2-ethylhexyl acrylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

2-EHA was negative in bacterial mutation tests, in the micronucleus assay in cultured human lymphocytes and in the gene mutations assay at the HPRT locus in V79 cells.

 

In-vitro studies: Bacterial systems

Three valid bacterial gene mutation assays are available (NTP 1982, 1984, Rohm & Haas 1979). Assays were negative at doses up to 10000 µg/plate with and without S-9 mix in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 97, TA 98, TA 100, TA 1535, respectively. All tests were conducted according to older test guidelines therefore testing in Salmonella typhimurium strain TA 102 or Escherichia coli strain WP2 uvrA was not included in any of the available studies. Nonetheless, since all three tests were negative with and without metabolic activation, there is no indication for a mutagenic potential of the test substance in bacterial cells.

 

In-vitro studies: Mammalian cell gene mutation test

In a key study, the potential of 2-EHA to induce gene mutations at the HPRT locus was investigated in V79 cells of the Chinese hamster (Sokolowski, 2019). The assay was performed in one experiment using two parallel cultures. The main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The maximum test item concentration of the pre-experiment and the main experiment (1843.0 µg/mL) was chosen with respect to the current OECD guideline 476 regarding the molecular weight of the test item. The test item was dissolved in ethanol. The main experiment was evaluated for mutagenicity at the following concentrations:

without metabolic activation: 14.4; 28.8; 57.6; and 115.2 µg/mL

with metabolic activation: 14.4; 28.8; 57.6; 115.2; and 230.4 µg/mL

Phase separation was noted after 4 hours treatment at 115.2 µg/mL and above in the absence of metabolic activation, and at 230.4 µg/mL and above in the presence of metabolic activation. No relevant cytotoxic effect indicated by an adjusted cloning efficiency I (referring to the mean values) below 50% occurred up to the highest concentration scored for gene mutations with and without metabolic activation. No biologically relevant increase in mutant colony numbers was observed in the main experiment up to the maximum concentration scored for gene mutations. The mutant frequencies obtained in the main experiment (mean values) with and without S9 mix for the solvent controls were in the range from 21.3 to 21.7 mutants per 10e6 cells. The values were well within the 95% confidence interval of our laboratory’s historical negative control data and, thus, fulfilled the requirements of the current OECD Guideline 476. The range of the mutant frequencies (mean values) of the groups treated with the test item was from 20.6 up to 28.8 mutants per 10e6 cells. Referring to the mean values no statistical significant linear regression was observed. Furthermore no increase in the number of mutant colonies (mean values) above the 95% confidence interval was observed. EMS (300 µg/mL) and DMBA (2.3 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

2-EHA is considered to be non-mutagenic in this HPRT assay.

 

Two former investigations were conducted on induction of HGPRT mutations in CHO cells. According to Union Carbide Co. (1980) 2-EHA did not induce HGPRT mutations after 5-hour treatment with or without S-9 mix. Doses of 3.13 to 50E-5 % (v/v; with S-9 mix) or 6.25 to 100E-5 % (without S-9 mix) were used; the maximum doses correspond to 5 and 10 nL/mL; only minor toxicity was seen.

In a 2nd investigation, cells were treated with doses of 5 to 80 µg/mL (monolayer assay) or 14 to 26 µg/mL (suspension assay) in the absence of S-9 mix (Moore et al. 1991). Whereas the suspension assay was clearly negative for all tested doses up to extreme toxicity, sporadic increases in mutation frequency were seen in the monolayer assay. In a 1st experiment, weak effects were seen at doses of 35 and 40 µg/mL which led to less than 20 % relative survival. In the 2nd experiment doses of 60 and 70 µg/mL resulted in slightly increased mutation frequencies, toxicity was moderate (33 % relative survival). Higher doses of 75 and 80 µg/mL were negative (40 and 7 % relative survival).

 

In conclusion, 2-EHA seems to have a low potential for induction of gene mutations in mammalian cells. Since the genetic effects were limited to doses with strong cytotoxicity, the potential will probably not be expressed in vivo.

 

Two former investigations were conducted on induction of TK mutationsin mouse lymphoma cells. Rohm & Haas (1984) reported on a mouse lymphoma assay which was weakly positive at doses in the toxic range. Doses of 15.6 to 150 nL/mL (with S-9 mix from Aroclor-induced rat livers) or 1.95 to 60 nL/mL (without S-9 mix) were tested in a 4-hour treatment. Effects were slightly more pronounced with S-9 mix. Here, combining the data from two experiments, more than a doubling of the mutation frequency was achieved at doses ranging from 70 to 150 nL/mL, increases were 2.2- to 4.6-fold and were accompanied by moderate to strong cytotoxicity (relative growth varied from 4.8 to 54.6 % without clear dose-dependency). Without S-9 mix, combining the data from 3 experiments, more than 2-fold increases in mutation frequencies were obtained for doses of 15.6 to 60 nL/mL, maximum increase was 2.9-fold at the highest dose with only 8.5 % relative survival.Another mouse lymphoma assay, with 4-hour treatment without S-9 mix, was reported by Dearfield et al. (1989). Slightly positive effects were obtained in high doses with strong cytotoxicity. Doses of 30, 31, 32, 33 and 34 nL/mL were tested in three independent experiments; according to combined data, mutation frequencies increased by factors of 1.6 to 1.9 without dose-effect relationship; relative survival ranged from 11 to 20.3 %. 2-Ethylhexyl acrylate produced an equivocal result for an increased mutant frequency. While a doubling of the mutant background frequency was seen at several tested concentrations, e.g. 31 ug/mL (experiment 2), other, sometimes higher concentrations and repeat experiments did not induce a consistent increase. No clear dose-response pattern was discerned.

 

In-vitro studies: chromosomal aberration and micronucleus tests

In a key study, 2-EHA, dissolved in ethanol (Exp. IA) or acetone (Exp. IB, IIA and IIB), was assessed for its potential to induce micronuclei in human lymphocytes in vitro in four independent experiments (Maumann, 2019).The following study design was performed:

In each experimental group two parallel cultures were analyzed. Per culture at least 1000 binucleated cells were evaluated for cytogenetic damage. The highest applied concentration in this study (1843µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 487.

Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item phase separation in accordance with OECD Guideline 487.

In Experiment IA in the absence and presence of S9 mix with ethanol as solvent, no cytotoxicity was observed up to the highest evaluated concentrations (138 and 241µg/mL, respectively), which showed phase separation. In Experiment IB in the absence of S9 mix with acetone as solvent, a non-dose responsive cytotoxicity was observed. Whereas a moderate level of cytotoxicity (39.0 % cytostasis) was observed after treatment with 14.7µg/mL, the next two higher tested concentrations (25.7 and 44.9µg/mL) showed no cytotoxicity (8.0 and 0.9 % cytostasis). The highest evaluated concentration (44.9µg/mL) showed phase separation. In Experiment IIA in the absence of S9 mix with acetone as solvent, moderate cytotoxicity (37.5% cytostasis) was observed at the highest evaluated concentration. The next higher tested concentration, however, which were separated by a smaller factor than requested by the guideline showed extensive cytotoxicity (76.2% cytostasis) and were not evaluable for cytogenetic damage. In Experiment IIB in the presence of S9 mix with acetone as solvent, no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation.

In Experiment IA in the absence of S9 mix, all values (1.35%, 1.88% and 1.58% micronucleated cells) exceeded the 95% control limit of the historical control data (0.06 – 1.19% micronucleated cells). The values of the two highest tested concentrations (78.6 and 138µg/mL) were statistically significantly increased. Dose dependency tested via trend test was not statistically significant. To verify the positive outcome in Experiment IA a confirmatory experiment was performed. In the confirmatory Experiment IB in the absence of S9 mix, two values (1.10% and 0.90%) after treatment with 8.4 and 44.9µg/mL were statistically significantly increased. Both values were clearly within the 95% control limit of the historical control data (0.06 – 1.19 % micronucleated cells) and dose dependency tested via trend test was not statistically significant. Therefore, this finding was regarded as biologically irrelevant. If Experiment IA and IB are considered together, the experimental arm pulse treatment without S9 mix is considered to be non-mutagenic.

In Experiment IA in the presence of S9 mix, the value of 1.50 % micronucleated cells after treatment with the lowest tested concentration (78.6µg/mL) exceeded the 95% control limit of the historical control data (0.08 – 1.38 % micronucleated cells). This value was statistically significantly increased but dose dependency tested via trend test was not statistically significant. Therefore, this finding was regarded as biologically irrelevant. To verify this result, again a confirmatory experiment was performed. In Experiment IIB in the presence of S9 mix after pulse treatment, no relevant increases in the numbers of micronucleated cells were observed after treatment with the test item. Therefore, the experimental arm pulse treatment with S9 mix is considered to be non-mutagenic.

In Experiment IIA in the absence of S9 mix after continuous treatment, no relevant increases in the numbers of micronucleated cells were observed after treatment with the test item and therefore considered to be non-mutagenic.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

In conclusion, under these experimental conditions, the test item did not induce micronuclei as determined by thein vitromicronucleus test in human lymphocytes.

Therefore, 2-EHA is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to phase separating or to the highest evaluable concentrations.

 

In mouse lymphoma cells, parallel investigations of structural chromosomal aberrations and micronuclei were performed by Dearfield et al. (1989). Treatment with doses of 20, 25, 31 and 34 µg/mL 2-EHA was done for 4 hours, without S-9 mix. All treatments resulted in strong cytotoxicity (27, 16, 12 and 12 % relative survival).

For analysis of chromosomal aberrations, cultures were exposed to BrdUrd after treatment and were sampled 14 to 15 hours after start of treatment. This was to enable the selection of 1st-division mitoses for analysis; however, co-treatment with BrdUrd, which is a genotoxin, is not recommended by the guidelines and makes findings difficult to interpret. 100 mitoses were analysed per experimental point. Aberration frequencies in treated cultures varied from 5 to 9 % (negative control, 4 %). Given the various methodological insufficiencies, the findings are evaluated as inconclusive.

For analysis of micronuclei, cultures were exposed to cytochalasin B (3 µg/mL) after treatment; sampling was 16 to 17 hours after start of treatment; 1000 cells were analysed per experimental point. The micronucleus frequency was 1.2 % in the negative control and varied from 0.8 to l.l % in the treated cultures, i.e. the result was negative.

In conclusion, there is no relevant evidence for clastogenicity of 2-EHA.

 

In vitro - mammalian cell indicator tests

A test for induction of unscheduled DNA synthesis (UDS) with primary rat hepatocytes was performed with the “liquid scintillation counting” methodology which is known to be quite insensitive (Union Carbide, 1980). A single experiment was conducted. The overall result for this test was negative for doses up to 100E-5 % (10 nL/mL).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Basic data given: scientifically acceptable study summary including result tabels; the test protocol can be viewed online. The quality of the NTP studies is generally good and accepted within the toxicological community. Neither Escherichia coli strain WP2 uvrA pKM101a nor S. typhimurium strain TA102 were employed in this study for the detection of oxidising mutagens and cross-linking agents.

Principles of method if other than guideline:

The study was conducted according to the Standard NTP protocol (Preincubation Assay) which is comparable to the method described in the publication by Mortelsman and Zeiger (2000).

Mortelmans K, Zeiger E (2000). The Ames Salmonella/microsome mutagenicity assay. Mutat Res. 455(1-2): 29-60

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidin auxotrophy

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 from Aroclor 1254-induced male Sprague-Dawley rat and Syrian hamster liver

Test concentrations with justification for top dose:

0, 3.3, 10, 33, 100, 333, 1000, 3333, 10000 ug/plate

Vehicle / solvent:

dimethylsulfoxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethylsulfoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without activation: for TA1535 sodium azide, for TA100 sodium azide, for TA98 4-nitrophenylenediamine, for TA1537 9-aminoacridine; With activation: for all strains 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation


METABOLIC ACTIVATION
The test was conducted with and without metabolic activation. In this study metabolic activation enzymes and cofactors from Aroclor 1254 -induced male Sprague-Dawley rat and Syrian hamster liver were used. The percentage of S9 in the S9 mixture that was then added to cultures, was 10 %.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: TA1537: 333 ug/plate; TA1535 and TA100: 10000 ug/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

TA1535:

			Mean number of revertants

Dose		No activation	10 % HLI	10 % RLI	
[ug/plate]	1st	2nd	1st	2nd	1st	2nd
--------------------------------------------------------------
0		13	30	13	23	8	19

100		11	23	12	18	13	21
333		9	22	11	15	9	20
1000		11	25	10	17	8	26
3333		13	20	8	14	9	24
10000		T	T	T	17	T	17

Pos. control	1071	1114	201	439	552	537
--------------------------------------------------------------

TA100:

			Mean number of revertants

Dose		No activation	10 % HLI	10 % RLI	
[ug/plate]	1st	2nd	1st	2nd	1st	2nd
--------------------------------------------------------------
0		130	166	159	238	164	193

100		138	171	118	255	134	227
333		126	163	103	223	165	202
1000		115	153	114	178	159	217
3333		112	152	108	183	176	193
10000		T	144	75	189	152	178

Pos. control	1632	358	1320	1321	906	2114
--------------------------------------------------------------

TA98:

			Mean number of revertants

Dose		No activation	10 % HLI	10 % RLI	
[ug/plate]	1st	2nd	1st	2nd	1st	2nd
--------------------------------------------------------------
0		19	15	28	33	27	21

100		15	15	31	33	21	28
333		14	14	26	33	25	28
1000		13	13	23	32	21	25
3333		16	10	21	31	17	25
10000		18	9	19	22	16	22

Pos. control	572	430	1058	924	1069	2119
--------------------------------------------------------------

TA1537:


			Mean number of revertants

Dose		No activation		10 % HLI	10 % RLI	
[ug/plate]	1st	2nd	3rd	1st	2nd	1st	2nd
---------------------------------------------------------------------
0		9	9	14	12	29	11	26

3.3			11	13
10			9	12
33			5	4
100		7	5	8	15	22	11	23
333		T	T	T	7	19	10	17
1000		T			7	15	13	19
3333		T			9	18	9	14
10000		T			6	19	8	14

Pos. control	199	730	1000	1341	413	382	196
---------------------------------------------------------------------
RLI: Rat Liver S9
HLI: Hamster Liver S9
s: slight toxicity
T: Toxic

Conclusions:

negative

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Basic data given: scientifically acceptable study summary including result tabels; the test protocol can be viewed online. The quality of the NTP studies is generally good and accepted within the toxicological community. Neither Escherichia coli strain WP2 uvrA pKM101a nor S. typhimurium strain TA102 were employed in this study for the detection of oxidising mutagens and cross-linking agents.

Principles of method if other than guideline:

The study was conducted according to the Standard NTP protocol (Preincubation Assay) which is comparable to the method described in the publication by Mortelsman and Zeiger (2000).

Mortelmans K, Zeiger E (2000). The Ames Salmonella/microsome mutagenicity assay. Mutat Res. 455(1-2): 29-60

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine auxotrophy

Species / strain / cell type:

other: Salmonella typhimurium TA 1535, TA 100, TA 97, TA 98

Metabolic activation:

with and without

Metabolic activation system:

S9 from Aroclor 1254-induced male Sprague-Dawley rat and Syrian hamster liver

Test concentrations with justification for top dose:

0, 0.3, 1.0, 3.3, 10, 20, 33, 100, and 200 ug/plate

Vehicle / solvent:

Vehicle: dimethylsulfoxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethylsulfoxide

True negative controls:

no

Positive controls:

yes

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation


METABOLIC ACTIVATION
The test was conducted with and without metabolic activation. In this study metabolic activation enzymes and cofactors from Aroclor 1254-induced male Sprague-Dawley rat and Syrian hamster liver were used. The percentage of S9 in the S9 mixture that was then added to cultures, was 30 % and 10 %, respectively.

Species / strain:

other: Salmonella typhimurium TA 1535, TA 100, TA 97, TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Without: 20 ug/plate; with: 100-200 ug/plate depending on the strain

Vehicle controls validity:

valid

Positive controls validity:

valid

TA1535:

					Mean number of revertants

Dose		No activation	10 % HLI	30 % HLI	10 % RLI	30 % RLI
[ug/plate]	1st	2nd
-----------------------------------------------------------------------------------------
0		27	18	8		12		13		14

0.3		29	24	
1.0		33	18
3.3		29	11	14		13		10		16
10		35	17	11		11		14		15
20		19s	6s		
33				10		9		16		12
100				11		10s		13		12
200				12s		13s		12s		12s

Pos. control	1002	899	89		125		97		88
-----------------------------------------------------------------------------------------

TA100:

					Mean number of revertants

Dose		No activation	10 % HLI	30 % HLI	10 % RLI	30 % RLI
[ug/plate]	1st	2nd
-----------------------------------------------------------------------------------------
0		89	89	84		102		80		88

0.3		79	84	
1.0		85	92
3.3		81	85	68		90		71		111
10		80	86	84		110		74		129
20		33s	70s		
33				70		108		61		107
100				74		96		68		105
200				54s		96		64s		121

Pos. control	808	911	803		861		978		351
-----------------------------------------------------------------------------------------

TA97:

					Mean number of revertants

Dose		No activation	10 % HLI	30 % HLI	10 % RLI	30 % RLI
[ug/plate]	1st	2nd
-----------------------------------------------------------------------------------------
0		110	101	133		160		132		158

0.3		103	73	
1.0		110	99
3.3		90	20	135		161		148		165
10		102	38s	129		169		129		178
20		82s	68s		
33				126		149		146		105
100				123		153s		150		97s
200				112s		116s		115s		111s

Pos. control	848	837	788		711		1027		406
-----------------------------------------------------------------------------------------

TA98:


					Mean number of revertants

Dose		No activation	10 % HLI	30 % HLI	10 % RLI	30 % RLI
[ug/plate]	1st	2nd
-----------------------------------------------------------------------------------------
0		19	42	33		48		35		47

0.3		17	35	
1.0		18	35
3.3		20	42	35		60		29		51
10		14	45	25		46		29		55
20		14s	28s		
33				33		56		33		52
100				31		46		38		50
200				32		40		33		52s

Pos. control	1710	1565	757		782		1176		242
-----------------------------------------------------------------------------------------
RLI: Rat Liver S9
HLI: Hamster Liver S9
s: slight toxicity

Conclusions:

negative

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

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 using the Hprt and xprt genes)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

HPRT (hypoxanthine-guanine phosphoribosyl transferase)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Phenobarbital/â-naphthoflavone induced rat liver S9
- method of preparation of S9 mix: An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. The S9 fraction will be used at concentrations of approx. 2% v/v in final test medium. S9 mix contained MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4).
- concentration or volume of S9 mix and S9 in the final culture medium: 50 µL/mL S9 mix
- quality controls of S9: Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test. The protein concentration of the S9 preparation was 32.7 mg/mL (Lot. No.: 270717) in the pre-experiment and in the main experiment

Test concentrations with justification for top dose:

14.4, 28.8, 57.6, 115.2, 230.4, 460.8, 921.5, 1843.0 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol, purity 99.99%
- Justification for choice of solvent/vehicle: , The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.
- Justification for percentage of solvent in the final culture medium: The final concentration of ethanol in the culture medium was 0.5% (v/v)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate)
- Number of independent experiments

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: Two to four days after sub-cultivation stock cultures were trypsinized at 37 °C for approximately 5 to 10 minutes. Then the enzymatic digestion was stopped by adding complete culture medium with 10% FBS and a single cell suspension was prepared. The trypsin concentration for all sub-culturing steps was 0.2% in saline.
Prior to the trypsin treatment the cells were rinsed with PBS. Approximately 0.7 to 1.2×107 cells were seeded in plastic flasks. The cells were grown for 24 hours prior to treatment.
- Exposure duration/duration of treatment: After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 µl/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. 4 hours after treatment, this medium was replaced with complete medium following two washing steps with PBS.
- Harvest time after the end of treatment (sampling/recovery times): Immediately after the end of treatment the cells were trypsinised as described above and sub-cultivated. At least 2.0x10e6 cells per experimental point (concentration series plus controls) were subcultured in 175 cm2 flasks containing 30 mL medium.
Two additional 25 cm² flasks were seeded per experimental point with approx. 500 cells each to determine the relative survival (RS) as measure of test item induced cytotoxicity. The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2.
The colonies used to determine the relative survival (RS) were fixed and stained 6 to 8 days after treatment as described below.
Three days after first sub-cultivation approximately 2.0×10e6 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium.

FOR GENE MUTATION:
- Selection time (if incubation with a selective agent): Following the expression time of 6 days five 75 cm² cell culture flasks were seeded with about 4 to 5×105 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability (cloning efficiency II).
The cultures were incubated at 37 °C in a humidified atmosphere with 1.5% CO2 for about 8 days.
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure.: 6-thioguanine, no other data
- Method to enumerate numbers of viable and mutants cells: The colonies were stained with 10% methylene blue in 0.01% KOH solution.
The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
A pre-test was performed in order to determine the toxicity of the test item. In addition the pH-value and the osmolarity were measured in the solvent control and in the highest concentration. The general culturing and experimental conditions in this pre-test were the same as described below for the mutagenicity experiment.
In this pre-test approximately 1.5 million cells were seeded in 25 cm² flasks 24 hours prior to treatment. After approximately 24 hours the test item was added and the treatment proceeds for 4 hours (with and without metabolic activation) (duplicate cultures per concentration level).
Immediately after treatment the test item was removed by rinsing with PBS. Subsequently, the cells were trypsinized and suspended in complete culture medium. After an appropriate dilution the cell density was determined with a cell counter. Toxicity of the test item is evident as a reduction of the cell density compared to a corresponding solvent control. A cell density of approximately 1.5 million cells in 25 cm² flasks is about the same as approximately 10 million cells seeded in 175 cm² bottles 24 hours prior to treatment with the main experiment.

Rationale for test conditions:

The pre-experiment was performed in the presence and absence (4 h treatment) of metabolic activation. Test item concentrations between 14.4 µg/mL and 1843.0 µg/mL were used. The highest concentration of the pre-experiment was chosen with respect to the current OECD guideline 476 (2016) regarding the molecular weight of the test item (184.28 g/mol).
No relevant cytotoxic effect, indicated by a relative cloning efficiency of approx. 50% or below was observed up to the highest concentration with and without metabolic activation.
In the pre-experiment the test medium was checked for precipitation or phase separation at the beginning and at the end of treatment (4 hours) prior to removal of the test item. No precipitation or phase separation occurred up to the highest concentration with and without metabolic activation.
There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.
According to the data generated in the pre-experiment the top dose of the main experiment was 1843.0 ìg/mL. The individual concentrations were spaced by a factor of 2.0.

Evaluation criteria:

A test item is classified as clearly mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).
A test item is classified as clearly non-mutagenic if, in all experimental conditions examined, all of the following criteria are met:
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the distribution of the historical negative control data (based 95% control limits).
In cases when the response is neither clearly negative nor clearly positive as described above, or in order to judge the biological relevance of a result, the data should be evaluated by expert judgement or further investigations.

Statistics:

A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
However, both, biological and statistical significance were considered together.

Linear Regression
experimental group p-value (mean of culture I and II)
without S9 mix 0.347
with S9 mix 0.475

A t-Test was not performed since all mean mutant frequencies were well within the 95% confidence interval of our laboratory’s historical negative control data.

Species / strain:

Chinese hamster Ovary (CHO)

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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The main experiment was evaluated for mutagenicity at the following concentrations:
without metabolic activation: 14.4; 28.8; 57.6; and 115.2 µg/mL
with metabolic activation: 14.4; 28.8; 57.6; 115.2; and 230.4 µg/mL
Phase separation was noted after 4 hours treatment at 115.2 µg/mL and above in the absence of metabolic activation, and at 230.4 µg/mL and above in the presence of metabolic activation.
No relevant cytotoxic effect indicated by an adjusted cloning efficiency I (referring to the mean values) below 50% occurred up to the highest concentration scored for gene mutations with and without metabolic activation.
No biologically relevant increase in mutant colony numbers was observed in the main experiment up to the maximum concentration scored for gene mutations.
The mutant frequencies obtained in the main experiment (mean values) with and without S9 mix for the solvent controls were in the range from 21.3 to 21.7 mutants per 10e6 cells. The values were well within the 95% confidence interval of the laboratory’s historical negative control data and, thus, fulfilled the requirements of the current OECD Guideline 476. The range of the mutant frequencies (mean values) of the groups treated with the test item was from 20.6 up to 28.8 mutants per 10e6 cells.
Referring to the mean values no statistical significant linear regression was observed. Furthermore no increase in the number of mutant colonies (mean values) above the 95% confidence interval was observed.
EMS (300 µg/mL) and DMBA (2.3 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Conclusions:

In conclusion, under these experimental conditions test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, 2-Ethylhexylacrylate is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The study was performed to investigate the potential of 2-Ethylhexylacrylate to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in one experiment using two parallel cultures. The main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The maximum test item concentration of the pre-experiment and the main experiment (1843.0 µg/mL) was chosen with respect to the current OECD guideline 476 regarding the molecular weight of the test item. The test item was dissolved in ethanol.

The main experiment was evaluated for mutagenicity at the following concentrations:

without metabolic activation: 14.4; 28.8; 57.6; and 115.2 µg/mL

with metabolic activation: 14.4; 28.8; 57.6; 115.2; and 230.4 µg/mL

Phase separation was noted after 4 hours treatment at 115.2 µg/mL and above in the absence of metabolic activation, and at 230.4 µg/mL and above in the presence of metabolic activation. No relevant cytotoxic effect indicated by an adjusted cloning efficiency I (referring to the mean values) below 50% occurred up to the highest concentration scored for gene mutations with and without metabolic activation. No biologically relevant increase in mutant colony numbers was observed in the main experiment up to the maximum concentration scored for gene mutations. The mutant frequencies obtained in the main experiment (mean values) with and without S9 mix for the solvent controls were in the range from 21.3 to 21.7 mutants per 10e6 cells. The values were well within the 95% confidence interval of our laboratory’s historical negative control data and, thus, fulfilled the requirements of the current OECD Guideline 476. The range of the mutant frequencies (mean values) of the groups treated with the test item was from 20.6 up to 28.8 mutants per 10e6 cells. Referring to the mean values no statistical significant linear regression was observed. Furthermore no increase in the number of mutant colonies (mean values) above the 95% confidence interval was observed. EMS (300 µg/mL) and DMBA (2.3 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

2-Ethylhexylacrylate is considered to be non-mutagenic in this HPRT assay.

Reference 4

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

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:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

other: Human lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Sex, age and number of blood donors: female donor (35 years old) for Experiment IA, female donor (31 years old) for Experiment IB, male donor (27 years old) for Experiment IIA and male donor (24 years old) for Experiment IIB.
- Whether whole blood or separated lymphocytes were used: whole blood
- Whether blood from different donors were pooled or not: no
- Mitogen used for lymphocytes: PHA

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: Blood cultures were established by preparing an 11 % mixture of whole blood in medium within 30 hrs after blood collection. The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 ìg/mL), the mitogen PHA (3 ìg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL).
All incubations were done at 37 °C with 5.5 % CO2 in humidified air.

Cytokinesis block (if used):

phytohemeagglutinine (PHA)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Phenobarbital/â-naphthoflavone induced rat liver S9
- method of preparation of S9 mix : An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. S9 mix contained MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4).
- concentration or volume of S9 mix and S9 in the final culture medium: approx. 2.5 % S9 fraction (50 ìL S9 mix/mL culture medium).
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): The protein concentration of the S9 preparation used for this study was 32.7 mg/mL (Lot no. 270717) for Experiment IA and 34.3 mg/mL (Lot no. 161117) for Experiment IIB. Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.

Test concentrations with justification for top dose:

8.4 to 1843 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol (Exp. IA) or acetone (Exp. IB, IIA and IIB)
- Justification for choice of solvent/vehicle: Stock formulations of the test item and serial dilutions were made in ethanol for Experiment IA or in acetone for Experiment IB, IIA and IIB. The change of the solvent during this study was made on request of the Sponsor. This change was justified by the Sponsor on the basis that after the initiation of this study, data were presented from other separate experiments that indicated acetone was the preferred vehicle for in vitro studies with 2-Ethylhexylacrylate. As indicated in the relevant OECD guidelines, acetone is an acceptable vehicle for use in in vitro genetic toxicology studies, although is generally only used as a vehicle in cases where water, DMSO, or ethanol, in that order, are not suitable.
- Justification for percentage of solvent in the final culture medium: 0.5%

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: demecolcine

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : 3 without S9, 2 with S9

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
- Test substance added in medium;

TREATMENT AND HARVEST SCHEDULE:
Without S9 mix With S9 mix
Exp. IA & IB Exp. IIA Exp. IA & IIB
Stimulation period 48 hrs 48 hrs 48 hrs
Exposure period 4 hrs 20 hrs 4 hrs
Recovery 16 hrs - 16 hrs
Cytochalasin B exposure 20 hrs 20 hrs 20 hrs
Total culture period 88 hrs 88 hrs 88 hrs

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance: Cytochalasin B (4 µg/mL) was added and the cells were cultured another approximately 20 hours until preparation
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored):
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): The micronuclei were counted in at least 1000 binucleate cells per culture showing a clearly visible cytoplasm area
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): not done
- Determination of polyploidy:
- Determination of endoreplication:

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.:cytokinesis-block proliferation index
- Any supplementary information relevant to cytotoxicity: To describe a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is expressed as % cytostasis. A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus.

Rationale for test conditions:

The highest treatment concentration in this study, 1843 µg/mL (approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test.

Evaluation criteria:

A test item can be classified as non-clastogenic and non-aneugenic if:
- None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
- There is no concentration-related increase
- The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data (95% control limit realized as 95% confidence interval).

A test item can be classified as clastogenic and aneugenic if:
- At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
- The increase is concentration-related in at least one experimental condition
- The results are outside the range of the laboratory historical solvent control data (95% control limit realized as 95% confidence interval).
When all of these criteria are met, the test item is then considered able to induce chromosome breaks and/or gain or loss in the test system.

Statistics:

Statistical significance was confirmed by the Chi square test (a < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.
A linear regression was performed using a validated test script of "R", a language and environment for statistical computing and graphics, to assess a possible dose dependency in the rates of micronucleated cells. The number of micronucleated cells obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
Both, biological and statistical significance were considered together.

Species / strain:

other: Human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

In Experiment IA, phase separation of the test item in the culture medium was observed at 138 µg/mL and above in the absence of S9 mix and at 241 µg/mL and above in the presence of S9 mix at the end of treatment. In addition, phase separation occurred in Experiment IB in the absence of S9 mix at 44.9 µg/mL and above, in Experiment IIA in the absence of S9 mix at 250 µg/mL and above and in Experiment IIB in the presence of S9 mix at 286 µg/mL and above at the end of treatment.
No relevant influence on osmolarity or pH was observed.
In Experiment IA in the absence and presence of S9 mix with ethanol as solvent, no cytotoxicity was observed up to the highest evaluated concentrations (138 and 241 µg/mL, respectively), which showed phase separation (Table 3). In Experiment IB in the absence of S9 mix with acetone as solvent, a non-dose responsive cytotoxicity was observed. Whereas a moderate level of cytotoxicity (39.0 % cytostasis) was observed after treatment with 14.7 µg/mL, the next two higher tested concentrations (25.7 and 44.9 µg/mL) showed no cytotoxicity (8.0 and 0.9 % cytostasis). The highest evaluated concentration (44.9 µg/mL) showed phase separation (Table 4). In Experiment IIA in the absence of S9 mix with acetone as solvent, moderate cytotoxicity (37.5% cytostasis) was observed at the highest evaluated concentration. The next higher tested concentration, however, which were separated by a smaller factor than requested by the guideline showed extensive cytotoxicity (76.2% cytostasis) and were not evaluable for cytogenetic damage (Table 5). In Experiment IIB in the presence of S9 mix with acetone as solvent, no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation (Table 6).

In Experiment IA in the absence of S9 mix, all values (1.35%, 1.88% and 1.58% micronucleated cells) exceeded the 95% control limit of the historical control data (0.06 – 1.19% micronucleated cells). The values of the two highest tested concentrations (78.6 and 138 µg/mL) were statistically significantly increased. Dose dependency tested via trend test was not statistically significant (Table 1, Table 9). To verify the positive outcome in Experiment IA a confirmatory experiment was performed. In the confirmatory Experiment IB in the absence of S9 mix, two values (1.10% and 0.90%) after treatment with 8.4 and 44.9 µg/mL were statistically significantly increased. Both values were clearly within the 95% control limit of the historical control data (0.06 – 1.19 % micronucleated cells) and dose dependency tested via trend test was not statistically significant. Therefore, this finding was regarded as biologically irrelevant (Table 1, Table 12). If Experiment IA and IB are considered together, the experimental arm pulse treatment without S9 mix is considered to be non-mutagenic.

In Experiment IA in the presence of S9 mix, the value of 1.50 % micronucleated cells after treatment with the lowest tested concentration (78.6 µg/mL) exceeded the 95% control limit of the historical control data (0.08 – 1.38% micronucleated cells). This value was statistically significantly increased but dose dependency tested via trend test was not statistically significant. Therefore, this finding was regarded as biologically irrelevant (Table 1, Table 10). To verify this result, again a confirmatory experiment was performed. In Experiment IIB in the presence of S9 mix after pulse treatment, no relevant increases in the numbers of micronucleated cells were observed after treatment with the test item (Table 1, Table 14, Table 16). Therefore, the experimental arm pulse treatment with S9 mix is considered to be non-mutagenic.
In Experiment IIA in the absence of S9 mix after continuous treatment, no relevant increases in the numbers of micronucleated cells were observed after treatment with the test item and therefore considered to be non-mutagenic (Table 1, Table 14).
Demecolcine (100 ng/mL), MMC (0.8 µg/mL) or CPA (15.0 or 17.5 µg/mL) were used as positive controls and showed distinct increases in cells with micronuclei.

Conclusions:

2-Ethylhexylacrylate is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to phase separation or to the highest evaluable concentrations.

Executive summary:

2-Ethylhexylacrylate, dissolved in ethanol (Exp. IA) or acetone (Exp. IB, IIA and IIB), was assessed for its potential to induce micronuclei in human lymphocytes in vitro in four independent experiments.The following study design was performed:

	Without S9 mix		With S9 mix
	Exp. IA & IB	Exp. IIA	Exp. IA & IIB
Stimulation period	48 hrs	48 hrs	48 hrs
Exposure period	4 hrs	20 hrs	4 hrs
Recovery	16 hrs	-	16 hrs
Cytochalasin B exposure	20 hrs	20 hrs	20 hrs
Total culture period	88 hrs	88 hrs	88 hrs

 

In each experimental group two parallel cultures were analyzed. Per culture at least 1000 binucleated cells were evaluated for cytogenetic damage. The highest applied concentration in this study (1843µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 487.

Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item phase separation in accordance with OECD Guideline 487.

In Experiment IA in the absence and presence of S9 mix with ethanol as solvent, no cytotoxicity was observed up to the highest evaluated concentrations (138 and 241µg/mL, respectively), which showed phase separation. In Experiment IB in the absence of S9 mix with acetone as solvent, a non-dose responsive cytotoxicity was observed. Whereas a moderate level of cytotoxicity (39.0 % cytostasis) was observed after treatment with 14.7µg/mL, the next two higher tested concentrations (25.7 and 44.9µg/mL) showed no cytotoxicity (8.0 and 0.9 % cytostasis). The highest evaluated concentration (44.9µg/mL) showed phase separation. In Experiment IIA in the absence of S9 mix with acetone as solvent, moderate cytotoxicity (37.5% cytostasis) was observed at the highest evaluated concentration. The next higher tested concentration, however, which were separated by a smaller factor than requested by the guideline showed extensive cytotoxicity (76.2% cytostasis) and were not evaluable for cytogenetic damage. In Experiment IIB in the presence of S9 mix with acetone as solvent, no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation.

In Experiment IA in the absence of S9 mix, all values (1.35%, 1.88% and 1.58% micronucleated cells) exceeded the 95% control limit of the historical control data (0.06 – 1.19% micronucleated cells). The values of the two highest tested concentrations (78.6 and 138µg/mL) were statistically significantly increased. Dose dependency tested via trend test was not statistically significant. To verify the positive outcome in Experiment IA a confirmatory experiment was performed. In the confirmatory Experiment IB in the absence of S9 mix, two values (1.10% and 0.90%) after treatment with 8.4 and 44.9µg/mL were statistically significantly increased. Both values were clearly within the 95% control limit of the historical control data (0.06 – 1.19 % micronucleated cells) and dose dependency tested via trend test was not statistically significant. Therefore, this finding was regarded as biologically irrelevant. If Experiment IA and IB are considered together, the experimental arm pulse treatment without S9 mix is considered to be non-mutagenic.

In Experiment IA in the presence of S9 mix, the value of 1.50 % micronucleated cells after treatment with the lowest tested concentration (78.6µg/mL) exceeded the 95% control limit of the historical control data (0.08 – 1.38 % micronucleated cells). This value was statistically significantly increased but dose dependency tested via trend test was not statistically significant. Therefore, this finding was regarded as biologically irrelevant. To verify this result, again a confirmatory experiment was performed. In Experiment IIB in the presence of S9 mix after pulse treatment, no relevant increases in the numbers of micronucleated cells were observed after treatment with the test item. Therefore, the experimental arm pulse treatment with S9 mix is considered to be non-mutagenic.

In Experiment IIA in the absence of S9 mix after continuous treatment, no relevant increases in the numbers of micronucleated cells were observed after treatment with the test item and therefore considered to be non-mutagenic.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

In conclusion, under these experimental conditions, the test item did not induce micronuclei as determined by thein vitromicronucleus test in human lymphocytes.

Therefore, 2-Ethylhexylacrylate is considered to benon-mutagenic in this in vitro micronucleus test, when tested up to phase separating or to the highest evaluable concentrations.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo, no genotoxic potential was observed in an UDS assay in rats.

 

An OECD 486-guideline study was conducted in compliance with GLP regulations on rats (BASF AG 2002). The substance 2-EHA was tested for its ability to induce DNA repair synthesis (unscheduled DNA synthesis; UDS) in vivo in rat hepatocytes. For this purpose, the test substance, dissolved in corn oil, was administered once orally to male Wistar rats at dose levels of 1000 mg/kg and 2,000 mg/kg body weight in a volume of 10 mL/kg body weight in each case. Hepatocytes were harvested 3 and 14 hours after administration of the test substance.

As a negative control, male rats were administered merely the vehicle, corn oil, by the same route, which gave frequencies of mean nuclear net grain counts within the historical control range.

The positive control chemical 2-acetylaminofluorene (2-AAF) administered once orally in a dose of 50 mg/kg body weight demonstrated the expected increase in unscheduled DNA synthesis. On the basis of the results from the present study, the single oral treatment with the test substance did not lead to an increase in the mean number of net nuclear grain counts at any dose level or exposure time in rat hepatocytes. Administration of 2-EHA did not lead to any signs of toxicity in the test animals.

Thus, under the experimental conditions of this assay, the test article 2-EHA was considered to be negative in the in vivo UDS assay using rat hepatocytes.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 June 2002 - 29 Nov 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline study conducted in compliance with GLP regulations

Qualifier:

according to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

unscheduled DNA synthesis

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Wistar (CrlGlxBrlHan:Wl)
- Source: Charles River Deutschland GmbH
- Age at study initiation: 10-12 weeks
- Weight at study initiation: about 229 g (mean)
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Housing: single
- Diet (ad libitum): Standardized pelleted feed (Ratte - Maus - Hamster Diaet, Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water (ad libitum): drinking water


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 30 - 70 %
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, corn oil was selected as the vehicle, which had been demonstrated to be suitable in the in vivo UDS assay and for which historical data are available.
- Concentration of test material in vehicle: 10 and 20 g/100 mL, respectively
- Amount of vehicle (if gavage or dermal): 10 mL

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
Dose volume was 10 mL/kg bw of a solution with a concentration of 10 g/100 mL and 20 g/100 mL, respectively.

Duration of treatment / exposure:

single

Frequency of treatment:

Male animals per sacrifice interval were treated once per gavage and livers were perfused 3 hours and 14 hours after treatment, respectively. No. of animals per dose: 3 animals per group; two groups per dose level and control. In one group perfusion of livers was performed 3 hours and in the other group 14 hours after treatment, respectively.

Post exposure period:

no

Remarks:

Doses / Concentrations:
1000 and 2000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

3

Control animals:

yes, concurrent vehicle

Positive control(s):

2-acetylaminofluorene (2-AAF)
- Route of administration: oral: gavage
- Doses / concentrations: 50 mg/kg bw suspended in corn oil in a volume of 10 mL/kg bw

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In a pretest for the determination of the acute oral toxicity, 2000 mg/kg body weight recommended as the highest dose according to the OECD Guideline were survived by all animals (male and female) without any clinical signs. Thus, only male animals were used for the main experiment and a doseof 2000 mg/kg body weight was selected as the highest dose; 1,000 mg/kg body weight were administered as further dose.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
Liver perfusion and preparation of the primary rat hepatocytes: The livers were perfused with ethylene glycol-bis(beta-amino ethyl ether) N,N,N',N',-tetraacetic acid (EGTA) solution followed by collagenase solution. After removal of the livers and isolation of the hepatocytes, the cells were washed with about 20 mL Williams medium E incomplete (WMEI) (about 20 °C) and mixed thoroughly with a pipette. The cell suspension was then filtered using sterile gauze. After centrifugation (500 rpm) for about 5 minutes, the supernatant was discarded and the pellet resuspended in about 20 ml WMEI to yield single cell suspensions.


DETERMINATION OF CELL VIABILITY
The viability of the hepatocytes was determined by the trypan blue exclusion method. In addition, the number of the isolated cells was determined. It was also examined whether significant morphological changes of the cells or a reduction of the cell material occurred after test substance treatment.


SEEDING AND ATTACHMENT
The isolated hepatocytes were seeded on coverslips on 1.9 cm2 wells containing 2 mL of attachment medium. About 400000 viable cells were seeded per well. 6 wells per animal were used for the UDS assay. After an attachment period of about 2 hours with 5 % CO2 at 37 °C and >= 90 % humidity, the medium was replaced by fresh medium to remove non-adherent cells.


LABELING
The medium was replaced by 2 mL labeling medium, and the cells were incubated with 5 % C02 at 37 °C and >= 90 % humidity for 4 hours. After the labeling period, cells were washed; then fresh medium containing 0.25 mM unlabeled thymidine was added and the cells were incubated for another 14 hours. The cells on the coverslips were then fixed with ethanol/acetic acid (3 : 1, v/v) for at least 30 minutes, rinsed 2-4 times with aqua dest. and air-dried. The dried coverslips were mounted cell side up on glass slides using Corbit-Balsam and dried overnight.


AUTORADIOGRAPHY
The slides were coated with KODAK NTB-2 photographic emulsion (at about 37 °C) for about 5-10 seconds. After drying at room temperature in the dark (lightproof boxes), the coated slides were stored in the dark with a desiccant at -20 °C for 3-12 days. Thereafter, the slide boxes were left at room temperature for at least 3 hours. The photographic emulsion was then developed with KODAK D-19 (about 15 °C), fixed in KODAK Acidofix for about 5 minutes, washed in water for about 5-10 minutes and stained with methyl green-pyronine Y. After rinsing with water and ethanol and air-drying, the slides were covered with a 2nd coverslip using Corbit-Balsam.


QUANTIFICATION OF UDS/MICROSCOPIC EVALUATION
The quantification of UDS was performed microscopically using 2 or 3 slides per test group. 25-50 cells in good morphological conditions were randomly selected per slide and examined to achieve a total number of 100 cells/animal.
For each cell, the following counts were performed with an automatic image analyzer:
- the nuclear grain (NG) count (= number of silver grains overlying the nucleus)
- the cytoplasmic grain (CG) count (= number of grains in two or three nucleusequivalent areas adjacent to the nucleus).

Evaluation criteria:

The following parameters were calculated:
-the net nuclear grain (NNG) count of each cell (= nuclear grain count minus cytoplasmic grain count; NG - CG)
- the mean nuclear grain (NG) count
- the mean cytoplasmic grain (CG) count - the mean net nuclear grain (NNG) count
- the percentage of cells in repair (cells showing net nuclear grain counts of >= 0) - the percentage of cells in repair (cells showing net nuclear grain counts of >= 5)

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The substance 2-Ethylhexylacrylate was tested for its ability to induce DNA repair synthesis (unscheduled DNA synthesis; UDS) in vivo in rat hepatocytes. For this purpose, the test substance, dissolved in corn oil, was administered once orally to male Wistar rats at dose levels of 1000 mg/kg and 2000 mg/kg body weight in a volume of 10 mL/kg body weight in each case. Hepatocytes were harvested 3 and 14 hours after administration of the test substance.

Clinical examinations:
The single oral administration of the vehicle in a volume of 10 mL/kg body weight was tolerated by all animals without any signs or symptoms. The administration of the test substance did not lead to signs of toxicity. The single administration of the positive control substance 2-AAF in a dose of 50 mg/kg body weight did not cause any evident signs of toxicity.

Cell viability:
Cell viability was not influenced by test substance treatment. Morphological changes of the cells were not obsereved, cell material was not reduced.

DNA repair activity:
As a negative control, male rats were administered merely the vehicle, corn oil, by the same route, which gave frequencies of mean nuclear net grain counts within the historical control range. The positive control chemical 2-acetylaminofluorene (2-AAF) administered once orally in a dose of 50 mg/kg body weight demonstrated the expected increase in unscheduled DNA synthesis. On the basis of the results from the present study, the single oral treatment with the test substance did not lead to an increase in the mean number of net nuclear grain counts at any dose level or exposure time in rat hepatocytes.

Perfusion 3 h after treatment:

						Test groups

Mean per group		vehicle		1000 mg/kg	2000 mg/kg	positive
 +/- SD			control						control
-------------------------------------------------------------------------------------
NG counts		6.65+/-0.86	6.14+/-0.52	5.93+/-0.71	22.89+/-5.15

CG counts		12.29+/-0.60	10.59+/-0.42	10.37+/-1.08	12.45+/-1.14

NNG counts		-5.64+/-0.27	-4.45+/-0.26	-4.44+/-0.43	10.44+/-4.43

% cells in repair	8.67+/-4.93	10.00+/-3.61	8.00+/-2.00	89.33+/-8.08
(NNG >= 0)

% cells in repair	0.33+/-0.58	0.00+/-0.00	0.00+/-0.00	72.67+/-17.21
(NNG >= 5)
-------------------------------------------------------------------------------------


Perfusion 14 h after treatment:

						Test groups

Mean per group		vehicle		1000 mg/kg	2000 mg/kg	positive
 +/- SD			control						control
-------------------------------------------------------------------------------------
NG counts		6.64+/-0.33	7.73+/-0.64	7.61+/-0.63	23.50+/-3.40

CG counts		11.45+/-1.41	11.14+/-1.25	12.60+/-0.35	11.76+/-1.14

NNG counts		-4.99+/-1.10	-3.41+/-1.30	-4.98+/-0.36	11.75+/-2.34

% cells in repair	6.33+/-3.21	18.00+/-10.39	9.33+/-0.58	94.00+/-2.00
(NNG >= 0)

% cells in repair	0.00+/-0.00	1.00+/-1.00	0.00+/-0.00	77.33+/-10.26
(NNG >= 5)
-------------------------------------------------------------------------------------
NG = nuclear grains
CG = cytoplasmic grains
NNG = net nuclear grains


Thus, under the experimental conditions of this assay, the test article 2-Ethylhexyl acrylate is considered to be negative in the in vivo UDS assay using rat hepatocytes.

Conclusions:

negative

Executive summary:

2-Ethylhexylacrylate was tested for its ability to induce DNA repair synthesis (unscheduled DNA synthesis; UDS) in vivo in rat hepatocytes. For this purpose, the test substance, dissolved in corn oil, was administered once orally to male Wistar rats at dose levels of 1,000 mg/kg and 2,000 mg/kg body weight in a volume of 10 ml/kg body weight in each case. Hepatocytes were harvested 3 and 14 hours after administration of the test substance. As a negative control, male rats were administered merely the vehicle, corn oil, by the same route, which gave frequencies of mean nuclear net grain counts within the historical control range. The positive control chemical 2-acetylaminofluorene (2-AAF) administered once orally in

a dose of 50 mg/kg body weight demonstrated the expected increase in unscheduled DNA synthesis.

On the basis of the results from the present study, the single oral treatment with the test substance did not lead to an increase in the mean number of net nuclear grain counts at any dose level or exposure time in rat hepatocytes.

2-Ethylhexylacrylat is considered to be negative in the in vivo UDS assay using rat hepatocytes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Conclusion

2-EHA was negative in bacterial mutation tests. Data from mammalian cells give no evidence for clastogenicity. In the absence of strong cytotoxicity, 2-EHA did not induce gene mutations in mammalian cells. The data from mammalian cell indicator tests did not add relevant information. In a study conducted in compliance with GLP and OECD TG 486, 2-ethylhexyl acrylate was negative in the in vivo UDS assay using rat hepatocytes. This in vivo test provides a method for investigating genotoxic effects of chemicals in the liver. The end-point measured is indicative of DNA damage and subsequent repair in liver cells. The liver is usually the major site of metabolism of absorbed compounds. It is thus an appropriate site to measure DNA damage in vivo. The lack of specific information on mutagenic activity provided by the UDS test is compensated for by the potential sensitivity of this endpoint because it is measured in the whole genome. Thus, taking the negative test result in vivo for 2-EHA into consideration, it can be assumed that 2-EHA will not cause any DNA damage, i.e. genotoxicity in vivo. Furthermore, cleavage products of 2-EHA 2-ethylhexanol and acrylic acid were also negative in in vivo mutagenicity tests.

Justification for classification or non-classification

EU classification according to Annex VI of Directive 67/548/EEC: no classification required

GHS classification (GHS UN rev.4, 2011) identical to REGULATION (EC) No 1272/2008: no classification required