alkoxy substituted aryl diazo heterocyclic aryl nitrile

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance has no mutagenic effects in the mammalian test system.

The mutagenic effects observed in the Ames test with the test item is a bacteria specific effect which is well investigated in nitro-compounds and is not relevant to mammalians.

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:

23 December 2014 to 27 April 2015

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:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial gene mutation assay

Target gene:

The test item Disperse Yellow DYLA 1306 was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 liver homogenate from induced rat (rat mixed induction).

Test concentrations with justification for top dose:

According to solubility, in Main Assay I: 248, 124, 62.0, 31.0 and 15.5 µg/plate.
Main Assay II: 248, 165, 110, 73.3, 48.9 and 32.6 μg/plate (TA100 +S9)

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

Toxicity and Main Assay I were performed using the plate incorporation method.
A second separate experiment were performed on the TA100 tester strain in order to confirm the positive results obtained in the first experiment.

Evaluation criteria:

For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.

Statistics:

Doubling rate (Chu et al. 1981).
Regression line.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

in the Main Assay 1, test result was questionable, in Main Assay II, there was a weak positive result

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test item induced increases in the number of revertant colonies, which were at least two-fold greater than the control value at the highest dose levels with TA100 tester strain in the presence of S9 metabolic activation.

Remarks on result:

not determinable because of methodological limitations

Remarks:

as the substance is a nitro-compound, the bacterial nitroreductase causes a bacteria-specific positive effect

Conclusions:

It is concluded that the test item Disperse Yellow DYLA 1306 might probably induce reverse mutation in Salmonella thyphimurium TA100 tester strain in the presence of S9 metabolism, under the reported experimental conditions.

Executive summary:

The test item Disperse Yellow DYLA 1306 was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone. The test item was used as a solution in dimethylsulfoxide (DMSO).

Toxicity test: Based on results obtained in a preliminary solubility trial, the test item Disperse Yellow DYLA 1306 was assayed in the toxicity test at a maximum concentration of 248 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 78.4, 24.8, 7.84 and 2.48 µg/plate. Precipitation of the test item was observed at the end of the incubation period at the two highest concentrations. No toxicity was observed with any tester strain in the absence or presence of S9 metabolism. Increases in revertant numbers were observed in the presence of S9 metabolism with TA100 tester strain.

Main Assay: On the basis of toxicity test results, in Main Assay I, using the plate incorporation method, the test item was assayed at the following dose levels: 248, 124, 62.0, 31.0 and 15.5 μg/plate.

Precipitation of the test item was observed at the end of the incubation period at the two highest concentrations both in the absence and presence of S9 metabolic activation.

No toxicity was observed with any tester strain at any dose level in the absence or presence of S9 metabolism. The test item induced increases in the number of revertant colonies in the presence of S9 metabolism with TA100 tester strain reaching a two-fold increase at the highest concentration tested, which showed precipitation of the test substance, compared to the vehicle control, but not compared to the untreated control (MF 1.8 -fold).

An additional confirmatory experiment (Main Assay II) was performed in which TA100 tester strain was treated in the presence of S9 metabolism at the dose levels of 248, 165, 110, 73.3, 48.9 and 32.6 μg/plate. Dose related increases in revertant numbers were observed. These increases were greater than twice the control values at the two highest dose levels, which were the dose levels with precipitation, thus indicating a possible positive response with TA100 tester strain in the presence of S9 metabolism.

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:

11 May 2015 - 28 July 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:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

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

Target gene:

The test item Disperse Yellow DYLA 1306 was examined for mutagenic activity by assaying for the induction of 6-thioguanine resistant mutants in Chinese hamster V79 cells after in vitro treatment. 6-thioguanine can be metabolised by the enzyme hypoxanthine-guaninphosphoribosyl-transferase (HPRT) into nucleotides, which are used in nucleic acid synthesis resulting in the death of HPRT-competent cells. HPRTdeficient cells, which are presumed to arise through mutations in the HPRT gene, cannot metabolise 6-thioguanine and thus survive and grow in its presence.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: EMEM medium supplemented with 10% Foetal Calf Serum (EMEM
complete)
- Properly maintained: yes; permanent stock of V79 cells are stored in liquid nitrogen and
subcoltures are prepared from the frozen stocks for experimental use.
- Periodically checked for Mycoplasma contamination: yes
- The karyotype, generation time, plating efficiency and mutation rates (spontaneous and induced)
have been checked in this laboratory.
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 tissue fraction: Species: Rat Strain: Sprague Dawley Tissue: Liver Inducing Agents: Phenobarbital – 5,6-Benzoflavone Producer: MOLTOX, Molecular Toxicology, Inc. Batch Number 3417

Test concentrations with justification for top dose:

A preliminary cytotoxicity assay was performed at the following dose levels: 100, 50.0, 25.0, 12.5, 6.25, 3.13,1.56, 0.781 and 0.391 μg/mL.
Two independent assays for mutation to 6-thioguanine resistance were performed using dose levels:
Main Assay I (+/- S9): 25.0, 12.5, 6.25, 3.13, 1.56 and 0.781 μg/mL.
Main Assay II (+/- S9): 10.0, 6.25, 3.91, 2.44 and 1.53

Vehicle / solvent:

Test item solutions/suspensions were prepared using dimethylsulfoxide (DMSO).

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

A preliminary cytotoxicity test was undertaken in order to select appropriate dose levels for the mutation assays. In this test a wide range of dose
levels of the test item was used. cell cultures were treated using the same treatment conditions as the mutation assays, and the survival of the cells
was subsequently determined. 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.
Two Mutation Assays were performed including negative and positive controls, in the absence and presence of S9 metabolising system. Duplicate cultures were prepared at each test point, with the exception of the positive controls which were prepared in a single culture. On the day before the experiment, sufficient numbers of 75 cm2 flasks were inoculated with 2 million freshly trypsinised V79 cells from a common pool. The cells were allowed to attach overnight prior to treatment. Following treatment, the cultures were incubated at 37°C for three hours. At the end of the incubation period, the treatment medium was removed and the cell monolayers were washed with PBS. Fresh complete medium was added to the flasks which were then returned to the incubator at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) to allow for expression of the mutant phenotype.
Determination of survival: The following day, the cultures were trypsinised and an aliquot was diluted and plated to estimate the viability of the cells.
Subculturing: At Day 1 a number of cells was replated in order to maintain the treated cell populations. On Day 3, the cell populations were subcultured in order to maintain them in exponential growth. When Day 8 is used as expression time, subculturing was performed on Day 4 and Day 6.
Determination of mutant frequency: A single expression time was used for each experiment: Day 6 in Main Assay I and Day 8 in Main Assay II. At the expression time, each culture was trypsinised, resuspended in complete medium and counted by microscopy. After dilution, an estimated 1 x 10^5 cells were plated in each of five 100 mm tissue culture petri dishes containing medium supplemented with 6-thioguanine. These plates were subsequently stained with Giemsa solutions and scored for the presence of mutants. After dilution, an estimated 200 cells were plated in each of three 60 mm tissue culture petri dishes. These plates were used to estimate Plating Efficiency (P.E.).

Evaluation criteria:

For a test item 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 item. 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:

The results of these experiments were subjected to an Analysis of Variance in which the effect of replicate culture
and dose level in explaining the observed variation was examined. For each experiment, the individual mutation
frequency values at each test point were transformed to induce homogeneous variance and normal distribution. The
appropriate transformation was estimated using the procedure of Snee and Irr (1981), and was found to be y = (x +
a)b where a = 0 and b = 0.275. A two way analysis of variance was performed (without interaction) fitting to two
factors:
- Replicate culture: to identify differences between the replicate cultures treated.
- Dose level: to identify dose-related increases (or decreases) in response, after allowing for the effects of replicate
cultures and expression time.
The analysis was performed separately with the sets of data obtained in the absence and presence of S9
metabolism.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Survival after treatment:
No relevant toxicity was observed at any concentration tested, in any experiment in the absence or presence of S9 metabolism.
Mutation results:
No relevant increases over the spontaneous mutation frequency were observed in any experiment, at any treatment level either in the absence or presence of
S9 metabolic activation.

Conclusions:

It is concluded that Disperse Yellow DYLA 1306 does not induce mutation in Chinese hamster V79 cells after in vitro treatment, in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Executive summary:

The test item Disperse Yellow DYLA 1306 was examined for mutagenic activity by assaying for the induction of 6-thioguanine resistant mutants in Chinese hamster V79 cells after in vitro treatment. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone. Test item solutions/suspensions were prepared using dimethylsulfoxide (DMSO). A preliminary cytotoxicity assay was performed. Based on solubility features, the test item was assayed, in the absence and presence of S9 metabolism, at a maximum dose level of 100 μg/mL and at a wide range of lower dose levels: 50.0, 25.0, 12.5, 6.25, 3.13, 1.56, 0.781 and 0.391 μg/mL. No relevant toxicity was observed at any concentration tested, in the absence or presence of S9 metabolism. Precipitation of the test item was noted starting from 12.5 μg/mL, both in the absence and presence of S9 metabolism. Two independent assays for mutation to 6-thioguanine resistance were performed using dose levels described in the following table:

Main Assay I (+/- S9): 25.0, 12.5, 6.25, 3.13, 1.56 and 0.781 μg/mL

Main Assay II (+/- S9): 10.0, 6.25, 3.91, 2.44 and 1.53 μg/mL

The dose range used in Main Assay II was modified taking into account precipitation observed by the end of treatment in the previous Main Assay. No reproducible five-fold increases in mutant numbers or mutant frequency were observed following treatment with the test item at any dose level, in the absence or presence of S9 metabolism. Negative and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolism. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system. It is concluded that Disperse Yellow DYLA 1306 does not induce gene mutation in Chinese hamster V79 cells after in vitro treatment, in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

Mutagenicity Assessment Disperse Yellow DYLA 1306

The test item Disperse Yellow DYLA 1306 was tested weakly positive in the Ames test with metabolic activation in Samonella typhimurium TA100 but negative in an HPRT assay in mammalian cells. This positive effect in the bacterial mutation assay is a bacteria-specific effect due to bacterial nitro-reductases, which are highly effective in these bacterial strains, but not in mammalian cells.

It is well-known for aromatic nitro compounds to be positive in the Ames assay resulting from metabolism by the bacteria-specific enzyme nitro-reductase [Tweats et al. 2012]. However, it has been demonstrated in various publications that this is a bacteria-specific effect and that these Ames positive substances are not mutagenic in mammalian assays.

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 Muta^TM 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. Thus, fexinidazole does not pose a genotoxic hazard to patients and represents a promising drug candidate for HAT.

 

 

The fact that this effect was only observed with metabolic activation and only in Samonella typhimurium TA100, is due to the fact that the metabolic activation of the aromatic amine group has been shown to be primarily prevented by steric hindrance [Klein et al 2000; Kazius et al 2005]. Hence, the nitroreductase could only activate the structure after the protective group has been removed by enzymes of the metabolising system. However this is only the case in one Salmonella strain with plasmid pKM101, whose mucAB gene products enhance SOS mutagenesis; which makes strain TA100 more sensitive for mutagen detection [Prival et Zeiger 1998].

 

 

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 Yellow DYLA 1306 is a bacteria specific effect and not relevant to mammalians.

 

 

References

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.

Kazius J, McGuire R, Bursi R: Derivation and validation of toxicophores for mutagenicity prediction. J Med Chem 2005;48:312–320.

Klein M, Voigtmann U, Haack T, Erdinger L, Boche G. From mutagenic to nonmutagenic nitroarenes: effect of bulky alkyl substituents on the mutagenic activity of 4-nitrobiphenyl in Salmonella typhimurium. Part I. Substituents ortho to the nitro group and in 2’-position. Mutat. Res. 2000;467:55-68.

Prival MJ, Zeiger E. Chemicals mutagenic in Salmonella typhimurium strain TA1535 but not in TA100, Mutat. Res. 1998;412:251–260.

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.

Additional information

The test item Disperse Yellow DYLA 1306 was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone. The test item was used as a solution in dimethylsulfoxide (DMSO).

Toxicity test: Based on results obtained in a preliminary solubility trial, the test item Disperse Yellow DYLA 1306 was assayed in the toxicity test at a maximum concentration of 248 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 78.4, 24.8, 7.84 and 2.48 µg/plate. Precipitation of the test item was observed at the end of the incubation period at the two highest concentrations. No toxicity was observed with any tester strain in the absence or presence of S9 metabolism. Increases in revertant numbers were observed in the presence of S9 metabolism with TA100 tester strain.

Main Assay: On the basis of toxicity test results, in Main Assay I, using the plate incorporation method, the test item was assayed at the following dose levels: 248, 124, 62.0, 31.0 and 15.5 μg/plate.

Precipitation of the test item was observed at the end of the incubation period at the two highest concentrations both in the absence and presence of S9 metabolic activation.

No toxicity was observed with any tester strain at any dose level in the absence or presence of S9 metabolism. The test item induced increases in the number of revertant colonies in the presence of S9 metabolism with TA100 tester strain reaching a two-fold increase at the highest concentration tested compared to the vehicle control, but not compared to the untreated control (MF 1.8 -fold).

Following the approval of a Protocol Amendment, an additional confirmatory experiment (Main Assay II) was performed in which TA100 tester strain was treated in the presence of S9 metabolism at the dose levels of 248, 165, 110, 73.3, 48.9 and 32.6 μg/plate.

Dose related increases in revertant numbers were observed. These increases were greater than twice the control values at the two highest dose levels, thus confiming the positive response with TA100 tester strain in the presence of S9 metabolism. No further experiment was undertaken.

Conclusion

It is concluded that the test item Disperse Yellow DYLA 1306 induces reverse mutation in Salmonella typhymurium TA100 tester strain in the presence of S9 metabolism, under the reported experimental conditions.

However, this effects was considered to be a bacteria-specific effect, as illustrated below and proven by the mutagenicity test in mammalian cells.

The test item Disperse Yellow DYLA 1306 was examined for mutagenic activity by assaying for the induction of 6-thioguanine resistant mutants in Chinese hamster V79 cells after in vitro treatment. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone. Test item solutions/suspensions were prepared using dimethylsulfoxide (DMSO). A preliminary cytotoxicity assay was performed. Based on solubility features, the test item was assayed, in the absence and presence of S9 metabolism, at a maximum dose level of 100 μg/mL and at a wide range of lower dose levels: 50.0, 25.0, 12.5, 6.25, 3.13, 1.56, 0.781 and 0.391 μg/mL. No relevant toxicity was observed at any concentration tested, in the absence or presence of S9 metabolism. Precipitation of the test item was noted starting from 12.5 μg/mL, both in the absence and presence of S9 metabolism. Two independent assays for mutation to 6-thioguanine resistance were performed using dose levels described in the following table:

Main Assay I (+/- S9): 25.0, 12.5, 6.25, 3.13, 1.56 and 0.781 μg/mL

Main Assay II (+/- S9): 10.0, 6.25, 3.91, 2.44 and 1.53 μg/mL

The dose range used in Main Assay II was modified taking into account precipitation observed by the end of treatment in the previous Main Assay. No reproducible five-fold increases in mutant numbers or mutant frequency were observed following treatment with the test item at any dose level, in the absence or presence of S9 metabolism. Negative and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolism. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system. It is concluded that Disperse Yellow DYLA 1306 does not induce gene mutation in Chinese hamster V79 cells after in vitro treatment, in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Hence, Disperse Yellow DYLA 1306 is not mutagenic.