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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:
13. Mar - 27. Mar 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP conform guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Directive 2000/32/EC, L1362000, Annex 4d, dated May 19, 2000
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Guidelines: Kanpoan No 287-Environment Protection Agency; Eisei No. 127-Ministry of Health&Welfare, Heisei 09/10/21 Kikyoku No. 2-Ministry of International Trade&Industry
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid

Method

Target gene:
His
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
33, 100, 333, 1000, 2500 and 5000 µg/plate
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine
Details on test system and experimental conditions:
METHOD OF APPLICATION: iin agar (plate incorporation); preincubation

DURATION
- Preincubation period: 100 µL test solution, 500 µL S9 mix/S9 mix substitution buffer and 100 µL bacterial suspension were mixed in a test tube and shaken at 37°C for 60 min.
- Exposure duration: 48 h at 37°C

SELECTION AGENT (mutation assays): His deficient medium

NUMBER OF REPLICATIONS: 3


DETERMINATION OF CYTOTOXICITY
- Method: background growth

Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An icrease exceeding the threshold at only one concentration is judged as biologically relevant if reporduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
No statistical evaluation of the data is required.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
minor toxic effects
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

During the described mutagenicity test and under the experimental conditions reported the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Executive summary:

The assay was performed in two independent experiments both with and without liver microsomal activation using Salmonella thyphimurium strains TA 1535, TA 1537, TA 98 and TA 100 and the Escherichia coli strain WP2 uvrA.

The test item was tested at the following concentrations:

33, 100, 333, 1000, 2500 and 5000 µg/plate

The plates incubated with the test item showed normal background up to 5000 µg/plate with and without S9 mix in both experiments.

Minor toxic effects (below the factor 0.5), evident as reduction in the number of revertants, were observed in strain TA 1535 at 333 and 1000 µg/plate without metabolic activation and at 5000 µg/plate with and without metabolic activation in experiment I. In experiment II, a minor toxic effect was observed at 5000 µg/plate without metabolic activation in strain TA 98.

No substantial increase in revertant colony number of any of the five tester strains was observed following the treatment with the test item at any dose level, neither in the presence nor 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 biological relevance.

In experiment I, with metabolic activation, the number of colonies did not quite reach the lower limit of the historical control data in strains TA 1535 (negative control) and TA 1537 (negative and solvent control). Sinced these deviationts are rather small, these effects are judged to be based upon statistical fluctuations and have no detrimantal impact on the outcome of the study. The historical range of positive controls was exceeded in strain TA 1535 (Exp. II) without metabolic activation. This effect indicates the sensitivity of the strains rather than compromising the assay.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.