Reaction mass of Trisodium 4-({4-chloro-6-[(4-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)-2-{[1-ethyl-2-hydroxy-4-methyl-6-oxo-5-(sulfonatomethyl)-1,6-dihydropyridin-3-yl]diazenyl}benzenesulfonate and Trisodium 4-({4-chloro-6-[(3-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)-2-{[1-ethyl-2-hydroxy-4-methyl-6-oxo-5-(sulfonatomethyl)-1,6-dihydropyridin-3-yl]diazenyl}benzenesulfonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

FAT 40000 was found to be not mutagenic in the Salmonella reverse mutation assays.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Study completion date - 06 November 1978.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

no tester strain to detect cross-linking mutagens has been included

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from rat liver

Test concentrations with justification for top dose:

25, 7 5, 225, 675, and 2025 µg / 0 .1 ml

Vehicle / solvent:

phosphate buffer

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without metabolic activation - for TA 1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without metabolic activation - for TA 1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: daunoblastin

Remarks:

Without metabolic activation - for TA 98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without metabolic activation - for TA 100 strain

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic acyivation - for TA 1535 strain

Details on test system and experimental conditions:

The bacteria on which the tests were performed were the histidine - auxotrophic TA 98, TA 100, TA 1535, and TA 1537 strains of
Salmonella typhimurium. Cultures were prepared from frozen stocks, and on the following days the Standard Plate Test was carried out with and without the addition of activation mixture (rat liver microsomes and co-factors).
The test was performed with the following concentrations of the trial substance with and without microsomal activation : 25, 75, 225, 675, and 2025 µg / 0.1 ml. The substance was dissolved in phosphate buffer. Phosphate buffer alone was used for the negative controls (the substances and vehicles used for the positive controls are indicated below). Each Petridish contained:
1) approx. 20 ml of minimum agar (Agar purified, "Difco" certified, Difco Laboratories, Detroit, Michigan, U.S.A., Art No.0560, plus salts (Vogel-Bonner Medium E) and glucose),
2) 0.1 ml of the solution of the test substance or the vehicle and 0.1 ml of a bacterial culture (in nutrient broth: Bacto Nutrient Broth dehydrated, Difco Laboratories, Detroit, Michigan, U.S.A., Art. No.0003 0.8% plus 0.5% NaCl) in 2.0 ml of soft agar. The soft agar was composed of: 100 ml of 0.6% agar solution (Agar Purified, "Difco" certified) with 0.6% NaCl and 10 ml of a solution of 1-histidine, 0.5 mM (Fluka, Buchs, Switzerland, Art.No .14400) and + biotin 0.5 mM (Fluka, Buchs, Switzerland, Art. No. 53320). In the experiments in which the substance was metabolically activated, 0.5 ml of an activation mixture was added also. 1 ml activation mixture contains : 0.3 ml S9 fraction of liver from rats induced with Aroclor 1254 (Analabs), 8 µmoles MgCl, 33 µmoles KCl, 5 µmoles glucose-6-phosphate, 4 µmoles NADP and 100 µmoles phosphate buffer, pH 7.4.

Positive control experiments were carried out simultaneously with the following substances:
1) for Strain TA 1535: N-methyl-N'-nitro-N-nitrosoguanidine (Fluka, Buchs, Switzerland, Art.No.68051), 3 and 5 µg/0.1 ml phosphate buffer;
2) for Strain TA 1537: 9(5)aminoacridine hydrochloride monohydrate (Fluka, Buchs, Switzerland, Art.No.06650), 25, 50, and 100 µg/0.1 ml DMSO; 3) for Strain TA 98: daunoblastin (Soc. Farmaceutici Italia, Milan, Italy), 2.5, 5, and 10 µg/0.1 ml phosphate buffer;
4) for Strain TA 100: 4-nitroquinoline-N-oxide (Fluka, Buchs, Switzerland, Art.No.73265), 0.0625, 0.125, and 0.25 µg/0.1 ml phosphate buffer. The activation mixture was tested with Strain TA 1535 and cyclophosphamide, 100 and 250 µg/0.1 ml phosphate buffer.

In the experiments with and without the addition of microsomal activation mixture three Petri dishes were prepared per strain and per group ( i.e. per concentration or per control group). In the positive control experiments two Petri dishes were used per strain and per group.
The plates were incubated for about 48 hours at 37 °C in darkness. When the colonies had been counted, the arithmetic mean was calculated. The test substance was considered to be non-mutagenic if the colony count in relation to the negative control was not doubled at any concentration .

Evaluation criteria:

When the colonies had been counted, the arithmetic mean was calculated. The test substance was considered to be non-mutagenic if the colony count in relation to the negative control was not doubled at any concentration.

Key result

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:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

The test item was determined to be "non-mutagenic" in the bacterial reverse mutation assay.

Executive summary:

A supporting study was performed according to method similar or equivalent to OECD test guideline 471, wherein preparation FAT 40000/B was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium.

The investigations were performed with the following concentrations of the trial substance with and without microsomal activation: 25, 75, 225, 675 and 2025 µg/0.1 ml. Since considerable fluctuations in the number of back-mutants produced were observed in the experiments with Strain TA 100, the experiments were repeated, in accordance with the usual practice in the laboratory.

In the experiments performed with and without microsomal activation, comparison of the numbers of back-mutant colonies in the controls and the cultures treated with the various concentrations of FAT 40000/B revealed no marked deviations. No evidence of the induction of point mutations by FAT 40000/B or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experiment start date - 24 June 1992; Experiment completion date - 10 July 1992; Study completion date - 10 August 1992.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

no tester strain to detect cross-linking mutagens has been included

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

no tester strain to detect cross-linking mutagens has been included

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Code number: FAT 40000/G
Batch number: 67/92
Purity: 79.5%
Appearance: solid
Solubility: miscible
Storage: room temperature
Expiration date: 01 June 1993.

Target gene:

Histidine

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Details on mammalian cell type (if applicable):

The bacterial strains were obtained from Dr. Heinz Trager, Knoll AG, D-6700 Ludwigshafen, F.R.G.
Regular checking of the properties of the strains with regard to membrane permeability and ampicillin resistance as well as normal spontaneous mutation rates is performed in CCR according to Ames et al. In this way it was ensured that the experi­ mental conditions set down by Ames were fulfilled.
The 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:

S9 (Preparation by CCR)
The S9 liver microsomal fraction was obtained from the liver of 8 - 12 weeks old male Wistar rats, strain WU (SAVO-Ivanovas, med.
Versuchstierzuchten GrnbH, D-7964 Kisslegg, F.R.G.; weight approx. 150 - 200 g) which received a single i.p. injection of 500 mg/kg b.w. Aroclor 1254 (Antechnika, D-7500 Karlsruhe, F.R.G.) in olive oil 5 days previously.

After cervical dislocation the livers of the animals were re­ moved, washed in 150 mM KCl and homogenised. The homogenate, diluted 1+3 in KCl was centrifuged cold at 9,000 g for 10 minutes. A stock of the supernatant containing the microsomes was frozen in ampoules of 2, 3 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 standardisation of the protein content was made using the analysis kit of Bio-Rad Laboratories, D-8000 Milnchen: Bio-Rad protein assay, Catalogue 500 000 6 (6).
The protein concentration in the S9 preparation was 32.4 mg/ml (lot 150491)

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:
8mM MgCl2
33mM KCl
5mM glucose-6-phosphate
5mM NADP
in 100mM sodium-ortho-phosphate-buffer, pH 7.4.

During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was pe formed according to Ames et al.

Test concentrations with justification for top dose:

10.0, 33.3, 100.0, 333.3, 1000.0 and 5000.0 µg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without metabolic activation - for TA 1535, TA 100 strains

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine, 4-NOPD

Remarks:

Without metabolic activation - for TA 1537, TA 1538, TA 98 strains

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 2-AA

Remarks:

With metabolic activation - for TA 1535, TA 1537, TA 1538, TA 98, TA 100 strains

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments : Two

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; in agar (plate incorporation);

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY : A test article is considered as mutagenic if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537, TA 1538, 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 poten­ tial of the test article regardless whether the highest dose induced the above described enhancement factors or not.

Evaluation criteria:

The generally accepted conditions for the evaluation of the results are: corresponding background growth on both negative control and test plates normal range of spontaneous reversion rates.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

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

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The plates incubated with the test article showed normal background growth up to 5000.0 µg/plate with and without S9 mix in all strains used.

No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with FAT 40'000/G at any dose level, either in the pres·ence or 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 significance.

Conclusions:

The test item was determined to be non-mutagenic in the bacterial reverse mutation assay.

Executive summary:

A key study was performed to investigate the potential of test item to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100. This study was conducted according to OECD test guideline 471 in a GLP certified laboratory.

The assay was performed in two independent experiments, using identical procedures, both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate.

The test article was tested at the following concentrations : 10.0, 33.3, 100.0, 333.3, 1000.0; and 5000.0 µg/plate. A slight toxic effect, evidenced by a reduction in the number of revertants, occurred only in strain TA 98 at 5000.0 µg/plate in the presence of S9 mix in experiment I. The plates incubated with the test article showed normal background growth up to 5000.0 µg/plate with and without S9 mix in all strains used. No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with test item at any dose level, either in the presence or 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 significance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce point mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The genetic toxicity database of FAT 40000 consists of two bacterial reverse mutation assays.

Bacterial reverse mutation assays with FAT 40000

FAT 40000 was evaluated for mutagenic potential in two Salmonella reverse mutation assays (1990 and 1978). The assays were performed with and without metabolic activation systems. No substantial increases in revertant colony numbers or tendency of higher mutation rates with increasing concentrations was observed. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. Hence it was concluded, that in these assays the target substance did not induce mutations in the bacterial cells.

Justification for classification or non-classification

Based on the above information, the substance does not warrant classification for genotoxicity as per the CLP (Regulation 1272/2008) criteria.