3,10-diamino-6,13-dichloro-2-((6-(((4-(1,1-dimethylethyl)phenyl)sulfonyl)amino)-2-naphthalenyl)sulfonyl)-4,11-triphenodioxazinedisulfonic acid, lithium potassium sodium salt

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:

January 2002: 08-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted July 21, 1997

GLP compliance:

yes

Type of assay:

bacterial forward mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

440-770-9

EC Name:

-

Cas Number:

371921-63-0

Molecular formula:

C38 H29 Cl2 N5 O12 S4 .x K .x Li .x Na

IUPAC Name:

3,10-diamino-2-{[6-(4-tert-butylbenzenesulfonamido)naphthalen-2-yl]sulfonyl}-6,13-dichloro-5,12-dioxa-7,14-diazapentacene-4,11-disulfonic acid lithium hydride potassium hydride sodium hydride

Test material form:

solid

Method

Target gene:

S. typhimurium TA1537: hisC3076; rfa-; uvrB-;
S. typhimurium TA 98: hisD3052; rfa-; uvrB-; R-factor
S. typhimurium TA1535: hisG46; rfa-; uvrB-;
S. typhimurium TA100: hisG46; rfa-; uvrB-; R-factor;
E. coli WP2 uvrA: trp-; uvrA-

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: uvrB deficient

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: uvrA deficient

Metabolic activation:

with and without

Metabolic activation system:

liver microsomal activation (S9 mix)

Test concentrations with justification for top dose:

Experiment I - TA98, TA 1535, TA 1537, TA 100, WP2 uvrA at 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without S9 mix
Experiment II - TA 1535, TA 1537, TA 100, WP2 uvrA at 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without S9 mix
Experiment II - TA 98 at 33, 100, 333, 1000, 2500 and 5000 µg/plate with S9 mix
Experiment II - TA 98 at 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate without S9 mix

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: the test item BLUE GS 5664.80 was dissolved in DMSO ((MERCK, D-64293 Darmstadt; purity > 99 %) and neutralised with 1N HCl.
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.

Controls

Untreated negative controls:

yes

Remarks:

Concurrent untreated and solvent controls were performed.

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: Experiment I was performed as a plate incorporation assay. Since a negative result was obtained in this experiment, experiment ll was performed as a pre-incubation assay

DURATION
- Preincubation period: 60 minutes (experiment II)
- Exposure duration: After solidification the plates were incubated upside down for at least 48 hours at 37' C in the dark.

NUMBER OF REPLICATIONS: Each concentration and the controls were tested in triplicate.

DETERMINATION OF CYTOTOXICITY
To evaluate the toxicity of the test item a pre-experiment was performed with strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA. Eight concentrations were tested for toxicity and mutation induction with three plates each. The experimental conditions in this pre-experiment were the same as described for the experiment I (plate incorporation test).
Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
The pre-experiment is reported as main experiment l, if the following criteria are met: Evaluable plates (>0 colonies) at five concentrations or more in all strains used.

Evaluation criteria:

The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce a significant increase in mutant colony frequencies

Statistics:

No statistical evaluation of the data is required

Results and discussion

Test results

Species / strain:

other: Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia colistrain WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

This study was performed to investigate the potential of BLUE GS 5664.80 to induce gene mutations according to the plate incorporation test (experiment l) and the pre-incubation test (experiment ll) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia colistrain WP2 uvrA following OECD TG 471 according to GLP.
It can be stated that during the mutagenicity test and under the experimental conditions reported, the test ítem did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, BLUE GS 5664.80 is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of BLUE GS 5664.80 to induce gene mutations according to the plate incorporation test (experiment l) and the pre-incubation test (experiment ll) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia colistrain WP2 uvrA following OECD TG 471 according to GLP.

The assay was performed in two independent experiments both with and without liver microsomal activation (S9 mix). Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Experiment I - all strains at 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without S9 mix

Experiment II - TA 1535, TA 1537, TA 100, WP2 uvrA at 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without S9 mix

Experiment II - TA 98 at 33, 100, 333, 1000, 2500 and 5000 µg/plate with S9 mix

Experiment II - TA 98 at 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate without S9 mix

The plates incubated with the test item showed normal background growth up to 1000 µg/plate with and without metabolic activation in both independent experiments. Due to the intense colour of the test item the background growth could not be assesed in the concentration range of 2500 and 5000 µg/plate.

Slight toxic effects, evident as a reduction in the number of revertants, were observed in strain TA 98 without 59 mix at 2500 and 5000 µg/plate in experiment I.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with BLUE GS 5664.80 at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

ln conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test ítem did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, BLUE GS 5664.80 is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.