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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

All the three required in vitro studies were performed on the reaction mass of decyl and octyl acrylate. The in vitro gene mutation study in bacteria, the in vitro cytogenicity / micronucleus study and the in vitro gene mutation study in mammalian cells showed negative results. Based on these results, the Reaction mass of decyl and octyl acrylate is considered as not mutagenic.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 August 2016 - 30 September 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
2014
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
Not applicable (not a gene mutation assay).
Species / strain / cell type:
other: mouse lymphoma L5178Y TK+/- cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium containing 10% (v/v) 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:
Since the test item was found cytotoxic and non-freely soluble in the preliminary test, the selection of the highest dose-level to be used in the main experiment was based on the level of cytotoxicity and/or on the presence of emulsion in the culture medium, according to the criteria specified in the international guidelines.

Experiment without S9 mix
With a treatment volume of 0.5% (v/v) in culture medium (24-hour treatment) or 1% (v/v) in culture medium (3-hour treatment), the dose-levels used for treatment were as follows:
- 5, 10, 20, 40, 60, 80, 100 and 120 µg/mL for the 3-hour treatment,
- 3.13, 6.25, 12.5, 25, 50, 100 and 300 µg/mL for the 24-hour treatment.

Experiment with S9 mix
With a treatment volume of 1% (v/v) in culture medium, the dose-levels used for treatment were 25, 50, 100, 200, 300, 400 and 500 µg/mL.
Vehicle / solvent:
- Vehicle used: ethanol
- Justification for choice: based on available solubility data, the test item was prepared as a solution in ethanol at the concentration of 500 mg/mL, which was expected to produce an emulsion 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 (-S9 mix); cyclophosphamide (+S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
Premiminary cytotoxicity test:
Without S9 mix:
3 h treatment + 24 h recovery
24 h treatment + 0 h recovery

With S9 mix: 3 h treatment + 24 h recovery

Main cytogenetic test
Without S9 mix:
3 h treatment + 24 h recovery
24 h treatment + 0 h recovery

With S9 mix: 3 h treatment + 24 h recovery

NUMBER OF CELLS EVALUATED: 2000 mononucleated cells/dose

DETERMINATION OF CYTOTOXICITY
- Method: population doubling
Evaluation criteria:
The biological relevance of the results was always taken into account when evaluating results.

Evaluation of a positive response: a test item is 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 demonstrated by a statistically significant trend test,
- 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.

Evaluation of a negative response: a test item is considered clearly negative if none of the criteria for a positive response was met.
Statistics:
yes
Key result
Species / strain:
other: mouse lymphoma L5178Y TK+/- 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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Evaporation from medium: none
- Emulsion:
* Experiment without S9 mix
An emulsion was observed in the culture medium at the end of the treatment period at dose-levels >= 80 µg/mL following the 3-hour treatment, and at the dose-level of 300 µg/mL following the 24-hour treatment.

* Experiment with S9 mix
An emulsion was observed in the culture medium at the end of the treatment period at dose-levels >= 100 µg/mL. It is to be noted that this emulsion did not prevent any scoring.

- Definition of acceptable cells for analysis:
Analysis was performed under a microscope (1000 x magnification), on the basis of the recommendations of Miller et al. (1995), according to the following criteria:
* micronuclei should be clearly surrounded by a nuclear membrane,
* the micronucleus area should be less than one-third of the area of the main nucleus,
* non-refractility of the micronuclei,
* micronuclei should not be linked to the main nucleus via nucleoplasmic bridges,
* micronuclei should be located within the cytoplasma of the cell,
* only mononucleated cells with a number of micronuclei <= 5 should be scored to exclude apoptosis and nuclear fragmentation.

- Other confounding effects: none.

RANGE-FINDING/SCREENING STUDIES:
Based on available solubility data, the test item was prepared as a solution in ethanol at the concentration of 500 mg/mL, which was expected to produce an emulsion in the culture medium.
Using this stock solution and a treatment volume of 1% (v/v) in culture medium, the highest recommended dose-level of 5000 µg/mL was achievable for the 3-hour treatment with and without S9 mix.
Taking into account the maximum practicable volume of 0.5% (v/v) of ethanol in culture medium for a continuous treatment, the highest achievable dose-level of 2500 µg/mL was reached for the 24-hour treatment without S9 mix.

The dose levels selected for the treatment of the preliminary test were the following:
- 0.1, 1, 10, 100, 500, 1000, 2500 and 5000 µg/mL for the 3-hour treatment with and without S9 mix,
- 0.01, 0.1, 1, 10, 100, 500, 1000 and 2500 µg/mL for the 24-hour treatment without S9 mix.

At the highest dose-levels of 2500 or 5000 µg/mL (depending on the treatment volume used in the culture medium), the pH of the culture media was approximately 7.1, as for the corresponding vehicle controls, whatever the treatment volume applied.
Using a treatment volume of 1% (v/v) in the culture medium, the osmolality at the highest dose-level of 5000 µg/mL was equal to 375 mOsm/kg H2O (482 mOsm/kg for the corresponding vehicle control).
Using a treatment volume of 0.5% (v/v) in the culture medium, the osmolality at the highest dose-level of 2500 µg/mL was equal to 334 mOsm/kg H2O (383 mOsm/kg for the corresponding vehicle control).

Therefore, none of the selected dose-levels was considered to produce extreme culture conditions. Thus, the highest recommended dose-level of 5000 µg/mL could be selected as the highest dose-level for the main experiment following the 3-hour treatments, and the dose-level of 2500 µg/mL could be selected as the highest dose-level for the main experiment following the 24-hour treatment.

An emulsion was observed in the culture medium at the end of the treatment periods at dose-levels >= 500 µg/mL.

Following the 3-hour treatment without S9 mix, a severe cytotoxicity was observed at dose-levels >= 100 µg/mL, as shown by a 100% decrease in the PD.
Following the 24-hour treatment without S9 mix, a slight cytotoxicity was noted at 0.1 µg/mL, then a severe cytotoxicity was noted at dose-levels >= 100 µg/mL, as shown by a 33 to 100% decrease in the PD.
Following the 3-hour treatment with S9 mix, a moderate to severe cytotoxicity was observed at dose-levels >= 500 µg/mL, as shown by a 47 to 100% decrease in the PD.


NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: see attached document


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see attached document
- Negative (solvent/vehicle) historical control data: see attached document

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Population Doubling
Conclusions:
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, either in the presence or 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 an increase in the frequency of micronucleated cells in the mouse cell line L5178Y TK+/-.

This study was conducted in compliance with OECD Guideline No. 487 and the principles of Good Laboratory Practices.

 

Methods

After a preliminary cytotoxicity test, the test item, diluted in ethanol, was tested in a single experiment, with a metabolic activation system (3 h treatment + 24 h recovery) and without a metabolic activation system (  3 h treatment + 24 h recovery, and,  24 h treatment + 0 h recovery), the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

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.

Then, after the final cell counting, the cells were washed and fixed. Then, cells from at least three dose-levels of the test item treated cultures were dropped onto clean glass slides. The slides were air-dried before being stained in 5% Giemsa. Slides from vehicle and positive controls cultures were also prepared as described above. All slides were coded before analysis, so that the analyst was unaware of the treatment details of the slide under evaluation ("blind" scoring). 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 dose).

Number of cells with micronuclei and number of micronuclei per cell were recorded separately for each treated and control culture.

 

Results

Since the test item was found cytotoxic and non-freely soluble in the preliminary test, the selection of the highest dose-level to be used in the main experiment was based on the level of cytotoxicity and/or on the presence of emulsion in the culture medium, according to the criteria specified in the international guidelines.

 

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

 

Experiment without S9 mix

With a treatment volume of 0.5% (v/v) in culture medium (24-hour treatment) or 1% (v/v) in culture medium (3-hour treatment), the dose-levels used for treatment were as follows:

.            5, 10, 20, 40, 60, 80, 100 and 120 µg/mL for the 3-hour treatment,

.            3.13, 6.25, 12.5, 25, 50, 100 and 300 µg/mL for the 24-hour treatment.

 

An emulsion was observed in the culture medium at the end of the treatment period at dose-levels superior or equal to 80 µg/mL following the 3-hour treatment, and at the dose-level of 300 µg/mL following the 24-hour treatment.


Cytotoxicity

Following the 3-hour treatment, a moderate to severe cytotoxicity was induced at dose-levels superior or equal to 60 µg/mL, as shown by a 49 to 100% decrease in the PD.

Following the 24-hour treatment, a moderate to severe cytotoxicity was induced at dose-levels superior or equal to 100 µg/mL, as shown by a 53 to 100% decrease in the PD.

 

Micronucleus analysis

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

.            20, 40 and 60 µg/mL for the 3-hour treatment, the latter inducing a 49% decrease in the PD (close to the recommended level of cytotoxicity (i.e. 55 ± 5% cytotoxicity)) and higher dose-levels being too cytotoxic,

.            25, 50 and 100 µg/mL for the 24-hour treatment, the latter inducing the recommended level of cytotoxicity (with 53% decrease in the PD).

 

Increases in the frequency of micronucleated cells were noted at 40 and 60 µg/mL after the 3-hour treatment. However, no dose-response relationship was evidenced, and none of these increases were statistically significant when compared to the corresponding vehicle control. Moreover, the frequencies of micronucleated cells of each replicate culture remained within the vehicle historical range.

Despite that the recommended level of cytotoxicity was not reached, considering the narrow dose-levels spacing used in this experiment, the available results were considered as suitable to allow a reliable interpretation. These results are thus considered to meet the criteria of a negative response.

 

No increase in the frequency of micronucleated cells were noted after the 24-hour treatment. Furthermore, no dose-response relationship was evidenced, and the frequencies of micronucleated cells of each replicate culture remained within the vehicle historical range. These results met the criteria of a negative response.

Experiment with S9 mix

With a treatment volume of 1% (v/v) in culture medium, the dose-levels used for treatment were 25, 50, 100, 200, 300, 400 and 500 µg/mL.

 

An emulsion was observed in the culture medium at the end of the treatment period, at dose-levels superior or equal to 100 µg/mL. It is to be noted that this emulsion did not prevent any scoring.

Cytotoxicity

A severe cytotoxicity was induced at dose-levels superior or equal to 200 µg/mL, as shown by a 100% decrease in the PD.

Micronucleus analysis

The dose-levels selected for micronucleus analysis were 25, 50 and 100 µg/mL, the latter corresponding to the lowest dose-level which induced an emulsion in the culture medium.

 

An increase in the frequency of micronucleated cells was noted at 25 µg/mL. At this dose-level, the frequencies of micronucleated cells of each replicate culture were slightly above the vehicle historical range. However, since this increase was neither statistically significant nor dose-related, the criteria of a positive response were only partially met and the overall results with S9 mix were considered to meet the criteria of a negative response.

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, either in the presence or absence of a rat liver metabolizing system.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October - December 1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
Ames Salmonella/Microsome assays using Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100.
The assays were conducted using one plate per dose level in the presence and absence of a metabolic activation system.
The procedure used in based on the paper published by Ames et al. (1975).
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
not applicable
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
rat liver induced by Aroclor 1254
Test concentrations with justification for top dose:
Doses were selected for the assay based on a preliminary dose range finding test with the strain TA100. 14 doses (0.02 µl to 150 µl) using two-fold dilutions from 10000 µg per plate for solides and 150 µl per plate for liquids were used in this dose selection assay. In this preliminary study, the test material was toxic to the indicator strain at 0.02 µl per plate and above as evidenced by the reduced number of revertants on the minimal media plates.

For the main assay, 8 doses were selected with the highest dose exhibiting 100% toxicity. The doses selected for the mutation assays ranged from 0.001 µl per plate to 1.0 µl per plate.
Vehicle / solvent:
DMSO
Water with sodium azide (one of positive control)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
sodium azide
other: 2-antramine, quinacrine mustard
Details on test system and experimental conditions:
One plate per dose level
Media: for daily use, an inoculum from stock culture plates is grown overnight at 37°C in Oxoid Media #2 (nutrient broth) and used in the mutagenicity test. The minimal media plates for the selection of histidine revertants consisted of the Vogel Bonner Medium E with 2% glucose and 1.5% bactoagar. The overlay agar contained the following per 100 ml volume; 0.6 grams of purified agar, 10 ml of 0.5 mM L-histidine-0.5mM biotin and 0.5 g NaCl according to the method of Ames et al . (1975).
Evaluation criteria:
Because the procedures used to evaluate the mutagenicity of the test material were semi quantitative, the criteria used to determine positive effects were inherently subjective and were based promarily on a historical data base.
For strains TA1535, TA1537, TA1538, data sets wiill be evaluated as positive if a dose response is observed over a minimum of three test concentrations and the increase in revertants is equal to or greater than three times the solvent control at the peak of the dose response. The solvent control value should be within the normal range for evaluating the results.
For strains TA98 and TA100, data sets will be evaluated as positive if a dose response is observed over a minimum of three test concentrations and the increase in revertants achieves a doubling of the solvent control at the peak of the dose response. The solvent control value should be within the normal range for evaluating the results.
The following ranges of revertants for solvent controls are generally considered acceptable: TA1535 (8-30), TA1537 (4-30), TA1538 (10-35), TA98 (20-75), TA100 (80-250).
Statistics:
no
Species / strain:
S. typhimurium, other: TA1535, TA1537, TA1538, TA98, TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The results of the assays conducted on the test material at dose levels ranged from 0.001 µl to 1.0 µl per plate in the absence and presence of metabolic activiation did not exhibit increased numbers of his+ revertant colonies.
The non activation assay with the strain TA1535 was repeated twice because of the increased number of revertants observed with the solvent control in the initial and first repeated assays. The activation assay with this strain was repeated because of the low number of revertants observed with the positive control compound.
The positive control treatments in both the non-activation and S9 acitivation assays induced large increases in the revertant numbers with all the indicator strains, which demonstrated the effectiveness of the S9 activation system and ability of the test system to detect known mutagens.
Conclusions:
The test item did not exhibit genetic activity in these assays and was considered not mutagenic under these test conditions according to the assay criteria.
Executive summary:

The mutagenic activity of the test item was examined in the Ames Salmonella/Microsome assays using Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100. The assays were conducted using one plate per dose level in the presence and absence of a metabolic activiation system. The test item did not exhibit genetic activity in these assays and was considered not mutagenic under these test conditions according to the assay criteria.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 2016 - February 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes
Type of assay:
other: in vitro gene mutation study in mammalian cells
Target gene:
hprt locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Dr Donald Clive, Burroughs Wellcome Co.
- Storage at Covance: as frozen stocks in liquid notrogen.
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 at 37+/-1°C. When the cells were growing well, subcutltures were established in an appropriate number of flasks.

MEDIA USED
- Type and identity of media: RPMI 1640 media containing L-glutamine and HEPES
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
liver rat
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 62.5 to 2000 µg/mL (the maximum
recommended concentration according to current regulatory test guidelines). The highest concentration to give =10% relative survival (RS) in the presence of S-9 was 125 µg/mL, which gave 13% RS. In the absence of S-9, all concentrations analysed for survival (62.5 to 125 µg/mL, limited by cytotoxicity) gave =2% RS.

In the Mutation Experiment eleven concentrations, ranging from 0.75 to 75 µg/mL, were tested in the absence of S-9 and twelve concentrations, ranging from 10 to 200 µg/mL, were tested in the presence of S-9. Seven days after treatment, the highest concentrations analysed to determine viability and 6TG resistance were 75 µg/mL in the absence of S-9 and 125 µg/mL in the presence of S-9, which gave 28% and 14% RS, respectively. In the absence of S-9, lower concentrations of 50 and 60 µg/mL gave 5% and 13% RS, respectively and both were included in the analysis.
Vehicle / solvent:
Ethanol
Preliminary solubility data indicated that Octyl Decyl acrylate was soluble in ethanol at concentrations up to at least 201.3 mg/mL. The solubility limit in culture medium was in excess of 2013 µg/mL, as indicated by a lack of visible precipitation at this concentration approximately 3 hours after test article addition with warming at 37°C. A maximum concentration of 2000 µg/mL was selected for the cytotoxicity Range-Finder Experiment in order that treatments were performed up to the maximum recommended concentration according to current regulatory test guidelines (OECD, 2016).
Negative (vehicle) controls comprised treatments with the vehicle ethanol diluted 100-fold in the treatment medium
Untreated negative controls:
yes
Remarks:
UTC (untreated control)
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
benzo(a)pyrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Preincubation period: 3h
- Exposure duration: 7d
- Expression time (cells in growth medium): 7d

NUMBER OF CELLS EVALUATED: At the end of the expression period, cell concentrations in the selected cultures were determined using a Coulter counter and adjusted to give 1 x 105 cells/mL in readiness for plating for 6TG resistance.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
- Any supplementary information relevant to cytotoxicity: Cloning Efficiency (CE) in any given culture is therefore: CE = P/No of cells plated per well, and as an average of 1.6 cells/well were plated on all survival and viability plates, CE = P/1.6.
Percentage Relative Survival (% RS) in each test culture was determined by comparing plating efficiencies in test and control cultures thus: % RS = [CE (test)/CE (control)] x 100.
To take into account any loss of cells during the 3 hour treatment period, percentage relative survival values for each concentration of test article were adjusted as follows: Adjusted % RS = [% RS x Post-treatment cell concentration for test article treatment] / Post-treatment cell concentration for vehicle control

- OTHER: metabolic activation system
The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was obtained from Molecular Toxicology Incorporated, USA where it is prepared from male Sprague Dawley rats induced with Aroclor 1254. The batches of S-9 were stored frozen in aliquots at <-50°C prior to use (Booth et al., 1980). Each batch was checked by the manufacturer for sterility, protein content, ability to convert known promutagens to bacterial mutagens and cytochrome P-450-catalyzed enzyme activities (alkoxyresorufin-O-dealkylase activities).
The S-9 mix was prepared in the following way: G6P (180 mg/mL), NADP (25 mg/mL), KCl (150 mM) and rat liver S-9 were mixed in the ratio 1:1:1:2. For all cultures treated in the presence of S-9, an aliquot of the mix was added to each cell culture to achieve the required final concentration of test article in a total of 20 mL. The final concentration of the liver homogenate in the test system was 2%.
Rationale for test conditions:
Acceptance Criteria: The assay was considered valid if the following criteria were met:
1. The MF in the concurrent negative control was considered acceptable for addition to the laboratory historical negative control database,
2. The MF in the concurrent positive controls induced responses that were compatible with those generated in the historical positive control database and give a clear, unequivocal increase in MF over the concurrent negative control,
3. The test was performed with and without metabolic activation,
4. Adequate numbers of cells and concentrations were analysable.
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 MF at one or more concentrations was significantly greater than that of the vehicle control (p=0.05)
2. There was a significant concentration-relationship as indicated by the linear trend analysis (p=0.05)
3. The results were outside the historical vehicle control range.
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis. Positive responses seen only at high levels of cytotoxicity required careful interpretation when assessing their biological relevance. Extreme caution was exercised with positive results obtained at levels of RS lower than 10%.
Statistics:
Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines (Robinson et al., 1990). The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Toxicity
In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9, ranging from 62.50 to 2000 µg/mL. Upon addition of the test article to the cultures, precipitate was observed at the highest four concentrations in the absence and presence of S-9 (250 to 2000 µg/mL). Following the 3 hour treatment incubation period, precipitate was observed at the highest two concentrations in the presence of S-9 only (1000 and 2000 µg/mL). The lowest concentration at which precipitate was observed at the end of the treatment incubation period in the presence of S-9 was retained and the higher concentration was discarded at the end of the treatment incubation period. In addition, the highest four concentrations in the absence of S-9 (250 to 2000 µg/mL) and the highest two remaining concentrations in the presence of S-9 (500 and 1000 µg/mL) were considered too toxic to be plated for survival. The highest concentration to give =10% relative survival (RS) in the presence of S-9 was 125 µg/mL, which gave 13% RS. In the absence of S-9, all concentrations analysed for survival (62.5 to 125 µg/mL) gave =2% RS.
No marked changes in osmolality or pH were observed in the Range-Finder at the highest concentrations analysed (2000 µg/mL in the absence of S-9 and 1000 µg/mL in the presence of S-9) as compared to the concurrent vehicle controls

In the Mutation Experiment eleven concentrations, ranging from 0.75 to 75 µg/mL, were tested in the absence of S-9 and twelve concentrations, ranging from 10 to 200 µg/mL, were tested in the presence of S-9. Upon addition of the test article to the cultures, precipitate was observed at the highest two concentrations (150 and 200 µg/mL) in the presence of S-9 only but following the 3 hour treatment incubation period, no evidence of precipitate was observed in the absence and presence of S-9.
Seven days after treatment, the highest three concentrations in the presence of S-9 (135 to 200 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance and the lowest three concentrations in the absence of S-9 (0.75 to 3 µg/mL) were not selected as there were sufficient concentrations to define the toxicity profile. All other concentrations were selected in the absence and presence of S-9. The highest concentrations analysed were 75 µg/mL in the absence of S-9 and 125 µg/mL in the presence of S-9, which gave 28% and 14% RS, respectively. In the absence of S-9, lower concentrations of 50 and 60 µg/mL gave 5% and 13% RS, respectively and both were included in the analysis.

Mutation
The acceptance criteria were met and the study was accepted as valid.
When tested up to highly toxic concentrations, no statistically significant increases in mean MF values were observed following treatment with Octyl Decyl acrylate at any concentration analysed in the absence and presence of S-9 and there were no statistically significant linear trends, indicating a negative result.
Conclusions:
It is concluded that Octyl Decyl acrylate did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to highly toxic concentrations for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9) under the experimental conditions described.
Executive summary:

Octyl Decyl acrylate 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 a Mutation Experiment, 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 ethanol, therefore untreated controls (containing culture medium) were included under each treatment condition. A 3 hour treatment incubation period was used for both experiments.

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 62.5 to 2000µg/mL (the maximum recommended concentration according to current regulatory test guidelines). The highest concentration to give=10% relative survival (RS) in the presence of S-9 was 125µg/mL, which gave 13% RS. In the absence of S-9, all concentrations analysed for survival (62.5 to 125µg/mL, limited by cytotoxicity) gave=2% RS. In the Mutation Experiment eleven concentrations, ranging from 0.75 to 75µg/mL, were tested in the absence of S-9 and twelve concentrations, ranging from 10 to 200µg/mL, were tested in the presence of S-9. Seven days after treatment, the highest concentrations analysed to determine viability and 6TG resistance were 75µg/mL in the absence of S-9 and 125µg/mL in the presence of S-9, which gave 28% and

14% RS, respectively. In the absence of S-9, lower concentrations of 50 and 60µg/mL gave 5% and 13% RS, respectively and both were included in the analysis.

Vehicle, untreated and positive control treatments were included in the 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.

When tested up to highly toxic concentrations, no statistically significant increases in mean MF values were observed following treatment with Octyl Decyl acrylate at any concentration analysed in the absence and presence of S-9 and there were no statistically significant linear trends, indicating a negative result.

It is concluded that Octyl Decyl acrylate did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to highly toxic concentrations for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9) under the experimental conditions described.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In vitro gene mutation study in bacteria (1986)

The mutagenic activity of the test item was examined in the Ames Salmonella/Microsome assays using Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100. The assays were conducted using one plate per dose level in the presence and absence of a metabolic activation system. The test item did not exhibit genetic activity in these assays and was considered not mutagenic under these test conditions according to the assay criteria.

in vitro cytogenicity / micronucleus study (Brient 2017)  

The objective of this study was to evaluate the potential of the test item to induce an increase in the frequency of micronucleated cells in the mouse cell line L5178Y TK+/-. This study was conducted in compliance with OECD Guideline No. 487 and the principles of Good Laboratory Practices.

 After a preliminary cytotoxicity test, the test item, diluted in ethanol, was tested in a single experiment, with a metabolic activation system (3 h treatment + 24 h recovery) and without a metabolic activation system (  3 h treatment + 24 h recovery, and,  24 h treatment + 0 h recovery), the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

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.

 Since the test item was found cytotoxic and non-freely soluble in the preliminary test, the selection of the highest dose-level to be used in the main experiment was based on the level of cytotoxicity and/or on the presence of emulsion in the culture medium, according to the criteria specified in the international guidelines.

In the experiment without S9 mix, an emulsion was observed in the culture medium at the end of the treatment period at dose-levels superior or equal to 80 µg/mL following the 3-hour treatment, and at the dose-level of 300 µg/mL following the 24-hour treatment. Following the 3-hour treatment, a moderate to severe cytotoxicity was induced at dose-levels superior or equal to 60 µg/mL, as shown by a 49 to 100% decrease in the PD. Following the 24-hour treatment, a moderate to severe cytotoxicity was induced at dose-levels superior or equal to 100 µg/mL, as shown by a 53 to 100% decrease in the PD. Increases in the frequency of micronucleated cells were noted at 40 and 60 µg/mL after the 3-hour treatment. However, no dose-response relationship was evidenced, and none of these increases were statistically significant when compared to the corresponding vehicle control. Moreover, the frequencies of micronucleated cells of each replicate culture remained within the vehicle historical range. Despite that the recommended level of cytotoxicity was not reached, considering the narrow dose-levels spacing used in this experiment, the available results were considered as suitable to allow a reliable interpretation. These results are thus considered to meet the criteria of a negative response. No increase in the frequency of micronucleated cells were noted after the 24-hour treatment. Furthermore, no dose-response relationship was evidenced, and the frequencies of micronucleated cells of each replicate culture remained within the vehicle historical range. These results met the criteria of a negative response.

In the experiment with S9 mix, an emulsion was observed in the culture medium at the end of the treatment period, at dose-levels superior or equal to 100 µg/mL. It is to be noted that this emulsion did not prevent any scoring. A severe cytotoxicity was induced at dose-levels superior or equal to 200 µg/mL, as shown by a 100% decrease in the PD. The dose-levels selected for micronucleus analysis were 25, 50 and 100 µg/mL, the latter corresponding to the lowest dose-level which induced an emulsion in the culture medium. An increase in the frequency of micronucleated cells was noted at 25 µg/mL. At this dose-level, the frequencies of micronucleated cells of each replicate culture were slightly above the vehicle historical range. However, since this increase was neither statistically significant nor dose-related, the criteria of a positive response were only partially met and the overall results with S9 mix were considered to meet the criteria of a negative response.

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, either in the presence or absence of a rat liver metabolizing system.

In vitro gene mutation study in mammalian cells - HPRT, OECD 476 (Lloyd 2017)

Octyl Decyl acrylate was assayed for the ability to induce mutation at thehypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG]resistance) in mouse lymphoma cells using a fluctuation protocol. The study consistedof a cytotoxicity Range-Finder Experiment followed by a Mutation Experiment,conducted in the absence and presence of metabolic activation by anAroclor 1254-induced rat liver post-mitochondrial fraction (S-9).The test article wasformulated in ethanol, therefore untreated controls (containing culture medium) wereincluded under each treatment condition.A 3 hour treatment incubation period was used for both experiments.

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in theabsence and presence of S-9 ranging from 62.5 to 2000µg/mL (the maximumrecommended concentration according to current regulatory test guidelines). Thehighest concentration to give=10% relative survival (RS) in the presence of S-9 was125µg/mL, which gave 13% RS. In the absence of S-9, all concentrations analysedfor survival (62.5 to 125µg/mL, limited by cytotoxicity) gave=2% RS.In the Mutation Experiment eleven concentrations, ranging from 0.75 to 75µg/mL,were tested in the absence of S-9 and twelve concentrations, ranging from 10 to200µg/mL, were tested in the presence of S-9. Seven days after treatment, the highestconcentrations analysed to determine viability and 6TG resistance were 75µg/mL inthe absence of S-9 and 125µg/mL in the presence of S-9, which gave 28% and

14% RS, respectively. In the absence of S-9, lower concentrations of 50 and60µg/mL gave 5% and 13% RS, respectively and both were included in the analysis.

Vehicle, untreated and positive control treatments were included in the MutationExperiment in the absence and presence of S-9. Mutant frequencies (MF) in vehiclecontrol cultures fell within acceptable ranges and clear increases in mutation wereinduced by the positive control chemicals 4-nitroquinoline 1-oxide (NQO) withoutS-9 and benzo(a)pyrene (B[a]P) with S-9. Therefore the study was accepted as valid.

When tested up to highly toxic concentrations, no statistically significant increases inmean MF values were observed following treatment with Octyl Decyl acrylate at anyconcentration analysed in the absence and presence of S-9 and there were nostatistically significant linear trends, indicating a negative result.

It is concluded that Octyl Decyl acrylate did not induce mutation at the hprt locus ofL5178Y mouse lymphoma cells when tested up to highly toxic concentrations for3 hours in the absence and presence of a rat liver metabolic activation system (S-9)under the experimental conditions described.

Justification for classification or non-classification

Based on the available data, no classification for genotoxicity is required for the Reaction mass of decyl and octyl acrylate according to the Regulation EC.N°1272/2008.