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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:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From November 16 to December 8, 1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Source study has reliability 1. Details on the read across are attached in section 13.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report date:
1993

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1983
Deviations:
yes
Remarks:
no statistical analysis was performed.
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
1992
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Similar Substance 02_DR89_1
IUPAC Name:
Similar Substance 02_DR89_1
Test material form:
solid: particulate/powder

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
61.7, 185.2, 555.6, 1666.7, 5000 µg/plate.
No toxic effect on the growth of bacteria.
Vehicle / solvent:
- Vehicle used: DMSO
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
Remarks:
without S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
other: 2-aminoanthracene
Remarks:
with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h, at 37 ± 1.5 °C in darkness

Evaluation criteria:
Assay acceptance criteria
A test is considered acceptable if the mean colony counts of the control values of all strains are within the acceptable ranges and if the results of the positive controls meet the criteria for a positive response.

Criteria for a positive response
The test substance is considered to be mutagenic in this test system if at least a reproducible meaningful increase of the mean number of revertants per plate above that of the negative control at any concentration for one or more of the following strains: S. typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537. Generally a concentration-related effect should be demonstrable.

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
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with
Genotoxicity:
ambiguous
Remarks:
marginal increase at 1666.7 and 5000 µg/plate
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: in orginal experiment

Any other information on results incl. tables

Toxicity test / range finding test

Six concentrations ranging from 20.6 to 5000 µg/plate were tested with strain S. typhimurium TA 100 to determine the highest concentration to be used in the mutagenicity assay. The experiments were performed with and without microsomal activation. The numbers of revertant colonies were not reduced. Normal back-ground growth was observed at all concentrations. The test material exerted no toxic effect on the growth of the bacteria . From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 5000 µg/plate without and with activation.

Mutagenicity test, original experiment

In the experiment performed without and with metabolic activation, treatment with test substance did not lead to an increase in the incidence of histidine-prototrophic mutants in comparison with the negative control.

Mutagenicity test, confirmatory experiment

In the experiment carried out without metabolic activation, again none of the tested concentrations led to an increase in the incidence of histidineprototrophic mutants by comparison with the negative control.

In the experiment performed with activation on strain TA 102, a marginal increase in the number of back-mutants was observed at the concentrations of 1666.7 and 5000 µg/ml. Since no such effects were registered in the original experiment conducted on this strain, the latter finding is not attributed to a mutagenic action of the test material. The marginal increase in the number of revertant counts probably may be due to spontaneously occurring back-mutants. Again, no effect was seen with the other strains.

In the mutagenicity tests without and with metabolic activation, normal back-ground growth was observed. The numbers of revertant colonies was not reduced. The test material exerted no toxic effect on the growth of the bacteria. The various mutagens, promutagens, sterility checks, sensitivity and resistance tests, etc., employed to ensure the test system was acceptable, all produced results within our established limits.

Applicant's summary and conclusion

Conclusions:
Based on test results and on standard evaluation criteria, it is concluded that test substance and its metabolites did not induce gene mutations in the strains of S. typhimurium used.
Executive summary:

Method

The test was conducted to detect gene mutations induced by test substance or its metabolites in histidine-requiring strains of Salmonella typhimurium.

The concentration range of test substance was determined in a preliminary toxicity test . Thus, it was tested for mutagenic effects without and with metabolic activation at five concentrations in the range of 61.7 to 5000 µg/plate. An independent repetition of the experiments was performed with the same concentrations.

Results

In the toxicity/range finding test, no toxic effect of the test material on the growth of the bacteria was observed with or without metabolic activation.

In the original mutagenicity test, none of the tested concentrations led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control both with and without metabolic activation.

Similarly, in the confirmatory mutagenicity test, no mutagenic effects were noted both without and with metabolic activation.

In the mutagenicity tests without and with metabolic activation, normal background growth was observed. The number of revertant colonies was not reduced. The test substance exerted no toxic effect on the growth of the bacteria.

Overall, test substance and its metabolites did not exhibit a mutagenic potential under test conditions.

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