Registration Dossier

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:
31 March 2014 - 14 April 2014
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:
2014
Report Date:
2014

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
Type:
impurity
Test material form:
liquid
Details on test material:
- Name of test material (as cited in the expert statement): Folenox
- Chemical name: 4,4,8,8-Tetramethyloctahydro-4a,7-methanonaphtho[1,8a-b]oxirene (IUPAC)
- Empirical formula: C15H24O
- Structural formula: See IUCLID section 1

Method

Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Species / strain:
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: 33, 100, 333, 1000, 3330 and 5000 µg/plate
Experiment 2 all five strains:
Without and with S9-mix: 100, 333, 1000, 3330 and 5000 µg/plate
Vehicle:
- 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 controls:
no
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 Migrated to IUCLID6: 650 µg/plate in DMSO for TA100
Negative controls:
no
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-nitrofluorene 10 µg/plate in DMSO for TA98
Remarks:
without S9
Negative controls:
no
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
other: ICR-191 2.5 µg/plate in DMSO for TA1537
Remarks:
without S9
Negative controls:
no
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
other: 4-nitroquinoline N-oxide 15 µg/plate in DMSO for WP2uvrA
Remarks:
without S9
Negative controls:
no
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9 Migrated to IUCLID6: 5 µg/plate in saline for TA1535
Negative controls:
no
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene in DMSO for all tester strains
Remarks:
with S9
Details on test system and 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 vehicle 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 vehicle 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:
yes
Remarks:
see additional information on results
Vehicle controls valid:
yes
Positive controls valid:
yes
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Remarks:
but tested up to limit concentrations
Vehicle controls valid:
yes
Positive controls valid:
yes
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, a slight to moderate reduction of the bacterial background lawn was observed at concentrations of 333 μg/plate and upwards in the absence of S9-mix and at 1000 μg/plate and upwards in the presence of S9-mix. A moderate reduction in the number of revertants was observed at the top dose of 5000 μg/plate in the absence of S9-mix only. In tester strain WP2uvrA, no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.

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:
In experiment 1, no toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate.
In experiment 2, Cytotoxicity, as evidenced by a slight to moderate reduction of the bacterial background lawn, was only observed in the absence of S9-mix in tester strain TA1535 at the highest tested concentration and in tester strain TA100 at 1000 to 5000 μg/plate. There was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all other tester strains in the absence and presence of S9-mix. No increase in the number of revertants was observed upon treatment with Folenox under all conditions tested.

Applicant's summary and conclusion

Conclusions:
Folenox is not mutagenic in the Salmonella typhimurium reverse mutation assay and Escherichia coli reverse mutation assay with and without S9-mix, performed according to OECD 471 guideline and GLP principles.
Executive summary:

The genetic toxicity of Folenox was assessed in the Salmonella typhimurium reverse mutation assay and Escherichia coli reverse mutation assay with and without S9-mix, performed according to OECD 471 guideline and GLP principles.

No precipitation was observed up to and including the top dose of 5000 µg/plate in two independently experiments. Slight toxicity was observed in strain TA100 with and without S9 -mix in experiment 1 and in strain TA1535 and TA100 without S9 -mix in experiment 2.

No increase in the number of revertants was observed upon treatment with Folenox under all conditions tested.

Based on the results it is concluded that Folenox is not mutagenic in the Salmonella typhimurium reverse mutation assay and Escherichia coli reverse mutation assay.