Reaction products of 2,4-diaminobenzensulfonic acid and 2,3-dibromopropanoyl chloride, diazotised, subsequently coupled with 4-amino-5-hydroxynaphthalene-2,7-disulfonic acid, reacted with reaction products of 2,4,6-trichloro-1,3,5-triazine and N-ethylaniline, sodium salts

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Test substance is equal to one component of the UVCB substance under registration and it only slightly differs from the other main identified components. Details on the read-across are available in section 13. Source study has reliability 1.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reactive Red 272 - ELINCS

IUPAC Name:

Reactive Red 272 - ELINCS

Method

Target gene:

Histidine for the S. typhimurium strains and Tryptophan for the E.coli strain

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

aroclor 1254 induced rat liver S9 mix

Test concentrations with justification for top dose:

Concentration range in the range finding test: 20.6 - 5000 µg/plate
Concentration ranges in the mutagenicity tests: original experiment 520.8 to 8333.0 µg/plate and confirmatory experiment: 1646.0 to 8333.0 µg/plate

Vehicle / solvent:

Aqua bidest.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

Strain TA 98, 100, 102, 1537 and E.coli WP2 uvrA, with metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Strain TA 1535. with metabolic activation

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Strain TA 100 and 1535, without metabolic activation

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Strain E.coli WP2 uvrA, without metabolic activation

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Strain TA 102, without metabolic activation

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Strain TA 98, without metabolic activation

Controls 7

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Strain 1537, without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- After solidification the plates were inverted and incubated for about 48 hours at 37± 1.5 °C in darkness.

DETERMINATION OF CYTOTOXICITY
- The toxicity will be assessed on the basis of reduction in the number of revertant colonies.

Evaluation criteria:

- A test is considered acceptable if the mean colony counts of the negative 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.
- The test substance will be considered to be positive in the test system if the following condition is met: 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: TA 98, TA 100, TA 102, TA 1535, TA 1537 and E. coli WP2 uvrA. Generally a concentration-related effect should be demonstrable.

Statistics:

A statistical analysis was not performed. At present the use of statistical methods concerning this particular test system is not generally recommended.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- Range finding test: the experiments were performed with and without metabolic activation. Normal background growth was observed with both strains. The numbers of revertant colonies were not reduced. From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 8333.0 µg/plate with and without metabolic activation.
- Mutagenicity test, original experiment (concentration range 520.8 to 8333.0 µg/plate): in the experiments carried out with and without metabolic activation on strain TA 100, a marginal increase in the number of revertant counts was seen at the concentration of 8333.0 µg/plate. No effect occurred on the other strains.
- Mutagenicity test, confirmatory experiment (concentration range 1646.0 to 8333.0 µg/plate): in the experiments performed with and without metabolic activation, treatment of strain TA 100 again led to a marginal increase in the number of revertant counts at the concentration of 8333.0 µg/plate. Similar effects were observed in the experiment with metabolic activation on strain TA 102 and in the experiment without activation on strain WP2 uvrA at the concentration of 8333.0 µg/plate each. Since the effects obtained with strains TA 102 and WP2 uvrA were not reproducible, the criteria for a positive response were not met with these strains. No effects occurred on the other strains. In the mutagenicity tests normal background growth was observed with all strains at all concentrations. The numbers of revertant colonies were not reduced with increasing concentration. Therefore, the test substance exerted no toxic effect on the growth of the bacteria.

Applicant's summary and conclusion

Conclusions:

No evidence of induction of gene mutations by the test substance and its metabolites in the strains of S. typhimurium and E. coli used was found.

Executive summary:

Method

In a mutagenicity test, according to OECD guideline 471, 5 Salmonella typhimurium strains (TA 1535, TA 1537, TA 98, TA 100, and 102) and one E. Coli strain (WP2 uvrA) were used to test the mutagenic potential of the test substance both with and without metabolic activation.

Results

In both experiments, performed with and without metabolic activation on strain S. typhimurium TA 100, test substance led to a minor increase in the number of back-mutants at the highest concentration of 8333.0 µg/plate only. These negligible effects are, however, not sufficient as indication of a mutagenic property of the test substance. An occasionally observed slight increase in the number of revertants with strains S. typhimurium TA 102 and E. coli WP2 uvrA was not reproducible and therefore did not fulfill the criteria for a positive response. No other differences were observed. From the results obtained, it is concluded that there is no evidence of induction of gene mutations by test substance and its metabolites in the strains of S. typhimurium and E. coli used.