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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1993
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:
1993
Report Date:
1993

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only four tester strains tested; no tester strain to detect cross-linking mutagens was included.
Principles of method if other than guideline:
Guideline followed
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid
Specific details on test material used for the study:
None

Method

Target gene:
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 ampicillin 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 tetracyclin resistance gene. TA 102 contains the ochre mutation in hisG gene.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Test concentrations with justification for top dose:
Experiment 1: 33.3, 100.0, 333.3, 1000.0, 2500.0 and 5000.0 µg/plate
Experiment 2: 500.0, 1000.0, 2000.0, 3000.0, 4000.0 and 5000.0 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: A.bidest.
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine
Details on test system and experimental conditions:
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.

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. 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 -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 München: Bio-Rad protein assay, Catalogue 500 000 6.

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

S9 MIX:
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution. The amount of S9 supernatant was 15 % v/v. The composition of the cofactor solution was concentrated to yield the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
5 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.

PRE-EXPERIMENT FOR TOXICITY:
To evaluate the toxicity of the test article a prestudy was performed with strains TA 98 and TA 100. 8 concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were the same as described below for the experiment I (plate incorporation
test).

Toxicity of the test article may be evidenced by a reduction in the number of spontaneous revertants, a clearing of the bacterial background lawn, or by degree of survival of treated cultures.

DOSE SELECTION:
According to the results of the pre-experiment the concentrations applied in the main experiments were chosen.
Experiment I was performed with the normal concentration grading. Due to the mutagenic effect obtained in experiment I, experiment II was performed as well as a plate incorporation assay. Additionally the concentration grading was designed to explore the linear part of the dose-response curve obtained in experiment I. Therefore, a more narrow concentration range (between 1000.0 and 5000.0 µg/plate) was selected in experiment II.

The maximum concentration was 5000.0 µg/plate in experiment I and II. The concentration range included two logarithmic decades in experiment I and one logarithmic decade in experiment II. In this study six adequately spaced concentrations were tested. Two independent experiments were performed.
Evaluation criteria:
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.
Statistics:
No appropriate statistical method is available

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
positive
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 in strain TA 1537 (without S9 mix) and in strain TA 102 (with S9 mix) at the highest investigated dose 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.

Up to the highest investigated dose a significant, dose-dependent increase in the number of revertants was observed in the strains TA 1535 and TA 100 with and without metabolic activation in experiment I and II.

No substantial increases in revertant colony numbers were observed following treatment with FAT 92'348/A (Lanasol Rot 2G roh trocken) at any dose level, either in the presence or absence of metabolic activation (S9 mix) in the strains TA 1537, TA 98 and TA 102.

A slight increase in revertant colony numbers was found in strain TA 98 (without S9 mix) at 5000.0 µg/plate in experiment I and in strain TA 102 at 3000.0 µg/plate (with S9 mix) in experiment II. These results are considered not to be biologically relevant since they could not be reproduced in the independent experiment.

Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced revertant colonies.
Remarks on result:
other: all strains/cell types tested

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
FAT 92'348/A (Lanasol Rot 2G roh trocken) is considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.
Executive summary:

This study was performed to investigate the potential of FAT 92348/A (Lanasol Rot 2G roh trocken) to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102.

This study followed the procedures indicated by the following internationally accepted guidelines and recommendations:

First Addendum to OECD Guidelines for Testing of Chemicals - Section 4, No. 471, "Salmonella typhimurium, Reverse Mutation Assay", adopted May 26, 1983 and EEC Directive 79/831, Annex V, B 14. The study was performed in compliance with GLP.

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:

Experiment I : 33.3; 100.0; 333.3; 1000.0; 2500.0 and 5000.0 µg/plate

Experiment II: 500.0; 1000.0; 2000.0; 3000.0; 4000.0 and 5000.0 µg/plate

Toxic effects, evidenced by a reduction in the number of revertants, occurred in strain TA 1537 (without S9 mix) and in strain TA 102 (with S9 mix) at the highest investigated dose 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. Up to the highest investigated dose a significant dose-dependent increase in the number of revertants was observed in the strains TA 1535 and TA 100 with and without metabolic activation in experiment I and II. 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 induced point mutations by base pair changes in the genome of the strains TA 1535 and TA 100. Therefore, FAT 92'348/A (Lanasol Rot 2G roh trocken) is considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.