Reaction mass of Tetrasodium 2-[{4-[{4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-5-sulfonatonaphthalen-1-yl}diazenyl]-7-sulfonatonaphthalen-1-yl}diazenyl]benzene-1,4-disulfonate and Tetrasodium 2-[{4-[{4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-5-sulfonatonaphthalen-1-yl}diazenyl]-6-sulfonatonaphthalen-1-yl}diazenyl]benzene-1,4-disulfonate

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:

1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

None

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not applicable

Principles of method if other than guideline:

None

GLP compliance:

yes

Type of assay:

bacterial forward mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of Tetrasodium 2-[{4-[{4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-5-sulfonatonaphthalen-1-yl}diazenyl]-7-sulfonatonaphthalen-1-yl}diazenyl]benzene-1,4-disulfonate and Tetrasodium 2-[{4-[{4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-5-sulfonatonaphthalen-1-yl}diazenyl]-6-sulfonatonaphthalen-1-yl}diazenyl]benzene-1,4-disulfonate

EC Number:

916-837-8

Molecular formula:

C29H16Na4ClN9O12S4

IUPAC Name:

Reaction mass of Tetrasodium 2-[{4-[{4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-5-sulfonatonaphthalen-1-yl}diazenyl]-7-sulfonatonaphthalen-1-yl}diazenyl]benzene-1,4-disulfonate and Tetrasodium 2-[{4-[{4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-5-sulfonatonaphthalen-1-yl}diazenyl]-6-sulfonatonaphthalen-1-yl}diazenyl]benzene-1,4-disulfonate

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Name: FAT 40032/E CIBACRON BRAUN P-6R ROHFEUCHT (Laborgetrocknet)
Batch No.: Op. 11
CAS No.: 70161-16-9
Aggregate State at RT: solid
Colour: brown
Purity: 75.7 % active ingredient

Method

Target gene:

None

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

liver microsomal activation (S9 mix)

Test concentrations with justification for top dose:

33.3; 100.0; 333.3; 1000.0; 2500.0 and 5000.0 ug/plate.

Vehicle / solvent:

DMSO

Details on test system and experimental conditions:

The strains are derived from S. typhimurium strain LT2 and due to a mutation in the histidine locus are histidine dependent. Additionally, due to the "deep rough" (rfa-minus) mutation they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation causes a reduction in the activity of an excision repair system. The latter alteration includes mutational processes in the nitrate reductase and biotin genes produced in a UV-sensitive area of the gene named "uvrB-minus". In the strains TA 98, TA 100 and TA 102 the R-factor plasmid pKM 101 carries the ampicilline resistance marker. The strain TA 102 does not contain the uvrB-mutation. Additionally, TA 102 contains the multicopy plasmid pAQl, which carries the hisG428 mutation and a tetracycline resistance gene. TA 102 contains the ochre mutation in hisG gene.

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

Storage
The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO in liquid nitrogen.

Pre-cultures
From the thawed ampoules of the strains 0.5 ml bacterial suspension was transferred to 250 ml Erlenmeyer flasks containing 20 ml nutrient medium. This nutrient medium contains per litre:

8 g Merck Nutrient Broth
5 g NaCl

The bacterial culture was incubated in a shaking water bath for 10 hours at 37 °C.

Selective Agar
2.0 % Vogel-Bonner-Glucose-Minimal-Agar was used as selective agar. Each petri dish was filled with 20 ml of this nutrient medium. Sterilisations were performed at 121 °C in an autoclave.

Overlay Agar
The overlay agar contains per litre:
6.0 g Merck Agar Agar
6.0 g NaCl
10.5 mg L-histidine x HCl x H20
12.2 mg biotin

Sterilisations were performed at 121 °C in an autoclave.
MAMMALIAN MICROSOMAL FRACTION S9 MIX
S9 (Preparation by C C R)

The S9 liver microsomal fraction was obtained from the liver of 8-12 weeks old male Wistar rats, strain WU (SAVO-Ivanovas, med.
Versuchstierzuchten GmbH, 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 removed, 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 -7 0° 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 München: Bio-Rad protein assay, Catalogue 500 000 6 (6).

The protein concentration in the S9 preparation was 33.6 mg/ml (lot 240292) and 31.6 mg/ml (lot 060792)

Rationale for test conditions:

None

Evaluation criteria:

None

Statistics:

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. 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 dose related and reproducible 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 dose related and reproducible 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 significant response is described as follows: A test article is considered as mutagenic if in strain TA 100 and TA 102 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 and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.

BIOMETRY:
No appropriate statistical method is available.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Toxic effects, evidenced by a reduction in the number of revertants, occurred only in strain TA 100 in experiment II (preincubation test) without metabolic activation at the highest investigated dose. The plates incubated with the test article showed normal background growth up to 5000 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 40032/E 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.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

FAT 40032/E is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Executive summary:

A study was performed to investigate the potential of FAT 40032/E to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed in two independent experiments 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: 33.3; 100; 333.3; 1000; 2500 and 5000 µg/plate. Toxic effects, evidenced by a reduction in the number of revertants, occurred only in strain TA 100 in experiment II (preincubation test) without metabolic activation at the highest investigated dose. The plates incubated with the test article showed normal background growth up to 5000 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 40032/E 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, FAT 40032/E is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.