown. Analysis of the chemical structure with standard analytical methods confirmed the presence of three nitro-groups in the molecule. Based on chemical analysis and the common mechanisms and findings published for substances with comparable structures in literature it is concluded that the mutagenic effects observed with the test item are due to the bacterial nitro-reductase activity leading to the formation of reactive oxygen species (ROS) and other reactive chemical breakdown products like nitrenium ions under the aerobic incubation conditions applied. Those reactive radicals will most likely result in DNA damage leading to forward mutations that are detected by the assay. Therefore, it is concluded that the positive results obtained with the Ames test is a bacterial specific effect with no relevance in mammalian speciesin vivo.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

no data

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only four strains were tested, no tester strain to detect cross-linking mutagens was included

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

yes

Remarks:

only four strains were tested, no tester strain to detect cross-linking mutagens was included

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine gene

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

The strains are derived from S. typhimurium strain LT2 and due to a mutation in the histidine locus are histidine dependent. Additionally due to the "deep rough" (rfa-minus) mutation they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation causes a reduction in the activity of an excision repair system. The latter alteration includes mutational processes in the nitrate reductase and biotin genes produced in a UV-sensitive area of the gene named "uvrB-minus". In the strains TA 98, TA 100 and TA 102 the R-factor plasmid pKM 101 carries the ampicillin resistance marker. Regular checking of the properties of the strains with regard to membrane permeability and ampicillin resistance as well as spontaneous mutation rates is performed in CCR according to Ames et al. In this way it was ensured that the experimental conditions set down by Ames were fulfilled.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

33.3, 100, 333.3, 1000, 2500 and 5,000 µg active ingredient/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Aqua bidest
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties

Untreated negative controls:

yes

Remarks:

(concurrent untreated)

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

(for TA 1535 and TA 100 without metabolic activation)

Untreated negative controls:

yes

Remarks:

(concurrent untreated)

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

(for TA 1537 and TA 98 without metabolic activation)

Untreated negative controls:

yes

Remarks:

(concurrent untreated)

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

(for all strains with metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation test (experiment I) and the pre-incubation test (experiment II). For each strain and dose level, including the controls, a minimum of three plates were used.

Experiment 1: The following materials were mixed in a test tube and poured onto the selective agar plates:
- 100 µL: Test solution at each dose level, solvent control, negative control, or reference mutagen solution (positive control),
- 500 µL: S9 mix (for test with metabolic activation) or S9 mix substitution-buffer (for test without metabolic activation),
- 100 µL: Bacteria suspension (cf. test system, pre-culture of the strains),
- 2000 µL: Overlay agar

Experiment 2: In the pre-incubation assay 100 µL test solution, 500 µL S9 mix /S9 mix substitution buffer and 100 µL bacteria suspension were mixed in a test tube and incubated at 37 °C for 60 minutes. After pre-incubation 2 mL overlay agar (45 °C) was added to each tube. The mixture was poured on minimal agar plates. After solidification the plates were incubated upside down for at least 48 h at 37 °C in the dark.

Evaluation criteria:

The generally accepted conditions for the evaluation of the results are corresponding background growth on both negative control and test plates as well as normal range of spontaneous reversion rates. Due to international guidelines a statistical evaluation of the results is recommended. However, no evaluated statistical procedure can be recommended for analysis of data from the bacterial assays at this time. A test substance is considered as positive if either a dose related or reproducible increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced. A test substance producing neither a dose related and reproducible increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system. A significant response is described as follows:
A test substance is considered mutagenic if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537 and TA 98 at least three times higher as compared to the spontaneous reversion rate. 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 of whether the highest dose induced the above-described enhancement factors or not.

Statistics:

No appropriate statistical method is available.

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

positive

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

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

(only in pre-incubation test)

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

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

not examined

Additional information on results:

Toxic effects, evidenced by a reduction in the number of revertants, occurred in strain TA 1535 at 1000, 2500 and 5000 µg/plate with S9 mix in experiment I as well as at 2500 (with S9 mix) and 5000 µg/plate (with and without S9 mix) in experiment II. The plates incubated with the test substance showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.
Up to the highest dose a significant dose-dependent increase in revertant colony numbers was obtained in tester strain TA 1537 in the presence of metabolic activation in experiment I. In strain TA 98 in experiment I higher values in revertant colony numbers were obtained at 2500 and 5000 µg/plate without S9 mix. Since the obtained effects were less distinct, experiment II was carried out as a pre-incubation assay in order to obtain results from a normally more sensitive assay. In the pre-incubation assay a significant increase in revertant colony numbers was obtained up to 5000 µg/plate with metabolic activation with a higher mutation factor at the highest investigated dose. In tester strain TA 98 a clear dose-dependent increase in revertant colonies was observed up to 5000 µg/plate without S9 mix in experiment II. The results obtained in both independent experiments indicate a mutagenic potential of the test substance in the tester strains TA 1537 and TA 98.

none

Conclusions:

The test substance is considered to be mutagenic in the Salmonella typhimurium reverse mutation assay.

Executive summary:

An in vitro study was performed to investigate the potential of the test substance (at ca. 80 % purity) to induce gene mutations according to OECD Guideline 471 and EU Method B.14 in compliance with GLP with deviation (only four strains were tested). The assay was performed in two independent experiments both with and without liver microsomal activation. Experiment I was performed as a plate incorporation assay and Experiment II as a pre-incubation assay using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100. Each concentration, including the controls, was tested in triplicate. The substance was tested up to 5000 µg/plate. Toxic effects, evidenced by a reduction in the number of revertants, occurred in strain TA 1535 at 1000, 2500 and 5000 µg/plate with S9 mix in Experiment I, as well as at 2500 (with S9 mix) and 5000 µg/plate (with and without S9 mix) in Experiment II. The plates incubated with the test substance showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. A significant dose-dependent increase in revertant colony numbers was observed in strain TA 1537 with S9 mix in Experiments I and II and in strain TA 98 without S9 mix in Experiment II. Based on the findings of the study, it was concluded that the test substance induced gene mutations by frameshifts in the genome of strains TA 1537 and TA 98. Therefore, it is considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 04 April, 1995 to 14 July, 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:

yes

Remarks:

(supernatant containing the microsomes was stored at -80 °C)

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

no data

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Large stocks of the V79 cell line are stored in liquid nitrogen in the cell bank of CCR allowing the repeated use of the same cell culture batch in experiments. Before freezing, the level of spontaneous mutants was depressed by treatment with HAT-medium. Each batch is screened for mycoplasma contamination and checked for karyotype stability and spontaneous mutant frequency. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells. Thawed stock cultures are propagated at 37 °C in 80 cm² plastic flasks. About 5x10E5 cells are seeded into each flask with 15 mL of MEM supplemented with 10 % foetal calf serum. The cells are subcultured twice weekly. The cell cultures are incubated at 37 °C in a 4.5 % carbon dioxide atmosphere (95.5 % air). For the selection of mutants the medium is supplemented with 11 µg/mL thioguanine.

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; 3; 10; 30**; 100** and 270** µg/mL
With S9 mix: 1; 3*; 10; 30; 100 and 270** µg/mL
Experiment II:
Without S9 mix: 1; 3*; 10; 15; 20 and 25** µg/mL
With S9 mix: 1; 3*; 30*; 100; 150 and 200 µg/mL
Experiment III:
Without S9 mix: 20*; 21*; 22*; 23* and 24 µg/mL

* Not evaluated, culture not continued; ** not evaluated, toxic effects

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. The final concentration of DMSO in the culture medium did not exceed 1 % v/v.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

(without metabolic activation)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

(with metabolic activation)

Details on test system and experimental conditions:

Seeding: Three days old exponentially growing stock cultures (more than 50 % confluent) were trypsinized at 37 °C for 5 min. Then the enzymatic digestion was stopped by adding complete culture medium and a single cell suspension was prepared. The trypsin concentration for all subculturing steps was 0.2 % in Ca-Mg-free salt solution. Prior to the trypsin treatment the cells were rinsed with Ca-Mg-free salt solution containing 200 mg/L EDTA (ethylene diamine tetraacetic acid). The cell suspension was seeded into plastic culture flasks. Approx. 1.5x10E6 (single culture) and 5x10E2 cells (in duplicate) were seeded in MEM with 10 % FCS (complete medium) for the determination of mutation rate and toxicity, respectively.

Treatment: After 24 h the medium was replaced with serum-free medium containing the test substance, either without S9 mix or with 50 µL/mL S9 mix. After 4 h this medium was replaced with complete medium after two washing steps with "saline G".

Experimental scheme:
Segment a): Procedure for determination of toxicity
Segment b): Procedure for determination of mutation rates
Day 1: Subculturing of a log-phase culture which showed an initial spontaneous mutation rate at the beginning of the experiment of 17.0 (experiment I) and 6.4 (experiment II) mutants per 10E6 cells.
a) About 500 cells in 5 mL medium/25 cm²-plastic-flask for cloning efficiency; in duplicate per experimental point
b) 1x10E6 cells in 30 mL medium/175 cm²-plastic-flask for the mutagenicity test, 1 flask per experimental point
Day 2: Treatment of a) and b)
Experiment I:
Day 5: Subculturing of b) in 175 cm²-plastic-flasks 1.5x10E6 cells in 30 mL medium/175 cm²- plastic-flasks
Experiment II:
Day 6: see day 5
Day 8: Fixation and staining of colonies in a)-flasks determination of concentration-related cloning efficiency
Day 9: Subculturing of b) in five 80 cm²-plastic-flasks containing selective medium: mutant selection (about 3-5x10E5 cells/flask); subculturing of b) in two 25 cm²-flasks for cloning efficiency (about 500 cells/flask)
Day 16: Fixation and staining of colonies in b) - derived flasks seeded on day 9 (cloning efficiency).
Day 18: Fixation and staining of colonies in b) - derived flasks seeded on day 9 (mutant selection).
The cultures were incubated at 37 °C in a humidified atmosphere with 4.5 % CO2. The colonies were stained with 10 % methylene blue in 0.01 % KOH solution. The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

Evaluation criteria:

A test substance 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 substance 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 substance 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 substance 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 corresponding negative control data. If there is by chance a low spontaneous mutation rate in the range normally found (0 - 45 mutants per 10E+6 cells) a concentration-related increase of the mutations within this range has to be discussed.

Statistics:

Since the distribution of mutant cells does not follow known statistical models, an adequate statistical method is not available.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Without metabolic activation strong toxic effects occurred already at concentrations as low as 20 µg/mL. With metabolic activation, the concentration of the test substance could be increased up to 200 µg/mL. The cloning efficiency of the cells was reduced below 30 % at the highest concentrations tested. The number of mutant colonies was not increased compared to the frequency of spontaneous mutations at any concentration of the test substance. There was also no indication of a concentration depend on increase of mutant colonies. In all experiments of this study (with and without S9 mix) the range of the negative controls was from 10.1 up to 16.5 mutants per 106 cells; the range of the groups treated with the test substance was from 3.5 up to 20.6 mutants per 10E6 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.

none

Conclusions:

The test substance was considered to be non-mutagenic in the HPRT assay.

Executive summary:

A study was performed to investigate the potential of the test substance ( at ca. 80 % purity) to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster according to OECD Guideline 476 in compliance with GLP. The assay was performed in three independent experiments, using identical procedures, two with and three without liver microsomal activation. Due to unexpectedly strong toxic effects, an additional experiment without metabolic activation was required to complete the data of Experiment I. Without metabolic activation, strong toxic effects occurred already at concentrations as low as 20 µg/mL. With metabolic activation, the concentration of the test substance could be increased up to 200 µg/mL. The cloning efficiency of the cells was reduced below 30 % at the highest concentrations tested. The number of mutant colonies was not increased compared to the frequency of spontaneous mutations at any concentration of the test substance. There was also no indication of a concentration depend increase of mutant colonies. Hence based on the above findings, it was concluded that the test substance did not induce gene mutations at the HPRT locus in V79 cells.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: other: clastogenicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-01-28 to 2014-07-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 of Wistar phenobarbital and ß-naphthoflavone-induced rat liver S9 mix

Test concentrations with justification for top dose:

Pre-experiment:
with and without metabolic activation: 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000, 2500 and 5000 µg/mL

Experiment I:
without metabolic activation: 7, 9 and 15 µg/mL
and with metabolic activation: 180, 200 and 220 µg/mL
Experiment II:
without metabolic activation: 2.5, 10 and 50 µg/mL
with metabolic activation: 190, 200 and 230 µg/mL

Vehicle / solvent:

-Vehicle (s)/solvent(s) used: cell culture medium
-Justification for choice of solvent/vehicle: Due to the nature of the test item it was dissolved in cell culture medium (MEM). The solvent was compatible with the survival of the cells and the S9 activity.

Untreated negative controls:

yes

Remarks:

(MEM)

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation (600 µg/mL)

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation (0.83 and 1.11 µg/mL)

Details on test system and experimental conditions:

TREATMENT TIME:
4 hours (Experiment I with and without metabolic activation, experiment II with metabolic activation);
20 hours (Experiment II without metabolic activation);

FIXATION INTERVAL: 20 hours (Experiment I and II with and without metabolic activation)
NUMBER OF REPLICATIONS: 2 independent experiments
NUMBER OF CELLS SEEDED: 1 x 10E4 - 5 x 10E4 cells
NUMBER OF CULTURES: two cultures per concentration
NUMBER OF CELLS SCORED: 200 cells per concentration (100 cells per culture) except for the dose group 6 in experiment I with metabolic activation (200 for the 1st and 100 for the 2nd culture)
DETERMINATION OF CYTOTOXICITY: Mitotic index, cell count

Evaluation criteria:

There are several criteria for determining a positive result:
- a clear and dose-related increase in the number of cells with aberrations,
- a biologically relevant response for at least one of the dose groups, which is higher than the laboratory negative control range (up to 4.0% aberrant cells without and 4.3% with metabolic activation)

Statistics:

A statistical evaluation was used as an aid for interpretation of the results. Statistical significance at the 5 % level (p <0.05) was evaluated by the Fischer´s exact test. The p value was used as a limit in judging for significance levels in comparison with the corresponding negative/solvent control.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Summary: Experiment I and II, without metabolic activation

	Dose Group	Concentration [µg/mL]	Relative Mitotic Index [%]	Relative Cell Count [%]	Mean % Aberrant Cells		Historical Laboratory Negative Control Range	Precipi-tation^a	Statistical Signifi-cance^b
					incl. Gaps	excl. Gaps
Experiment I 4 h treatment, 20 h preparation interval	C	0	100	100	5.0	3.0	0.0 % - 4.0 % aberrant cells	-	/
	3	7	99	83	4.5	2.0		-	-
	4	9	73	82	3.5	1.5		-	-
	6	15	50	74	7.0	4.0		-	-
	EMS	600	108	87	10.5	8.5		-	+
Experiment II 20 h treatment, 20 h preparation interval	C	0	100	100	2.5	1.0	0.0 % - 4.0 % aberrant cells	-	/
	1	2.5	89	87	4.5	2.0		-*	-
	3	10	68	75	8.0	2.5		-*	-
	5	50	42	78	5.5	3.0		-*	-
	EMS	600	57	64	26.0	22.0		-	+

  Summary: Experiment I and II, with metabolic activation

	Dose Group	Concentration [µg/mL]	Relative Mitotic Index  [%]	Relative Cell Count [%]	Mean % Aberrant Cells		Historical Laboratory Negative Control Range	Precipi-tation^a	Statistical Signifi-cance^b
					incl. Gaps	excl. Gaps
Experiment I 4 h treatment, 20 h preparation interval	C	0	100	100	5.0	2.0	0.0 % - 4.0 % aberrant cells	-	/
	5	180	98	72	7.0	3.0		-	-
	6	200	59	66	5.0	3.7		-	-
	7	220	43	57	3.5	1.5		-	-
	CPA	0.83	81	100	11.0	9.5		-	+
Experiment II 4 h treatment, 20 h preparation interval	C	0	100	100	7.0	4.0	0.0 % - 4.0 % aberrant cells	-	/
	4	190	88	69	7.0	3.5		-	-
	5	200	70	55	7.5	2.5		-	-
	6	230	53	44	5.5	1.0		-	-
	CPA	1.11	107	53	13.5	11.0		-	+

Conclusions:

FAT 20060/F is considered to be non-clastogenic in this chromosome aberration test.

Executive summary:

To investigate the potential of FAT 20060/F to induce structural chromosome aberrations in Chinese hamster V79 cells, an in vitro chromosome aberration assay was carried out. The metaphases were prepared 20 h after start of treatment with the test item. The treatment interval was 4 h without and with metabolic activation in experiment I. In experiment II, the treatment interval was 20 h without and 4 h with metabolic activation. Duplicate cultures were treated at each concentration. 100 metaphases per culture were scored for structural chromosomal aberrations (for exceptions, see Tables). Based on the results of the solubility test MEM cell culture medium was used as solvent (MEM + 0 % FBS). The following concentrations were evaluated for the microscopic analysis of chromosomal aberrations:

Experiment I:

without metabolic activation: 7, 9 and 15 µg/mL

with metabolic activation: 180, 200 and 220 µg/mL

Experiment II:

without metabolic activation: 2.5, 10 and 50 µg/mL

with metabolic activation: 190, 200 and 230 µg/mL

In experiment I without and with metabolic activation and II with metabolic activation, no precipitation of the test item was noted in all dose groups evaluated. In experiment II without metabolic activation no precipitation, but residuals of the test item were observed in the cell culture medium. The medium was coloured red from the dark red colour test item.

In experiment I without metabolic activation, cytotoxic effects of the test item were noted at a concentration of 15 µg/mL as indicated by the relative mitotic index.According the OECD Guideline 473 the maximum of cytotoxicity should not exceed the limit of 55 % ± 5 %. Higher levels of cytotoxicity may induce chromosome damage as a secondary effect of cytotoxicity. According to laboratory experience a culture showing reduced cell viability (more than 30 % rel. cytostasis) of the negative control displays cytotoxicity. Due to this the acceptable limit of cytotoxicity is ≤70 %. This corresponds to ≥30 % of rel. cytostasis.The relative cell count was not decreased. With metabolic activation cytotoxic effects of the test item were seen at concentrations of 200 µg/mL and higher as indicated by the relative mitotic index. The relative cell count was decreased below 70 % rel. cell count at concentrations of 200 µg/mL and higher.

In experiment II without metabolic activation, cytotoxic effects of the test item were observed at concentrations of 10 µg/mL and higher as indicated by the relative mitotic index. The relative cell count was not decreased below 70 % rel. cell count. With metabolic activation, cytotoxic effects of the test item were noted at a concentration of 230 µg/mL considering the relative mitotic index. The relative cell count was decreased below 70 % rel. cell count at all dose groups evaluated for microscopic analysis.

In both experiments, no biologically relevant increase of the aberration rates was noted after treatment with the test item without and with metabolic activation. The aberration rates of all dose groups treated with the test item were within the historical control data of the negative control.

In the experiments I and II without and with metabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item as compared to the negative controls. The positive controls induced the appropriate responses. There was no evidence of chromosome aberration induced over background. This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5375; OECD 473 for in vitro cytogenetic mutagenicity data. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial reverse mutation assay:

The bacterial reverse mutation assay was performed in two independent experiments both with and without liver microsomal activation. Experiment I was performed as a plate incorporation assay and Experiment II as a pre-incubation assay using Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100. Toxic effects, evidenced by a reduction in the number of revertants, occurred in strain TA 1535 at 1000, 2500 and 5000 µg/plate with S9 mix in Experiment I, as well as at 2500 (with S9 mix) and 5000 µg/plate (with and without S9 mix) in Experiment II. The plates incubated with the test substance showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. A significant dose-dependent increase in revertant colony numbers was observed in strain TA 1537 with S9 mix in Experiments I and II and in strain TA 98 without S9 mix in Experiment II. Based on the findings of the study, it was concluded that the test substance induced gene mutations by frameshifts in the genome of strains TA 1537 and TA 98. Therefore, it is considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.

 

In vitro mammalian cell gene mutation assay:

A study was performed to investigate the potential of the test substance ( at ca. 80 % purity) to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster according to OECD Guideline 476 in compliance with GLP. The assay was performed in three independent experiments, using identical procedures, two with and three without liver microsomal activation. Due to unexpectedly strong toxic effects, an additional experiment without metabolic activation was required to complete the data of Experiment I. Without metabolic activation, strong toxic effects occurred already at concentrations as low as 20 µg/mL. With metabolic activation, the concentration of the test substance could be increased up to 200 µg/mL. The cloning efficiency of the cells was reduced below 30 % at the highest concentrations tested. The number of mutant colonies was not increased compared to the frequency of spontaneous mutations at any concentration of the test substance. There was also no indication of a concentration- dependent increase of mutant colonies. Hence, based on the above findings, it was concluded that the test substance did notinduce gene mutations at the HPRT locus in V79 cells.

 

In vitro chromosomal aberration assay:

Clastogenic potential of FAT 20060/F was evaluated in an in vitro chromosomal aberration assay performed with Chinese hamster V79 cells. The assay included two experiments both with and without metabolic activation. In both experiments, no biologically relevant increase of the aberration rates was noted after treatment with the test item without and with metabolic activation. The aberration rates of all dose groups treated with the test item were within the historical control data of the negative control. In the experiments I and II without and with metabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item as compared to the negative controls. The positive controls induced the appropriate responses. There was no evidence of chromosome aberration induced over background.

 

Conclusion:

The test substance was evaluated for genotoxic potential in the standard Ames/salmonella gene mutation test. Follow-up tests in mammalian cells included the in vitro mammalian cell gene mutation test (HPRT-locus) and the in vitro cytogenicity chromosome aberration study. The test substance was found to induce mutations in bacterial cells, however, it was not mutagenic in the in vitro mammalian cell gene mutation test. Hence, the substance is considered to be not mutagenic. Further, the test substance was found to be in the in vitro mammalian chromosome aberration test. Based on the above results, it can be concluded that Acid Red 315 is neither mutagenic nor clastogenic, hence not genotoxic.

Justification for classification or non-classification

Based on the above stated assessment of the genotoxic potential of the substance (Ames test positive,in vitro mammalian cell gene mutation test negative; in vitro mammalian chromosome aberration test negative) the substance is deemed non-genotoxic and accordingly does not need to be classified according to Council Directive 2001/59/EC (28th ATP of Directive 67/548/EEC) and according to CLP (Regulation (EC) No 1272/2008 Of The European Parliament And Of The Council) as implementation of UN-GHS in the EU.