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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:
2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study, compliant with OECD guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 21st July, 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
dated May 30, 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
BSL Bioservice Scientific Laboratories GmbH, Planegg, Germany
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Method

Target gene:
his and trp operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Mammalian microsome enzyme activation mixture (supplemented S9 fraction from liver of untreated hamster and from liver of rats induced with phenobarbital and beta-naphthoflavone)
Test concentrations with justification for top dose:
3.16, 10, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
Vehicle / solvent:
The test item was suspended in DMSO and diluted prior to treatment. The solvent was compatible with the survival of the bacteria and the S9 activity.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Remarks:
A. dest.
Positive controls:
yes
Positive control substance:
other: Sodium azide (TA 100, TA 1535), 4-nitro-o-phenylene-diamine (4-NOPD; TA 98, TA 1537), Methyl methane sulfonate (MMS; E. coli)
Remarks:
without metabolic activation
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (all strains)
Remarks:
with metabolic activation (rat liver)
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2-AA; TA 1535, TA 1537, E. coli), Congo Red (TA 98, TA100)
Remarks:
with metabolic activation (hamster liver)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) (experiment I); preincubation (experiment II)

DURATION
- Preincubation period: 30 minutes at 30°C
- Exposure duration: at least 48 hours at 37°C

NUMBER OF REPLICATIONS: three plates

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
Evaluation of Cytotoxicity
Cytotoxicity can be detected by a clearing or rather diminution of them background lawn or a reduction in the number of revertants down to a mutation factor of approximately < 0.5 in relation to the solvent control.

Evaluation of Mutagenicity
The Mutation Factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values are used for calculation).

A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.

A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and E. coli WP2 uvrA the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher
as compared to the reversion rate of the solvent control.

A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

A test is considered acceptable if for each strain:
-the bacteria demonstrate their typical responses to ampicillin (TA 98, TA 100)
- the control plates without S9 mix are within the following ranges (mean values of the spontaneous reversion frequency are within the historical control data range (TA98: 16-46, TA 100: 77-174, TA 1535: 5 -29, TA 1537: 5 -28, E. coli WP2 uvrA: 36-81)
- corresponding background growth on both negative control aod test plates is observed.
- the positive controls show a distinct enhancement of revertant rates over the control plate
Statistics:
According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
except in tester strain TA 1537 in the pre-incubation assay at 5000µg/plate with metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Was observed in all tester strains used in experiment I and II (with and without metabolic activation). In experiment I precipitation of the test item was found at concentrations of 316 µg/plate and higher (with and without metabolic activation), in experiment II precipitation of the test item was found at concentrations of 1000 µg/plate and higher (with and without metabolic activation).

ADDITIONAL INFORMATION ON CYTOTOXICITY: No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated with and without metabolic activation in experiment I and II, with the exception of a toxie effect observed in tester strain TA 1537 in experiment II at a concentration of 5000 µg/plate (with metabolic activation). The reduction in the number of revertants down to a mutation factor of 0.5 found in experiment II in tester strain E. coli WP2 uvrA at a concentration of 10.0 µg/plate (without metabolic activation) was regarded as not biologically relevant due to lack of a dose-response relationship.
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

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the substance did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.