Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 August 2006 - 07 September 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

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
Test material form:
solid
Details on test material:
Sponsor's identification: FSM-005W
Description: white solid
Batch number: 2005-01

Specific details on test material used for the study:
Sponsor's identification: FSM-005W
Description: White Solid
Batch number: #2005-01
Date received: 23 August 2005
Storage conditions: room temperature in the dark under nitrogen over silica gel

The integrity of supplied data relating to the identity, purity and stability of the test material is the responsibility of the Sponsor.

Method

Target gene:
Histidine locus
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
rat liver homogenate metabolising system (10% liver S9 in standard co-factors)
Test concentrations with justification for top dose:
Preliminary Toxicity Test
0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Mutation test - Experiment 1
Salmonella strains (with and without S9): 5, 15, 50, 150, 500, 1500 µg/plate.
E.coli strain WP2uvrA· (with and without S9): 5, 15, 50,150,500, 1500, 5000 µg/plate.

Mutation test - Experiment 2
Salmonella strains (with and without S9): 5, 15, 50, 150, 500, 1500 µg/plate.
E.coli strain WP2uvrA· (with and without S9): 5, 15, 50,150,500, 1500 µg/plate.

At 500 µg/plate the test material caused a visible reduction in the growth of the bacterial background lawn of all of the tester strains tested either with or without S9-mix. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate or the toxic limit, depending on bacterial strain type and experiment number. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: tetrahydrofuran
- Justification for choice of solvent/vehicle: the test material was insoluble in dimethyl sulpoxide, acetone, DMSO and acetonitrile. The test material was hydroscopic; therefore water was not evaluated as a vehicle in this system.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Remarks:
2 µg/plate for WP2uvrA, 3 µg/plate for TA100 and 5 µg/plate for TA1535.
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Remarks:
80 µg/plate for TA1537
Positive control substance:
9-aminoacridine
Remarks:
Without S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Remarks:
0.2 µg/plate for TA98
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Without S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Remarks:
1 µg/plate for TA100. 2 µg/plate for TA1535 and TA1537. 10 µg/plate for WP2uvrA
Positive control substance:
other: 2-Aminoanthracene (2AA)
Remarks:
with S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Remarks:
5 µg/plate for TA98
Positive control substance:
benzo(a)pyrene
Remarks:
with S9 mix
Details on test system and experimental conditions:
Preliminary Toxicity Test
In order to select appropriate dose levels for use in the main test, a preliminary assay was canied out to determine the toxicity of the test material. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. The assay was performed by mixing 0.1 ml of bacterial culture(TA100or WP2uvrA"), 0.025 ml of test material formulation, 0.5 ml of S9-rnix or phosphate buffer and 2 ml of molten, trace histidine or tryptophan supplemented, top agar and overlaying onto sterile plates of Vogel-Bonner Minimal agar(30 ml/plate).  Ten concentrations of the test material and a vehicle control (tetrahydrofuran) were tested. In addition, 0.025 ml of the maximum concentration of the test material and 2 ml of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Nutrient agar plate in order to assess the sterility of the test material. After approximately 48 hours incubation at 37°C the plates were assessed for numbers of revertant colonies using a Domino colony counter andexaminedfor effects on the growth of the bacterial background lawn.

Mutation Test-Experiment 1 (Range-findingTest)
Up to seven concentrations ofthetest material were assayed in triplicate against each tester strain, using the direct plate incorporation method. Dose ranges wereallocatedas follows:
Salmonella strains (with and without S9): 5, 15, 50, 150, 500,1500 µg/plate.
E.coli strain WP2uvrA· (with and without S9): 5, 15, 50,150,500, 1500, 5000 µg/plate.
Additional dose levels were included to allow for test materialinducedtoxicity,ensuring that at least four non-toxic doses were achieved.
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of testtubesfollowed by 2.0 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.025 ml of the vehicle or test material fonnulation or 0.1 ml of positive control and either 0.5 ml of S9-mix or phosphate buffer. The contents of each test tube were mixed andequallydistributed 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 Test-Experiment 2 (MainTest)
The second experiment was performed using methodology as described for the range-finding test, using fresh bacterial cultures, test material and control solutions. The test materialdose range was amended slightly, following the results from the range-finding test, and was 5 to 1500 µg/plate.
An additional dose level was again included to allow for test material induced toxicity, ensuring that at least four non-toxic doses were achieved.
Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS (5) can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.

A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Preliminary toxicity test:
The test material was toxic at and above 500 and 1500 µg/plate to the strains of bacteria used TA100 and WP2uvrA-. The test material formulation and S9-mix used in this experiment were both shown to be sterile.

Mutation Test
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile.
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
At 500 µg/plate the test material caused a visible reduction in the growth of the bacterial background lawn of all of the tester strains tested either with or without S9-mix. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate or the toxic limit, depending on bacterial strain type and experiment number. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

No significant increases in the frequency of reverta.nt colonies were recorded for any of the bacterial strains, at any dose level 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 bacteria] strains.

Applicant's summary and conclusion

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

Introduction. The method was designed to conform to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets 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 typhimuriumstrains TA1535, TA1537, TA98 and 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 the range-finding test was determined in a preliminary toxicity assay and was 5 to 1500 µg/plate for the Salmonella strains and 5 to 5000 µg/plate for Escherichia coli strain WP2uvrA-. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test material formulations. The test material dose range was amended slightly, following the results of the range-finding test, and was 5 to 1500 µg/plate for all of the bacterial strains.

Additional dose levels were included to allow for test material induced toxicity, ensuring that at least four non-toxic doses were achieved.

Results. The vehicle (tetrahydrofuran) 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 or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. At 500 µg/plate the test material caused a visible reduction in the growth of the bacterial background lawn of all of the tester strains tested either with or without S9-mix. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate or the toxic limit, depending on bacterialstraintype and experiment number. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

 

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.