Trisodium 5-[[4-chloro-6-(methylphenylamino)-1,3,5-triazin-2-yl]amino]-4-hydroxy-3-[(2-sulphonatophenyl)azo]naphthalene-2,7-disulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In two Ames tests performed with the source substance, the source substance did not induce mutations with or without metabolic activation

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that the source and the target substance have very similar physicochemical and (eco)toxicological properties because their chemical structures are nearly identical. An analogue approach has thus been employed. The target substance is Reactive Red 24 carrying a methyl group, while the source chemical is Reactive Red 24:1 carrying an ethyl group.
The presence of sulphonate groups make both dyes highly water soluble and therefore less critical for human health and environmental issues. Based on their chemical similarity, similar properties are expected in both humans and the environment.
2. SOURCE AND TARGET CHEMICAL(S)
Source: Reactive Red 24:1 (CAS# 72829-25-5 / EC# 276-911-4)
Target: Reactive Red 24 (CAS# 70210-20-7 / EC# 274-417-3)
3. ANALOGUE APPROACH JUSTIFICATION
see attachment under 4.12 Auto flammability

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA98, TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

The substance is non-mutagenic in the bacterial reverse mutation assay.

Executive summary:

In two Ames tests performed with the source substance, the source substance did not induce mutations with or without metabolic activation.

The structurally related target substance will show the same behaviour and will therefore be not mutagenic in the Ames test.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1979

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

None

Qualifier:

according to guideline

Guideline:

other: AMES, B.N., J. McCANN, and E. YAMASAKI (1975), Methods for Detecting Carcinogens and Mutagens with the Salmonella/ Mammalian-Microsome Mutagenicity Test. Mut. Res. 31, 347-364.

Deviations:

not specified

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

None

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

None

Target gene:

Histidine auxotrophic strains of Salmonella typhimurium

Species / strain / cell type:

other: Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of liver from rats induced with Arochlor and cofactors

Test concentrations with justification for top dose:

None

Vehicle / solvent:

phosphate buffer

Untreated negative controls:

yes

Remarks:

Phosphate buffer

Positive controls:

yes

Positive control substance:

other: With metabolic activation: Strain TA 1535 with cyclophosphamide,

Details on test system and experimental conditions:

The tests were carried out in accordance with the method described by AMES et al. The bacteria on which the tests were performed were the histidineauxotrophic TA 98, TA 100, TA 1535, TA 15 37 and TA 1538, strains of Salmonella typhimurium.
The test was performed with the following concentrations of the trial substance without and with microsomal activation: 15, 45, 135, 405 and 1215 microg/0.1 ml. A repetition of the experiments was performed with the concentrations of 25, 75, 225, 6 75 and 2025 microg/ 0.1 ml. In these experiments tests on Strain TA 15 38 were included. The substance was dissolved in phosphate buffer. Phosphate buffer alone was used for the negative controls. In the experiments in which the substance was metabolically activated, activation mixture was added also. 1 ml activation mixture contains: 0.3 ml S9 fraction of liver from rats induced with Aroclor 1254 and 0.7 ml of a solution of co-factors.

Positive control experiments were carried out simultaneously with the following substances: 1) for Strain TA 98: daunorubicin-HCl (DAUNOBLASTINR), 5 and 10 microg/0.1 ml phosphate buffer; 2) for Strain TA 100: 4-nitroquinoline-N-oxide, 0.125 and 0.25 microg/0.1 ml phosphate buffer; 3) for Strain TA 1535: N-methyl-N1-nitro-N-nitrosoguanidine, 3 and 5 microg/0.1 ml phosphate buffer; 4) for Strain TA 15 37: 9(5) aminoacridine hydrochloride monohydrate, 50 and 100 microg/0.1 ml DMSO; 5) for Strain TA 15 38: 2-nitrofluorene, 5 and 10 microg/0.1 ml DMSO. The activation mixture was tested with Strain TA 15 35 and cyclophosphamide (ENDOXAN-ASTA ), 250 microg/0.1 ml phosphate buffer.
In the experiments with and without the addition of microsomal activation mixture three Petri dishes were prepared per strain and per group (i.e. per concentration or per control group).
The plates were incubated for about 48 hours at 37 deg C in darkness. When the colonies had been counted, the arithmetic mean was calculated.
A test substance is generally considered to be non-mutagenic if the colony count in relation to the negative control is not doubled at any concentration .

1.AMESf B.N., F.D. LEE, and W.E. DURSTON (1973), An Improved Bacterial Test System for the Detection and Classification of Mutagens and Carcinogens. Proc. Natl. Acad. Sei. USA 70, 782-786.
2.AMES, B.N., W.E. DURSTON, E. YAMASAKI, and F.D. LEE (1973), Carcinogens are Mutagens: A Simple Test System Combining Liver Homogenates for Activation and Bacteria for Detection.
Proc. Natl. Acad. Sei. USA 7_0, 2281-2285.
3.AMES, B.N., J. McCANN, and E. YAMASAKI (1975), Methods for Detecting Carcinogens and Mutagens with the Salmonella/ Mammalian-Microsome Mutagenicity Test. Mut. Res. 31, 347-364.

Rationale for test conditions:

None

Evaluation criteria:

None

Statistics:

None

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

In the experiments performed with and without microsomal activation, comparison of the number of histidine-prototrophic mutants in the controls and after treatment with FAT 40034/B revealed no marked differences.

Remarks on result:

other: all strains/cell types tested

None

Conclusions:

FAT 40034/B was determined to be non-mutagenic in the bacterial reverse mutation assay.

Executive summary:

FAT 40034/B was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium which included TA 98, TA 100, TA 1535, TA 15 37 and TA 1538. The investigations were performed with the following concentrations of the trial substance with and without microsomal activation: 15, 45, 135, 405 and 1215 microg/0.1 ml. A repetition of the experiments was performed with the concentrations of 25, 75, 225, 675 and 2025 microg/0.1 ml. In these experiments tests on Strain TA 1538 were included.

In the experiments performed with and without microsomal activation, comparison of the number of back-mutant colonies in the controls and the cultures treated with the various concentrations of FAT 40034/B revealed no marked deviations.

Based on the findings of the study, no evidence of the induction of point mutations by FAT 40034/B or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments. Hence, FAT 40034/B was determined to be non-mutagenic in the bacterial reverse mutation assay.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Justification for type of information:

None

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only 4 tester strains tested; no tester strain to detect crosslinking mutagens was included

Principles of method if other than guideline:

None

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

None

Target gene:

None

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from rat liver

Test concentrations with justification for top dose:

0.2, 2, 20, 200, 2000 µg/petri dish

Vehicle / solvent:

DMSO

Details on test system and experimental conditions:

Growing and Confirmation of Bacterial Test Strains:
The bacterial strains are kept as frozen broth cultures, in aliquots of 0.5 ml, at -70°C with 8.0* dimethylsulfoxide (DMSO). Fresh cultures are prepared by adding 0.1 ml of a thawed stock culture to 15 ml of nutrient broth (0.8* Difco nutrient broth, 0.5* NaCl). A nutrient agar (2.5% Difco nutrient broth, 1.2* Difco agar) is also streaked with the scrapings. The broth is incubated in the dark in a shaking water bath at 37 deg C for 16 hours, whilst the plate is incubated at 37 C overnight. Broths and plates are then transferred to the refrigerator where they can be kept for one week.

Checking out Tester Strains:
All strains are tested for the presence of their mutations.
a) The mutation in the histidine operon, basic to the test system is tested by checking for growth in the presence of absence of histidine on a minimal medium-agar base.
Bacteria of the nutrient agar plate are streaked on a minimal medium plate supplemented with 0.1 ml of 0.1 mM L-histidine and 0.1 ml of 0.5 mM biotin as a growth requirement.
b) Sensitivity to crystal violet is a check for the presence of the deep rough (rfa) mutation, the loss of the lipopolysaccharide coat on the bacterial surface. Three drops of the broth culture are spread on the surface of a
nutrient agar plate. A sterile filter paper disc containing crystal violet (10 microlitre of a 1 mg/ml solution) is placed on this surface. A zone of inhibition around the disc, after 24 hours incubation, shows the presence of the (rfa) mutation.
c) Two of the tested strains (98 and 100) contain a plasmid expressing resistance to ampicillin (R factor). To check for its presence, a sterile filter paper disc containing Ampicillin (10 microlitre of 8 mg/ml in 0.02 N NaOH) is placed on a nutrient agar plate spread with the broth. In strains 1535 and 1537 a zone of inhibition occurs around the disc, but for 98 and 100 where the R factor is present, no zone of inhibition is seen.
d) The uvrB deletion, the loss of the excision repair system, makes the bacteria sensitive to UV irradiation. One drop of the broth is cross-streaked on a nutrient agar plate. One half of the plate is irradiated for 20 seconds under a 15 watt UV lamp at approximately 30 cm. After 24 hours' incubation, growth was found only on the unirradiated part of the streak for all strains.

Induction of rat liver enzymes:
Three male Sprague-Dawley rats weighing approximately 200 grams are given an I.P. injection of Aroclor 125^ (Monsanto) in peanut oil at a
dose of 500 mg/kg.
Five days after the injection, the rats are anaesthetized with ether, decapitated and the livers removed sterilely. The livers are homogenized in 2.0 ml/g tissue sterile 0.15 M KCl, pooled together and centrifuged for 15 min at 9000 x g (Beekman ultracentrifuge). The supernatant (termed the S-9 fraction) is aliquoted into sterile tubes and frozen. These are stored at -70 C.

Rationale for test conditions:

None

Evaluation criteria:

None

Statistics:

None

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

None

Remarks on result:

other: all strains/cell types tested

None

Conclusions:

FAT 40034 was not mutagenic for Salmonella typhimurium strains TA 1535, TA1537, TA 98 and TA 100.

Executive summary:

A bacterial reverse mutation assay was performed to determine the mutagenic potential of FAT 40034. Salmonella typhimurium strains TA 1535, TA1537, TA 98 and TA 100 were exposed to the test substance with and without metabolic activation. However, under the conditions employed and using the doubling of the spontaneous reversion rate as a criterion of mutagenicity, FAT 40034/A did not induce significant reverse mutations with and without metabolic activation. Hence, based on the findings of the study, it can be concluded that FAT 40034 was not mutagenic in the bacterial reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The two in vitro studies performed with the source substance, showed that the substance did not induce mutations in bacteria with or without metabolic activation.

The structurally related target substance will show similar behaviour and therefore it is anticipated that it will not be mutagenic in the Ames test either.

Justification for classification or non-classification

Based on the above mentioned results the substance does not need to be classified according to CLP regulation (Regulation EC No.1272/2008).