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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:
weight of evidence
Study period:
1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
Identity: H109363
Batch number: BASF 277/1B
Purity: 85.07 g/100g (main component)
Appearance: Red brown powder
Storage: Room temperature
Specific details on test material used for the study:
Identity: H109363
Batch number: BASF 277/1B
Purity: 85.07 g/100g (main component)
Appearance: Red brown powder
Storage: Room temperature

Method

Target gene:
Histidine
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: See remarks
Remarks:
rfa: deep rough (defective lipopolysaccharide cellcoat); gal: mutation in galacto se metabolism; chl: mutation in nitrate reductase; bio defective biotin synthesis; uvrB: loss of the excision repair system (deletion of the ultraviolet-repair B gene)
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: See remarks
Remarks:
rfa: deep rough (defective lipopolysaccharide cellcoat); gal: mutation in galacto se metabolism; chl: mutation in nitrate reductase; bio: defective biotin synthesis; uvrB: loss of the excision repair system (deletion of the ultraviolet-repair B gene)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S-9 mix and uninduced hamster liver S-9 mix.
Test concentrations with justification for top dose:
Ames standard plate test with and without rat liver S-9 mix: 0, 23.6, 1118, 590, 2950 and 5950 µg/plate, no toxicity was observed at highest dose also.
Prival preincubation test with and without hamster liver S-9 mix: 0, 23.6, 1118, 590, 2950 and 5950 µg/plate, slight decrease in the number of revertants was observed depending on the experiment at doses > 2950 µg/plate.
Vehicle / solvent:
Water for test substance and DMSO for positive controls
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
congo red
other: See remarks
Details on test system and experimental conditions:
The test was performed in two tests:
1) Ames standard plate test (with and without induced Rat liver system)
2) Prival preincubation test (With andd without uninduced hamster liver system
Rationale for test conditions:
Based on the most recent OECD and EC guidelines.
Evaluation criteria:
Evaluation criteria:

The test substance is considered positive in this assay if the following criteria met:
A dose related and reproducible increases in the number of revertant colonies, that is about doubling of the spontaneous mutations rate in at least one tester strain either with or without metabolic activation.

A test substance is generally considered non-mutagenic in this test if:

The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.
Statistics:
No formal hypothesis testing was done.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
other: Ames standard plate test
Remarks:
TA1535, TA100, TA 1537, T98 and E.coli WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
other: Prival preincubation test
Remarks:
TA1535, TA100, TA1537, TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
negative
Remarks:
Slightly elevated at 5900 µg/plate
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
negative
Remarks:
Slightly elevated figures (factor < 2.0) without any dose dependency
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
ambiguous
Remarks:
Mutagenicity observed from 590 µg/plate (1st test factor 2.7), 118 µg/plate (2nd test, factor 2.1) onwards with a maximum increase in the number of revertant colonies by a factor of 5.3 at 2950 µg/plate (1st test) or of 3.1 at 590 µg/plate (2nd test)
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
Even though mutagenic potential was observed in TA 98 strain in the presence of a hamster metabolic activation system, the results are not sufficient to classify the test substance as mutagenic. Under the study conditions, the mutagenic potential of the test substance was therefore inconclusive.
Executive summary:

A study was conducted to determine the in vitro genetic toxicity of the test substance according to OECD Guideline 471 and EU Method B.13/14, in compliance with GLP. Salmonella typhimurium strains TA1535, TA1537, TA100 and TA98 and Escherichia coli strain WP2uvrA were exposed to the substance at concentrations of 0, 23.6, 118, 590, 2950 and 5950 μg/plate, and also to negative or positive controls. Two types of studies were run: 1) an Ames standard plate test with and without metabolic activation (Arochlor-induced rat liver S9 mix), 2) a Prival preincubation assay with and without metabolic activation (uninduced hamster liver S9 mix).

In experiment 1, the Ames standard plate test, no increase in the number of his* or trp* revertants occurred under any study conditions.

In experiments 2 and 3 conducted according to the Prival preincubation plate assay method, no increase in the number of his* revertants was seen in any tester strain in the absence of metabolic activation. However when hamster liver S9 mix was added, the following observations were made:

-         TA 1535: slightly elevated figures at 5900 µg/plate (factor 2.1),

-         TA 100: no increase in the number of his* revertants,

-         TA 1537: slightly elevated figures (factor <2.0) without any dose-dependency

-         TA 98: mutagenicity was observed from about 590 µg/plate (1st test, factor 2.7) onward or 118 µg/plate (2nd test, factor 2.1) onwards with a maximum increase in the number of revertant colonies by a factor of 5.3 at 2950 µg/plate (1st test) or of 3.1 at 590 µg/plate (2nd test).

No bacteriotoxic effects were reported in the Ames standard plate test. In the Prival preincubation assay, a slight decrease in the number of revertant occurred in TA 98 at doses ≥ 2950 µg/plate.

Even though mutagenic potential was observed in TA 98 strain in the presence of a hamster metabolic activation system, the results are not sufficient to classify the test substance as mutagenic. Under the study conditions, the mutagenic potential of the test substance was therefore inconclusive (Engelhardt, 1998).