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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 August 25, 2010 to June 01, 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD Guideline 471 and OPPTS 870.5100, in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH Guidelines S2A and S2B (ICH, 1995, 1997)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
3-phenyl-5-(2-thienyl)-1,2,4-oxadiazole
EC Number:
810-533-8
Cas Number:
330459-31-9
Molecular formula:
C12H8N2OS
IUPAC Name:
3-phenyl-5-(2-thienyl)-1,2,4-oxadiazole
Test material form:
solid: pellets
Details on test material:
- Name of test material (as cited in study report): MON 102100

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
Initial mutagenicity assay: 1.60, 5.00, 16.0, 50.0, 160, 500, 1600, and 5,000 µg/plate with and without S9
Confirmatory assay: 10.0, 50.0, 100, 500, 1000, and 5,000 µg/plate with and without S9
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Without S9 (TA98)
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without S9 (TA100, TA1535)
Negative solvent / vehicle controls:
yes
Positive control substance:
other: ICR-191
Remarks:
Without S9 (TA1537)
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Without S9 (WP2uvrA)
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With S9 (TA98)
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With S9 (TA100, TA1535, TA1537, WP2uvrA)
Details on test system and experimental conditions:
TEST SYSTEM RATIONALE:
The bacterial reverse mutation assay has been shown to be a sensitive, rapid, and accurate indicator of the mutagenic activity of many materials including a wide range of chemical classes. By using several different tester strains, both base pair substitution and frameshift mutations can be detected. Salmonella and E. coli strains used in this assay are histidine and tryptophan auxotrophs, respectively, by virtue of conditionally lethal mutations in the appropriate operons. When these histidine (his–) or tryptophan (trp–) dependent cells are exposed to the test substance and grown under selective conditions (minimal media with a trace amount of histidine or tryptophan), only those cells which revert to histidine (his+) or tryptophan (trp+) independence are able to form colonies. Trace amounts of histidine or tryptophan added to the media allow all the plated bacteria to undergo a few cell divisions, which is essential for mutagenesis to be fully expressed. his+ or trp+ revertants are readily discernable as colonies against the limited background growth of his– or trp– cells.

SOURCE OF TESTER STRAINS:
Salmonella tester strains were received from Dr. Bruce Ames, Department of Biochemistry, University of California. E. coli tester strain WP2uvrA was received from The National Collection of Industrial Bacteria, Torrey Research Station, Scotland (United Kingdom).

PLATING PROCEDURES:
Each plate was labelled with a code that identified the test substance, test phase, tester strain, activation condition, and dose level. Dilutions of the test substance were prepared immediately prior to their use. Treatments were performed by adding 100 µL tester strain and 50 µL of test or control substance to 2.5 mL of molten selective top agar (maintained at 45±2°C). After the required components had been added, the mixture was vortexed and overlaid onto the surface of 25 mL minimal bottom agar in a 15 x 100 mm petri dish. After the overlay solidified, the plates were inverted and incubated for 52±4 h at 37±2°C. Cultures were treated in the presence of S9 in an identical manner, except using 2.0 mL undiluted molten selective top agar and adding 500 µL S9 mix.

- In an initial mutagenicity assay, all doses of the test substance, as well as the concurrent positive and vehicle controls, were evaluated in duplicate plates. In an independent confirmatory assay, all test and control substances were evaluated in triplicate plates.

SCORING THE PLATES:
Plates which were not evaluated immediately following the incubation period were held at >0 to 10°C until such time that colony counting and bacterial background lawn evaluation could take place.

BACTERIAL BACKGROUND LAWN EVALUATION:
The condition of the bacterial background lawn was evaluated macroscopically and microscopically (using a dissecting microscope) for indications of cytotoxicity and test substance precipitate. Evidence of cytotoxicity was scored relative to the vehicle control plate and was recorded along with the revertant counts for all plates at that dose level. Lawns were scored as normal (N), reduced (R), obscured by precipitate (O), macroscopic precipitate present (P), absent (A), or enhanced (E); contaminated plates (C) were also noted as applicable.

COUNTING REVERTANT COLONIES:
Revertant colonies were counted by automated colony counter and/or by hand.
Evaluation criteria:
ASSAY EVALUATION CRITERIA:

Criteria for a Positive Response:
A test substance is considered to have produced a positive response if it induces a dose dependent increase in revertant frequency that is ≥2.0 fold vehicle control values for tester strains TA98, TA100, and WP2uvrA, or ≥3.0 fold vehicle control values for tester strains TA1535 and TA1537. In addition, any response should be reproducible.

Criteria for a Negative Response:
A test substance is considered to have produced a negative response if no dose dependent, ≥2.0 fold or ≥3.0 fold increases are observed in tester strains TA98, TA100, and WP2uvrA, or TA1535 and TA1537, respectively.

Criteria for an Equivocal Response:
Even after repeated trials, a test substance may produce results that are neither clearly positive nor clearly negative (e.g., responses that do not meet the dose-dependency or fold increase requirements but are reproducible). In those rare instances, the test substance may be considered to have produced an equivocal response.

- Other criteria also may be used in reaching a conclusion about the study results (e.g. comparison to historical control values, biological significance, etc.).

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 / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
MUTAGENICITY ASSAYS
In the initial mutagenicity assay, normal growth was observed in all five tester strains at all doses evaluated with and without S9. However, the test substance was incompletely soluble in the aqueous top agar at the highest three or four doses evaluated with and without S9. Revertant frequencies for all doses of test substance, in all tester strains with and without S9, approximated or were less than those observed in the concurrent vehicle control cultures.

In the confirmatory mutagenicity assay, all doses of the test substance, normal growth again was observed in all five tester strains at all doses evaluated with and without S9. In addition, the test substance again was incompletely soluble at the highest three or four doses evaluated with and without S9. Revertant frequencies for all doses of test substance, in all tester strains with and without S9, again approximated or were less than control values. All positive and vehicle control values were within acceptable ranges. The positive control response of TA98 with S9 in initial mutagenicity assay was above 2009 historical positive control range. Since the high response confirmed the sensitivity of the test system in this study, the response above 2009 historical positive control range was accepted and will be used for 2010 historical positive control. There was, therefore, no impact on the study. All criteria for a valid study were met.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
The substance was not mutagenic with or without metabolic activation under the conditions of this bacterial reverse mutation assay.
Executive summary:

The test substance was investigated for the ability to induce reverse mutations at the histidine locus in several strains of Salmonella (TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of E. coli strain WP2uvrA according to OECD Guideline 471 and OPPTS 870.5100, in compliance with GLP. The assay was performed in two phases, using the plate incorporation method. The first phase, the initial mutagenicity assay was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test substance. The studies were performed in the presence and absence of Aroclor-induced rat liver S9. In the confirmatory mutagenicity assay, normal growth was observed in all five tester strains at all doses evaluated with and without S9. In addition, the test substance was incompletely soluble at the highest three or four doses evaluated with and without S9. Revertant frequencies for all doses of test substance, in all tester strains with and without S9, approximated or were less than control values. All positive and vehicle control values were within acceptable ranges, and all criteria for a valid study were met. The substance was not mutagenic with or without metabolic activation under the conditions of this bacterial reverse mutation assay (Xu Y, 2011).