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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:
04 April 2013 -- 14 May 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: colorless viscous liquid

Method

Target gene:
Histidine operon
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
20.6, 61.7, 185.2, 555.6, 1666.7 and 5000 µg/plate for the five strains, in both experiments with and without S9 mix.
Vehicle / solvent:
- Vehicle used: dimethylsulfoxide (DMSO), batch K48474850 145
- Justification for choice: according to solubility assays performed at CiToxLAB France, the highest dose-level of 5000 µg/plate was achievable using a test item solution at 100 mg/mL under the treatment volume of 50 µL/plate
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide, 9-aminoacridine, 2-nitrofluorene, mitomycin C (-S9 mix); 2-anthramine, benzo(a)pyrene (+S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 to 72 hours

DETERMINATION OF CYTOTOXICITY
- Method: decrease in number of revertant colonies and/or thinning of the bacterial lawn
Evaluation criteria:
A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account.
Statistics:
no

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:
cytotoxicity
Remarks:
at 5000 µg/plate in the TA 1537 strain only
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
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:
Experiments without S9 mix
In both experiments, a moderate to strong precipitate and/or emulsion was observed in the Petri plates when scoring the revertants at dose-levels = 1666.7 µg/plate in the five tested strains. Except for a slight decrease in the number of revertants noted at 5000 µg/plate in the TA 1537 strain in the first experiment, no noteworthy toxicity was noted at any tested dose-levels, towards the five strains used. Slight increases in the number of revertants were noted in the first experiment at 555.6 µg/plate in the TA 98 strain, and at 61.7 and 185.2 µg/plate in the TA 100 strain (1.7-fold the vehicle control value). These increases did not exceed the positive threshold (2-fold the vehicle control value) and were observed without dose-response relationship. Furthermore, no similar effect was noted in the second experiment performed in the same experimental conditions. Consequently, these increases were not considered to be biologically relevant. Slight increases in the number of revertants were noted in the second experiment at 20.6 and 61.7 µg/plate in the TA 1535 strain, and at 1666.7 µg/plate in the TA 1537 strain (up to 2.6-fold the vehicle control value). These increases did not exceed the positive threshold (3-fold the vehicle control value) and were observed without dose-response relationship. Furthermore, no similar effect was noted in the first experiment performed in the same experimental conditions. Consequently, these increases were not considered to be biologically relevant.

Experiments with S9 mix
No precipitate was observed in the Petri plates when scoring the revertants at any dose-levels.
In both experiments, a moderate to strong emulsion was observed in the Petri plates when scoring the revertants at dose-levels = 1666.7 µg/plate in the five tested strains. Except for a moderate toxicity (thinning of the bacterial lawn) noted at 5000 µg/plate in the TA 1537 strain in both experiments and in the TA 102 strain in the second experiment only, no noteworthy toxicity was noted at any tested dose-levels towards the four other tested strains. A slight increase in the number of revertants was noted in the first experiment (direct plate incorporation method) at 5000 µg/plate in the TA 98 strain (1.6-fold the vehicle control value) and at 61.7 µg/plate in the TA 100 strain (1.7-fold the vehicle control value). These increases did not exceed the positive threshold (2-fold the vehicle control value), and no dose-response relationship was observed. Furthermore, no
similar effect was noted in the second experiment, performed following the pre-incubation method. Consequently, these increases were not considered to be biologically relevant.

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions of this study, the test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium either in the presence or in the absence of a rat liver metabolizing system.

Executive summary:

The objective of this study was to evaluate the potential of the test item to induce reverse mutations in Salmonella typhimurium.

This study was conducted in compliance with OECD Guideline No. 471, Council Regulation (EC) No. 440/2008 of 30 May 2008, laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), Annex, Part B.13/14 p. 248 and the principles of Good Laboratory Practices.

 

Methods

 

A preliminary toxicity test was performed to define the dose-levels ofthe test itemto be used for the mutagenicity study. The test item was then tested in two independent experiments, both with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254.

 

Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the pre-incubation method (60 minutes, 37°C).

 

Five strains of bacteria Salmonella typhimurium were used: TA 1535, TA 1537, TA 98, TA 100 and TA 102. Each strain was exposed to six dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

 

The test item was dissolved indimethylsulfoxide (DMSO).

 

Results

 

The number of revertants for the vehicle and positive controls met the acceptance criteria. Also, there were six analysable dose-levels for each strain and test condition. The study was therefore considered to be valid.

 

Since the test item was found to be poorly soluble and slightly toxic in the preliminary test, the selection of the highest dose-level to be used in the main experiments was based on the level of precipitate and toxicity, according to the criteria specified in the international guidelines.

 

The treatment-levels were 20.6, 61.7, 185.2, 555.6, 1666.7 and 5000 µg/plate for the five strains, in both experiments with and without S9 mix.

 

Experiments without S9 mix

In both experiments, a moderate to strong precipitate and/or emulsion was observed in the Petri plates when scoring the revertants at dose-levels superior or equal to 1666.7 µg/plate in the five tested strains.

 

Except for a slight decrease in the number of revertants noted at 5000 µg/plate in the TA 1537 strain in the first experiment, no noteworthy toxicity was noted at any tested dose-levels, towards the five strains used.

Slight increases in the number of revertants were noted in the first experiment at 555.6 µg/plate in the TA 98 strain, and at 61.7 and 185.2 µg/plate in the TA 100 strain (1.7-fold the vehicle control value). These increases did not exceed the positive threshold (2-fold the vehicle control value) and were observed without dose-response relationship. Furthermore, no similar effect was noted in the second experiment performed in the same experimental conditions. Consequently, these increases were not considered to be biologically relevant.

 

Slight increases in the number of revertants were noted in the second experiment at 20.6 and 61.7 µg/plate in the TA 1535 strain, and at 1666.7 µg/plate in the TA 1537 strain (up to 2.6-fold the vehicle control value). These increases did not exceed the positive threshold (3-fold the vehicle control value) and were observed without dose-response relationship. Furthermore, no similar effect was noted in the first experiment performed in the same experimental conditions. Consequently, these increases were not considered to be biologically relevant.

 

Experiments with S9 mix

No precipitate was observed in the Petri plates when scoring the revertants at any dose-levels.

 

In both experiments, a moderate to strong emulsion was observed in the Petri plates when scoring the revertants at dose-levels superior or equal to 1666.7 µg/plate in the five tested strains.

Except for amoderate toxicity (thinning of the bacterial lawn) noted at 5000 µg/plate in the TA 1537 strain in both experiments and in the TA 102 strain in the second experiment only, no noteworthy toxicity was noted at any tested dose-levels towards the four other tested strains.

 

A slight increase in the number of revertants was noted in the first experiment (direct plate incorporation method) at 5000 µg/plate in the TA 98 strain (1.6-fold the vehicle control value) and at 61.7 µg/plate in the TA 100 strain (1.7-fold the vehicle control value). These increases did not exceed the positive threshold (2-fold the vehicle control value), and no dose-response relationship was observed. Furthermore, no similar effect was noted in the second experiment, performed following the pre-incubation method. Consequently, these increases were not considered to be biologically relevant.

Conclusion

Under the experimental conditions of this study, the test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium either in the presence or in the absence of a rat liver metabolizing system.