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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From June 20th, 1989 to October 20th, 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with international test guidelines (Japan/EU) and GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
with the exception of statistical analysis
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
with the exception of statistical analysis
Qualifier:
according to
Guideline:
EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Deviations:
yes
Remarks:
with the exception of statistical analysis
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
yes
Remarks:
with the exception of statistical analysis
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Appearance: Yellow liquid

Method

Target gene:
Histidine (his- to his+) and tryptophan (tryp- to tryp+) genes in Salmonella typhimurium and Escherichia coli respectively
Species / strain
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of liver from rats induced with Aroclor 1254 and cofactors.
Test concentrations with justification for top dose:
313, 625, 1250, 2500 and 5000 µg/0.1 mLl (with and without microsomal activation)
Vehicle / solvent:
Acetone (p.a., Merck, Darmstadt, Germany)
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
see vehicle control
Negative solvent / vehicle controls:
yes
Remarks:
Acetone alone was used for the negative vehicle control
True negative controls:
no
Positive controls:
yes
Remarks:
without S9 mix
Positive control substance:
other: Strain TA 98: 2-nitrofluorene, 10 µg/0.1 mL ; Strain TA 100 and TA 1535: sodium azide 2 µg/0.1 mL ; Strain TA 1537: (5)-aminoacridine hydrochloride monohydrate, 150 µg/0.1 mL; Strain E.coli WP2uvrA: 4-nitroquinoline-N-oxide 1 µg/0.1 mL.
Remarks:
2-nitrofluorene, (5)-aminoacridine hydrochloride monohydrate and 4-nitroquinoline-N-oxide were dissolved in DMSO; sodium azide was dissolved in bidistilled water.
Positive controls:
yes
Remarks:
with S9 mix
Positive control substance:
other: Strain TA 98 and TA 100: 2-aminoanthracene, 2.5 µg/0.1 mL; Strain TA 1535: cyclophosphamide monohydrate, 400 µg/0.1 mL bidistilled water; Strain TA 1537: 2-aminoanthracene, 5 µg/0.1 mL ; Strain E.coli WP2uvrA: 2- aminoanthracene, 50 µg/0.1 mL.
Remarks:
2-aminoanthracene was dissolved in DMSO; cyclophosphamide monohydrate was dissolved in bidistilled water.
Details on test system and experimental conditions:
The tests are carried out in accordance with the method described by Ames et al. (Ames BN et al. (1973) Proc. Natl. Acad. Sci. USA 70, 782-786; Ames BN et al. (1973) Proc. Natl. Acad. Sci. USA 20, 2281-2285; Ames BN et al. (1975) Mutation Res. 31, 347-364).
The test concentrations were selected on the basis of a preliminary toxicity test which had been carried out with concentrations ranging from 20 to 5000 µg/0.1 mL; accordingly, the concentration of 5000 µg/0.1 mL was retained as the highest in the mutagenicity test and the remaining test concentrations were set at 313, 625, 1250 and 2500 µg/mL.
Each Petri dish contained: 1) approx. 20 mL of minimum agar, 2) 0.1 mL of a solution of the test substance or the vehicle and 0.1 mL of a bacterial culture (in 2.5% nutrient broth) in 2.0 mL of soft agar. For the Salmonella strains the soft agar was supplemented with histidine; for E. coli, the soft agar was supplemented with tryptophan.
In the experiments without microsomal activation 0.5 mL of sodium phosphate buffer was added. In the experiments in which the substance is metabolically activated, the buffer is replaced by 0.5 ml of an activation mixture (S9 mix), which, as already indicated, consisted of the S9 fraction of liver from Sprague-Dawley rats induced with Aroclor 1254 and a solution of co-factors.
In the experiments without and with the addition of microsomal activation mixture three Petri dishes were prepared per strain and per group (i.e. per concentration or per control group). The plates were incubated for about 48 hours at 37 +/-0.5 °C in darkness. At test ending, the number of counted back-mutant colonies in the cultures treated with the various concentrations was compared with controls.
Evaluation criteria:
The test substance is considered to be positive in this test system if one or both of the following conditions are met:
- at least a reproducible doubling of the mean number of revertants per plate above that of the negative control at any concentration level for one or more of the tester strains TA 98, TA 1535, TA 1537 and E. coli WP2uvrA;
- a reproducible increase of the mean number of revertants per plate for any concentration above that of the negative control by at least a factor of 1.5 for strain TA 100. Generally a concentration-related effect should be demonstrable.
Statistics:
The data reported include individual numbers of revertants per plate, mean values and standard deviation for each bacterial strain and each concentration.

Results and discussion

Test results
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
(at the highest test concentration of 5000 µg/0.1 mL the substance precipitated in soft agar)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In the experiments performed without and with microsomal activation, comparison of the number of back-mutant colonies in the controls and the cultures treated with the various concentrations revealed no marked deviations. No evidence of the induction of point mutations by the substance or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium and Escherichia coli used in these experiments.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

SUMMARY OF RESULTS

Experiment I

TA 1535 TA 100 TA 1537 TA 98 WP2 uvra
Dose (µg/plate) -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
Control 9 7 137 139 4 7 14 27 15 19
313 9 6 125 133 4 7 17 22 15 16
625 12 9 139 128 6 5 21 35 18 20
1250 8 5 101 120 3 9 19 33 19 19
2500 9 5 112 118 4 10 16 34 19 18
5000 7 6 108 123 3 7 19 28 16 16
positive control 510 234 580 646 446 123 1107 1584 240 480

Experiment II

TA 1535 TA 100 TA 1537 TA 98 WP2 uvra
Dose (µg/plate) -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
Control 11 18 126 151 6 16 18 30 27 24
313 11 16 109 162 5 20 16 37 23 25
625 13 14 118 151 8 22 20 35 26 22
1250 11 10 127 159 6 16 21 36 34 24
2500 15 14 137 145 6 13 25 36 22 25
5000 7 13 136 168 6 13 20 24 24 23
positive control 201 352 737 944 1198 311 963 1430 397 558

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

No evidence of the induction of point mutations by the test item or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium and Escherichia coli used in these experiments.
Executive summary:

The test substance was tested for mutagenic effects on histidine-auxotrophicmutants of Salmonella typhimurium (strain TA 1535, TA 1537, TA 98 and TA 100) and on a tryptophan-auxotrophic strain of E. coli (WP2 uvrA) according to the OECD TG 471 (1983). The investigations were performed with the following concentrations of the trial substance without and with microsomal activation: 313, 625, 1250, 2500 and 5000 µg/0.1 mL. In order to confirm the results, the experiments were repeated.

For all tested concentrations, without and with microsomal activation,comparison of the number of back-mutant colonies in the controls and the treated bacteria cultures revealed no marked deviations. No evidence of the induction of point mutations by the substance or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium and Escherichia coli used in these experiments. No cytotoxicity was observed up to the highest tested concentration of 5000 µg/0.1 mL, which precipitated in soft agar.