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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:
08-Dec-2011 to 15-Dec-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
Details on test material:
- Name of test material (as cited in study report): FAT 41043/A TE
- Substance type: Red powder
- Physical state: Solid
- Storage condition of test material: At room temperature in the dark


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:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Mutation Experiment
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: 3, 10, 33, 100 and 333 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: A homogeneous suspension could be in DMSO and DMSO is 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 S

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: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

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

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:
other: TA1537, TA98, TA100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: TA1535, WP2uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation was observed at dose levels of 100 µg/plate and above


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

- Mutagenicity:

TA100: FAT 41043/A TE induced up to 1.6- and 1.5-fold dose related increases in the number of revertant colonies compared to the solvent controls in the absence and presence of S9-mix, respectively.

TA1537: FAT 41043/A TE induced up to 9.7- and 9.8-fold dose related, increases in the number of revertant colonies compared to the solvent control in the absence and presence of S9-mix, respectively.

TA98: FAT 41043/A TE induced up to 65- and 39-fold dose related, increases in the number of revertant colonies compared to the solvent control in the absence and presence of S9-mix, respectively.

TA1535 and WP2uvrA, no increase in the number of revertants was observed upon treatment with FAT 41043/A TE.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive

FAT 41043/A TE is mutagenic in the Salmonella typhimurium reverse mutation assay and that FAT 41043/A TE is not mutagenic in the Escherichia coli reverse mutation assay.
Executive summary:

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.

 

In the absence of S9-mix, FAT 41043/A TE induced dose related increases in two out of the five tester strains (TA1537 and TA98). The increases observed in these tester strains were abovethelaboratory historical control data range and 9.7- and 65-fold the concurrent control. In addition in tester strain TA100, increases above the historical control data range were observed. However, these increases are just above the historical control data range and not more than a 1.6-fold increase was observed.

 

In the presence of S9-mix, FAT 41043/A TE induced dose related increases in two out of the five tester strains (TA1537 and TA98). The increases observed in these tester strains were abovethelaboratory historical control data range and 9.8- and 39-fold the concurrent control. In addition in tester strain TA100, increases just above the historical control data range were observed. However, these increases are just above the historical control data range and not more than a 1.5-fold increase was observed.

 

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