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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:
23 April 2013 - 17 June 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
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
Test material form:
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:
With a treatment volume of 1% (v/v) in culture medium, the dose-levels used for treatments, were as follows:
. 0.16, 0.31, 0.63, 1.25, 2.5, 5, 10 and 20 µg/mL in the first experiment without S9 mix,
. 0.31, 0.63, 1.25, 2.5, 5, 10, 20 and 40 µg/mL in the second experiment without S9 mix,
. 0.63, 1.25, 2.5, 5, 10, 20, 40 and 80 µg/mL in both experiments with S9 mix.
Vehicle / solvent:
- Vehicle used: dimethylsulfoxide (DMSO), batch No. K42474850 145.
- Justification for choice according to solubility assays performed, the highest recommended dose-level of 5000 µg/plate was achievable using a test item solution of 100 mg/mL under a treatment volume of 50 µL/plate.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
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 test item is considered to have shown a mutagneic activity if:
- 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 mean number of revertants compared with the vehicle controls is observed, at any dose-level,
- and/or a reproducible dose-response relationship is evidenced.
In all case, biological relevance (such as reproducibility and reference to historical data) are taken into consideration when evaluating the results.
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:
with S9 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:
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 in the preliminary test, the selection of the highest dose-level to be used in the main experiments was based on the level of emulsion, according to the criteria specified in the international guidelines.

Experiments without S9 mix
A moderate emulsion was observed in the Petri plates in all strains when scoring the revertants at dose-levels superior or equal to 625 µg/plate (first experiment), superior or equal to 1250 µg/plate (second experiment, depending on the strain, and third experiment) and
superior or equal to 2500 µg/plate (second experiment, depending on the strain).
No noteworthy toxicity was noted in the five tested strains, either with or without S9 mix.
In the first experiment, an increase in the number of revertant was observed at 5000 µg/plate in the TA 1537 strain. This increase exceeded the positive threshold of 3-fold the vehicle control value. The corresponding value obtained for the mean number of revertants was above the maximum value observed in historical data, but heterogeneity was noted between the corresponding individual revertant colony counts. Moreover, this effect was not reproduced either in the second or in the third experiments, performed in the same experimental conditions. Consequently, this effect was not considered to be biologically relevant.
In the first experiment, a slight increase in the number of revertants was noted at 5000 µg/plate in the TA 98 strain. This increase did not exceed the positive threshold (2-fold the vehicle control value) and no similar effect was noted in the second experiment. Consequently, this increase did not meet the criteria for a positive response.

Experiments with S9 mix
A moderate emulsion was observed in the Petri plates in all strains when scoring the revertants at dose-levels superior or equal to 1250 µg/plate in the first and second experiments.
Decreases in the number of revertants (cytotoxicity) were noted in the first experiment in the TA 1537 strain at dose-levels superior or equal to 2500 µg/plate.
A moderate toxicity (thinning of the bacterial lawn) was observed at 5000 µg/plate in the TA 98 strain, and at dose-levels superior or equal to 1250 µg/plate in the TA 1535 and TA 1537 strains.
A strong toxicity (decrease in the number of revertants and thinning of the bacterial lawn) was noted in the TA 98 strain at 1250 and 2500 µg/plate.
The test item did not induce any noteworthy increase in the number of revertants, in any of the five tested strains.

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions of this study, 1,10-decanediol diacrylate 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 1,10-decanediol diacrylate to induce reverse mutations in Salmonella typhimurium.

This study was conducted in compliance with OECD Guideline No. 471 and the principles of Good Laboratory Practices.

 

Methods

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

All 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, 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 in dimethylsulfoxide (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 in the preliminary test, the selection of the highest dose-level to be used in the main experiments was based on the level of emulsion, according to the criteria specified in the international guidelines.

In the first experiment, the treatment-levels were 156.3, 312.5, 625, 1250, 2500 and 5000 µg/plate for the five strains, both with and without S9 mix.

In the second experiment, the treatment-levels were:

. 92.59, 277.8, 833.3, 2500, 3750 and 5000 µg/plate for the TA 1535, TA 1537 and TA 98 strains, without S9 mix,

. 156.3, 312.5, 625, 1250, 2500 and 5000 µg/plate for the TA 100 and TA 102 strains without S9 mix, and for the five strains with S9 mix.

In the third experiment, the treatment-levels were 156.3, 312.5, 625, 1250, 2500 and 5000 µg/plate for the TA 1537 strain without S9 mix.

Experiments without S9 mix

A moderate emulsion was observed in the Petri plates in all strains when scoring the revertants at dose-levels superior or equal to 625 µg/plate (first experiment), superior or equal to 1250 µg/plate (second experiment, depending on the strain, and third experiment) and

superior or equal to 2500 µg/plate (second experiment, depending on the strain).

No noteworthy toxicity was noted in the five tested strains, either with or without S9 mix.

In the first experiment, an increase in the number of revertant was observed at 5000 µg/plate in the TA 1537 strain. This increase exceeded the positive threshold of 3-fold the vehicle control value. The corresponding value obtained for the mean number of revertants was above the maximum value observed in historical data, but heterogeneity was noted between the corresponding individual revertant colony counts. Moreover, this effect was not reproduced either in the second or in the third experiments, performed in the same experimental conditions. Consequently, this effect was not considered to be biologically relevant.

In the first experiment, a slight increase in the number of revertants was noted at 5000 µg/plate in the TA 98 strain. This increase did not exceed the positive threshold (2-fold the vehicle control value) and no similar effect was noted in the second experiment. Consequently, this increase did not meet the criteria for a positive response.

Experiments with S9 mix

A moderate emulsion was observed in the Petri plates in all strains when scoring the revertants at dose-levels superior or equal to 1250 µg/plate in the first and second experiments.

Decreases in the number of revertants (cytotoxicity) were noted in the first experiment in the TA 1537 strain at dose-levels superior or equal to 2500 µg/plate.

A moderate toxicity (thinning of the bacterial lawn) was observed at 5000 µg/plate in the TA 98 strain, and at dose-levels superior or equal to 1250 µg/plate in the TA 1535 and TA 1537 strains.

A strong toxicity (decrease in the number of revertants and thinning of the bacterial lawn) was noted in the TA 98 strain at 1250 and 2500 µg/plate.

The test item did not induce any noteworthy increase in the number of revertants, in any of the five tested strains.

 

Conclusion

Under the experimental conditions of this study, 1,10-decanediol diacrylate 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.