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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 March 1997 to 20 March 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1983
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Version / remarks:
1983
Deviations:
no
Qualifier:
according to
Guideline:
other: Proposal for Replacement of Guidelines 471 and 472: Bacterial Reverse Mutation Test, Revised Draft Document, OECD
Version / remarks:
1995
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

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

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Species / strain / cell type:
E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9 mix induced by Aroclor 1254
Test concentrations with justification for top dose:
First series: 5, 15.8, 50, 158, 500, 1580, and 5000 µg/plate (maximum recommended concentration according to Guideline)
Second series: 5, 15.8, 50, 158, and 500 µg/plate (highest concentration was choosen due to precipitation at 5000 µg/plate)
Vehicle / solvent:
- Vehicle/solvent used: DMSO
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: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 2 independent experiments (3 plates per concentration)

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of background lawn of non-revertant bacteria



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 streng precipitation or cytotoxicity, and the effects are reproduced at the same concentration level in the same test system.

In all further cases should a third test series with the bacterial strain in question be performed. lf 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.

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:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
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
Positive controls validity:
valid
Key result
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
Positive controls validity:
valid
Key result
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
Positive controls validity:
valid
Key result
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
Positive controls validity:
valid
Key result
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
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at concentrations >= 500 µg/plate

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The positive control compounds should induce a "clear increase" in the number of revertants.
- Negative (solvent/vehicle) historical control data: TA98: 15- 60, TA 1535: 3- 37, TA 100: 75 - 200, TA 1537: 4 - 31, TA 102: 200 - 450, WP2: 50 - 200

Applicant's summary and conclusion

Conclusions:
With and without addition of S9-Mix as the external metabolizing system, the test item was not mutagenic in the Bacterial Reverse Mutation Assay.
Executive summary:

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 between 5 and 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.