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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:
May 12, 2006 - June 22, 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
, 2000
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2005-11-21
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name: BIO 1031/1
Substance type: pure active substance
Physical state: solid
Storage condition of test material: ambient temp., dark, dry, in original container

Method

Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
post mitochondrial fraction (S9)
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
post mitochondrial fraction (S9)
Test concentrations with justification for top dose:
Concentration range in the main test (experiment I and experiment II): 50, 150, 500, 1500 and 5000 µg/plate (direct plate incubation method)
Vehicle:
Dimethylsulfoxide
Remark: The test material was fully soluble at 50mg/ml in DMSO.
Controlsopen allclose all
Negative controls:
yes
Remarks:
2AA: 1 µg/plate for TA100 and 2 µg/plate for TA1535 and TA1537, BP: 5 µg/plate for TA98 and DAN: 10 µg/plate for TA102
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Migrated to IUCLID6: 0.5 µg/plate for TA102
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Migrated to IUCLID6: 80 µg/plate for TA1537
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Migrated to IUCLID6: 0.2µg/plate for TA98
Details on test system and conditions:
MICROSOMAL ENZYME FRACTION: S9 was prepared from the livers of male Sprague-Dawley rats weighing approx. 250g. These had each orally received 3 consecutive daily doses of phenobarbitone/beta-naphthoflavone (80/100 mg/kg/day) prior to S9 preparation.

PRELIMINARY TOXICITY TEST: In order to select appropirate dose levels for use in the main test, 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 5000µg/plate. The test was performed by mixing 0.1ml of bacterial culture, 2ml of molten, trace histidine supplemented, top agar, 0.1ml of test material formulation, 0.5ml of S9-mix or phosphate buffer and overlaying onto sterile plates of Vogel-Bonner Minimal agar. After 48 hours incubation at 37°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.

MAIN MUTATION TESTS 1 AND 2:
METHOD OF APPLICATION: Measured aliquts (0.1ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0ml of molten, trace histidine supplemented, top agar, 0.1ml of the test material formulation, vehicle or positive control and either 0.5ml of S9-mix or phosphate buffer.The contents of each 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.
DURATION
All of the plates were incubated at 37°C for approx. 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.

ACCEPTANCE 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. Acceptable ranges are presented in the standard test method section 3.
- The appropirate characteristics for each tester strain have been confirmed.
- All tester strain cultures should be in approx. range of 1 to 9.9 x 10^9 bacteria/ml.
- Each mean positive control value should be at least two times the respective vehicle contol value for each strain, thus the demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
- There should be a minimum of 4 non-toxic test material dose levels.
- There should be no evidence of excessive contamination.
Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Statistics:
not applicable

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
(> 5000 µg/plate)
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
(> 5000 µg/plate)
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.
Additional information on results:
An opaque film was observed from 1500 µg/plate upwards which did not prevent the scoring of revertant colonies.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The results indicate that the test substance under the experimental conditions described, is not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence and absence of a metabolising system.