Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6 MAR 2000 - 16 JUN 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline

Method

Target gene:
histidine (S. typhimurium strains), tryptophan (E.coli strain)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
other: please refer to 'Any other information on materials and methods'
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Additional strain / cell type characteristics:
other: This strain lacks excision repair (uvrA), so that it is more readily mutated by treatment with UV, for instance. Additionally, the presence of the R-factor causes much greater sensitivity to a number of carcinogens and resistance to ampicillin.
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : The batch of the liver homogenate fraction (S9) used in this study was prepared at the Institute of Toxicology of MERCK KGaA, Darmstadt, from June 28 to July 05, 1999. It was obtained from Aroclor 1254 treated male Chbb:Thom (Wistar) rats (aged 6-8 weeks).
- method of preparation of S9 mix :

Components Quantitiy per mL S9 mix
1st Series 2nd Series
Liver homogenate (S9) 0.10 mL 0.30 mL
MgCl2/KCl aqueous solution (0.4 M/1.64 M) 0.02 mL 0.02 mL
Glucose-6-phosphate 1.50 mg 1.50 mg
NADP 3.15 mg 3.15 mg
Sodium phosphate buffer (0.2 M, pH 7.4) 0.50 mL 0.50 mL
Distilled water 0.38 mL 0.18 mL

- concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Every S9-batch is tested for its metabolic activity by the use of specific substrates, requiring different enzymes of the P450-isoenzyme family. The mutagenicity of 2-aminoanthracene, benzo[a]pyrene, and 3-methylcholanthrene is thus determined once for every S9-batch. Clear increases in the number of revertants for S. typhimurium TA 98, TA 100, and TA 1537 with all positive controls and for TA 1535 with 2-aminoanthracene are used as an acceptance criterion for each S9-batch. In this study 2-aminoanthracene, and benzo[a]pyrene for TA 102, are used as the concurrent positive controls for the different strains in the presence of S9 mix.
Test concentrations with justification for top dose:
The test material should be investigated in the first experimental series at seven concentrations, separated by half-log intervals, ranging from 5 to 5000 µg per plate. In the second experimental series, usually 5 concentrations including at least 4 non-toxic concentrations should be tested. Signs of toxicity to the bacteria are a reduction in the number of spontaneous revertants or a clearing of background lawn of non-revertant bacteria. Precipitation of the test material, if it occurs, should not interfere with scoring of the colonies. Depending upon the precipitation characteristics of the test material and toxicity data ascertained in the first series, high concentrations may be omitted or lower concentrations may additionally be tested in the second experimental series.
On the basis of the criteria stated above, the following test material concentrations were used

1st series: 5.00, 15.8, 50.0, 158, 500, 1580 and 5000 µg per plate
2nd series: 5.00, 15.8, 50.0, 158 and 500 µg per plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: Preferentially distilled water or dimethyl sulfoxide (DMSO), alternatively acetone or ethanol, are used as solvents.

- Justification for percentage of solvent in the final culture medium: Analysis of the historical data of the laboratory and experience of other research groups (Maron et al. 1981) showed that the amounts of the selected solvents used have no influence on the number of spontaneous revertants of any strain. For this reason only the respective solvent control was used as the negative control in this study. Since on the one hand organic solvents may have diverse effects on e.g. gene regulation and, on the other hand, high amounts of water (added as the solvent) will dilute the top agar, usually the maximum amount of solvent is limited to 100 µL per plate for water and 10 µL per plate for DMSO, ethanol, acetone or other organic solvents. Only the highest test material concentration may be plated with either 316 µL water or 31.6 µL organic solvent.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
cumene hydroperoxide
other: Daunomycin, 2-Aminoanthracene
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : triplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition; the presence of a background lawn of non-revertant cells was checked for each plate

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Revertant colonies were either scored using an Artek MiniCount colony counter or manually.
Rationale for test conditions:
The applied in vitro technique, described by Ames et al. (1975), Green and Muriel (1976), Maron and Ames (1983) and Utesch et al. (1987), is an internationally accepted standard procedure.
Evaluation criteria:
A test material is defined as non-mutagenic in this assay if
• "no" or "weak increases" occur in the first and second series of the main experiment. ("Weak increases" randomly occur due to experimental variation.)
A test material is defined as mutagenic in this assay if
• a dose-related (over at least two test material concentrations) increase in the number of revertants is induced, the maximal effect is a "clear increase", and the effects are reproduced at similar concentration levels in the same test system;
• "clear increases" occur at least at one test material concentration, higher concentrations show strong precipitation or cytotoxicity, and the effects are reproduced at the same concentration level in the same test system.
In all further cases, a third test series with the bacterial strain in question should be performed. If the criteria for a positive test result are not fulfilled in at least two out of the three series, the test material is defined as being non-mutagenic in this test system.
Statistics:
Tables of individual and mean values are generated by use of a validated, automated data processing program (COLONY V2.31, York Electronic Research, York, UK).

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: Precipitation of the test material on the agar plates occurred at concentrations ≥ 500 µg/plate

RANGE-FINDING/SCREENING STUDIES:
The test material should be investigated in the first experimental series at seven concentrations, separated by half-log intervals, ranging from 5 to 5000 µg per plate. In the second experimental series, usually 5 concentrations including at least 4 non-toxic concentrations should be tested. Signs of toxicity to the bacteria are a reduction in the number of spontaneous revertants or a clearing of background lawn of non-revertant bacteria. Precipitation of the test material, if it occurs, should not interfere with scoring of the colonies. Depending upon the precipitation characteristics of the test material and toxicity data ascertained in the first series, high concentrations may be omitted or lower concentrations may additionally be tested in the second experimental series.
On the basis of the criteria stated above, the following test material concentrations were used

1st series: 5.00, 15.8, 50.0, 158, 500, 1580 and 5000 µg per plate
2nd series: 5.00, 15.8, 50.0, 158 and 500 µg per plate

STUDY RESULTS
- Concurrent vehicle negative and positive control data : Each treatment with the substances used as positive controls led to a clear increase in revertant colonies, thus showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used (for details on results please refer to tables attached under 'Attached background material').

Ames test:
- Mean number of revertant colonies per plate and standard deviation : please refer to tables attached under 'Attached background material'

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data:
The positive control compounds should induce a "clear increase" in the number of revertants.
- Negative (solvent/vehicle) historical control data: According to the publications of Levin et al. (1982) and Kier et al. (1986) and the historical controls of the laboratory, usually the following mean numbers of revertants are acceptable as negative (or solvent) controls for the bacterial strains used:
TA98 : 15 - 60
TA 1535: 3 - 37
TA 100: 75 -200
TA 1537: 4- 31
TA 102: 200-450
WP2: 50-200

Any other information on results incl. tables

for details on results, please refer to tables under 'Attached background material'

Applicant's summary and conclusion

Conclusions:
With and without addition of S9 mix as the external metabolizing system, the test item was not mutagenic under the experimental conditions described.
Executive summary:

The mutagenic potential of the test item was tested in an Ames test according to OECD Guideline 471 following GLP. The investigations for mutagenic potential were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA pkM101. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed. The test item was dissolved in dimethyl sulfoxide and tested at concentrations ranging from 5 to 5000 µg/plate. Precipitation of the test material on the agar plates occurred at concentrations ≥ 500 µg/plate. Toxicity to the bacteria was not observed. Daunomycin, N-ethyl-N'-nitro-N-nitrosoguanidine, 9-aminoacridine and cumene hydroperoxide served as strain specific positive control compounds in the absence of S9 mix. 2-Aminoanthracene and benzo[a]pyrene were used for testing the bacteria and the activity of the S9 mix. Each treatment with the substances used as positive controls led to a clear increase in revertant colonies, thus showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used. With and without addition of S9 mix as the external metabolizing system, the test item was not mutagenic under the experimental conditions described.