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

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

None

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1982

Report date:

1982

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only four tester strains tested; no tester strain to detect cross-linking mutagens was included.

Principles of method if other than guideline:

None

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Trisodium 5-({4-chloro-6-[ethyl(phenyl)amino]-1,3,5-triazin-2-yl}amino)-4-hydroxy-3-[(1-sulfonatonaphthalen-2-yl)diazenyl]naphthalene-2,7-disulfonate

Cas Number:

84045-65-8

Molecular formula:

C31H21Na3ClN7O10S3

IUPAC Name:

Trisodium 5-({4-chloro-6-[ethyl(phenyl)amino]-1,3,5-triazin-2-yl}amino)-4-hydroxy-3-[(1-sulfonatonaphthalen-2-yl)diazenyl]naphthalene-2,7-disulfonate

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

None

Method

Target gene:

Histidine-auxotrophic strains of Salmonella typhimurium

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction from rats

Test concentrations with justification for top dose:

0.2, 2, 20, 200 and 2000 µg (or nl) per Petri dish

Vehicle / solvent:

distilled-water

Controls

Untreated negative controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: Without S9: TA 98 daunomycine (1 µg); TA 100 and TA 1535: MNNG (1.6 µg); and TA 1537: 9-aminoacridine (50 µg). With S9: all strains 2-anthramine.

Details on test system and experimental conditions:

GROWING AND CONSERVATION OF BACTERIAL TEST STRAINS
The bacterial strains were kept as frozen broth cultures, in aliquots of 0.5 ml at -70 °C with 8.0 % dimethylsulfoxide (DMSO). Fresh cultures were prepared by adding 0.1 ml of a thawed stock culture to 15 ml of nutrient broth (normal Difco nutrient broth for strains TA 1535 and TA 100, and double strength Difco nutrient broth for TA 1537 and TA 98, in 0.5 % NaCI). A nutrient agar (2.5 % Difco nutrient broth, 1.2% Difco agar) was also streaked. The broth was incubated in the dark in a shaking water bath at 37 °C for 16 hours, while the plate was incubated at 37 °C overnight. Broths and plates were then transferred
to the refrigerator, for up to one week.

PREPARATION OF MATERIALS
The minimal-glucose agar medium base was prepared from a 1.5 % Bacto-Difco agar in Vogel & Bonner E medium with 2 % glucose. The top agar is a 0.6 % Bacto-Difco agar, 0.6 % NaCI solution. Before use, the agar was melted in a boiling water bath and then left to equilibrate to 45 °C. Ten ml of sterile 0.5 mM L-histidine - 0.5 mM biotin were added per 100 ml of top agar.
The compounds to be tested were prepared fresh daily in DMSO or in sterile water. The appropriate dilutions were made from a 20 mg (or nl)/ml stock. The S-9 mix was prepared fresh daily from sterile stocks. 0.5 ml contains:
4.0 µ moles MgCl2
16.5 µ moles KCl
2.5 µ moles G-6-P
2.0 µ moles NADP
50.0 µ moles Phosphate Buffer, pH 7.4
150.0 µl liver homogenate.

Evaluation criteria:

The criteria of mutagenicity used in this test are a doubling of the spontaneous reversion rate and a dose-effect relationship.

Results and discussion

Test results

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Any other information on results incl. tables

RESULTS

The lack of evidence for mutagenicity existed both in the absence and presence of a liver microsomal enzyme preparation (S-9 mix) from male rats pretreated with Aroclor 1254 and over a concentration range of 0.2 to 2000 µg of product per Petri dish.

Applicant's summary and conclusion

Conclusions:

FAT 40147/D is considered to be non-mutagenic in the bacterial reverse mutation assay.

Executive summary:

A study was performed to investigate the potential of FAT 40147/D to induce gene mutations according to the bacterial reverse mutation assay using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100. The test article was tested at the following concentrations:  0.2, 2, 20, 200 and 2000 µg (or nl) per Petri dish. The lack of evidence for mutagenicity existed both in the absence and presence of a liver microsomal enzyme preparation (S-9 mix) from male rats pretreated with Aroclor 1254 and over a concentration range of 0.2 to 2000 µg of product per Petri dish. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce gene mutations. Therefore, FAT 40147/D is considered to be non-mutagenic in this bacterial reverse mutation assay.