Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 November 1997 - 12 December 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: bulk
Details on test material:
brown crystalline solid
Specific details on test material used for the study:
(R)-1-Acetyl-3-(1-Methyl-2-pyrrolidinyl methyl)-512-(phenylsulphonyl)viny1]-1Hindole
Batch Number: 1160441Q/171X1/1
Purity ca. 90%

Method

Target gene:
uvrB -Salmonella strains and uvrA - E.co/i strain
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 prepared in-house from the livers of male Sprague-Dawley rats following a single i.p. injection of Aroclor 1254 at 500 mg/kg, five days before S9 preparation.
Test concentrations with justification for top dose:
A range finding study was conducted with a test substance concentration of 0,5, 15, 50, 150, 500, 1500 and 5000ug/plate. The test material was toxic at and above 500 and 1500ug/plate in the strains of bacteria used (TA100 and WP2uvrA) respectively. For the main study concentrations of 0, 5, 15, 50, 150, 500 and 1500 ug/plate. The test material caused a visible reduction in the growth of the bacterial
background lawn to all of the tester strains initially at 500 pg/plate without metabolic activation and 1500 yg/plate with metabolic activation. A further confirmatory experiment was performed both with and
without a pre-incubation modification and a tightened test material dose range in some of the strains, TA100, TA98, Wp2uvrA.

Vehicle / solvent:
dimethyl sulphoxide
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
dimethyl sulphoxide
True negative controls:
yes
Remarks:
2-Aminoanthracene; 1 ug/plate for TA100, 2 ug/plate for TA1535 and TA1537 10 ug/plate for WP2uvrA, 0.5 ug/plate for TA98
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
Details on test system and experimental conditions:
The Salmonella typhimurium strains were obtained from the University of California at Berkeley on culture discs on 4 August 1995 whilst Escherichia coli strain WP2uvrA- was obtained from the British Industrial Biological Research Association on 17 August 1987. All of the strains were stored at -196°C in a Statebourne liquid nitrogen freezer. Prior to the master strains being used, characterisation checks were carried out to determine the amino-acid requirement, presence of rfa, Rfactors, uvrB or uvrA mutation and the spontaneous reversion rate. In this assay, overnight sub-cultures of the appropriate coded stock cultures
were prepared in nutrient broth and incubated at 37°C for approximately 10 hours.

Preparation of Test and Control Materials
The test material was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer for ten minutes
and son ication for 15 minutes at room temperature 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 1/16 inch pellets with a nominal pore diameter of 4A.

Vehicle and positive controls were used in parallel with the test material. A solvent treatment group was used as the vehicle control and the positive control materials were as follows:
N-ethyl-N'-nitro-N-nitrosoguanidine (EN NG) : 2 ug/plate for WP2uvrA-,
3 ug/plate for TA100 and 5 ug/plate for TA1535
9-Aminoacridine (9AA) : 80 ,ug/plate for TA1537
4-Nitroquinoline-1-oxide (4NQ0) : 0.2 ug/plate for TA98
In addition, the material 2-Aminoanthracene (2AA), which is non-mutagenic in
the absence of metabolising enzymes was used in the S9 series of plates at the
following concentrations:
1 ug/plate for TA100
2 ug/plate for TA1535 and TA1537
10 ug/plate for WP2uvrA
0.5 ug/plate for TA98

Preliminary Toxicity Study
In order to select appropriate dose levels for use in the main study, a preliminary test was carried out to determine the toxicity of the test material. A mixture of0.1 ml of bacterial culture (TA100 or WP2uvrA-), 0.1 ml of test material formulation, 0.5 ml phosphate buffer and 2 ml of molten, trace histidine/tryptophan supplemented, top agar was overlaid onto sterile plates of Vogel-Bonner Minimal agar (30 ml/plate). Five doses of the test material and a vehicle control (dimethyl sulphoxide) were tested in duplicate. In addition, 0.1 ml of the maximum concentration of the test material and 2 ml of molten, trace histidine/tryptophan supplemented, top agar was overlaid onto sterile Vogel-Bonner Minimal agar plates 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 and examined for effects on the growth of the bacterial background lawn.

Mutation Study - Experiment 1 (Range-finding Study)
Seven concentrations of the test material were assayed in triplicate against each tester strain, using the direct plate incorporation method. 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/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 (Main Study)
The second experiment was performed using methodology as described for experiment 1, using fresh bacterial cultures, up to six concentrations of test material per tester strain and control solutions.

Mutation Study - Experiment 3 (Confirmatory Study)
A third confirmatory experiment was performed in response to small but statistically significant increases in revertant colony frequency observed in both Experiments 1 and 2. The experiment was performed using only those strains that had shown increases in Experiments 1 and 2, with the test material dose range tightened for each strain in an effort to both confirm and possibly enhance the responses. Additionally, a pre-incubation modification was also performed where 0.5 ml of 59-mix or phosphate buffer, 0.1 ml of tester strain and 0.1 ml of each test material formulation were incubated for 20 minutes at 37 C prior to addition of top agar and plating out
Rationale for test conditions:
The study was based on the in vitro technique described by Ames and his co-workers and Garner et al in which mutagenic activity is assessed by exposing histidine auxotrophs of Salmonella typhimurium to various concentrations of the test material.
Evaluation criteria:
For a substance to be considered positive in this test system, it should have induced a dose-related and statistically(5) significant increase in the revertant count in one or more strains of bacteria in the presence and/or absence of S9 in both experiments at sub-toxic dose levels. To be considered negative, the number of revertants at each dose level should be less than two fold that of the vehicle control frequency.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no 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:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
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. 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 Study.
The individual plate counts, the mean number of revertant colonies and the standard deviations for the test material, vehicle and positive controls both with and without metabolic activation.
Small but reproducible and statistically significant increases in revertant colony frequency were observed in tester strains TA100, TA98 and WP2uvrA- in both Experiments 1 and 2. A confirmatory experiment was performed both with and without a pre-incubation modification and a tightened test material dose range. The responses in this case were again reproducible and exhibited a doserelated relationship at sub-toxic dose levels of the test material. Although a two fold increase in revertant colony frequency over the concurrent solvent control was not achieved, the responses were extremely reproducible both in terms of fold increases and statistical values and were sufficiently above inhouse ranges to suggest that the test material was inducing a weak mutagenic response

Applicant's summary and conclusion

Conclusions:
Although a twofold increase in revertant colony frequency over the concurrent solvent control was not achieved, the responses were extremely reproducible both in terms of fold increases and statistical values and were sufficiently above in-house ranges to suggest that the test material was inducing a weak mutagenic response. The test material was, therefore, considered to be mutagenic with a weak response under the conditions of this test.
Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia con 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). This method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including MITI, MHW, MOL and MAFF. It also meets the requirements of the OECD, EC and USA, EPA (TSCA) guidelines. The dose range for the first experiment was determined in a preliminary toxicity assay and was 1.5 to 1500 yg/plate and 5 to 5000 ,ug/plate without and with S9-mix respectively. 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. Extra dose levels were incorporated into both experiments to allow for the toxicity of the test material and to ensure there were a minimum of four non-toxic doses plated out. Additionally, in an effort to enhance/confirm a weak mutagenic response observed in Experiments 1 and 2, a confirmatory experiment was performed both with and without a pre-incubation modification and a tightened dose range using tester strains TA100, TA98 and WP2uvrA.

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.

The test material caused a visible reduction in the growth of the bacterial background lawn to all of the tester strains initially at 500 ,ug/plate without metabolic activation and 1500 pg/plate with metabolic activation.

Small but reproducible and statistically significant increases in revertant colony frequency were observed in tester strains TA100, TA98 and WP2uvrA- in both Experiments 1 and 2. A confirmatory experiment was performed both with and without a pre-incubation modification and a tightened test material dose range. The responses in this case were again reproducible and exhibited a dose-related relationship at sub-toxic dose levels of the test material. Although a twofold increase in revertant colony frequency over the concurrent solvent control was not achieved, the responses were extremely reproducible both in terms of fold increases and statistical values and were sufficiently above in-house ranges to suggest that the test material was inducing a weak mutagenic response. The test material was, therefore, considered to be mutagenic with a weak response under the conditions of this test.