Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
4th November 1997 - 30th January 1998.
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)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Test material form:
solid: bulk
Details on test material:
Buff-colored
Specific details on test material used for the study:
Sponsor's identification: UK-114,958
Chemical name: (R)-3-(1-methyl-2-pyrrolidinylmethyl)-5- [2-(phenylsulphonyl)viny1]-1H-indole
Batch number: 116044/D/16/X2/1
Purity: ca. 94 - 95%
Description: buff solid
Sponsor's description: pale yellow to light gold powder
Date received: 28 October 1997
Storage conditions: room temperature in the dark

Method

Target gene:
uvrB - Salmonella strains and uvrA - E.coli strain
Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
10% liver S9 in standard co-factors
Test concentrations with justification for top dose:
The dose range was determined in a preliminary toxicity assay and was 5 to 5000 pg/plate in the first experiment. The experiment was repeated on a separate day using a dose
range of 15 to 5000 pg/plate with S9-mix and 15 to 1500 ,ug/plate without S9-mix with fresh cultures of the bacterial strains and fresh test material formulations.

Extra dose levels were included in both experiments to allow for the toxicity of the test material and to ensure that there were at least four non-toxic dose levels plated out.
Vehicle:
dimethyl sulphoxide (DMSO)
Controls
Negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
other: 4-Nitroquinoline-1-oxide (4NQ0)
Details on test system and conditions:
Tester Strains:
Salmonella typhimurium: TA1535, TA1537, TA98 and TA100
Escherichia coli: WP2uvrA-

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, model SXR 34. Prior to the master strains being used, characterisation checks were carried out to determine the amino-acid requirement, presence of rfa, R factors, 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 and sonication for 10 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 µg/plate for WP2uvrA , 3 µg/plate for TA100 and 5 µg/plate for TA1535
9-Aminoacridine (9AA): 80 µg/plate for TA1537
4-Nitroquinoline-1-oxide (4NQ0): 0.2 µg/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 µg/plate for TA100
2 µg/plate for TA1535 and TA1537
10 µg/plate for WP2uvrA-
0.5 µg/plate for TA98

Microsomal Enzyme Fraction:
S9 was prepared in-house on 17/09/97 from the livers of male Sprague Dawley rats weighing ~200g. These had each received a single i.p. injection of Aroclor 1254 at 500 mg/kg, five days before S9 preparation. Prior to use, all batches of S9 were checked for suitability using the indirect mutagen 2AA. The S9 was stored at -196°C.

S9-Mix and Agar:
The S9-mix was prepared at 4°C as follows:
S9: 5.0 ml
1.65 M KCl/0.4 M MgCl 2: 1.0 ml
0.1 M Glucose-6-phosphate: 2.5 ml
0.1 M NADPH: 2.0 ml
0.1 M NADH: 2.0 ml
0.2 M Sodium phosphate buffer (pH 7.4): 25.0 ml
Sterile distilled water: 12.5 ml

A 0.5 ml aliquot of S9-mix and 2 ml of molten, trace histidine/tryptophan supplemented, top agar was overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.

Top agar was prepared using 0.6% Difco Bacto agar and 0.5% sodium chloride with 5 ml of 1.0 mM histidine/1.0 mM biotin or 1.0 nnM tryptophan solution added to each 100 ml of top agar. Vogel-Bonner Minimal agar plates were prepared using 1.6% Oxoid Agar Technical No.3 with Vogel-Bonner Medium E and 20 mg/ml D-glucose.

Test Procedure:
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 of 0.1 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 seix concentrations of test material per strain and control solutions.

Mutation Study - Experiment 3 (Confirmatory Study):
Further work was performed with tester strain TA1535 (without S9-mix) after small but reproducible increases in revertant colony frequency had been observed in experiments 1 and 2. The experiment was carried out using a tightened test material dose range of 200, 300, 400, 500, 600, 700 and 800 pg/plate with and without a 20 minute pre-incubation
where the test material, tester strain and buffer were incubated at 37°C prior to addition of top agar and plating out.

Acceptance Criteria:
The reverse mutation assay may be considered of value if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.

The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pkM101 plasmid R-factor etc.

All tester strain cultures were in the range of 1 to 9.9 x 109 bacterial per ml.

Each mean positive control value was at least two times the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix with historical control ranges for 1997

A minimum of four non-toxic dose levels were achieved.
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:
The test material may be considered to be 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) significant increase in the revertant count in at least one strain of bacteria. If a greater than twofold increase in revertant count is observed in two experiments then this is taken as evidence of a positive response.
Statistics:
Dunnett's method of linear regression

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Vehicle controls valid:
yes
Positive controls valid:
yes
Remarks on result:
mutagenic potential (based on QSAR/QSPR prediction)
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
positive
Vehicle controls valid:
yes
Positive controls valid:
yes
Remarks on result:
mutagenic potential (based on QSAR/QSPR prediction)
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
positive
Vehicle controls valid:
yes
Positive controls valid:
yes
Remarks on result:
mutagenic potential (based on QSAR/QSPR prediction)
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Vehicle controls valid:
yes
Positive controls valid:
yes
Remarks on result:
mutagenic potential (based on QSAR/QSPR prediction)
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
positive
Vehicle controls valid:
yes
Positive controls valid:
yes
Remarks on result:
mutagenic potential (based on QSAR/QSPR prediction)
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).

The test material caused a visible reduction in the growth of the bacterial background lawn to all of the tester strains both with and without S9-mix at and above 1500 µg/plate. The test material was much more toxic after a 20 minute pre-incubation at 37°C with weakened lawns observed at and above 400 µg/plate.

Any other information on results incl. tables

Small, statistically significant increases in revertant colony frequency were observed with tester strain TA1535 (without S9-mix) in experiments 1 and 2 at a test material dose level of 500 µg/plate. Because the responses were so small an additional experiment was performed as outlined in section 1 of the summary. In this case, statistically significant increases were observed in the plate incorporation assay at all doses between 300 and 800 µg/plate with the greatest response again observed at 500 µg/plate. There were no meaningful results obtained after pre-incubation because of toxicity. Although a twofold increase in revertant colony frequency over the concurrent solvent control was only achieved in one of the three experiments, the responses were highly reproducible at a test material dose of 500 µg/plate.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies and the activity of the S9 fraction was shown to be satisfactory.

Applicant's summary and conclusion

Conclusions:
The test material was considered to be mutagenic with a weak response under the conditions of this test.
Executive summary:

This study was conducted according to Safepharm Standard Method Number 700.02 and was designed to assess the mutagenic potential of the test material using a bacterial/microsome test system. The study was based on the in vitro technique described by Ames and his co-workers (1, 2, 3) and Garner et al (4) in which mutagenic activity is assessed by exposing histidine auxotrophs of Salmonella typhimurium to various concentrations of the test material.

These mutant strains of Salmonella are incapable of synthesising histidine and are, therefore, dependent for growth on an external source of this particular amino acid. When exposed to a mutagenic agent these bacteria may undergo a reverse mutation to histidine independent forms which are detected by their ability to grow on a histidine deficient medium. Using various strains of this organism, revertants produced after exposure to a chemical mutagen may arise as a result of base-pair substitution in the genetic material (miscoding) or frame-shift mutation in which genetic material is either added or deleted. In order to make the bacteria more sensitive to mutation by chemical and physical agents, several additional traits have been introduced. These include a deletion through the excision repair gene (uvrB - Salmonella strains and uvrA - E.coli strain) which renders the organism incapable of DNA excision repair and deep rough mutation (rfa) which increases the permeability of the cell wall. A mutant strain of Escherichia coli (WP2uvrA-), which requires tryptophan and which can be reverse mutated by base substitution to tryptophan independence, was used to complement the Salmonella strains. Since many compounds do not exert a mutagenic effect until they have been metabolised by enzyme systems not available in the bacterial cell, the test material and the bacteria are also incubated in the presence of a liver microsomal fraction (S9) prepared from rats pre-treated with a compound known to induce an elevated level of these enzymes.

The test material was considered to be mutagenic with a weak response under the conditions of this test.