Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The three in vitro recommended tests were performed on 1,10-decanediol diacrylate. The Ames test, HPRT test and in vitro micronucleus test showed negative results in presence and in absence of metabolic activation.

Bacterial reverse mutation assay (Brient 2013):

The objective of this study was to evaluate the potential of 1,10-decanediol diacrylate to induce reverse mutations inSalmonella typhimurium.

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

The test item was dissolved in dimethylsulfoxide (DMSO).

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.

Under the experimental conditions of this study, 1,10-decanediol diacrylate did not show any mutagenic activity in the bacterial reverse mutation test withSalmonella typhimuriumeither in the presence or in the absence of a rat liver metabolizing system.

In vitro mammalian cell micronucleus test (Brient 2013):

The objective of this study was to evaluate the potential of 1,10-decanediol diacrylate to induce an increase in the frequency of micronucleated cellsin the mouse cell line L5178YTK+/-. This study conducted in compliance with OECD Guideline No. 487 and the principles of Good Laboratory Practices.

After a preliminary toxicity test, the test item was tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254, as follows:

-First experiment: 3 h treatment + 24 h recovery (without and with S9 mix),

-Second experiment :24 h treatment + 20 h recovery (without S9 mix), and3 h treatment + 24 h recovery (with S9 mix).

Each treatment was coupled to an assessment of cytotoxicity at the same dose-levels. Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells and quality of the cells on the slides has also been taken into account.

For each main experiment (with or without S9 mix), micronuclei were analyzed for three dose-levels of the test item, for the vehicle and the positive controls, in 1000 mononucleated cells per culture (total of 2000 mononucleated cells per concentration). 

The test item was dissolved in dimethylsulfoxide (DMSO).

 Experiments without S9 mix: Following the first experiment,a severe toxicity was induced at the highest tested dose-level of 20 µg/mL, as shown by a 100% decrease in the PD. The immediately lower dose-level of 10 µg/mL induced a slight but acceptable toxicity, as shown by a 37% decrease in the PD. Following the second experiment,a severe toxicity was induced at the highest tested dose-level of 40 µg/mL, as shown by a 100% decrease in the PD. The immediately lower dose-level of 20 µg/mL induced no toxicity, as shown by no noteworthy decrease in the PD.

In the first experiment, a statistically significant increase in the frequency of micronucleated cells was noted at the highest dose-level of 10 µg/mL. However, no dose-response relationship was noted, and only one replicate of the two cultures used for this dose-level showed a frequency of micronucleated cells above the corresponding vehicle control historical data range. These results were thus considered to be equivocal, and the second experiment without S9 mix was performed following a long treatment period. During the second experiment, no statistically significant increase in the frequency of micronucleated cells was noted. Consequently, the increase observed during the first experiment was not reproduced, and was thus not considered to be biologically relevant.

Experiments with S9 mix: Following the first experiment,a marked toxicity was induced at the highest tested dose-level of 80 µg/mL, as shown by a 77% decrease in the PD. The immediately lower dose-level of 40 µg/mL induced a slight but acceptable toxicity, as shown by a 38% decrease in the PD. Following the second experiment, a slight toxicity was induced at the highest tested dose-level of 80 µg/mL, as shown by a 28% decrease in the PD.

In the first experiment, a dose-response relationship was noted, but no statistically significant increase in the frequency of micronucleated cells was observed. In the second experiment performed in the same experimental conditions, some increases in the frequency of micronucleated cells were noted at both higher doses (40 and 80 µg/mL). However, these increases were not statistically significant, and the corresponding frequencies of micronucleated cells remained within the historical data of the vehicle control. Consequently, these increases did not meet the criteria for a positive response and were thus considered as non-biologically relevant.

 

Under the experimental conditions of the study, the test item did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, in the absence or in the presence of a rat metabolising system.

 

In vitro mammalian cell gene mutation assay

1,10-decanediol diacrylate was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by two independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9). The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO).

A 3 hour treatment incubation period was used for all experiments.

In Experiment 1, thirteen concentrations, ranging from 2.5 to 150 µg/mL in the absence of S-9 and from 25 to 400 µg/mL in the presence of S-9, were tested. Seven days after treatment, the highest concentrations analysed to determine viability and 6TG resistance were 40mg/µL in the absence of S-9 and 330 µg/mL in the presence of S-9, limited by toxicity, which gave 17% and 12% RS, respectively.

In Experiment 2 twelve concentrations, ranging from 5 to 80 µg/mL in the absence of S-9 and from 50 to 400 µg/mL in the presence of S-9, were tested.The highest concentrations analysed to determine viability and 6TG resistance were 30 µg/mL in the absence of S-9 and 270 µg/mL in the presence of S-9, which gave 14% and 12% RS, respectively.

In Experiments 1 and 2 no statistically significant increases in MF were observed following treatment with1,10-decanediol diacrylate at any concentration tested in the absence and presence of S-9 and there were no significant linear trends.

It is concluded that 1,10-decanediol diacrylate did not induce mutation at thehprtlocus of L5178Y mouse lymphoma cells when tested up to toxic concentrations in two independent experiments, in the absence and presence of a rat liver metabolising system (S-9)



Link to relevant study records

Referenceopen allclose all

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
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
Target gene:
Histidine operon
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.
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
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.
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.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 April 2013 - 18 June 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD Guideline No. 487 (In vitro mammalian cell micronucleus test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
Not applicable (not a gene mutation assay).
Species / strain / cell type:
other: L5178Y TK+/- mouse lymphoma cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium containing 10% (v/v) heat-inactivated horse serum, L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium pyruvate (200 µg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
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: the vehicle was dimethylsulfoxide (DMSO), batch No. K42474850 145.
- Justification for choice according to solubility assays performed at CiToxLAB France: highest recommended dose-level of 5000 µg/mL was achievable using a test item solution at 500 mg/mL under a treatment volume of 1% (v/v) in the culture medium.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: mitomycin C, colchicine (without S9 mix); cyclophosphamide (with S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
-First experiment: 3 h treatment + 24 h recovery (without and with S9 mix),
-Second experiment : 24 h treatment + 20 h recovery (without S9 mix), and 3 h treatment + 24 h recovery (with S9 mix).

NUMBER OF CELLS EVALUATED: 2000 mononucleated cells/dose

DETERMINATION OF CYTOTOXICITY
- Method: population doubling
Evaluation criteria:
A test item was considered to have clastogenic and/or aneugenic potential, if all the following criteria were met:
- a dose-related increase in the frequency of micronucleated cells was observed,
- for at least one dose-level, the frequency of micronucleated cells of each replicate culture was above the corresponding vehicle historical range,
- a statistically significant difference in comparison to the corresponding vehicle control was obtained at one or more dose-levels.

The biological relevance of the results was considered first.
If the criteria of a positive response are only partially met, results will be evaluated on a case by case basis, taking into account other parameters such as reproducibility between experiments. If results remain inconclusive, or when the highest analyzable dose-level does not exhibit about 55% toxicity (in case of toxic items), additional confirmatory experiments may be needed.
Statistics:
no
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Experiments without S9 mix
Cytotoxicity
Following the first experiment,a severe toxicity was induced at the highest tested dose-level of 20 µg/mL, as shown by a 100% decrease in the PD. The immediately lower dose-level of 10 µg/mL induced a slight but acceptable toxicity, as shown by a 37% decrease in the PD.
Following the second experiment,a severe toxicity was induced at the highest tested dose-level of 40 µg/mL, as shown by a 100% decrease in the PD. The immediately lower dose-level of 20 µg/mL induced no toxicity, as shown by no noteworthy decrease in the PD.
 
Micronucleus analysis
The dose-levels selected for micronucleus analysis were as follows:
. 2.5, 5 and 10 µg/mL for the 3-hour treatment, the higher being too cytotoxic,
. 5, 10 and 20 µg/mL for the 24-hour treatment, the higher being too cytotoxic.
 
In the first experiment, a statistically significant increase in the frequency of micronucleated cells was noted at the dose-level of 10 µg/mL. However, no dose-response relationship was noted, and only one replicate of the two cultures used for this dose-level showed a frequency of micronucleated cells above the corresponding vehicle control historical data range. These results were thus considered to be equivocal, and the second experiment without S9 mix was performed following a long treatment period. During the second experiment, no statistically significant increase in the frequency of micronucleated cells was noted. Consequently, the increase observed during the first experiment was not reproduced, and was thus not considered to be biologically relevant.

Experiments with S9 mix
Cytotoxicity
Following the first experiment,a marked toxicity was induced at the highest tested dose-level of 80 µg/mL, as shown by a 77% decrease in the PD. The immediately lower dose-level of 40 µg/mL induced a slight but acceptable toxicity, as shown by a 38% decrease in the PD.
Following the second experiment, a slight toxicity was induced at the highest tested dose-level of 80 µg/mL, as shown by a 28% decrease in the PD.
 
Micronucleus analysis
The dose-levels selected for micronucleus analysis were as follows:
. 10, 20 and 40 µg/mL for the first experiment, the higher being too cytotoxic,
. 20, 40 and 80 µg/mL for the second experiment, the latter showing a precipitate at the end of the treatment period.
 
In the first experiment, a dose-response relationship was noted, but no statistically significant increase in the frequency of micronucleated cells was observed. In the second experiment performed in the same experimental conditions, some increases in the frequency of micronucleated cells were noted at 40 and 80 µg/mL. However, these increases were not statistically significant, and the corresponding frequencies of micronucleated cells remained within the historical data of the vehicle control. Consequently, these increases did not meet the criteria for a positive response and were thus considered as non-biologically relevant.
Conclusions:
Under the experimental conditions of the study, 1,10-decanediol diacrylate did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, in the absence or in the presence of a rat metabolising system.

Executive summary:

The objective of this study was to evaluate the potential of 1,10-decanediol diacrylate to induce an increase in the frequency of micronucleated cellsin the mouse cell line L5178YTK+/-. This study conducted in compliance with OECD Guideline No. 487 and the principles of Good Laboratory Practices.

 

Methods

After a preliminary toxicity test, the test item was tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254, as follows:

-First experiment: 3 h treatment + 24 h recovery (without and with S9 mix),

-Second experiment : 24 h treatment + 20 h recovery (without S9 mix), and 3 h treatment + 24 h recovery (with S9 mix).

Each treatment was coupled to an assessment of cytotoxicity at the same dose-levels. Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells and quality of the cells on the slides has also been taken into account.

For each main experiment (with or without S9 mix), micronuclei were analyzed for three dose-levels of the test item, for the vehicle and the positive controls, in 1000 mononucleated cells per culture (total of 2000 mononucleated cells per concentration).

 

The test item was dissolved in dimethylsulfoxide (DMSO).

 

Results

 

The mean PD and mean frequencies of micronucleated cells for the vehicle control cultures were as specified in the acceptance criteria. Positive control cultures showed clear statistically significant increases in the frequency of micronucleated cells. The study was therefore considered to be valid.

 

Since the test item was found to be cytotoxic and 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 precipitate/emulsion and/or cytotoxicity, according to the criteria specified in the international guidelines.

 

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.

 

A precipitate was observed at the end of the treatments performed with S9 mix at the highest tested dose-level of 80 µg/mL.

Experiments without S9 mix

Cytotoxicity

Following the first experiment,a severe toxicity was induced at the highest tested dose-level of 20 µg/mL, as shown by a 100% decrease in the PD. The immediately lower dose-level of 10 µg/mL induced a slight but acceptable toxicity, as shown by a 37% decrease in the PD.

Following the second experiment,a severe toxicity was induced at the highest tested dose-level of 40 µg/mL, as shown by a 100% decrease in the PD. The immediately lower dose-level of 20 µg/mL induced no toxicity, as shown by no noteworthy decrease in the PD.

 

Micronucleus analysis

The dose-levels selected for micronucleus analysis were as follows:

. 2.5, 5 and 10 µg/mL for the 3-hour treatment, the higher being too cytotoxic,

. 5, 10 and 20 µg/mL for the 24-hour treatment, the higher being too cytotoxic.

 

In the first experiment, a statistically significant increase in the frequency of micronucleated cells was noted at the dose-level of 10 µg/mL. However, no dose-response relationship was noted, and only one replicate of the two cultures used for this dose-level showed a frequency of micronucleated cells above the corresponding vehicle control historical data range. These results were thus considered to be equivocal, and the second experiment without S9 mix was performed following a long treatment period. During the second experiment, no statistically significant increase in the frequency of micronucleated cells was noted. Consequently, the increase observed during the first experiment was not reproduced, and was thus not considered to be biologically relevant.

Experiments with S9 mix

Cytotoxicity

Following the first experiment,a marked toxicity was induced at the highest tested dose-level of 80 µg/mL, as shown by a 77% decrease in the PD. The immediately lower dose-level of 40 µg/mL induced a slight but acceptable toxicity, as shown by a 38% decrease in the PD.

Following the second experiment, a slight toxicity was induced at the highest tested dose-level of 80 µg/mL, as shown by a 28% decrease in the PD.

 

Micronucleus analysis

The dose-levels selected for micronucleus analysis were as follows:

. 10, 20 and 40 µg/mL for the first experiment, the higher being too cytotoxic,

. 20, 40 and 80 µg/mL for the second experiment, the latter showing a precipitate at the end of the treatment period.

 

In the first experiment, a dose-response relationship was noted, but no statistically significant increase in the frequency of micronucleated cells was observed. In the second experiment performed in the same experimental conditions, some increases in the frequency of micronucleated cells were noted at 40 and 80 µg/mL. However, these increases were not statistically significant, and the corresponding frequencies of micronucleated cells remained within the historical data of the vehicle control. Consequently, these increases did not meet the criteria for a positive response and were thus considered as non-biologically relevant.

 

Conclusion

Under the experimental conditions of the study, the test item did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, in the absence or in the presence of a rat metabolising system.

 

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 April 2013 to 20 August 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT gene
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
The master stock of L5178Y tk+/- (3.7.2°C) mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co. Cells supplied to Covance Laboratories Ltd. were stored as frozen stocks in liquid nitrogen. Each batch of frozen cells was purged of mutants and confirmed to be mycoplasma free. For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated in a humidified atmosphere of 5 ± 1% v/v CO2 in air. When the cells were growing well, subcultures were established in an appropriate number of flasks.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation were from male Sprague Dawley rats induced with Aroclor 1254
Test concentrations with justification for top dose:
In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 88.13 to 2820 µg/mL (equivalent to 10 mM at the highest concentration tested).

In Experiment 1 thirteen concentrations, ranging from 2.5 to 150 µg/mL in the absence of S-9 and from 25 to 400 µg/mL in the presence of S-9, were tested.
In Experiment 2 twelve concentrations, ranging from 5 to 80 µg/mL in the absence of S-9 and from 50 to 400 µg/mL in the presence of S-9, were tested.

Positive controls
4-nitroquinoline 1-oxide (NQO), stock solution: 0.015 and 0.020 mg/mL and final concentration: 0.15 and 0.20 µg/mL, no metabolic activation
Benzo[a]pyrene (B[a]P), stock solution: 0.200 and 0.300 mg/mL and final concentration: 2.00 and 3.00 µg/mL with metabolic activation
Vehicle / solvent:
DMSO diluted 100 fold in the treatment medium
Untreated negative controls:
yes
Remarks:
DMSO diluted 100 fold in the treatment medium
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
benzo(a)pyrene
Remarks:
For positive control concentrations see test concentration section
Details on test system and experimental conditions:
DURATION
- Preincubation period: Not applicable
- Exposure duration: 3-hour exposure followed by 7-day incubation period
Evaluation criteria:
For valid data, the test article was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
1. The mutant frequency at one or more concentrations was significantly greater than that of the negative control (p < 0.05).
2. There was a significant concentration relationship as indicated by the linear trend analysis (p < 0.05).
3. The effects described above were reproducible.
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.
Statistics:
Not applicable
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 88.13 to 2820 µg/mL (equivalent to 10 mM at the highest concentration tested). The highest concentration to provide >10% relative survival (RS) in the presence of S-9 was 176.3 µg/mL, which gave 96% RS. Extreme or complete toxicity was observed at the remaining higher concentrations analysed in the presence of S-9 (352.5 to 705 µg/mL). In the absence of S-9, complete toxicity (0% RS) was observed at all concentrations analysed (88.13 to 705 µg/mL).
Following consultation with the Study Monitor, an extended range of closely spaced concentrations was tested in the absence of S-9 in Experiment 1.
In Experiment 1, thirteen concentrations, ranging from 2.5 to 150 µg/mL in the absence of S-9 and from 25 to 400 µg/mL in the presence of S-9, were tested. Seven days after treatment, the highest concentrations analysed to determine viability and 6TG resistance were 40 µg/mL in the absence of S-9 and 330 µg/mL in the presence of S-9, limited by toxicity, which gave 17% and 12% RS, respectively.
In Experiment 2, twelve concentrations, ranging from 5 to 80 µg/mL in the absence of S-9 and from 50 to 400 µg/mL in the presence of S-9, were tested. The highest concentrations analysed to determine viability and 6TG resistance were 30 µg/mL in the absence of S-9 and 270 µg/mL in the presence of S-9, which gave 14% and 12% RS, respectively.
In both Experiments 1 and 2, no statistically significant increases in mutant frequency were observed following treatment with 1,10-decanediol diacrylate at any concentration tested, in the absence or presence of S-9, and there were no significant linear trends. Mutant frequencies in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals. The study was therefore accepted as valid.

Table 1: %RS Values –Range-Finder Experiment

Treatment

(µg/mL)

-S-9

% RS

+S-9

% RS

0

100

100

88.13

0

122

176.3

0

96

352.5 P

0

3

705.0 P, PP

0

0

1410 P, PP

NP

NP

2820 P, PP

NP

NP

P Precipitation observed at time of treatment

PP Precipitation observed following treatment incubation period

NP Not plated

 

Table 2: Summary of Mutation Data

Experiment 1 (3-hour treatment in the absence and presence of S-9)

Treatment

(mg/mL)

-S-9

Treatment

(mg/mL)

+S-9

%RS

MF§

%RS

MF§

0

100

5.38

0

100

2.79

2.5

103

1.29

NS

50

101

2.39

NS

5

101

2.34

NS

100

77

1.71

NS

10

73

3.45

NS

150

$$, P

51

(4.76)

20

59

2.46

NS

200

P

39

2.45

NS

30

31

2.91

NS

230

P

29

4.18

NS

40

17

4.28

NS

250

P

22

4.00

NS

270

$$, P

18

(6.36)

300

P

10

2.18

NS

330

P

12

1.32

NS

Linear trend

NS

Linear trend

NS

NQO

B[a]P

0.15

63

31.36

2

79

21.52

0.2

43

47.63

3

57

56.34

 

Experiment 2 (3-hour treatment in the absence and presence of S-9)

Treatment

(µg/mL)

-S-9

Treatment

(µg/mL)

+S-9

%RS

MF§

%RS

MF§

0

100

3.31

0

100

2.06

5

78

3.20

NS

50

92

5.28

NS

7.5

76

1.52

NS

75

84

1.55

NS

10

64

1.86

NS

100

65

2.83

NS

20

31

1.88

NS

150

43

2.67

NS

25

18

2.15

NS

200

23

3.36

NS

30

14

3.01

NS

250

P

14

3.23

NS

270

P

12

2.00

NS

Linear trend

NS

Linear trend

NS

NQO

B[a]P

0.15

71

54.02

2

58

23.88

0.2

61

47.44

3

22

53.43

§ 6-TG resistant mutants/106viable cells 7 days after treatment

%RS Percent relative survival adjusted by post treatment cell counts

$$ Treatment excluded from analysis due to excessive heterogeneity for mutation

Data in parentheses indicates marked heterogeneity observed

P Precipitation noted at time of treatment only

NS Not significant

 

Conclusions:
It is concluded that 1,10-decanediol diacrylate did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to toxic concentrations in two independent experiments, in the absence and presence of a rat liver metabolising system (S-9).
Executive summary:

1,10-decanediol diacrylate was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by two independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9). The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO).

A 3 hour treatment incubation period was used for all experiments.

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9, ranging from 88.13 to 2820 µg/mL (equivalent to 10 mM at the highest concentration tested). The highest concentration to provide >10% relative survival (RS) in the presence of S-9 was 176.3 µg/mL, which gave 96% RS. Extreme or complete toxicity was observed at the remaining higher concentrations analysed in the presence of S-9 (352.5 to 705 µg/mL). In the absence of S-9 complete toxicity (0% RS) was observed at all concentrations analysed (88.13 to 705 µg/mL). Following consultation with the Study Monitor, an extended range of closely spaced concentrations was tested in the absence of S-9 in Experiment 1.

In Experiment 1 thirteen concentrations, ranging from 2.5 to 150 µg/mL in the absence of S-9 and from 25 to 400 µg/mL in the presence of S-9, were tested. Seven days after treatment, the highest concentrations analysed to determine viability and 6TG resistance were 40mg/µL in the absence of S-9 and 330 µg/mL in the presence of S-9, limited by toxicity, which gave 17% and 12% RS, respectively.

In Experiment 2 twelve concentrations, ranging from 5 to 80 µg/mL in the absence of S-9 and from 50 to 400 µg/mL in the presence of S-9, were tested.The highest concentrations analysed to determine viability and 6TG resistance were 30 µg/mL in the absence of S-9 and 270 µg/mL in the presence of S-9, which gave 14% and 12% RS, respectively.

Negative (vehicle) and positive control treatments were included in each Mutation Experiment in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (NQO) (without S-9) and benzo(a)pyrene (B[a]P) (with S-9). Therefore the study was accepted as valid.

In Experiments 1 and 2 no statistically significant increases in MF were observed following treatment with1,10-decanediol diacrylate at any concentration tested in the absence and presence of S-9 and there were no significant linear trends.

It is concluded that 1,10-decanediol diacrylate did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to toxic concentrations in two independent experiments, in the absence and presence of a rat liver metabolising system (S-9).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on the negative results in all three regulatory in vitro genotoxicity tests, no classification for 1,10 -decanediol diacrylate is required for genotoxicity according to the Regulation EC n°1272/2008.