3,3'-[ethylenebis[(3-sulpho-p-phenylene)azo]]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonic acid] hexasodium salt AND 3,3'-[ethylenebis[(3-sulpho-p-phenylene)azo]]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonic] acid, compound with 2,2',2''-nitrilotriethanol (1:6)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study, non-GLP, strain for AT substitution was not included

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

No strain to detect AT substitution is included in the study design

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3,3'-[ethylenebis[(3-sulpho-p-phenylene)azo]]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonic] acid, compound with 2,2',2''-nitrilotriethanol (1:6)

Cas Number:

75701-36-9

IUPAC Name:

3,3'-[ethylenebis[(3-sulpho-p-phenylene)azo]]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonic] acid, compound with 2,2',2''-nitrilotriethanol (1:6)

Test material form:

solid: particulate/powder

Details on test material:

Name of test substance (as cited in the study report): Fastusol Blau PR 8516 (gefriergetrocknet)
Storage: at 4 °C

Method

Target gene:

histidine

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 fraction from Aroclor 1254 induced rat liver (prepared on Dec 7 1987)

Test concentrations with justification for top dose:

exp 1 (plate incorporation): S. typhimurium TA 1535, TA 1537, TA 98 and TA 100: 0, 20, 100, 500, 2500 and 5000 ug/plate
exp 2 (preincubation) S. typhimurium TA 1535, TA 1537, TA 98 and TA 100: 0, 20, 100, 500, 2500 and 5000 ug/plate
exp 3 (preincubation) S. typhimurium TA 98: 0, 20, 100, 500, 2500 and 5000 ug/plate

Vehicle / solvent:

aqua dist.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

yes

Remarks:

sterility control

Positive controls:

yes

Positive control substance:

other: N-methyl-N-nitro-N-nitrosoguanidine

Remarks:

for TA 100 and TA 1535 without S9

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

yes

Remarks:

sterility control

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylendiamine

Remarks:

for TA98 without S9

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

yes

Remarks:

sterility control

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

for TA 1537 without S9

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

yes

Remarks:

sterility control

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

for all strains with S9 (no additional control is included)

Details on test system and experimental conditions:

METHOD OF APPLICATION: exp 1 plate incorporation; exp 2 and 3 preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 hours at 37°C

NUMBER OF REPLICATIONS: 3 per concentration

DETERMINATION OF CYTOTOXICITY
- Method: titer/bacterial backgound lawn

S-9 fraction: ratio 3/7 as S9 fraction/cofactors prepared on the day of the experiment
concentration of cofactors:
MgCl 8 mM
KC1 33 mM
glucose-6-phosphate 5 mM
NADP 4 mM
phosphate buffer(

Evaluation criteria:

positive if:
-doubling of the spontaneous mutation rate (control)
-dose-response relationship
-reproducibility of the results

Statistics:

NA

Results and discussion

Test results

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:

cytotoxicity

Remarks:

slight reduction of the titer in some of the strains at 5000 ug/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The 3rd experiment was performed because in the 2nd experiment a decrease in bacterial background lawn was observed at all concentrations.

Applicant's summary and conclusion

Conclusions:

negative without metabolic activation
negative with metabolic activation

The substance is negative in an Ames test with and without metabolic activation

Executive summary:

The substance was tested at concentrations between 0 and 5000 ug/plate in an Ames test with and without metabolic activation. Both a plate incorporation and pre-incubation assay were perfomed. The substance did not induce mutations in Salmonella Typhimurium strains TA 98, TA100, TA1535 and TA1537. Therefore it is concluded that the substance is not mutagenic in bacterial cells.