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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:
September - October 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Conducted to GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Description: off-white powder
Date received: 14 August 2002
Storage conditions: room temperature in the dark

Method

Target gene:
Histidine locus
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbitone/Beta-naphthoflavone induced rat liver S9.
Test concentrations with justification for top dose:
Preliminary Toxicity Study:
The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 500, 1500 and 5000 microg/plate.
Mutation Study 1 and 2:
Five concentrations of the test material (50, 150, 500, 1500 and 5000 migrog/plate) were assayed.
Vehicle / solvent:
Dimethyl sulphoxide was selected as the vehicle of choice because it formed a good partial solution/suspension with the test material at 50mg/ml.
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
A solvent treatment group was used as vehicle control
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Migrated to IUCLID6: 3 microg/plate for TA100 and 5 microg/plate for TA1535
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Migrated to IUCLID6: 80 microg/plate for TA1537
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Migrated to IUCLID6: 0.5 microg/plate for TA102
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Migrated to IUCLID6: 0.2 microg/plate for TA98
Positive control substance:
other: 2-Aminoanthracene 1 microg/plate for TA100 and 2 microg/plate for TA 1535 and TA 1537
Positive control substance:
benzo(a)pyrene
Remarks:
Used in the S9 series of plates

Migrated to IUCLID6: 5 microg/plate for TA98
Positive control substance:
other: 1,8-Dihydroxyanthraquinone 10 microg/plarte for TA102
Remarks:
Used in the S9 series of plates
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. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 microg/plate. The test was performed by mixing 0.1ml of bacterial culture (TA100), 2ml of molten, trace histidine supplemented, top agar, 0.1ml of test material fomulation, 0.5ml of S9-mix or phosphate buffer and overlaying onto sterile plates of Volgel-Bonner Minimal agar (30 ml/plate). Ten doses of the test material and a vehicle control (dimethyl suplhoxide) were tested. In addition, 0.1ml of the maximum concentration of the test material and 2ml of molten, trace histidine supplemented, top agar was overlaid onto a sterile Nutrient agar plate in order to assess the sterility of the test material. After approximately 48 hours incubation at 37 deg 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:
Five concentrations of the test material 50, 150, 500, 1500 and 5000 microg/plate 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 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 the plates were incubated at 37 degC 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 in Experiment 1, using fresh bacterial cultures, test material and control solutions. The test material dose range was the same as Experiment 1.
Evaluation 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.
The appropriate characteristics for each tester strain have been confirmed, e.g. rfa cell-wall mutation and pkM101 plasmid R-factor etc.
All tester strain cultures should be in the approximate range of 1 to 9.9E9 bacteria per ml.
Each mean positive control value should be at least two times the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strain to mutagenic exposure and the integrity of the S9-mix.

The test material may be considered positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statistically significant increase in the revertant count in at least one strain of bacteria.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level, although a decrease in the frequency of revertant colonies was noted at 5000 microg/plate. The test material was, therefore, tested up to the maximum recommended dose level of 5000 microg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence of absence of S9-mix.
No significant increases in the frequency of revertant colonies were recorded for any of the strains of Salmonella, at any dose level either with or without metabolic activation.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

ZMB2 is considered to be non-mutagenic under the conditions of this test.