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Toxicological information

Genetic toxicity: in vitro

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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

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of tetrasodium 2-[[4-[[4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-5-sulphonato-1-naphthyl]azo]-6-sulphonato-1-naphthyl]azo]benzene-1,4-disulphonate and tetrasodium 2-[[4-[[4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-5-sulphonato-1-naphthyl]azo]-7-sulphonato-1-naphthyl]azo]benzene-1,4-disulphonate
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-sulphonato-1-naphthyl]azo]-6-sulphonato-1-naphthyl]azo]benzene-1,4-disulphonate and tetrasodium 2-[[4-[[4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-5-sulphonato-1-naphthyl]azo]-7-sulphonato-1-naphthyl]azo]benzene-1,4-disulphonate
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
Key result
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100 and 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
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.