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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:
1985-1986
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
publication
Title:
Salmonella mutagenicity tests: II. Results from the testing of 270 chemicals.
Author:
Mortelmans K.
Year:
1986
Bibliographic source:
Environmental Mutagenesis Volume8, Supplement 7:1-119
Report Date:
1986

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Test material form:
solid

Method

Target gene:
- S. typhimurium: Histidine gene
Species / strain
Species / strain / cell type:
other: TA1535, TA1537, TA98 and TA100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat and hamster metabolic activation system.
Test concentrations with justification for top dose:
- Dose range finding test:
The test item was initially tested with strain TA100 in the presence and absence of metabolic activation over a wide dose range with an upper limit of 10 mg/plate. As a rule, at least one toxic dose was incorporated into the first mutagenicity test.

Five concentration were used in this study, ranging from 0.1 to 6.6 mg/plate.
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent: the test substance was found to be soluble in DMSO
Controls
Untreated negative controls:
yes
Remarks:
(untreated plates)
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Remarks:
potassium chloride
Positive controls:
yes
Remarks:
2-aminoanthracene was used with all strains with metabolic activation
Positive control substance:
9-aminoacridine
sodium azide
other: 4-nitro-o-phenylenediamine
Details on test system and experimental conditions:
Preincubation assay
Mutagenicity was tested in the preincubation assay. The mixture of S-9 mix or buffer, the overnight culture and the solvent of test chemical was allowed to incubate for 20 min at 37'C, at which time molten top agar was added, supplemented with L-histidine or D-biotin. The content of the tubes was mixed and poured onto minimal glucose bottom agar in petri dishes. When the top agar solidified, the plates were incubated at 37'C for 48 h.
Concurrent solvent and positive controls were tested with and without metabolic activation. Five dose levels of test item were tested, with three plates per dose level.

In this study E. coli was not used, neither TA102 strain. Therefore, this study is used in combination with another study in a WoA approach.
Evaluation criteria:
The criteria used for data evaluation, were as follows: 1) mutagenic response: a dose-related, reproducible increase in the number of revertants over background, even if the increase was less than twofold; 2) nonmutagenic response: when no increase in the number of revertants was elicited by the chemical.

Results and discussion

Test results
Key result
Species / strain:
other: TA1535, TA1537, TA98 and TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose of 6.6 mg/plate.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- In all strains toxicity was observed at the highest dose of 6.6 mg/plate.

5 concentrations were used for each strain, which resulted in at least 4 analyzable concentrations up to 3333 ug/plate (in some cases the highest dose of 6666 ug/plate was toxic).

Applicant's summary and conclusion

Conclusions:
The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay performed according to OECD 471.
Executive summary:

The mutagenic activity of the substance was evaluated in accordance with OECD 471. The test was performed in a pre-incubation assay, both in the absence and presence of S9-mix. The dose levels were selected based on observed cytotoxicity in dose setting experiment. Adequate negative and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in any of the four S. typhimurium tester strains (TA1535, TA1537, TA98 and TA100), neither in the absence nor in presence of S9-metabolic activation. Based on the results of this study, it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay.