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Genetic toxicity in vitro

Description of key information

STUDY SPONSOR : SEPPIC

STUDY TITLE : REVERSE MUTATION ASSAY "AMES TEST"

USING SALMONELLA TYPHIMURIUM

AND ESCHERICHA COLI

TEST MATERAL : SEPICALM VG

1. 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). 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 was determined in a preliminary toxicity assay and ranged from 5 to 5000 Aug/plate in the first experiment. 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 included to allow for the toxicity of the test material to the bacterial strains used and to ensure that there were at least four non-toxic doses plated out.

2. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonieswithin the normal range.

3. 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.

4. The test material caused a visible reduction in the growth of the bacterial lawn to the Salmonella strains only both with and without metabolic activation, whilst Escherichia Coli strain WP2uvra remained unaffected. The first indication of a toxic response was observed at 500 pug/plate. The test material was, therefore, either tested up to its toxic limit or the maximum recommended dose depending on bacterial strain type and presence or absence of S9-mix.

5. No biologically 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. Small, statistically significant increases in revertant colony frequency were observed in tester strain TA1537 (with S9-mix). The response was considered spurious as twofold increases over the vehicle were not achieved and it was non-reproducible. The test material was, therefore, considered to be non mutagenic under the Conditions of this test.


Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18.June.1998-24.July.1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Principles of method if other than guideline:
This study was conducted according to Safepharm Standard Method 700.03 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 all (4) in which mutagenic activity is assessed by exposing histidine auxotrophs of Salmonella typhimurium and tryptophan auxotrophs of Escherichia coli to various concentrations of the test material. The method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including MIT, MHW, MOL and MAFF. It also meets the requirements of the OECD, EC and USA, EPA (TSCA) guidelines. A copy of the Certificate of Compliance with GLP, issued by the UK Department of Health, is included as Appendix Ill.
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) 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. This strain also has a deletion in an excision repair gene (uvrA).
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 preparation (S9-mix) prepared from rats pre-treated with a compound known to induce an elevated level of these enzymes.
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Test item:
Sponsor's identification : SEP|CALM VG
Batch number : 98035300
Description : pale straw coloured paste
Sponsor's description : liquid, clear at 40'C
Date received : 4 June 1998
Storage Conditions : room temperature in the dark

Target gene:
his D, his C, his G, tryp E
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
The Salmonella strains were obtained from the University of California at Berkeley on culture discs on 4 August 1995 whilst the 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 confirm 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 (Oxoid limited; lot number 201416 1/03) and incubated at 37' C for approximately 10 hours.
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
0, 5, 15, 50, 150, 500, 1500, 5000 µg/plate
Vehicle / solvent:
dimethyl sulphoxide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-aminoanthracene (2AA)
Details on test system and experimental conditions:
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), 2 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of test material formulation and 0.5 ml of S9-mix or phosphate buffer was overlaid onto sterile plates of Vogel-Bonner Minimal agar (30 ml/plate). Ten concentrations of the test material formulation and a vehicle control (dimethylsulphoxide) were tested. In addition, 0.1 ml of the maximum concentration of the test material and 2 ml of molten, trace histidine or 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):
Up to 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 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, up to six Concentrations of the test material and control solutions.
Evaluation criteria:
Acceptance criteria:
The reverse mutation assay may be considered valid 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. Acceptable ranges are presented in the standard test method section 3.2 with historical control ranges for 1997 presented in Appendix I.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pkM 101 plasmid R-factor etc.
All tester strain cultures were in the range of 1 to 9.9 x 10° bacteria 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. The historical control ranges for 1997 are presented in Appendix I.
A minimum of four non-toxic dose levels were achieved.
No evidence of excessive contamination.

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.
Key result
Species / strain:
other: all cell type tested
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
The test material was toxic at and above 500 microg/plate to tester strain TA100 only.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
The test material was considered to be non-mutagenic under the conditions of this test.
Executive summary:

STUDY SPONSOR : SEPPIC

STUDY TITLE : REVERSE MUTATION ASSAY "AMES TEST"

USING SALMONELLA TYPHIMURIUM

AND ESCHERICHA COLI

TEST MATERAL : SEPICALM VG

1. 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). 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 was determined in a preliminary toxicity assay and ranged from 5 to 5000 Aug/plate in the first experiment. 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 included to allow for the toxicity of the test material to the bacterial strains used and to ensure that there were at least four non-toxic doses plated out.

2. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range.

3. 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.

4. The test material caused a visible reduction in the growth of the bacterial lawn to the Salmonella strains only both with and without metabolic activation, whilst Escherichia Coli strain WP2uvra remained unaffected. The first indication of a toxic response was observed at 500 pug/plate. The test material was, therefore, either tested up to its toxic limit or the maximum recommended dose depending on bacterial strain type and presence or absence of S9-mix.

5. No biologically 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. Small, statistically significant increases in revertant colony frequency were observed in tester strain TA1537 (with S9-mix). The response was considered spurious as twofold increases over the vehicle were not achieved and it was non-reproducible. The test material was, therefore, considered to be non mutagenic under the Conditions of this test.


Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

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

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