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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:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
yes
Remarks:
In this study, the S9-fraction was obtained from Trinova Biochem; The temperature was recorded to be outside the range of 37.0 ± 1.0°C as specified in the protocol for approximately 3 hours in the dose range finding test assay (with a maximum of 39.9°C)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: F-12676, PDC lot 20
- Expiration date of the lot/batch: 03 September 2013 (allocated by WIL Research Europe B.V., 1 year after receipt of the test substance)
- Purity test date: 99.96%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature in the dark
- Solubility and stability of the test substance in the solvent/vehicle: DMSO: soluble, but not quantified. Stability >96 hours

FORM AS APPLIED IN THE TEST (if different from that of starting material):
No correction was made for the purity/composition of the test compound. The test substance was dissolved in DMSO

Method

Target gene:
Histidine operon (S. typhimurium) and tryptophan (E. coli)
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:
Rat liver microsomal enzymes (S9 homogenate); from phenobarbital and ß-naphthoflavone dosed rats
Test concentrations with justification for top dose:
Experiment 1 (dose-finding study): 3, 10, 33, 100, 333, 1000, 3330, and 5000 ug/plate (with and without metabolic activation; 5% v/v)
Experiment 2 (mutation assay): 100, 333, 1000, 3330, and 5000 ug/plate (with and without metabolic activation; 5% v/v) (strains TA1535, TA1537, TA98)
Repeat Experiment 2 (mutation assay): 100, 333, 1000, 3330, and 5000 ug/plate (with and without metabolic activation; 10% v/v) (All strains)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Based on protocol.
Controlsopen allclose all
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
other: ICR-191
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): 10^9 cells/mL

DURATION
- Exposure duration: 48 ± 4 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: To determine the toxicity of the test article, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.
Rationale for test conditions:
Test conditions were based on the protocols in OECD Guideline 471 (Bacterial Reverse Mutation Assay) and EU Method B. 13/14 (Mutagenicity-Reverse Mutation Test Using Bacteria).
Evaluation criteria:
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The negative control data (number of spontaneous revertants per plate) should be within the laboratory historical range for each tester strain.
b) The positive control chemicals should produce responses in all tester strains, which are within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at least three times the concurrent vehicle control group mean.
c) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
Statistics:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent vehicle control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
other: E. coli WP2uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
other: TA98, TA100, TA1535, TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Dose-range test: a slight reduction of the bacterial background lawn was observed at 5000 ug/plate and slight to extreme reductions in the number of revertant colonies were observed at 3330 and 5000 ug/plate with and without S9 in tester strain TA100
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
The test article was tested in the tester strains TA100 and WP2uvrA with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 μg/plate in the absence and presence of S9-mix. Precipitation of the test article on the plates was not observed at the start or at the end of the incubation period in both tester strains. To determine the toxicity of the test article, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined. In tester strain WP2uvrA, no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed. In tester strain TA100 a slight reduction of the bacterial background lawn was observed at the highest concentration tested and slight to extreme reductions in the number of revertant colonies were observed at the concentrations of 3330 and 5000 μg/plate in the absence and presence of S9-mix. In the dose range finding test, no increase in the number of revertants was observed upon treatment with the test article under all conditions tested.

Applicant's summary and conclusion

Conclusions:
Based on the results of this study it is concluded that the test article is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with and without metabolic activation (S9-mix).  
Executive summary:

The mutagenic potential of the test article was evaluated in the Bacterial Reverse Mutation Assay with S. typhimurium strains TA98, TA100, TA1535, and TA1537 and E. coli strain WP2 uvrA in the presence and absence of a metabolic activation system (S9-mix rat liver induced by Phenobarbital and B-napthoflavone). The study was performed in compliance with OECD GLP (1997). The test method was based on OECD No. 471 (1997) and EC No. 440/2008 Part B (2008). The test article was dissolved in dimethyl sulfoxide. A dose range-finding test was performed with concentration up to 5000 ug/plate in the absence and presence of metabolic activation in strains TA100 and WP2uvrA. Based on the results of the dose range finding test, the test article was dosed at concentrations of 100, 333, 1000, 3330, and 5000 ug/plate in the TA1535, TA1537 and TA98 strains in the presence and absence of 5% S9 mix. A second assay was performed in all strains with the same doses in the presence and absence of 10% S9 mix. Strain specific positive controls and negative (vehicle) controls were tested in parallel. All treatments were performed in triplicate. Top agar was melted and the following solutions were added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (10^9 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test material in DMSO and either 0.5 mL S9-mix (in case of activated assays) or 0.5 mL of 0.1 M phosphate buffer (in case of non-activated assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification, plates were inverted and incubated in the dark at 37 ± 1.0°C for 48 ± 4 hours. After this period, revertant colonies (His+) for S. typhimurium and (Trp+) for E. coli were counted. In tester strain TA100 a slight reduction of the bacterial background lawn was observed at the highest concentration tested and slight to extreme reductions in the number of revertant colonies were observed at the concentrations of 3330 and 5000 μg/plate in the absence and presence of S9-mix indicating cytotoxicity. The test article did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. All criteria for a valid test were met as described in the protocol. Based on the results of this study it is concluded that the test article is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with and without metabolic activation (S9-mix).