In vitro genotoxicity studies: n-Butyl acrylate L5178Y mouse lymphoma (TK+/− locus assay), 2-Ethylhexyl acrylate gene mutation assay in Chinese hamster V79 cells, and 2-Ethylhexyl acrylate micronucleus test in human lymphocytes

Available point mutation tests have shown inconsistent results with various acrylates. Most of those tests were performed prior to OECD guidelines and appropriate data regarding cytotoxicity are not given. Data from three current OECD guideline compliant experiments conducted under GLP are provided. They include (a) an in vitro mouse lymphoma (TK+/−) assay (OECD 490) [3], (b) an in vitro HPRT locus gene mutation assay utilizing cultures of Chinese hamster V79 cells (OECD 476) [1], and (c) an in vitro micronucleus test in human lymphocytes (OECD 487) [2]. Test materials were not mutagenic under these experimental conditions, adding to the weight-of-evidence of non-genotoxicity for this group of chemicals.


a b s t r a c t
Available point mutation tests have shown inconsistent results with various acrylates. Most of those tests were performed prior to OECD guidelines and appropriate data regarding cytotoxicity are not given. Data from three current OECD guideline compliant experiments conducted under GLP are provided. They include (a) an in vitro mouse lymphoma (TKþ / À ) assay (OECD 490) [3], (b) an in vitro HPRT locus gene mutation assay utilizing cultures of Chinese hamster V79 cells (OECD 476) [1], and (c) an in vitro micronucleus test in human lymphocytes (OECD 487) [2]. Test materials were not mutagenic under these experimental conditions, adding to the weight-of-evidence of non-genotoxicity for this group of chemicals. &

Value of the data
Genotoxicity is an important determinant in the mode of action of a chemical and important in human hazard assessments, such as that recently conducted on 2-Ethylhexyl acrylate-induced skin tumorigenesis [1].
Older genotoxicity tests showed inconsistent results with various acrylates. Most of those tests were performed prior to OECD guidelines and appropriate data regarding cytotoxicity are not given.
Three new in vitro genotoxicity studies conducted according to current OECD guidelines (i.e., mouse lymphoma-TG 490 [2], HPRT-TG 476 [3], and micronucleus-TG 487 [4]) did not show genotoxic activity under these experimental conditions, adding to the weight-of-evidence of nongenotoxicity for this group of chemicals.
1. Data, experimental design, materials, and methods

n-Butyl acrylate mouse lymphoma assay
An in vitro gene mutation test in L5178Y mouse lymphoma cells was conducted under GLP according to OECD Guideline 490 [2], to evaluate the ability to induce gene mutations at the thymidine kinase (TK) locus or structural chromosome aberrations at chromosome 11 in L5178Y TKþ/ À mouse lymphoma cells with the microwell method.
Metabolic activation: Phenobarbital and β-naphthoflavone induced rat S9 fraction was prepared at the laboratory and frozen until needed; fresh S9 mix was prepared prior to each experiment.
Cell culture: L5178Y TKþ /À mouse lymphoma cells were removed from frozen stock; thawed and incubated in medium for a day, resuspended in fresh medium and incubated until used.
Treatment: Cells were treated with the test substance for 4 and 24 h without S9 and 4 h with S9. Cells were then cultured for an expression period of about 48 h and then cultured in selection medium for approx. 10 days, after which the number of large and small colonies was determined.
In this study all incubations were performed at 37°C with a relative humidity of Z 90% in a 5% (v/v) CO 2 atmosphere.
Scoring and data analysis: The number of empty wells of the 96-well plates was scored and recorded. To account for any loss of cells during the 4-h treatment, a relative growth during treatment factor (RGDT, %) was calculated by comparing the growth of each treated culture relative to the control. The colonies were classified into large colonies (indication of gene mutation) and small colonies (indication of chromosome breakage). An appropriate statistical method to test for linear trend was performed to assess a possible linear dose-relation in mutant frequencies and judged statistically significant whenever the one-sided p-value (probability value) was o 0.05 and the slope 4 0. Both biological and statistical significance have been considered together.
The cloning efficiency (CE, %) was calculated for each test group as follows: CEx ¼ À ln ½total number of empty wells=total number of seeded wells number of seeded cells per well Â 100 RCEx ¼ ½CEx of the test group=CEx of the vehicle control Â 100 Positive and negative controls reacted as expected supporting test validity. Cytotoxicity indicated by reduced relative total growth (RTG) of below 20% of control was observed in all experiments in the absence and presence of metabolic activation, except in the first experiment with metabolic activation. A third experiment was added to comply with current guidelines. nBA did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after adding a metabolizing system in three experiments performed independently of each other. Linear dose response relationships determined to be statistically significant were biologically irrelevant if the corrected mutant frequencies did not exceed the corresponding global evaluation factor. (Tables 1-3). Positive controls: Ethylmethane sulfonate 99% (EMS) (CAS# 62-50-0) in nutrient medium; 7,12dimethylbenz(a)anthracene Z 95% (DMBA) (CAS# 57-97-6) in DMSO (CAS # 67-68-5) vehicle (final concentration in nutrient medium 0.5%).
Metabolic activation: phenobarbital/β-naphthoflavone induced rat liver S9 supernatant mixed with S9 cofactor solution for a final protein concentration of 0.75 mg/mL in the cultures.
Cell cultures: The V79 cell line was obtained from the stored cell bank of the test laboratory. For seeding of the cell cultures, the complete culture medium was minimal essential medium containing Hanks salts, neomycin (5 mg/mL), 10% fetal bovine serum (FBS), and amphotericin B (1%). During treatment no FBS was added to the medium. For the selection of mutant cells the complete medium was supplemented with 11 mg/mL 6-thioguanine. All incubations were done at 37°C with 1.5% carbon dioxide (CO 2 ) in humidified air.
Doses were selected based on pretests in the presence and absence (4 h treatment) of metabolic activation.
Scoring and data analysis: A linear regression 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.
Treatment: Test item concentrations between 14.4 mg/mL and 1843.0 mg/mL ( $ 10 mM) were used. Due to phase separation the following test concentrations were selected: without metabolic activation: 14.4, 28.8, 57.6, and 115.2 mg/mL; with metabolic activation: 14.4, 28.8, 57.6, 115.2, and 230.4 m g/mL. No relevant cytotoxic effect occurred up to the highest concentration with and without metabolic activation. A single cell suspension was prepared from phosphate buffered saline (PBS) rinsed cells, then trypsinized in complete culture medium with 10% FBS. The cells were grown for 24 h prior to treatment. After 24 h the medium was replaced with serum-free medium containing the test item, with or without S9. Concurrent solvent and positive controls were treated in parallel. 4 h after treatment, this medium was replaced with complete medium following two washing steps with PBS. Immediately after treatment the cells were trypsinized and sub-cultivated, and then again in 3 days before final seeding of cultures that were stained and evaluated. Cloning efficiency was evaluated after each subcultivation using additional flasks seeded 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 for about 8 days. The colonies were stained with 10% methylene blue in 0.01% KOH solution. The stained colonies with more than 50 cells were counted.
No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiment. Positive controls induced a distinct increase in mutant colonies and, Values indicating a statistically significant trend are printed in bold characters. * The linear trend-test testing for an increased mutant frequency is significant (significance level of 5%), if the one-sided pvalue is lower than 0.05 and the slope is greater than 0.
thus, showed the sensitivity of the test system and the activity of the metabolic activation system Tables 4-6.

2-Ethylhexyl acrylate in vitro mammalian cell micronucleus test in human lymphocytes
An in vitro genotoxicity experiment was conducted using 2EHA, under GLP and in accordance with OECD Guideline 487 [4]: in vitro mammalian cell micronucleus test assay to investigate the potential to induce micronuclei associated with chromosome damage in cultures of human lymphocyte cells.
Metabolic activation: Phenobarbital/β-naphthoflavone induced rat liver S9 supernatant mixed with S9 cofactor solution for a final protein concentration of 0.75 mg/mL in the cultures.
Treatment: All incubations were done at 37°C with 5.5% CO 2 in humidified air. Preparation time for all trials was 40 h: 4 h þ16 h recovery, or 20 h continuous exposure, followed by 20 h of Cytochalasin B (CAS# 14930-96-2) exposure.
Scoring and data analysis: At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. The frequency of micronucleated cells was reported as % micronucleated cells. 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.   groups compared to the solvent controls. A trend is judged as significant whenever the p-value (probability value) is below 0.05. Biological and statistical significance were considered together. Interpretation of results: Test substance is not clastogenic and non-aneugenic if: (a) no concentration exhibits a statistically significant increase compared with the concurrent solvent control; (b) there is no concentration-related increase; and (c) results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control.
It is clastogenic and aneugenic if: (a) at least one test item concentration is statistically significantly increased compared with the concurrent solvent control; (b) a concentration-related increase occurs in at least one experiment trial; and (c) the results are outside the range of the laboratory historical solvent control data.
Results: Toxicity tests conducted using 10 concentrations showed phase separation occurred at the end of treatment in experimental trials: 4 h exposure time point Z 44.9 mg/mL (À S9) and Z 241 mg/ mL ( þ S9) (Table 7); 20 h exposure time point Z 250 mg/mL ( À S9) (Table 8). No cytotoxicity was observed in the 4 h trials up to highest evaluated concentration which showed phase separation. In    the 20 h experiment in the À S9, moderate cytotoxicity was observed at the highest evaluated concentration. No clear cytotoxic effects were able to be evaluated for cytogenetic damage. The micronuclei evaluation (Table 9) showed that in the absence of S9, two values at the 4 h time point were statistically significantly increased (8.4 and 44.9 mg/mL). 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. At the 20 h time point in the absence of S9 after continuous treatment, and at the 4 h time point in the presence of S9 after pulse treatment, no relevant increases in the numbers of micronucleated cells were observed after treatment with 2-ethylhexyl acrylate.

Transparency document. Supplementary material
Transparency document associated with this article can be found in the online version at https:// doi.org/10.1016/j.dib.2018.06.008.