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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

1,12 -dodecanediol bismethacrylate is considered to be not mutagenic in the Ames test based on the weight-of-evidence approach.

A bacterial reverse mutation assay was performed on 1,10-decanediol diacrylate, an analogue of 1,12 -dodecanediol bismethacrylate. This Ames test showed negative results in presence and in absence of metabolic activation.

A QSAR (VEGA / CAESAR) predicts also that 1,12 -dodecanediol bismethacrylate has no mutagen activity in the Ames test.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
23 April 2013 - 17 June 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
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 information:
Composition 1
Target gene:
Histidine operon
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Species / strain:
S. typhimurium TA 102
Additional strain 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:
- 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.
Negative controls:
no
Solvent 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 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:
yes
Remarks:
with S9 only
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other:
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other:
Remarks:
Migrated from field 'Test system'.
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 gene mutation study in bacteria
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
accepted calculation method
Justification for type of information:
1. SOFTWARE
VEGA
It is implemented inside the VEGA online platform, accessible at:http://www.vega-qsar.eu/
The model extends the original CAESAR model, freely available at: http://www.caesarproject.eu/software/

2. MODEL (incl. version number)
Mutagenicity model (CAESAR) (version 2.1.12)

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
SMILE used : CC(=C)C(=O)OCCCCCCCCCCCCOC(=O)C(C)=C

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
[Explain how the model fulfils the OECD principles for (Q)SAR model validation. Consider attaching the QMRF or providing a link]
QMRF is attached in this Robust Study Summary.

- Defined endpoint: The model provides a qualitative prediction of mutagenicity on Salmonella typhimurium (Ames test).

- Unambiguous algorithm:
An integrated model was arranged cascading two models: Model A, a trained Support Vector Machine (SVM) classifier, and an additional Model B for false negatives (FNs) removal based on Structural Alerts (SAs) matching. Model B works with two sets of SAs: the first one is related to mutagenicity activity, if no matching is found the second set is checked, and if some matches are found, the prediction is "suspect mutagen". Full reference and details of the used formulas can be found in:
Ferrari T., Gini G. An open source multistep model to predict mutagenicity from statistical analysis and relevant structural alerts. Chemistry Central Journal (2010), 4 (Suppl 1):S2 Structural Alerts have been taken from the Benigni/Bossa rulebase for mutagenicity and carcinogenicity (implemented as a module of Toxtree software).
Full reference and details of the used SAs can be found in:
Benigni R., Bossa C., Jeliazkova N.G., Netzeva T.I., Worth A.P. The Benigni/Bossa rulebase for mutagenicity and carcinogenicity - a module of toxtree. Technical Report EUR 23241 EN, European
Commission - Joint Research Centre 2008.

- Defined domain of applicability:
The applicability domain of predictions is assessed using an Applicability Domain Index (ADI) that has values from 0 (worst case) to 1 (best case). The ADI is calculated by grouping several other indices (see below), each one taking into account a particular issue of the applicability domain. Most of the indices are based on the calculation of the most similar compounds found in the training and test set of the model, calculated by a similarity index that consider molecule's fingerprint and structural aspects (count of atoms, rings and relevant fragments).
For each index, including the final ADI, three intervals for its values are defined, such that the first interval corresponds to a positive evaluation, the second one corresponds to a suspicious evaluation and the last one corresponds to a negative evaluation.

- Appropriate measures of goodness-of-fit and robustness and predictivity:
See the attached QMRF for details on each index. Measured applicability domain scores :
1-Similar molecules with known experimental value. This index takes into account how similar are the first two most similar compounds found. Values near 1 mean that the predicted compound is well represented in the dataset used to build the model, otherwise the prediction could be an extrapolation.
2-Accuracy of prediction for similar molecules. This index takes into account the classification accuracy in prediction for the two most similar compounds found. Values near 1 mean that the predicted compounds falls in an area of the model's space where the model gives reliable predictions (no misclassifications), otherwise the lower is the value, the worse the model behaves.
3-Concordance for similar molecules . This index takes into account the difference between the predicted value and the experimental values of the two most similar compounds. Values near 0 mean that the prediction made disagrees with the values found in the model's space, thus the prediction could be unreliable.
4-Atom Centered Fragments similarity check. This index takes into account the presence of one or more fragments that aren't found in the training set, or that are rare fragments. First order atom centered fragments from all molecules in the training set are calculated, then compared with the first order atom centered fragments from the predicted compound; then the index is calculated as following: a first index RARE takes into account rare fragments (those who occur less than three times in the training set), having value of 1 if no such fragments are found, 0.85 if up to 2 fragments are found, 0.7 if more than 2 fragments are found; a second index NOTFOUND takes into account not found fragments, having value of 1 if no such fragments are found, 0.6 if a fragments is found, 0.4 if more than 1 fragment is found.
5-Model descriptors range check. This index checks if the descriptors calculated for the predicted compound are inside the range of descriptors of the training and test set. The index has value 1 if all descriptors are inside the range, 0 if at least one descriptor is out of the range.
6-Global AD Index. The final global index takes into account all the previous indices, in order to give a general global assessment on the applicability domain for the predicted compound.

MODEL STATISTICS:
Following, statistics obtained applying the model to its original dataset:
-Training set: n = 3240; Accuracy = 0.91; Specificity = 0.83; Sensitivity = 0.96
- Test set: n = 804; Accuracy = 0.89; Specificity = 0.82; Sensitivity = 0.93
From these statistics, compounds predicted as Suspicious Mutagen have been omitted. Structural alerts for suspect mutagenicity have been found as follows:
- In the training set: n = 127 (36 mutagen compounds, 91 non-mutagen compounds)
- In the test set: n = 30 (6 mutagen compounds, 27 non-mutagen compounds)
Furthermore, the statistics for the test set considering the Applicability Domain (AD) index is here reported; the AD index is used, as in the final model's assessment, in order to divide results in three
groups (into AD, possibly out of AD, out of AD), showing that compounds considered into AD have better performance than the others:
- Test set with AD index greater than 0.9 (compounds into the AD): n = 442; Accuracy = 0.93; Specificity = 0.85; Sensitivity = 0.97
- Test set with AD index between 0.9 and 0.7 (compounds could be out of AD): n = 250; Accuracy = 0.78; Specificity = 0.73; Sensitivity = 0.84
- Test set with AD index lower than 0.7 (compounds out of the AD): n = 113; Accuracy = 0.50; Specificity = 0.47; Sensitivity = 0.55

5. APPLICABILITY DOMAIN [Explain how the substance falls within the applicability domain of the model]
Predicted substance is into the Applicability domain of the model (ADI > 0.9). AD index of 0.951.
The list of 6 most similar compounds found in the training and test set of the model was reported in the prediction report, with their description and relevant in information (predicted and experimental data).
Similar molecules with known experimental value : Similarity index = 0.904. Strongly similar compounds with known experimental values in the training set have been found.
Concordance for similar molecules: Concordance index = 1 : Similar molecules found in the training set have experimental values that agree with the predicted value.
Accuracy of prediction for similar molecules: Accuracy index = 1 : Accuracy of prediction for similar molecules fould in the training set is good.
Atom centered fragments similarity check: ACF matching index = 1 : All atom centered fragment of the compound have been found in the compounds of the training set.
Model descriptors range check: Descriptors range check = true : Descriptors for this compound have values inside the descriptor range of the compounds of the training set.

6. ADEQUACY OF THE RESULT [Explain how the prediction fits the purpose of classification and labelling and/or risk assessment]
The QSAR prediction is that the substance is not mutagen.
This prediction has high reliability. Predicted substance in into the Applicability Domain of the model.
The results of this QSAR is used in the Weight of Evidence approach to consider the registered substance as not mutagen in the Ames test.
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
other: REACH Guidance on QSARs R.6
Version / remarks:
2008
Deviations:
not applicable
Test material information:
Composition 1
Specific details on test material used for the study:
SMILE : CC(=C)C(=O)OCCCCCCCCCCCCOC(=O)C(C)=C
Remarks on result:
no mutagenic potential (based on QSAR/QSPR prediction)
Conclusions:
The QSAR prediction is that the substance is not mutagen.
This prediction has high reliability. Predicted substance in into the Applicability Domain of the model.
The results of this QSAR is used in the Weight of Evidence approach to consider the registered substance as not mutagen in the Ames test.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Bacterial reverse mutation assay (Brient 2013)/ read-across :

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.

QSAR: Mutagenicity model VEGA/CAESAR :

The model provides a qualitative prediction of mutagenicity on Salmonella typhimurium (Ames test).

The QSAR prediction is that the substance is not mutagen.

This prediction has high reliability. Predicted substance in into the Applicability Domain of the model.

The results of this QSAR is used in the Weight of Evidence approach to consider the registered substance as not mutagen in the Ames test.

Justification for classification or non-classification

Based on the weight-of-evidence approach, no classification of 1,12 -dodecanediol bismethacrylate is required for genotoxicity according to the Regulation EC n°1272/2008.