Octasodium 7,7'-[(2,2'-disulphonato[1,1'-biphenyl]-4,4'-diyl)bis[imino(6-chloro-1,3,5-triazine-4,2-diyl)imino[2-(carbamoylamino)]-4,1-phenylene]azo]]bis(naphthalene-1,3,6-trisulphonate)

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:

From May 23 to June 30, 2017

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)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reactive Yellow 084

IUPAC Name:

Reactive Yellow 084

Test material form:

solid: particulate/powder

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Toxicity testing in strain TA98: 10, 100, 500, 1000, 2500, 5000 µg/plate
First mutagenicity assay - all strains: 50, 150, 500, 1500, 5000 µg/plate
Second mutagenicity assay with preincubation - TA100, E. coli WP2 uvrA: 50, 150, 500, 1500, 5000 µg/plate
Second mutagenicity assay with preincubation - TA98, TA1537: 15, 50, 150, 500, 1500 µg/plate
Second mutagenicity assay with preincubation - TA1535: 15, 50, 150, 500, 1500, 5000 µg/plate

Vehicle / solvent:

Water for injection

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

untreated control, no solvent

Negative solvent / vehicle controls:

yes

Remarks:

0.1 ml of water for injection

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 4-nitro-o-phenylendiamine (TA98), N-methyl-N-nitro-N-nitrosoguanidine (E.coli)

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

yes

Remarks:

untreated control, no solvent

Negative solvent / vehicle controls:

yes

Remarks:

0.1 ml of water for injection

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

other: 2-aminoanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

Preparation and using of S9
Metabolic activation was performed by S9 fraction of rat liver homogenate and mixture of cofactors. The liver homogenate was prepared from Wistar male rats weighing approximately 200 g, previously induced with Delor 106 (mixture of PCBs). Delor 106 was diluted with olive oil to a concentration of 200 mg/ml, and each rat was administered a single injection of 500 mg/kg 5 days before S9 preparation. The liver was removed from each animal and washed in ice cold 0.15M KCl. The livers washed were mixed with another 0.15 M KCl (3 ml/g wet liver) homogenised in a grinder, and the tissue suspension was centrifuged for 10 min at 9000 g. Aliquots of the supernatant (S9) were stored in plastic tubes using sterile technique at a temperature below –70 °C. Cofactors (NADP and glucoso-6-phosphate) were dissolved in buffer.
Each plate in all experiments with metabolic activation contained 0.5 ml of buffer with NADP and glucoso-6-phosphate and 30 or 100 µl S9 (the concentration of S9 in the S9mix was 5.7 or 19 %). In experiments without metabolic activation only buffer was added to the top agar.

Plate incorporation test
First experiments and toxicity test
100 µl of test substance of required concentration, 100 µl of 16-18 h culture of tester strain of density 10^8 -10^9 CFU/ml, 0.5 ml relevant buffer and S9 post-mitochondrial fraction (in case of test with metabolic activation) were added to the 2 ml of molten top agar (with trace of histidine or tryptophan) kept in a test tube at 45±3 °C. After shaking the mixture was poured into a minimal glucose agar plate.

Second experiments
0.5 ml of relevant buffer, 100 µl of test substance of required concentration, 100 µl of 16-18 h culture of tester strain of density 10^8-10^9 CFU/ml and 30/100 µl of S9 post mitochondrial fraction were mixed and shaken at 37±1 °C for 30 minutes. Then, 2 ml of molten top agar (with trace of histidine or tryptophan) was added and the mixture was poured into a minimal glucose agar plate.
Petri dishes were incubated of 72 h at 37±1 °C, the number of revertant colonies on the plate was counted manually with exception of positive controls, which were counted by an AccuCount 1000.
For an adequate estimate of variation, triplicate plating was used at each dose level. The toxicity test, which serves for finding of optimal concentrations the mutagenicity test, was performed in strain TA 98 and two Petri dishes were used for every concentration.

Selection of doses/toxicity
Solubility of 58.25 g per litre is fully sufficient for preparing the highest concentration 5 mg per ml recommended by OECD guideline 471. Test substance was dissolved in water for injection in the concentration 5000 μg per 0.1 ml. For toxicity experiment the highest concentration was diluted to the other 5 concentrations in 3 digit places interval. No problems with solubility or precipitation occurred at preparation of the concentration row.
The concentration row was tested for toxicity in strain TA 98 without metabolic activation.
No toxicity or precipitation was observed in any dose. The concentration of 5000 µg/0.1 ml was used as maximum in the first mutagenicity experiments. Further doses were diluted with factor approximately 2-√10.
In some cases decreased number of revertants was observed in the first experiments, whereas no diminution of bacterial background was observed. In these cases, concentrations for the second experiment were decreased, such that the highest dose was omitted and the concentration of 15 μg per plate was added (Salmonella typhimurium TA 98 and TA 1537) or 6 concentrations including 15 and 5000 μg per plate were used (S. typhimurium TA 1535).
To modify experimental conditions and to increase the sensitivity of the assay, the second mutagenicity experiments with metabolic activation were performed with pre-incubation (30 minutes, 37±1 °C, shaking). The same concentrations were used.
Fresh solutions of test substance were prepared before each experiment. Concentrations of test substance solution were dosed in the volume of 0.1 ml per plate.

Evaluation criteria:

The main criterion for evaluation of results was modified two-fold increase rule, which is compatible with the application of statistical methods. After this rule the result is positive, if a reproducible dose-response effect occurs and/or a doubling of the ratio Rt/Rc is reached.
An increase is considered as ”biologically relevant“:
- if the number of reversions is at least twice as high as that in the solvent control for the strains having spontaneous reversion >10;
- if the number of reversions is at least three times as high as that in the solvent control for the strains having spontaneous reversion ≤10;
A test substance producing neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.
According to OECD guideline 471, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium, other: TA98, TA1535, TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 µg

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium, other: TA98, TA100, TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

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:

valid

Positive controls validity:

valid

Remarks on result:

other: first exp.

Applicant's summary and conclusion

Conclusions:

Non mutagenic.

Executive summary:

Method

Mutagenic potential of test substance was assessed according to OECD guideline 471.

Four Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and one E. coli WP2 uvrA strain were used. Test substance was diluted in water for injection and tested in doses of 50 - 5000 µg/plate, which were applied to plates in volume of 0.1 ml.

First mutagenicity experiments were performed without and with metabolic activation using a supernatant of rat liver (30 μl or 100 μl per plate) and a mixture of cofactors by plate incorporation test with a dose range of 50 – 5000 µg/plate.

To modify experimental conditions and to increase sensitivity of the assay, second mutagenicity experiments with metabolic activation were performed with pre-incubation.

The concurrent positive controls verified the sensitivity of the assay and the metabolising activity of the liver preparations.

Results

Average revertant colony counts for vehicle controls were within the current historical control range for the laboratory.

Test substance was non-mutagenic for all strains without as well as with metabolic activation.