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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report date:
1996

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1983
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Similar Substance 02
Molecular formula:
n.a
IUPAC Name:
Similar Substance 02

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Remarks:
and TA 1538
Metabolic activation:
with and without
Metabolic activation system:
Mammalian microsomal fraction S9 mix
Test concentrations with justification for top dose:
According to the results of the pre-experiment the concentrations applied in the main experiments were chosen.The maximum concentration was 5000.0 µg/plate in experiment I and 1000.0 µg/plate in experiment IA and II; the concentration range included two logarithmic decades. In this study six adequately spaced concentrations were tested in experiment I and eight concentrations were tested in experiment IA and II.
Vehicle / solvent:
Bistilled waterThe solvent was chosen because of its solubility properties.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
other: 4-nitro-o-phenylene-diamine and 2-aminoanthracene
Details on test system and experimental 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. A solution of 20 µl ampicillin was added to the strains TA 98 and TA 100. This nutrient medium contains per litre: 8 g Merck Nutrient Broth (MERCK, D-64293 Darmstadt) 5 g NaCl (MERCK, D-64293 Darmstadt). The bacterial culture were incubated in a shaking water bath for 8 hours at 37 °C.
Selective Agar: The plates with the minimal agar were obtained from E. Merck, D-64293 Darmstadt, lot n° 66998.
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 H2012.2 mg BiotinSterilisations were performed at 121° C in an autoclave S9 mix (Preparation by CCR): The S9 liver microsomal fraction was obtained from the livers of 8 - 12 weeks old male Wistar rats, strain Hanlbm (BRL, CH-4414 Füllinsdorf, weight approx. 220 - 320 g) which received a triple i.p. injection of 80 mg/kg b.w. phenobarbital (Desitin, D-22335 Hamburg)and ß-naphtoflavone (Aldrich, D-89555 Steinheim) orally, in corn oil 1 - 3 days previously. After cervical dislocation the livers of the animals were removed, washed in 150 mM KCl and homogenised. The homogenate was diluted 1+3 in KCl and centrifliged at 9,000 g for 10 minutes at 4 °C. A stock of the supernatant containing the microsomes was frozen in ampoules of 2, 3 or 5 ml and stored at -80 °C. Small numbers of the ampoules are kept at -20 °C for up to several weeks before use. The standardisation of the protein content was made using the analysis kit of Bio-Rad Laboratories, D-80939 München: Bio-Rad protein assay, Catalogue 500 000 6.
The protein concentration in the S9 preparation was 25.2 mg/ml (lot 150296).Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 15 % v/v. The composition of the co-factor solution was chosen to yield the following concentrations in the S9 mix: 8 mM MgCl233 mM KCl5mM 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.
Colony counting and scoring of the plates: Colonies were counted electronically with an Artek counter.
The results were sent on line to a computer. They were checked on a random basis by the operator. Observations indicating precipitates of the test substance in the top agar or a reduced or absent bacterial background lawn were registered additionally. Means and standard deviations for all mutagenicity assays were calculated by a previously validated computer program.
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.A test article is considered positive if either a dose related and reproducible increase in thenumber of revertants or a relevant and reproducible increase for at least one test concentrationis induced.

A test article producing neither a dose related and reproducible increase in the number of revertants nor a relevant and reproducible positive response at any one of the test points is considered non-mutagenic in this system. A relevant response is described as follows:A test article is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate in strains TA 98 and TA 100 or thrice in TA 1535 and TA 1537.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 criteria described above or not.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium, other: TA1535, TA1537, TA98 and TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Distinct toxic effects, evidenced by a reduction in the number of revertants, occurred in the test groups at higher concentrations with and without metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Distinct toxic effects, evidenced by a reduction in the number of revertants, occurred in the test groups at higher concentrations with and without metabolic activation.The background growth was reduced at higher concentrations with and without S9 mix in all strains used.

Applicant's summary and conclusion

Conclusions:
Based on the results of these experiments, it is concluded that the test substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Executive summary:

The test substance was tested for mutagenic effects in vitro in histidine-requiring strains of Salmonella typhimurium, according to the OECD Guideline 471 (1983).

The test was performing without and with metabolic activation in the range of concentration of 33 to 5000 µg/plate, using strains of Salmonella typhimurium (TA 98, TA 100, TA 1535 and TA 1537). Each concentration and control were tested in triplicate.

Distinct toxic effects, evidenced by a reduction in the number of revertants, occurred in the test groups at higher concentrations with and without metabolic activation.

The background growth was reduced with and without S9 mix in all strains used.

No substantial increase in revertant colony numbers of any of the four tester strains was observed following treatment with the test substance at any concentration level, neither in the presence nor absence of metabolic activation.

Based on the results of these experiments, it is concluded that the test substance did not induce gene mutations in the strains of S. typhimurium used, both with and without metabolic activation.

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