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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
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)
Deviations:
no
Remarks:
Conducted according to guideline in effect at time of study conduct
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Remarks:
Conducted according to guideline in effect at time of study conduct
Qualifier:
according to
Guideline:
other: EC Methods for the Determination of Toxicity and Other Health Effects Method B.13/14 Directive 440/2008/EC
Deviations:
no
Remarks:
Conducted according to guideline in effect at time of study conduct
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: 22.82% solids in water

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:
33.3, 66.7, 100, 333, 667, 1000, 3333, and 5000 μg/plate
A correction factor based on the percent solids was used for preparation of the dosing concentrations.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Sterile water.
- Justification for choice of solvent/vehicle: Based on the solubility of the test substance and compatibility with the target cells.
Due to evidence of bacterial contamination of the test substance in the first toxicity-mutation test a 100 mg/mL stock solution was prepared in ethanol (ETOH) and allowed to age for approximately 30 minutes prior to formulation with sterile water. This method formulated a top dose of 50 mg/mL that contained approximately 30% ETOH and formed a clear pale amber solution. Each lower dose level contained proportionally decreasing amounts of ETOH. The negative control used on this study was a 30% ETOH solution prepared in sterile water to approximately match the amount of ETOH in the top dose level.
Controls
Untreated negative controls:
yes
Remarks:
30% ethanol in sterile water
Negative solvent / vehicle controls:
yes
Remarks:
30% ethanol in sterile water
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (TA1535, TA1537, TA100, WP2 uvrA +S9), benzo[a]pyrene (TA98 +S9), 2-nitrofluorene (TA98 -S9), sodium azide (TA100 and TA1535 -S9), Acridine mutagen ICR-191 (TA1537 –S9), 4-nitroquinoline-N-oxide (WP2 urvA -S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: The plate incorporation method was applied. Treatment without activation was conducted by adding 100 μL of an overnight culture containing approximately 10E9 bacteria per millilitre to top agar supplemented with L-histidine and D-biotin or L-tryptophan. The components were mixed and poured onto a plate containing minimal agar. Treatments with activation were conducted as those without activation except that S9 mix was added to the bacteria/top agar mixture before it was poured onto a minimal glucose agar plate. The plates were incubated at approximately 37±2°C for approximately 51 to 69 hours.

DURATION
- Exposure duration: 51 to 69 hours

NUMBER OF REPLICATIONS: The mutagenicity test was conducted 2 times. In the initial trial (E-1) the negative control values for WP2uvrA in the presence of S9 activation were outside the acceptable range. This condition was retested in the second trial (E-2). The data collected for WP2uvrA in the presence of activation in trial E-1 is considered invalid and is not included in the study report.
Evaluation criteria:
For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate unless observed at the top dose level only. Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the highest numerical dose response was ≥ 3.0-fold the mean concurrent negative control value (vehicle control). Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the highest numerical dose response was ≥ 2.0-fold the mean concurrent negative control value (vehicle control).
Statistics:
For each selected tester strain, the mean number of revertants and the standard deviation at each concentration with and without S9 activation were calculated.

Results and discussion

Test resultsopen allclose all
Species / strain:
other: TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative 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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In the toxicity-mutagenicity test, test substance precipitation was observed starting at 3333 μg/plate for all test conditions with the exception of TA1537 in the absence of S9 activation where precipitation was observed starting at 1000 μg/plate and for WP2uvrA in the presence of S9 activation where precipitation was observed starting at 667 μg/plate. Two types of precipitation were observed in the mutagenicity test: a microscopic precipitation that was consistent with the precipitation observed in the toxicity-mutation trial, and a precipitation that appeared as a subtle change in the appearance of the top agar that was visible to the naked eye. The microscopic precipitation was observed for all conditions in the top two dose levels. Test substance precipitation was observed as a change in the appearance of the top agar in the absence of S9 activation starting at1000 μg/plate for TA1357, and 667 μg/plate for TA98, TA100, TA1535, and WP2uvrA; and in the presence of S9 activation starting at 667 μg/plate for WP2uvrA, and 333 μg/plate for TA98, TA100, TA1535, and TA1537.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative in the absence and presence of S9 activation

The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
Negative when tested in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and in Escherichia coli WP2 uvr A in the absence and presence of S9 activation.
Executive summary:

The test substance was evaluated for mutagenicity in the Bacterial Reverse Mutation Test using the plate incorporation method. Salmonella typhimuriumstrains TA98, TA100, TA1535, and TA1537 andEscherichia colistrain WP2 uvr A were tested in the absence and presence of an exogenous metabolic activation system (Aroclor-induced rat liver S9). The test was performed in 2 phases. The first phase was the toxicity-mutation test, which established the dose range for the mutagenicity test, and provided a preliminary mutagenicity evaluation. The second phase was the mutagenicity test, which evaluated and confirmed the mutagenic potential of the test substance. Based on the toxicity-mutation test, the maximum dose evaluated in the mutagenicity test was 5000 μg/plate. The mutagenicity test was conducted in two trials. WP2uvrA was retested based on invalid negative control values in the first trial. In the initial toxicity-mutation test contaminating colonies were observed on the sterility plates for the most concentrated test substance dilution (50 mg/mL). The test substance was decontaminated with ETOH prior to use on any of the subsequent tests and no other contaminating colonies were observed.

No positive mutagenic responses were observed at any dose level or with any tester strain in either the absence or presence of S9 metabolic activation. No toxicity was observed at any dose level with any tester strain in either the absence or presence of S9. Two types of precipitation were observed: a microscopic precipitation that was consistent with precipitation observed in the toxicity-mutation trial, and a precipitation that appeared as a subtle change in the appearance of the top agar that was visible to the naked eye. The microscopic precipitation was observed for all conditions in the top two dose levels. Test substance precipitation was observed as a change in the appearance of the top agar in the absence of S9 activation starting at 1000 μg/plate for TA1357, and 667 μg/plate for TA98, TA100, TA1535, and WP2uvrA; and in the presence of S9 activation starting at 667 μg/plate for WP2uvrA, and 333 μg/plate for TA98, TA100, TA1535, and TA1537. All criteria for a valid study were met. Under the conditions of this study, the test substance showed no evidence of mutagenicity in the Bacterial Reverse Mutation Test either in the absence or presence of Aroclor-induced rat liver S9. It was concluded that the test substance was negative in thisin vitrotest.