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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline, GLP study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Test material form:
solid
Details on test material:
Sponsor's identification : LCE01089
Description : white solid
Batch number : #01116001
Label : Product name: SEPIFEEL ONE Batch No. 01 116 001
Manufacturing date: 26/04/2001
Validity date: 25/04/2004
Date received : 24 December 2001
Storage conditions : room temperature, in the dark
Specific details on test material used for the study:
Sponsor's identification : LCE01089
Description : white solid
Batch number : #01116001
Label : Product name: SEPIFEEL ONE Batch No. 01 116 001
Manufacturing date: 26/04/2001
Validity date: 25/04/2004
Date received : 24 December 2001
Storage conditions : room temperature, in the dark

Method

Target gene:
histidine auxotrophs of Salmonella typhimurium and tryptophan auxotrophs of Escherichia coli
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
Details on mammalian cell type (if applicable):
uvrA-
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Preliminary Toxicity Study (TA100 or WP2uvrA-)
The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate.

Mutation Study
All Salmonella tester strains (with and without S9-mix): 1.5, 5, 15, 50, 150 and 500 μg/plate
Escherichia coli (with and without S9-mix): 50, 150, 500, 1500 and 5000 μg/plate
Vehicle / solvent:
dimethyl sulphoxide
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
(dimethyl sulphoxide)
Positive controls:
yes
Remarks:
2AA at 1 μg/plate for TA100 - 2AA at 2 μg/plate for TA1535 and TA1537 - BP at 5 μg/plate for TA98 - 2AA at 10 μg/plate for WP2uvrA-
Remarks:
with S9-mix
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
N-ethyl-N'-nitro-N-nitrosoguanidine(ENNG): 3 μg/plate for TA100, 5 μg/plate for TA1535 and 2 μg/plate for WP2uvrA- 9-Aminoacridine (9AA): 80 μg/plate for TA1537 - 4-Nitroquinoline-1-oxide (4NQO): 0.2 μg/plate for TA98
Remarks:
without S9-mix
Details on test system and experimental conditions:
Preparation of Test and Control Materials
The test material was accurately weighed and approximate half-log dilutions prepared in dimethylsulphoxide by mixing on a vortex mixer and sonication for 5 minutes at 40°C on the day of each experiment. Analysis for concentration, homogeneity and stability of the test material formulations is not a requirement of the test guidelines and was, therefore, not determined. Prior to use, the solvent was dried using molecular sieves (sodium alumino-silicate) ie 2 mm pellets with a nominal pore diameter of 4 x 10-4 microns.

Vehicle and positive controls were used in parallel with the test material.

mutation study:
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes
followed by 2.0 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of the
test material formulation, vehicle or positive control and either 0.5 ml of S9-mix or phosphate
buffer. The contents of each test tube were mixed and equally distributed onto the surface of
Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in
triplicate, for each bacterial strain and for each concentration of test material both with and
without S9-mix.
All of the plates were incubated at 37°C for approximately 48 hours and the frequency of
revertant colonies assessed using a Domino colony counter.

Mutation Study - Experiment 2
The second experiment was performed using methodology as described for Experiment 1, using
fresh bacterial cultures, test material and control solutions. The test material dose range was the
same as Experiment 1 except for TA1535 (without S9-mix only). The dose range for this strain
was amended slightly to 0.5 to 500 μg/plate after excessive toxicity had been observed in
Experiment 1.
Evaluation criteria:
The test material may be considered positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statistically (Dunnett's
method of linear regression(5)) significant increase in the revertant count in at least one strain of
bacteria.
Statistics:
Dunnett's method of linear regression

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:
The test material caused either a visible reduction in the growth of the bacterial background lawn and/or a substantial decrease in the frequency of revertant colonies in all of the Salmonella tester strains at 150 μg/plate.
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
No toxicity was observed in Escherichia coli strain WP2uvrA- at any test material dose level either with or without metabolic activation
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test material caused either a visible reduction in the growth of the bacterial background lawn
and/or a substantial decrease in the frequency of revertant colonies in all of the Salmonella tester
strains at 150 μg/plate. No toxicity was observed in Escherichia coli strain WP2uvrA- at any test
material dose level either with or without metabolic activation. The test material was, therefore,
tested up to the maximum recommended dose level of 5000 μg/plate. A globular precipitate was
observed at 5000 μg/plate, this did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the
bacterial strains, with any dose of the test material, either with or without metabolic activation.

All of the positive control chemicals used in the test induced marked increases in the frequency of
revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial
strains.
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

The test material was considered to be non-mutagenic under the conditions of this test.
Executive summary:

Introduction.

The method was designed to meet the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Directive 2000/32/EC and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Methods.

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA- were treated with the test material using the Ames plate incorporation method at up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was determined in a preliminary toxicity assay and was 1.5 to 500 μg/plate for all of the Salmonella tester strains and 50 to 5000 μg/plate for Escherichia coli strain, WP2uvrA-. The experiment was repeated on a separate day using a similar dose range to Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations. Additional dose levels were included for all of the Salmonella tester strains to allow for test material induced toxicity and to ensure there were a minimum of four non-toxic doses plated out.

Results.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused either a visible reduction in the growth of the bacterial background lawn and/or a substantial decrease in the frequency of revertant colonies in all of the Salmonella tester strains at 150 μg/plate. No toxicity was observed in Escherichia coli strain WP2uvrA- at any test material dose level either with or without metabolic activation. The test material was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. A globular precipitate was observed at 5000 μg/plate, this did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

Conclusion.

The test material was considered to be non-mutagenic under the conditions of this test.