Isodecyl acrylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The Ames test, HPRT and in vitro micronucleus test were performed according to the OECD guideline on isodecyl acrylate. Negative results were observed in all the tests with and without metabolic activation. No further test is required for the genotoxicity endpoint.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 August 2012 to 25 January 2013

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)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

Mouse lymphoma L5178Y cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The master stock of L5178Y tk+/- (3.7.2C) 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:

mammalian liver post-mitochondrial fraction (S-9), prepared from male Sprague Dawley rats induced with Aroclor 1254

Test concentrations with justification for top dose:

Experiment 1: thirteen concentrations, ranging from 5 to 80 µg/mL in the absence of S-9 and 10 to 140 mg/mL in the presence of S-9, were tested.
Experiment 2: twelve concentrations, ranging from 10 to 50 µg/mL in the absence of S-9 and fourteen concentrations, ranging from 20 to 140 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

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9, ranging from 31.25 to 1000 mg/mL (limited by solubility in culture medium).

Vehicle / solvent:

ethanol diluted 100 fold in the treatment medium

Untreated negative controls:

no

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 suspension

DURATION
- Preincubation period: NA
- Exposure duration: 7 days

Cultures were maintained in flasks for a period of 7 days during which the hprt- mutation would be expressed. Sub-culturing was performed as required with the aim of not exceeding 1 x 106 cells/mL and, where possible, retaining at least 6 x 106 cells/flask. From observations on recovery and growth of the cultures during the expression period, the cultures were selected to be plated for viability and 6TG resistance.
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. Samples from these were diluted to 8 cells/mL.
Using a multichannel pipette, 0.2 mL of the final concentration of each culture was placed into each well of 2 x 96-well microtitre plates (192 wells averaging 1.6 cells/well). The plates were incubated at 37±1ºC in a humidified incubator gassed with 5±1% v/v CO2 in air until scoreable (8 to 10 days). Wells containing viable clones were identified by eye using background illumination and counted.
At the end of the expression period, the cell densities in the selected cultures were adjusted to 1 x 105 cells/mL. 6TG (1.5 mg/mL) was diluted 100-fold into these suspensions to give a final concentration of 15 µg/mL. Using a multichannel pipette, 0.2 mL of each suspension was placed into each well of 4 x 96-well microtitre plates (384 wells at 2 x 104 cells/well). Plates were incubated at 37±1ºC in a humidified incubator gassed with 5±1% v/v CO2 in air until scoreable (12 to 13 days) and wells containing clones were identified as above and counted.

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.
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis.

Statistics:

Not required

Key result

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:

Range-finder study :
In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S 9 ranging from 31.25 to 1000 µg/mL (limited by solubility in culture medium). Upon addition of the test article to the cultures, precipitate was observed at the highest three concentrations in the absence and presence of S-9 (250 to 1000 µg/mL). No evidence of precipitation was observed following the 3 hour incubation period therefore all cultures were retained. Complete or extreme toxicity was observed at 62.5 µg/mL and above in the absence of S-9 and at 125 µg/mL and above in the presence of S-9. The highest concentrations to give >10% relative survival (RS) were 31.25 µg/mL in the absence of S-9 and 62.5 µg/mL in the presence of S-9, which gave 83% and 45% RS, respectively.
No marked changes in osmolality or pH were observed in the Range-Finder at the highest concentration tested (1000 µg/mL) as compared to the concurrent vehicle controls.

Main experiments :
In Experiment 1, thirteen concentrations, ranging from 5 to 80 µg/mL in the absence of S-9 and 10 to 140 µg/mL in the presence of S-9, were tested. The highest seven concentrations tested in the absence of S-9 (45 to 80 µg/mL) were not plated for survival due to excessive toxicity. Seven days after treatment, the highest remaining concentration in the absence of S-9 (40 µg/mL) and the highest five concentrations in the presence of S-9 (90 to 140 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. In addition an intermediate concentration of 30 µg/mL in the presence of S-9 was not plated as there were sufficient non toxic concentrations available for analysis. All other concentrations were selected in the absence and presence of S-9. The highest concentrations analysed were 35 µg/mL in the absence of S-9 and 80 µg/mL in the presence of S-9, which gave 2% and 7% RS, respectively. Steep concentration-related toxicity was observed between 30 and 35 µg/mL (37% and 2% RS, respectively) in the absence of S-9 and between 70 and 80 µg/mL (33% and 7%, respectively) in the presence of S-9, therefore both concentrations were analysed in each case.
In Experiment 2, twelve concentrations, ranging from 10 to 50 µg/mL in the absence of S-9 and fourteen concentrations, ranging from 20 to 140 µg/mL in the presence of S-9, were tested. Seven days after treatment, the highest concentration tested in the absence of S-9 (50 µg/mL) and the highest three concentrations (110 to 140 µg/mL) in the presence of S-9 were considered too toxic for selection to determine viability and 6TG resistance. In addition intermediate concentrations of 15 and 27.5 µg/mL in the absence of S-9 and 75, 85 and 90 µg/mL in the presence of S-9 were not plated as there were sufficient non toxic concentrations available for analysis. All other concentrations were selected in the absence and presence of S-9. The highest concentrations analysed were 40 µg/mL in the absence of S-9 and 105 µg/mL in the presence of S-9, gave 19% and 20% RS, respectively.

Table 1: RS values: Range-Finder Experiment

Treatment (µg/mL)	-S-9 % RS	+S-9 % RS
0	100	100
UTC	118	73
31.25	83	81
62.5	1	45
125	0	0
250 P	NP	0
%RS- Percentage Relative Survival adjusted by post treatment cell counts P - Precipitation observed at time of treatment NP - Not plated for survival UTC - Untreated control

 

Table2: Summary of mutation data

Experiment 1 (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	6.62		0		100	6.75
UTC		100	4.49	NS	UTC		100	8.09	NS
5		104	8.02	NS	10		89	4.32	NS
10		86	5.59	NS	20		91	5.59	NS
20		74	5.31	NS	40		77	3.10	NS
30		37	6.18	NS	50		73	3.35	NS
35		2	3.75	NS	60		67	3.96	NS
					70		33	2.34	NS
					80		7	3.10	NS
Linear trend			NS		Linear trend			NS
NQO					B[a]P
0.15		61	45.16		2		49	49.13
0.2		37	42.84		3		19	74.69
§                     6-TG resistant mutants/10^6viable cells 7 days after treatment %RS                Percent relative survival adjusted by post treatment cell counts NS                   Not significant UTC                 Untreated controls

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	2.38		0		100	9.30
UTC		101	2.86	NS	UTC		96	8.22	NS
10		87	3.24	NS	20		93	6.41	NS
20		71	4.68	NS	40		75	6.86	NS
25		85	1.73	NS	60		71	2.62	NS
30		79	4.88	NS	70		62	6.24	NS
32		84	4.51	NS	80		49	10.68	NS
34		70	2.46	NS	95		32	5.31	NS
36		77	2.68	NS	100		17	5.41	NS
38		42	2.43	NS	105		20	4.37	NS
40		19	3.10	NS
Linear trend			NS		Linear trend			NS
NQO					B[a]P
0.15		65	42.01		2		74	19.21
0.2		49	83.41		3		49	35.80
§                     6-TG resistant mutants/10^6viable cells 7 days after treatment %RS                Percent relative survival adjusted by post treatment cell counts NS                   Not significant UTC                 Untreated controls

 

Conclusions:

It is concluded that Isodecyl Acrylate did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to toxic concentrations in two independent experiments in the absence and presence of a rat liver metabolic activation system (S-9).

Executive summary:

Isodecyl 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 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 ethanol.

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 31.25 to 1000 mg/mL (limited by solubility in culture medium). The highest concentrations to give >10% relative survival (RS) were 31.25 µg/mL in the absence of S-9 and 62.5 µg/mL in the presence of S-9, which gave 83% and 45% RS, respectively.

Accordingly, for Experiment 1, thirteen concentrations, ranging from 5 to 80 µg/mL in the absence of S-9 and 10 to 140 mg/mL in the presence of S-9, were tested. Seven days after treatment the highest concentrations analysed to determine viability and 6TG resistance were 35 µg/mL in the absence of S-9 and 80 µg/mL in the presence of S-9, which gave 2% and 7% RS, respectively. Steep concentration-related toxicity was observed between 30 and 35 µg/mL (37% and 2% RS, respectively) in the absence of S-9 and between 70 and 80 mg/mL (33% and 7%, respectively) in the presence of S-9, therefore both concentrations were analysed in each case.

In Experiment 2, twelve concentrations, ranging from 10 to 50 µg/mL in the absence of S-9 and fourteen concentrations, ranging from 20 to 140 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 105 µg/mL in the presence of S-9, which gave 19% and 20% RS, respectively.

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

In Experiments 1 and 2 no statistically significant increases in mutant frequency were observed following treatment with Isodecyl Acrylate at any concentration tested in the absence and presence of S-9 and there were no significant linear trends. Due to the steep toxicity profile observed, highly toxic concentrations giving <10% RS were analysed in Experiment 1 in the absence and presence of S-9. However, in the absence of any significant increase in mutant frequency at these concentrations or significant linear trends, the data were considered valid and the integrity of the study unaffected.

It is concluded that Isodecyl Acrylate did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to toxic concentrations in two independent experiments in the absence and presence of a rat liver metabolic activation system (S-9).

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 August 2012 to 24 January 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine operon

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S-9 from male Sprague Dawley rats induced with Aroclor 1254

Test concentrations with justification for top dose:

First experiment : 5, 15.81, 50, 1581 and 5000 µg/plate
Second experiment : 156.3 - 5000 µg/plate

Vehicle / solvent:

Ethanol

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-aminoanthracene

Details on test system and experimental conditions:

For all assays, bacteria were cultured at 37±1°C for 10 hours in nutrient broth, containing ampicillin (TA98, TA100) or ampicillin and tetracycline (TA102) as appropriate, to provide bacterial cultures in the range of approximately 108 to 109 cells/mL, based on cell count data from testing of each strain batch. Incubation was carried out with shaking in an anhydric incubator, set to turn on using a timer switch. All treatments were completed within 6 hours of the end of the incubation period.
Isodecyl acrylate was tested for mutation (and toxicity) in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102), in two separate experiments, at the concentrations detailed previously, using triplicate plates without and with S-9. Negative (vehicle) controls were included in quintuplicate, and positive controls were included in triplicate in both assays without and with S-9. These platings were achieved by the following sequence of additions to 2.5 mL molten agar at 46±1°C:
• 0.1 mL bacterial culture • 0.1 mL test article solution or control • 0.5 mL 10% S-9 mix or buffer solution
followed by rapid mixing and pouring on to Vogel-Bonner E agar plates. When set, the plates were inverted and incubated at 37±1°C in the dark for 3 days. Following incubation, these plates were examined for evidence of toxicity to the background lawn, and where possible revertant colonies were counted.
As the results of Experiment 1 were negative, treatments in the presence of S-9 in Experiment 2 included a pre-incubation step. Quantities of test article or control solution (reduced to 0.05 mL), bacteria and S-9 mix detailed above, were mixed together and incubated for 20 minutes at 37±1°C, before the addition of 2.5 mL molten agar at 46±1°C. Plating of these treatments then proceeded as for the normal plate-incorporation procedure. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected in the assay.
Volume additions for the Experiment 2 pre-incubation treatments were reduced to 0.05 mL due to the vehicle (ethanol) employed in this study and positive control vehicle (DMSO). These, and some other organic vehicles, are known to be near to toxic levels when added at volumes of 0.1 mL in this assay system when employing the pre-incubation methodology. By reducing the addition volume to 0.05 mL per plate, it was hoped to minimise or eliminate any toxic effects of the vehicle that may have otherwise occurred.

Evaluation criteria:

For valid data, the test article was considered to be mutagenic if:
1.When assessed using Dunnett's test, an increase in revertant numbers gave a significant response (p < 0.01) which was concentration related.
2.The positive trend/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.
Results which only partially satisfied the above criteria were dealt with on a case by case basis. Biological relevance was taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments.

Statistics:

Not reported

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and 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:

In experiment 1 treatments, of all tester strains were performed in the absence and in the presence of S-9, using final concentrations of Isodecyl acrylate at 5, 15.81, 50, 1581 and 5000 µg/plate, plus negative (vehicle) and positive controls. Follwoing these treatments, evidence of toxicity in the form of a slight thinning of the background bacterial lawn was observed at 5000 µg/plate in strains TA 98, TA 1535, TA 1537 and TA 102 in the presence of S-9 only.
In experiment 2, treatments of all the tester strains were performed in the absence and in the presence of S-9. The maximum test concentration of 5000 µg/plate was retained for all strains. Narrowed concentration intervals were employed covering the range 156.3 - 5000 µg/plate, in order to examine more closely those concentrations of Isodecyl acrylate approaching the maximum test concentration and considered therfore most likely to provide evidence of any mutagenic activity. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre-incubation step. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system. Following these treatments, no clear evidence of toxicity was observed.
No precipitation was observed on the test plates following incubation in both experiments.

Conclusions:

It was concluded that Isodecyl acrylate did not induce mutation in five histidine requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 µg/plate (the maximum recommended concentration according to current regulatory guidelines), in the absence and in the presence of a rat liver metabolic activation system (S-9).

Executive summary:

Isodecyl acrylate was assayed for mutation in five histidine requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation by an Aroclor 1254 induced rat liver post-mitochondrial fraction (S-9), in two separate experiments. All Isodecyl acrylate treatments in this study were performed using formulations prepared in ethanol, and all concentrations are expressed in terms of pure compound using a correction factor of 1.03. Experiment 1 treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of Isodecyl acrylate at 5, 15.81, 50, 158.1, 500, 1581 and 5000 µg/plate, plus negative (vehicle) and positive controls. Following these treatments, evidence of toxicity was observed at 5000 µg/plate in strains TA98, TA1535, TA1537 and TA102 in the presence of S-9 only. Experiment 2 treatments of all the tester strains were performed in the absence and in the presence of S-9. The maximum test concentration of 5000 µg/plate was retained for all strains. Narrowed concentration intervals were employed covering the range 156.3 – 5000 µg/plate, in order to examine more closely those concentrations of Isodecyl acrylate approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre incubation step to increase the range of mutagenic chemicals that could be detected using this assay system. Following these treatments, no clear evidence of toxicity was observed. No precipitation was observed on the test plates following incubation in both experiments. Negative (vehicle) and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies all fell within acceptable ranges for negative control treatments, and were significantly elevated by positive control treatments. Following Isodecyl acrylate treatments of all the test strains in the absence and presence of S-9, no increases in revertant numbers were observed that were statistically significant when the data were analysed at the 1% level using Dunnett’s test. This study was considered therefore to have provided no evidence of any Isodecyl acrylate mutagenic activity in this assay system. It was concluded that Isodecyl acrylate did not induce mutation in five histidine requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested at concentrations up to 5000 µg/plate (the maximum recommended concentration according to current regulatory guidelines), in the absence and in the presence of a rat liver metabolic activation system (S-9).

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 August 2012 to 24 January 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD guideline 487 (OECD, 2010)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: Human lymphocyte cultures

Details on mammalian cell type (if applicable):

Blood from two healthy, non-smoking male volunteers from a panel of donors at Covance was used for each experiment in this study.
No volunteer was suspected of any virus infection or exposed to high levels of radiation or hazardous chemicals. All volunteers are non-smokers and are not heavy drinkers of alcohol. Donors were not taking any form of medication.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S-9 from male Sprague Dawley rats unduced with Arcolor 1254

Test concentrations with justification for top dose:

3+21 hour treatments -S9 : 10-250 µg/ml (1st trial), 10-100 µg/ml (2nd trial)
24+24 hour treatments -S9: 10-250 µg/ml (1st trial), 10-60 µg/ml (2nd trial)
3+21 hour treatments +S9 : 10-250 µg/ml (1st trial)

Vehicle / solvent:

Ethanol

Untreated negative controls:

yes

Remarks:

untreated control (UTC)

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Vinblastine

Details on test system and experimental conditions:

No volunteer was suspected of any virus infection or exposed to high levels of radiation or hazardous chemicals. All volunteers are non-smokers and are not heavy drinkers of alcohol. Donors were not taking any form of medication. The measured cell cycle time of the donors used at Covance falls within the range 13 ± 2 hours. For each experiment, an appropriate volume of whole blood was drawn from the peripheral circulation into heparinised tubes within one day of culture initiation. Blood was stored refrigerated and pooled using equal volumes from each donor prior to use.
Whole blood cultures were established in sterile disposable centrifuge tubes by placing 0.4 mL of pooled heparinised blood into 9.0 mL pre-warmed (in an incubator set to 37 ± 1°C) HEPES-buffered RPMI medium containing 10% (v/v) heat inactivated foetal calf serum and 0.52% penicillin/streptomycin, so that the final volume following addition of S-9 mix/KCl and the test article in its chosen vehicle was 10 mL. The mitogen Phytohaemagglutinin (PHA, reagent grade) was included in the culture medium at a concentration of approximately 2% of culture to stimulate the lymphocytes to divide. Blood cultures were incubated at 37 ± 1°C for 48 hours and rocked continuously.

Evaluation criteria:

For valid data, the test article was considered to induce clastogenic and/or aneugenic events if:
1. A statistically significant increase in the frequency of MNBN cells at one or more concentrations was observed.
2. An incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates was observed.
3. A concentration-related increase in the proportion of MNBN cells was observed.
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:

Results which only partially satisfied the above criteria were dealt with on a case by case basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result (Scott et al., 1990).

Key result

Species / strain:

lymphocytes: Human lymphocyte cultures

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Treatment of cells with Isodecyl acrylate in the absence and presence of S-9 resulted in frequencies of MNBN cells which were similar to and not significantly (p = 0.05) higher than those observed in concurrent vehicle controls for all concentrations analysed (all treatments). The MNBN cell frequency of the majority of Isodecyl acrylate treated cultures fell within normal ranges. The single exception to this was noted at the lowest concentration analysed (25 µg/mL) post 3+21 hour +S-9 treatment (Micronucleus Experiment, Trial 1) where a single culture demonstrated numbers of MNBN cells that marginally exceeded the normal range. As this increase was not observed in the replicate culture, or for any other concentration analysed (all treatments), this was considered spurious and of no biological importance.

Conclusions:

It is concluded that Isodecyl acrylate did not induce micronuclei in cultured human peripheral blood lymphocytes following treatment in the absence and presence of an Aroclor induced rat liver metabolic activation system (S 9). Maximum concentrations analysed resulted in 51%, 54% and 66% cytotoxicity for 3+21 hour –S-9, 3+21 hour +S-9 and 24+24 hour –S-9 treatments respectively.

Executive summary:

Isodecyl acrylate was tested in an in vitro micronucleus assay using duplicate human lymphocyte cultures prepared from the pooled blood of two male donors in a single experiment. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9) from Aroclor 1254 induced animals. The test article was formulated in ethanol and the highest concentrations tested in the Micronucleus Experiment, (limited by toxicity), were determined following a preliminary cytotoxicity Range Finder Experiment.

Treatments were conducted (as detailed in the following summary table) 48 hours following mitogen stimulation by phytohaemagglutinin (PHA). The test article concentrations for micronucleus analysis were selected by evaluating the effect of Isodecyl acrylate on the replication index (RI). In the Micronucleus Experiment, micronuclei were analysed at three concentrations.

Appropriate negative (vehicle and untreated) control cultures were included in the test system under each treatment condition. The proportion of micronucleated binucleate cells (MNBN) in these cultures fell within (or very close to) the current historical vehicle control (normal) ranges and were considered acceptable. Untreated control cultures analysed for the 3+21 hour + S-9 treatment (Trial 1) demonstrated MNBN cell values within normal ranges and similar to those in the concurrent vehicle control. It was therefore not considered necessary to further analyse untreated control cultures from Micronucleus Experiment Trial 2.

Mitomycin C (MMC) and Vinblastine (VIN) were employed as clastogenic and aneugenic positive control chemicals respectively in the absence of rat liver S-9. Cyclophosphamide (CPA) was employed as a clastogenic positive control chemical in the presence of rat liver S-9. Cells receiving these were sampled in the Micronucleus Experiment at 24 hours (CPA, MMC) or 48 hours (VIN) after the start of treatment. All positive control compounds induced statistically significant increases in the proportion of cells with micronuclei.

The study was therefore considered as valid.

Treatment of cells with Isodecyl acrylate in the absence and presence of S-9 resulted in frequencies of MNBN cells which were similar to and not significantly (p = 0.05) higher than those observed in concurrent vehicle controls for all concentrations analysed (all treatments). The MNBN cell frequency of the majority of Isodecyl acrylate treated cultures fell within normal ranges. The single exception to this was noted at the lowest concentration analysed (25 µg/mL) post 3+21 hour +S-9 treatment (Micronucleus Experiment, Trial 1) where a single culture demonstrated numbers of MNBN cells that marginally exceeded the normal range. As this increase was not observed in the replicate culture, or for any other concentration analysed (all treatments), this was considered spurious and of no biological importance.

It is concluded that Isodecyl acrylate did not induce micronuclei in cultured human peripheral blood lymphocytes following treatment in the absence and presence of an Aroclor induced rat liver metabolic activation system (S-9). Maximum concentrations analysed resulted in 51%, 54% and 66% cytotoxicity for 3+21 hour –S-9, 3+21 hour +S-9 and 24+24 hour –S-9 treatments respectively.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Additional information from genetic toxicity in vitro:

Reverse mutation test in bacteria (Ames test, OECD 471):

Isodecyl acrylate was assayed for mutation in five histidine requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation by an Aroclor 1254 induced rat liver post-mitochondrial fraction (S-9), in two separate experiments. All Isodecyl acrylate treatments in this study were performed using formulations prepared in ethanol. Experiment 1 treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of Isodecyl acrylate at 5, 15.81, 50, 158.1, 500, 1581 and 5000 µg/plate, plus negative (vehicle) and positive controls. Following these treatments, evidence of toxicity was observed at 5000 µg/plate in strains TA98, TA1535, TA1537 and TA102 in the presence of S-9 only. Experiment 2 treatments of all the tester strains were performed in the absence and in the presence of S-9. The maximum test concentration of 5000 µg/plate was retained for all strains. Narrowed concentration intervals were employed covering the range 156.3 – 5000 µg/plate, in order to examine more closely those concentrations of Isodecyl acrylate approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre incubation step to increase the range of mutagenic chemicals that could be detected using this assay system. Following these treatments, no clear evidence of toxicity was observed. No precipitation was observed on the test plates following incubation in both experiments. Negative (vehicle) and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies all fell within acceptable ranges for negative control treatments, and were significantly elevated by positive control treatments. Following Isodecyl acrylate treatments of all the test strains in the absence and presence of S-9, no increases in revertant numbers were observed that were statistically significant when the data were analysed at the 1% level using Dunnett’s test. This study was considered therefore to have provided no evidence of any Isodecyl acrylate mutagenic activity in this assay system. It was concluded that Isodecyl acrylate did not induce mutation in five histidine requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested at concentrations up to 5000 µg/plate (the maximum recommended concentration according to current regulatory guidelines), in the absence and in the presence of a rat liver metabolic activation system (S-9).

In vitro gene mutation test in mammalian cells (HPRT, OECD 476):

Isodecyl 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 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 ethanol.

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

Accordingly, for Experiment 1, thirteen concentrations, ranging from 5 to 80 µg/mL in the absence of S-9 and 10 to 140 mg/mL in the presence of S-9, were tested. Seven days after treatment the highest concentrations analysed to determine viability and 6TG resistance were 35 µg/mL in the absence of S-9 and 80 µg/mL in the presence of S-9, which gave 2% and 7% RS, respectively. Steep concentration-related toxicity was observed between 30 and 35 µg/mL (37% and 2% RS, respectively) in the absence of S-9 and between 70 and 80 mg/mL (33% and 7%, respectively) in the presence of S-9, therefore both concentrations were analysed in each case.

In Experiment 2,twelve concentrations, ranging from 10 to 50 µg/mL in the absence of S-9 and fourteen concentrations, ranging from 20 to 140 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 105 µg/mL in the presence of S-9, which gave 19% and 20% RS, respectively.

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

In Experiments 1 and 2 no statistically significant increases in mutant frequency were observed following treatment with Isodecyl Acrylate at any concentration tested in the absence and presence of S-9 and there were no significant linear trends. Due to the steep toxicity profile observed, highly toxic concentrations giving <10% RS were analysed in Experiment 1 in the absence and presence of S-9. However, in the absence of any significant increase in mutant frequency at these concentrations or significant linear trends, the data were considered valid and the integrity of the study unaffected.

It is concluded that Isodecyl Acrylate did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to toxic concentrations in two independent experiments in the absence and presence of a rat liver metabolic activation system (S-9).

In vitro cytogenicity test in mammalian cells (micronucleus assay, OECD 487):

Isodecyl acrylate was tested in an in vitro micronucleus assay using duplicate human lymphocyte cultures prepared from the pooled blood of two male donors in a single experiment. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9) from Aroclor 1254 induced animals. The test article was formulated in ethanol and the highest concentrations tested in the Micronucleus Experiment, (limited by toxicity), were determined following a preliminary cytotoxicity Range Finder Experiment.

Treatments were conducted 48 hours following mitogen stimulation by phytohaemagglutinin (PHA). The test article concentrations for micronucleus analysis were selected by evaluating the effect of Isodecyl acrylate on the replication index (RI). In the Micronucleus Experiment, micronuclei were analysed at three concentrations.

Appropriate negative (vehicle and untreated) control cultures were included in the test system under each treatment condition. Mitomycin C (MMC) and Vinblastine (VIN) were employed as clastogenic and aneugenic positive control chemicals respectively in the absence of rat liver S-9. According to the results of negative and positive control groups, the study was therefore considered as valid.

Treatment of cells with Isodecyl acrylate in the absence and presence of S-9 resulted in frequencies of MNBN cells which were similar to and not significantly (p = 0.05) higher than those observed in concurrent vehicle controls for all concentrations analysed (all treatments). The MNBN cell frequency of the majority of Isodecyl acrylate treated cultures fell within normal ranges. The single exception to this was noted at the lowest concentration analysed (25 µg/mL) post 3+21 hour +S-9 treatment (Micronucleus Experiment, Trial 1) where a single culture demonstrated numbers of MNBN cells that marginally exceeded the normal range. As this increase was not observed in the replicate culture, or for any other concentration analysed (all treatments), this was considered spurious and of no biological importance.

It is concluded that Isodecyl acrylate did not induce micronuclei in cultured human peripheral blood lymphocytes following treatment in the absence and presence of an Aroclor induced rat liver metabolic activation system (S-9). Maximum concentrations analysed resulted in 51%, 54% and 66% cytotoxicity for 3+21 hour –S-9, 3+21 hour +S-9 and 24+24 hour –S-9 treatments respectively.

Justification for selection of genetic toxicity endpoint
All three in vitro recommended tests were performed on isodecyl acrylate. All these tests were reliable and showed negative results.

Justification for classification or non-classification

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