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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:
16-Jan-2012 to 23-Feb-2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

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 (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
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:
Exp 1: Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone / exp2 and 3: hamster liver S9-mix, uninduced male Golden Syrian Hamster
Test concentrations with justification for top dose:
Experiment 1
Preliminary test (without and with S9) TA100 and WP2uvrA: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 100, 333, 1000, 3330 and 5000 µg/plate
Experiment 2: TA1535, TA1537, TA98, TA100 and WP2uvrA
Without and with S9-mix: 100, 333, 1000, 3330 and 5000 µg/plate
Experiment 3:
TA100 and WP2uvrA
With S9-mix: 100, 333, 1000, 3330 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle:
Test compound was stable in water and water has been accepted and approved by authorities and international guidelines
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9

Migrated to IUCLID6: 650 µg/plate in DMSO for TA100
Positive control substance:
2-nitrofluorene
Remarks:
without S9

Migrated to IUCLID6: 10 µg/plate in DMSO for TA98 and 15 µg/plate for TA1537
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9

Migrated to IUCLID6: 10 µg/plate in DMSO for WP2uvrA
Positive control substance:
sodium azide
Remarks:
without S9

Migrated to IUCLID6: 5 µg/plate in saline for TA1535
Positive control substance:
other: 2-aminoanthracene in DMSO for all tester strains
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Experiment 1: in agar (plate incorporation)
Experiment 2 and 3: preincubation for 30 minutes at 37°C

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.
Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) A two-fold (TA100) or more or a three-fold (TA1535, TA1537, TA98, WP2uvrA) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
in the experiment using a preincubation step
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: S. typhimurium TA1535, TA1537, TA98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
in the experiment using a preincubation step
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle 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 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES:
- No toxicity was observed up to and including the top dose of 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No toxicity was observed up to and including the top dose of 5000 µg/plate

Any other information on results incl. tables

The mean plate counts of the solvent control of TA100 in the presence of S9-mix (second and third experiment) were not within the laboratory historical range.

Evaluation:Although the values (38 and 45) were below the limit of the range (58), clear positive responses were observed both in this tester strain and tester strain WP2uvrA, thereforethis deviation in the mean plate countsof the solvent controlhad no effect on the results of the study.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive

FAT 40854/A TE is mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay. The mutagenicity was confined only to incubations with a pre-incubation step.
Executive summary:

In the absence of S9-mix, FAT 40854/A TE induced a 3.3-fold dose related increase in tester strain WP2uvrA after the pre-incubation treatment. The increase observed was above the laboratory historical control data range and was more than three times the concurrent control.

 

In the presence of S9-mix, FAT 40854/A TE induced dose related increases in two tester strains (TA100 and WP2uvrA) after the pre-incubation treatment. The increases observed in tester strain WP2uvrA were abovethelaboratory historical control data range, in two independently repeated experiments and were up to 6.4-fold the concurrent controls. The increases observed in tester strain TA100 were abovethelaboratory historical control data range in two independently repeated experiments and were up to 6.8-fold the concurrent controls. The increases observed in tester strain TA100 were only in the third experiment above the laboratory historical control data range, however the increase was more than two times the concurrent control in both experiments.

 

Since 3.3- to 6.8-fold, dose related increases were observed in two tester strains, both in the absence and presence of S9-mix (WP2uvrA) and in the presence of S9-mix (TA100) and the results obtained in the presence of S9-mix were reproducible in the repeat assay, these increases are considered biologically relevant, FAT 40854/A TE is mutagenic in the absence and presence of S9-mix.

 

All other bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, increase in the number of revertants in two independently repeated experiments.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly, except for TA100 in the presence of S9-mix (second and third experiment; solvent control) and WP2uvrA in the presence of S9-mix (third experiment; positive control). Although these values were without the limit of the range, clear mutagenic responses after treatment with FAT 40854/A TE were observed in these tester strains, therefore the validity of the test was considered to be not affected.