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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:
March 14, 2011 - April 04, 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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:
(1997)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
(2008)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): FDCA
- Substance type: White to off-white powder

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:
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
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test compound was soluble in DMSO and DMSO has been accepted and approved by authorities and international guidelines
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9: 650 µg/plate in DMSO for TA100
Negative solvent / vehicle controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without S9: 10 µg/plate in DMSO for TA98 and 15 µg/plate for TA1537
Negative solvent / vehicle controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9: 10 µg/plate in DMSO for WP2uvrA
Negative solvent / vehicle controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9: 5 µg/plate in saline for TA1535
Negative solvent / vehicle controls:
yes
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. 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 (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent control.
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 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the first mutation experiment, a reduction of the bacterial background lawn, was only observed in tester strain TA100 in the presence of S9. In the second experiment, toxicity was observed in all strains at the concentration of 3330 and 5000 μg/plate.
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the first mutation experiment, there was no reduction of the bacterial background lawn. In the second experiment, toxicity was observed in the presence of S9 mix at the concentration of 5000 μg/plate.
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:
- In tester strain TA100, toxicity was observed at dose levels of 3330 and 5000 μg/plate in the presence of S9-mix. In tester strain WP2uvrA and TA100 without S9-mix, no toxicity was observed up to and including the top dose of 5000 µg/plate.
- No mutagenicity 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 the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly, except the response for TA1537 in the absence of S9-mix (first and second experiment; positive control). Evaluation: The values (874 and 859) were above the limit of the range (756), however these values were more than 3 times greater than the concurrent solvent control values, this deviation in the mean plate counts of the positive control had no effect on the results of the study.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Second experiment:
TA1535, TA1537, TA98 and TA100: without S9 and with S9: 3330 and 5000 µg/plate
WP2uvrA: with S9: 5000 µg/plate
- No mutagenicity was observed up to and including the top dose of 5000 µg/plate

Applicant's summary and conclusion

Conclusions:
A Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and an Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA) with FDCA was performed according to OECD 471 guideline and GLP principles in two independent experiments. It is concluded that FDCA is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

A Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and an Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA) with FDCA was performed according to OECD 471 guideline and GLP principles in two independent experiments.

In the first mutation experiment, a reduction of the bacterial background lawn, was only observed in tester strain TA100 at dose levels of 3330 and 5000 μg/plate in the presence of S9-mix. In the second experiment, toxicity was observed in all strains at the concentration of 3330 and 5000 μg/plate, except in tester strain WP2uvrA in the absence of S9-mix.

FDCA did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

It is concluded that FDCA is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.