Trisodium 3-[[4-[[6-(anilino)-1-hydroxy-3-sulphonato-2-naphthyl]azo]-5-methoxy-o-tolyl]azo]naphthalene-1,5-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:

19 October 2016 to 25 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

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 OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Principles of method if other than guideline:

includes the Prival and Mitchell (1982) modification to assess the mutagenic activity of azo dyes.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Trisodium 3-[[4-[[6-(anilino)-1-hydroxy-3-sulphonato-2-naphthyl]azo]-5-methoxy-o-tolyl]azo]naphthalene-1,5-disulphonate

EC Number:

228-327-6

EC Name:

Trisodium 3-[[4-[[6-(anilino)-1-hydroxy-3-sulphonato-2-naphthyl]azo]-5-methoxy-o-tolyl]azo]naphthalene-1,5-disulphonate

Cas Number:

6227-20-9

Molecular formula:

C34H27N5O11S3.3Na

IUPAC Name:

trisodium 3-[[4-[[6-(anilino)-1-hydroxy-3-sulphonato-2-naphthyl]azo]-5-methoxy-o-tolyl]azo]naphthalene-1,5-disulphonate

Test material form:

solid: flakes

Specific details on test material used for the study:

the concentrations were corrected for 8.6% water

Method

Target gene:

histidine

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: University of California, Berkeley, on culture discs, on 04 August 1995
- Methods for maintenance in cell culture if applicable: stored at -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34

Metabolic activation:

with and without

Metabolic activation system:

test 1 : phenobarbital/beta-naphthaflavone induced rat liver S9; Test 2: uninduced hamster liver S9

Test concentrations with justification for top dose:

Test 1 (pre-incubation with rat S-9): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Test 2 (pre-incubation with hamster S9): 15, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: substance fully soluble up to 50 mg/L

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

congo red

mitomycin C

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 30 min at 37 ± 3 °C
- Exposure duration: 48 h at 37 ± 3 °C

NUMBER OF REPLICATIONS: 3/concentration

DETERMINATION OF CYTOTOXICITY
- Method: visible reduction in the growth of the bacterial background lawn

Evaluation criteria:

a substance is considered positive when:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester
strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).

Statistics:

Dunnetts Regression Analysis

Results and discussion

Test results

Key result

Species / strain:

other: TA1535, TA1537, TA98, TA102 and TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The mean positive control value of Congo Red used in conjunction with TA100 in the second mutation test (Privall Mitchell modification) showed an 1.9-fold ncrease over the concurrent vehicle control. This was considered acceptable as the test concentrations were all within 84 to 125% of vehicle controls (without dose response).

Applicant's summary and conclusion

Conclusions:

Based on the findings in this test the substance is considered non-mutagenic in bacteria

Executive summary:

In an Ames test the substance was tested in salmonella typhimurium strains TA1535, TA1537, TA98, TA102 and TA100 in presence and absence of metabolic activation. As the substance is an azo-dye the Prival Mitchell modification was included in the test design. No increase in mutant frequency was reported in any of the strains tested both in presence and absence of metabolic activation. The substance is considered to be non-mutagenic under the conditions of this test.