2-propylheptyl methacrylate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

June 17, 2008 to August 20, 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-ethylhexyl methacrylate

EC Number:

211-708-6

EC Name:

2-ethylhexyl methacrylate

Cas Number:

688-84-6

IUPAC Name:

2-ethylhexyl methacrylate

Details on test material:

- Name of test material (as cited in study report): 2-Ethylhexyl methacrylate
- Physical state: liquid
- Analytical purity: 99.08 %
- Purity test date: 2008-04-16
- Lot/batch No.: 2027090327
- Expiration date of the lot/batch: October 21, 2008
- Storage condition of test material: At room temperature (+15 to +25 °C), protected from light

Method

Target gene:

HPRT

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM (minimal essential medium; SEROMED, 12247 Berlin, Germany) supplemented with 10 % fetal calf serum (FCS; PAA Laboratories GmbH, 35091 Cölbe, Germany).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Experiment I:
without S9 mix: 0.3; 0.5; 1.0; 2.0; and 4.0 μg/mL
with S9 mix: 62.5; 125; 250; 500; and 1000 μg/mL

Experiment II:
without S9 mix: 3.8; 7.5; 15.0; 30.0; and 45.0 μg/mL
with S9 mix: 62.5; 125; 250; 500; and 1000 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its non-toxicity to the cells. The final concentration of ethanol in the culture medium did not exceed 0.5 % v/v.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without S9 activation: Ethyl methanefulfonate (EMS); With S9 activation: 7,12-dimethylbenz(a)anthracene (DMBA)

Evaluation criteria:

The gene mutation assay is considered acceptable if it meets the following criteria:
- the numbers of mutant colonies per 106 cells found in the negative and/or solvent con-trols fall within the laboratory historical control data range of 2001 – 2007).
- the positive control substances must produce a significant increase in mutant colony frequencies.
- the cloning efficiency II (absolute value) of the negative and/or solvent controls must exceed 50 %.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT®11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant colonies 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. However, both, biological and statistical significance were considered together.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiments up to the maximum concentration.

Appropriate reference mutagens were used as positive controls and showed a distinct in-crease in induced mutant colonies and thus showed the sensitivity of the test system and the activity of the S9 mix.

The evaluated experimental points and the results are summarized in Table 2

Table 2: Summary of results

			relative	relative	mutant		relative	relative	mutant
	Conc. ug/mL	S9 mix	Cloning eff. I %	Cloning eff. II %	Colonies per 10E5 cells	Induction factor	Cloning eff. I %	Cloning eff. II %	Colonies per 10E5 cells	Induction factor
EXP I 4 hr			CULTURE I				CULTURE II
Solvent control (EtOH)	150	-	100.0	100.0	11.6	1.0	100.0	100.0	23.3	1.0
Pos control	0.1	-	14.2	71.7	133.0	11.5	51.9	58.5	162.7	7.0
Test item	0.3	-	116.3	Culture was not continued*			108.4	Culture was not continued*
Test item	0.5	-	95.8	104.4	4.3	0.4	100.4	54.1	12.3	0.5
Test item	1.0	-	110.4	104.6	2.0	0.2	103.3	67.9	9.5	0.4
Test item	2.0	-	90.3	99.6	26.7	2.3	90.8	61.2	3.0	0.1
Test item	4.0	-	67.4	66.7	15.4	1.3	20.0	65.1	28.2	1.2
Test item	8.0	-	17.4	87.4	1.3	0.1	4.5	64.9	8.0	0.3
Test item	16.0	-	0.0	Culture was not continued**			0.0	Culture was not continued**
Test item			0.0	Culture was not continued**			0.0	Culture was not continued**
Solvent control (EtOH)		+	100.0	100.0	10.2	0.9	100.0	100.0	14.4	1.0
Pos control DMBA	1.3	+	33.3	103.5	617.6	53.2	6.2	105.1	515.8	35.9
Test item	62.5	+	79.7	97.9	17.1	1.5	87.7	72.8	22.2	1.5
Test item	125.0	+	54.7	100.4	13.1	1.1	76.5	84.3	9.6	0.7
Test item	250.0	+	71.3	104.1	15.8	1.4	80.9	79.0	18.4	1.3
Test item	500.0	+	70.4	104.6	8.8	0.8	75.6	91.4	31.5	2.2
Test item	1000.0	+	21.8	92.4	11.2	1.0	9.0	99.7	19.2	1.3
Test item	2000.0	+	3.6	Culture was not continued**			1.5	Culture was not continued**
EXP II 24 hr			CULTURE I				CULTURE II
Solvent control (EtOH)		-	100.0	100.0	18.1	1.0	100.0	100.0	13.4	1.0
Pos control	75.0	-	80.1	104.5	118.3	6.5	75.6	82.6	261.8	19.6
Test item	3.8	-	91.3	118.8	18.9	1.0	90.0	102.1	26.0	1.9
Test item	7.5	-	81.0	122.1	5.7	0.3	86.0	86.2	10.7	0.8
Test item	15.0	-	76.1	118.4	13.9	0.8	80.2	116,6	17.6	1.3
Test item	30.0	-	79.8	111.2	8.8	0.5	71.7	100.8	10.3	0.8
Test item	45.0	-	35.7	125.9	8.9	0.5	0.0	74.5	6.9	0.5
Test item	60.0	-	0.0	Culture was not continued**			0.0	Culture was not continued**
Exp II 4 hr			CULTURE I				CULTURE II
Solvent control (EtOH)		+	100.0	100.0	29.4	1.0	100.0	100.0	29.3	1.0
Pos control DMBA	1.3	+	25.3	61.3	1610.2	54.8	21,8	88.0	970.6	33.1
Test item	62.5	+	99.5	81.8	47.5	1.6	90.3	82.2	21.0	0.7
Test item	125.0	+	101.3	76.8	9.9	0.3	79.9	89.5	26.3	0.9
Test item	250.0	+	70.6	62.9	20.7	0.7	69.1	89.4	20.4	0.7
Test item	500.0	+	97.4	82.7	20.6	0.7	74.5	109.9	17.8	0.6
Test item	1000.0	+	5.3	96.1	5.5	0.2	50.7	116.7	17.3	0.6
Test item	2000.0	+	7.7	Culture was not continued**			41.9	Culture was not continued**

*Culture was not continued since a minimum of only four analyzable concentrations is required.

**Culture not continued due to exceedingly strong toxic effects

Statistical Analysis

Experimental Group	P-value
First experiment, culture 1 without S9	0.704
First experiment, culture 2 without S9	0.602
First experiment, culture 1 with S9	0.451
First experiment, culture 2 with S9	0.495
Second experiment, culture 1 without S9	0.241
Second experiment, culture 2 without S9	0.183
Second experiment, culture 1 with S9	0.174
Second experiment, culture 2 with S9	0.086

Applicant's summary and conclusion

Conclusions:

2-Ethylhexyl methacrylate did not induce gene mutations at the HPRT locus in V79 cells with and without metabolic activation. Therefore, 2-Ethylhexyl methacrylate is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The potential of 2-Ethylhexyl methacrylate to induce gene mutations at the HPRT locus in V79 tells of the Chinese hamster was investigated in an OECD guideline 476 and GLP study (Harlan, 2008). The assay was performed in two independent experiments. The cells were exposed to the test item for 4 hours in the first experiment with and without metabolic activation. The second experiment was performed with a treatment period of 24 hours in the absence and 4 hours in the presence of metabolic activation. The maximum dose of the pre-test was 2000 µg/mL corresponding to a molar concentration of about 10 mM. The concentration range of the main experiments was limited by cytotoxic effects and was 0.1 - 16.0 (1^stexperiment) and 3.8 – 60.0 (2^ndexperiment) µg/ml without S9 and 62.5 – 2000 µg/ml with S9 (1^stand 2^ndexperiments). No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiments up to the maximum concentration. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies and thus showed the sensitivity of the test system and the activity of the S9 mix. In conclusion, 2-ethylhexyl methacrylate did not induce gene mutations at the HPRT locus in V79 cells.