Benzoic acid, 2-hydroxy-5-[[4-[[4-[[8-hydroxy-7-[[4-[(8-hydroxy-3,6-disulfo-1-naphthalenyl)azo]-2-methoxy-5-methylphenyl]azo]-3,6-disulfo-1-naphthalenyl]amino]-6-(phenylamino)-1,3,5-triazin-2-yl]amino]phenyl]azo]-, pentasodium salt

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Non mutagenic in the AMES assay.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From November 16 to December 8, 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Deviations:

yes

Remarks:

no statistical analysis was performed.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

61.7, 185.2, 555.6, 1666.7, 5000 µg/plate.
No toxic effect on the growth of bacteria.

Vehicle / solvent:

- Vehicle used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

Remarks:

without S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: 2-aminoanthracene

Remarks:

with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h, at 37 ± 1.5 °C in darkness

Evaluation criteria:

Assay acceptance criteria
A test is considered acceptable if the mean colony counts of the control values of all strains are within the acceptable ranges and if the results of the positive controls meet the criteria for a positive response.

Criteria for a positive response
The test substance is considered to be mutagenic in this test system if at least a reproducible meaningful increase of the mean number of revertants per plate above that of the negative control at any concentration for one or more of the following strains: S. typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537. Generally a concentration-related effect should be demonstrable.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

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

Vehicle controls validity:

not applicable

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

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

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:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with

Genotoxicity:

ambiguous

Remarks:

marginal increase at 1666.7 and 5000 µg/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: in orginal experiment

Toxicity test / range finding test

Six concentrations ranging from 20.6 to 5000 µg/plate were tested with strain S. typhimurium TA 100 to determine the highest concentration to be used in the mutagenicity assay. The experiments were performed with and without microsomal activation. The numbers of revertant colonies were not reduced. Normal back-ground growth was observed at all concentrations. The test material exerted no toxic effect on the growth of the bacteria . From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 5000 µg/plate without and with activation.

Mutagenicity test, original experiment

In the experiment performed without and with metabolic activation, treatment with test substance did not lead to an increase in the incidence of histidine-prototrophic mutants in comparison with the negative control.

Mutagenicity test, confirmatory experiment

In the experiment carried out without metabolic activation, again none of the tested concentrations led to an increase in the incidence of histidineprototrophic mutants by comparison with the negative control.

In the experiment performed with activation on strain TA 102, a marginal increase in the number of back-mutants was observed at the concentrations of 1666.7 and 5000 µg/ml. Since no such effects were registered in the original experiment conducted on this strain, the latter finding is not attributed to a mutagenic action of the test material. The marginal increase in the number of revertant counts probably may be due to spontaneously occurring back-mutants. Again, no effect was seen with the other strains.

In the mutagenicity tests without and with metabolic activation, normal back-ground growth was observed. The numbers of revertant colonies was not reduced. The test material exerted no toxic effect on the growth of the bacteria. The various mutagens, promutagens, sterility checks, sensitivity and resistance tests, etc., employed to ensure the test system was acceptable, all produced results within our established limits.

Conclusions:

Based on test results and on standard evaluation criteria, it is concluded that test substance and its metabolites did not induce gene mutations in the strains of S. typhimurium used.

Executive summary:

Method

The test was conducted to detect gene mutations induced by test substance or its metabolites in histidine-requiring strains of Salmonella typhimurium.

The concentration range of test substance was determined in a preliminary toxicity test . Thus, it was tested for mutagenic effects without and with metabolic activation at five concentrations in the range of 61.7 to 5000 µg/plate. An independent repetition of the experiments was performed with the same concentrations.

Results

In the toxicity/range finding test, no toxic effect of the test material on the growth of the bacteria was observed with or without metabolic activation.

In the original mutagenicity test, none of the tested concentrations led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control both with and without metabolic activation.

Similarly, in the confirmatory mutagenicity test, no mutagenic effects were noted both without and with metabolic activation.

In the mutagenicity tests without and with metabolic activation, normal background growth was observed. The number of revertant colonies was not reduced. The test substance exerted no toxic effect on the growth of the bacteria.

Overall, test substance and its metabolites did not exhibit a mutagenic potential under test conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A genetic toxicity study in bacteria (Ames test) was conducted following OECD guideline 471, using 5 Salmonella typhimurium strains:

- TA 1535, TA 100 and TA 102 to detect base-pair substitutions,

- TA 98 and TA 1537 to detect frame-shifts.

No cytotoxicity, evident as reduction in the number of revertants, was reported. Moreover, no mutagenic effects were noted, in both the original and the confirmatory experiment; in particular, positive findings with TA 102 strain in the confirmatory experiment were considered as incidental.

Based on these experimental evidences, a mutagenic potential of test item could be reasonably excluded.

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), a mutation means a permanent change in the amount or structure of the genetic material in a cell. The term ‘mutation’ applies both to heritable genetic changes that may be manifested at the phenotypic level and to the underlying DNA modifications when known.

The more general terms ‘genotoxic’ and ‘genotoxicity’ apply to agents or processes which alter the structure, information content, or segregation of DNA, including those which cause DNA damage by interfering with normal replication processes, or which in a non- physiological manner (temporarily) alter its replication.

For the purpose of classification for germ cell mutagenicity, substances are allocated to one of two categories:

 

Category 1: substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans.

Category 1A: based on positive evidence from human epidemiological studies.

Category 1B: based on:

- positive result(s) from in vivo heritable germ cell mutagenicity tests in mammals; or

- positive result(s) from in vivo somatic cell mutagenicity tests in mammals, in combination with some evidence that the substance has potential to cause mutations to germ cells.

- positive results from tests showing mutagenic effects in the germ cells of humans, without demonstration of transmission to progeny; for example, an increase in the frequency of aneuploidy in sperm cells of exposed people.

 

Category 2: substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans, based on positive evidence obtained from experiments in mammals and/or in some cases from in vitro experiments, obtained from:

- somatic cell mutagenicity tests in vivo, in mammals; or

- other in vivo somatic cell genotoxicity tests which are supported by positive results from in vitro mutagenicity assays

 

Based on negative result in the available experimental study, a mutagenic potential for Direct Green 026 was excluded.

Accordingly, the substance is not classified under the CLP Regulation (EC 1272/2008).