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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:
2002-08-05 to 2002-08-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline compliant

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)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
(2000)
Deviations:
no
GLP compliance:
yes
Remarks:
statement
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid, fine scales

Method

Species / strain
Species / strain / cell type:
other: TA 97a, TA98, TA100, TA102, TA1535
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix of Aroclor 1254 induced rat liver
Test concentrations with justification for top dose:
The concentrations for the first experiment were set according to a preliminary toxicity test. The test substance was not toxic up to 5000 µg/ plate. However, a precipitate of the test substance was observed at concentrations of 556 µg/ plate and above, which was not to distinguish from the bacterial colonies. It was therefore decided to use 1000 µg/ plate as the highest concentration to obtain a useful evaluation. Each of the other 4 concentrations was root mean square of 3 of the preceding one. For the second experiment the concentrations were changed according to the results of the first one. The granulous precipitate of the test substance impeded the counting of the colonies at 1000 µg/plate. Therefore the concentrations were put back one step.
Concentration range (with metabolic activation): 12, 37, 111, 333, 1000 µg/plate.
Concentration range (without metabolic activation): 12, 37, 111, 333, 1000 µg/plate
Vehicle / solvent:
Solvent: DMSO
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 1,8-Dihydroxy-anthraquinone (DHA)
Remarks:
with metabolic activation (S9 mix): TA102
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activation (S9 mix): TA97a
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2-AA)
Remarks:
with metabolic activation (S9 mix): TA98; TA100; TA1535
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-Nitro-o-phenylenediamine (4-NOPD)
Remarks:
without metabolic activation (S9 mix): TA97a
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: t-Butyl-hydroperoxide (tBHPO)
Remarks:
without metabolic activation (S9 mix): TA102
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without metabolic activation (S9 mix): TA98
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without metabolic activation (S9 mix): TA100; TA1535
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 2 days at 37°C

SELECTION AGENT: histidine

NUMBER OF REPLICATIONS: 2

OTHER:
Concentration of the test substance resulting in precipitation: 333 µg/plate
Evaluation criteria:
In the Ames test a substance is considered to induce mutations if a reproducible increase in the number of revertants to more than the threshold values for at least one of the concentrations occurs.
The criteria for a positive result are:
A reproducible increase of the number of revertants to more than the following threshold values for at least one of the concentrations:
- For the strains with a low spontaneous revertant rate, i.e. TA98 and TA1535: The 2.5- fold of the amount of the spontaneous revertants.
- For the strains with a high spontaneous revertant rate, i.e. TA97a, TA100 and TA102: The 1.6- fold of the amount of the spontaneous revertants.
These threshold values were derived from the variations in the control samples of the laboratory's historic data of the Ames test.
Statistics:
Means and standard deviation were calculated for the number of mutants in every concentration group.

Results and discussion

Test results
Species / strain:
other: TA 97a, TA98, TA100, TA102, TA1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
up to 1000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
A dose range finding study was performed using concentrations up to 5000 µg/plate. No toxicity was observed to any of the strains of bacteria used. Therefore the highest concentration was selected to be 1000 µg/plate for the main study.

SOLUBILITY:
A precipitate was visible when the test item was mixed with the agar at the 1000 and 333 µg/plate samples. The precipitate was still visible when the colonies were counted and impeded the counting of the 1000 µg/plate samples.


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

There was no such increase in the number of mutants in any of the tested bacterial strains at any of the tested concentrations with or without metabolic activation. According to these results, the test item is not mutagenic in the Ames test with the strains of Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 up to 1000 µg/ plate.
Executive summary:

The aim of the study was to investigate the potential of the test item to induce gene mutations in S. typhimurium with and without metabolic activation. The study was performed according to the OECD 471, adopted in 1983. The test item was dissolved in DMSO. The dose range was determined in a preliminary toxicity assay using 50 to 5000 µg/ plate. Cytotoxicity was excluded according to normal growth. For the main test, concentrations ranging from 4 µg to 1000 µg/ plate using the direct plate incorporation method with and without external metabolisation (S9- mix) were used. The bacterial strains S. typhimurium TA97a, TA98, TA100, TA102 and TA1535 were used. Negative and positive controls were included and remained within the historical range. An independent repetition of the experiment was performed. No toxicity of the test substance to the bacteria was observed up to 1000 µg/plate. A precipitate was visible when the test substance was mixed with the agar at the 1000 and 333 µg/ plate samples. The precipitate was still visible when the colonies were counted and impeded the counting of the 1000 µg/ plate samples. In none of the concentrations tested and with none of the strains used an increase of the mutation frequency to more than the threshold values (250 % of the controls for strains TA98 and TA1535, 167 % of the controls for strains TA97a, TA100 and TA102) was obtained. Metabolic activation did not change these results. Under the selected test conditions the test item is considered to be non-mutagenic in the Ames test neither with nor without metabolic activation.