Reaction mass of 3,3'-(1E,1'E)-(4,4'-(6-(bis(2-hydroxyethyl)amino)-1,3,5-triazine-2,4-diyl)bis(azanediyl)bis(3-methoxy-4,1-phenylene))bis(diazene-2,1-diyl)dibenzenesulfonic acid and disodium 3,3'-[[6-[bis(2-hydroxyethyl)amino]-1,3,5-triazine-2,4-diyl]bis[imino(3-methoxy-4,1-phenylene)azo]]bis[benzenesulphonate]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

From May 17 to June 05, 1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Remarks:

Source study has reliability 1. Details on the read across are attached in section 13.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted on May 26, 1983

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Similar Substance 02

IUPAC Name:

Similar Substance 02

Test material form:

solid

Method

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELL CHECK
Regular checking of the properties of the Salmonella strains with regard to membrane permeability and ampicillin resistance as well as normal spontaneous mutation rates is performed in testing laboratory, according to Ames et al. In this way it was ensured that the experimental conditions set down by Ames were fulfilled.

STORAGE
Strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO in liquid nitrogen.

Metabolic activation:

with and without

Metabolic activation system:

hamster liver microsomal fraction

Test concentrations with justification for top dose:

10.0, 100.0, 333.3, 1000.0 and 5000.0 µg/plate
According to results of the pre-experiment, concentrations applied in main experiments were chosen.

Vehicle / solvent:

- Vehicle: bidest. water.
- Justification for choice of vehicle: solubility properties and non-toxicity to bacteria.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

congo red

other: 4-nitro-o-phenylene-diamine

Details on test system and experimental conditions:

METHOD OF APPLICATION: pre-incubation.
The following materials were mixed in a test tube and gently shaken at 30 °C for 30 min.: 100 µl test solution at each dose level, solvent control, negative control, or reference mutagen solution (positive control); 500 µl S9 mix (for test with metabolic activation) or S9 mix substitution-buffer (for test without metabolic activation); 100 µl Bacteria suspension (cf. test system, pre-culture of the strains).

After pre-incubation 2.0 ml of molten 45 °C overlay agar was added to each tube. The mixture was poured on minimal agar plates.
After solidification the plates were incubated upside down for 72 hours at 37 °C in the dark.

NUMBER OF REPLICATIONS: the assay was performed in two independent experiments, using identical procedures. Each concentration, including the controls, was tested in triplicate.

CONDITIONS
- Precultures: from the thawed ampoules of the strains 0.5 ml bacterial suspension was transferred to 250 ml Erlenmeyer flasks containing 20 ml nutrient medium.
- Nutrient medium: the nutrient medium contains per litre 8 g Difco Nutrient Broth and 5 g NaCl. - Incubation: the bacterial culture was incubated in a shaking water bath for 6 hours at 37 °C.
- Selective Agar: 2.0 % Vogel-Bonner-Glucose-Minimal-Agar was used.
- Dished preparation: each petri dish was filled with 20 ml of this nutrient medium. Sterilizations were performed at 121 °C in an autoclave.
- Overlay Agar: overlay agar contains per litre 6.0 g Difco Bacto Agar, 6.0 g NaCl, 10.5 mg L-histidine x HCl x H2O and 12.2 mg biotin. Sterilizations were performed at 121° C in an autoclave.

PRE-EXPERIMENT FOR TOXICITY
To evaluate the toxicity of the test article a prestudy is performed with strains TA 98 and TA 100. 8 concentrations are tested for toxicity and mutation induction with each 3 plates.
The experimental conditions in the pre-experiment are the same as described for the main experiments.
Toxicity of the test article may be evidenced by a reduction in the number of spontaneous revertants, a clearing of the bacterial

DATA RECORDING
The colonies were counted using the BIOTRAN III counter. The counter was connected to an IBM XT compatible PC with printer which printed out the individual values and the means from the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates. If precipitation of the test article precluded automatic counting the revertant colonies were counted by hand.

MAMMALIAN MICROSOMAL FRACTION S9 MIX
The S9 liver microsomal fraction was obtained from the liver of 7 - 8 weeks old male Syrian golden hamsters. After cervical dislocation the livers of the animals were removed, washed in 0.1 M sodium phosphat buffer pH 7.4, 0.25 M sucrose and 1 mM disodium EDTA in bidestilled water and homogenised. The homogenate, diluted 1:3 in sodium phosphat buffer was centrifuged cold at 9000 g for 10 minutes. A stock of the supernatant containing the microsomes was frozen in ampoules of 2 or 5 ml and stored at -70 °C. Small numbers of the ampoules are kept at -20 °C for only several weeks before use. The protein concentration in the S9 preparation is usually between 20 and 45 mg/ml.

S9 Mix
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution in a ratio 3:7.
The composition of the cofactor solution was concentrated to yield the following concentrations in the S9 mix: 33 mM KCl, 8 mM MgCl2, 20 mM glucose 6-phosphate, 2.8 units/ml glucose 6-phosphate dehydrogenase, 4 mM NADP, 2 NADH, 2 FMN in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath. The preparation was performed according to Ames et al. and and Mitchell.

Evaluation criteria:

The generally accepted conditions for evaluation of results are:
- corresponding background growth on both negative control and test plates
- normal range of spontaneous reversion rates.
Due to international guidelines a statistical evaluation of the results is recommended. However, no evaluated statistical procedure can be recommended for analysis of data from the bacterial assays at this time.
A test article is considered as positive if either a significant dose-related increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced.
A test article producing neither a significant dose-related increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.

A test article is considered as mutagen if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537, and TA 98 it is at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of test article regardless whether the highest dose induced the above described enhancement factors or not.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

weak toxic effects

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Only weak toxic effects, evidenced by a reduction in the number of spontaneous revertants, occurred at the highest investigated dose in TA 1537 with metabolic activation (exp. I) and in TA 98 at 10.0 and 5000.0 µg/plate without metabolic activation and at 5000.0 µg/plate with metabolic activation (exp. I).
The plates incubated with test article showed normal back-ground growth up to 5000.0 µg/plate with and without S9 mix in all strains used.
Up to the highest investigated dose, no significant and reproducible dose-dependent increase in revertant colony numbers was obtained in any of the Salmonella typhimurium strains used. The presence of liver microsomal activation did not influence these findings.
Weak increases of revertant colony numbers in strain TA 1535 (exp. I, without S9 mix at 10.0 and 1000.0 µg/plate), in TA 98 (exp. I, with S9 mix at 10.0 µg/plate) and in TA 1537 (exp. II, with S9 mix at 333.3 µg/plate) are considered not to be relevant. The obtained effects were not reproduced in the independent experiment.

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

PRE-EXPERIMENT FOR TOXICITY
The plates with test article showed normal background growth up to 5000.0 µg/plate in strain TA 98 and TA 100, respectively.

Applicant's summary and conclusion

Conclusions:

Non-mutagenic in the Salmonella typhimurium reverse mutation assay.

Executive summary:

The potential of test substance to induce gene mutation in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 was assessed.

The assay was run as pre-icubation test in two independent experiments, using identical procedures, both with and without liver microsomal activation. Each concentration, including controls, was tested in triplicate. Test article was tested at concentrations of 10.0, 100.0, 333.3, 1000.0 and 5000.0 µg/plate.

Only weak toxic effects, evidenced by a reduction in the number of spontaneous revertants, occurred at the highest investigated dose in TA 1537 with metabolic activation (exp. I) and in TA 98 at 10.0 and 5000.0 µg/plate without metabolic activation and at 5000.0 µg/plate with metabolic activation (exp. I).

Plates incubated with test substance showed normal back-ground growth up to 5000.0 µg/plate with and without S9 mix in all strains used.

Test substance induced a slight increase in number of revertants in strain TA 1535 at 10.0 and 1000.0 µg/plate without metabolic activation (exp. I), in TA 98 at 10.0 µg/plate (exp. I) and in TA 1537 at 333.3 µg/plate (exp. II) both with metabolic activation.

Up to the highest investigated dose, no significant and reproducible dose-dependent increase in revertant colony numbers was obtained in any of the Salmonella typhimurium strains used. Metabolic activation did not influence these findings.

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

In conclusion, test substance did not induce point mutations by base pair changes or frameshifts in the genome of the strains used.