2-ethylhexyl methacrylate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

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

Molecular formula:

C12H22O2

IUPAC Name:

2-ethylhexyl methacrylate

Test material form:

liquid

Method

Species / strain

Species / strain / cell type:

other: Human lymphocytes

Details on mammalian cell type (if applicable):

One healthy, non-smoking human volunteer (male) was used in this study. The donor was not suspected of any virus infection nor had been exposed to high levels of radiation or hazardous chemicals. An appropriate volume of whole blood was drawn from the peripheral circulation on the day of culture (Trial 1) or one day prior to culture (Trial 2). Blood was stored refrigerated until use. Whole blood cultures were established in sterile disposable centrifuge tubes by placing 0.4 mL heparinised blood into 9.0 mL Hepes-buffered RPMI medium containing 20% (v/v) foetal calf serum and 50 µg/mL gentamycin. Phytohaemagglutinin (PHA, reagent grade) was included at a concentration of approximately 10 µg per mL of culture to stimulate the lymphocytes to divide. Blood cultures were incubated for approximately 48 hours at 37°C and rocked continuously.

Metabolic activation:

with and without

Metabolic activation system:

rat liver post-mitochondrial fraction (S-9) from Aroclor 1254 induced animals

Test concentrations with justification for top dose:

Trial 1: 79.90, 114.1, 163.1, 232.9, 332.8, 475.4, 679.1, 970.2, 1386 and 1980 µg/ml
Trial 2: 17.18, 21.47, 26.84, 33.55, 41.94, 52.43, 65.54, 81.92, 102.4, 128.0, 160.0 and 200.0 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: solubility
The highest dose level used, 1980 µg/mL was equivalent to a 10 mM concentration and exceeded the limit of solubility.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-Nitroquinoline 1-oxide (-S9) and cyclophosphamide (+S9)

Details on test system and experimental conditions:

DURATION
- Fixation time (start of exposure up to fixation or harvest of cells): Treatment in the absence of S-9 was continuous for 20 or 44 hours (20+0, 44+0). Treatment in the presence of S-9 was for 3 hours only followed by a 17 or 41 hour recovery period (3+17, 3+41). The test article dose levels for chromosome analysis were selected by evaluating the effect of 2-ethylhexyl methacrylate on mitotic index. Following 20+0 hour treatments, -S-9 and 3+17 hour treatments, +S-9, chromosome aberrations were analyzed at three consecutive dose levels.
The effects of single concentrations only, (41.94 µg/mL, without and 1980 µg/mL with S-9) were investigated at the delayed (44+0, 3+41) sampling time.

SPINDLE INHIBITOR (cytogenetic assays): Approximately 1½ hours prior to harvest, colchicine was added to give a final concentration of approximately 1µg/mL to arrest dividing cells in metaphase.

STAIN (for cytogenetic assays):
After the slides had dried the cells were stained for 5 minutes in 4% (v/v) filtered Giemsa stain in pH 6.8 buffer. The slides were rinsed, dried and mounted with coverslips.

NUMBER OF CELLS EVALUATED:
Twenty-five cells from each of the selected NQO and CPA positive control cultures were analysed to ensure that the system was operating satisfactorily. Where possible 100, metaphases from each test concentration were analysed for chromosome aberrations.

DETERMINATION OF CYTOTOXICITY
Slides were examined, uncoded, for mitotic index (MI) or percentage of cells in mitosis. Slides from enough dose levels from each treatment regime were scored to determine if chemically induced mitotic inhibition had occurred. This is defined as a clear decrease in mitotic index compared with negative controls (based on at least 1000 cells counted), preferably dose-related.

OTHER EXAMINATIONS:
Only cells with 44-46 chromosomes were considered acceptable for analysis of structural aberrations. Any cell with more than 46 chromosomes, that is polyploid, endoreduplicated and hyperdiploid cells, observed during this search was noted and recorded separately.

Evaluation criteria:

The test article was to be considered as positive in this assay if:
1) a statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) occurred at one or more concentrations, and
2) the proportion of cells with structural aberrations at such doses exceeded the normal range.

A positive result only at the delayed harvest was to be taken as evidence of clastogenicity provided criteria 1 and 2 were met. Increases in numbers of cells with gaps or increases in the proportions of cells with structural aberrations, not exceeding the normal range or occurring only at very high or very toxic concentrations, were likely to be concluded as "equivocal". Full assessment of the biological importance of such increases is likely only to be possible with reference to data from other test systems. Cells with exchange aberrations or cells with greater than one aberration were to be considered of particular biological significance.

Statistics:

Fisher's exact test

Results and discussion

Test results

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

Positive controls validity:

valid

Additional information on results:

The highest concentrations chosen for analysis, 65.54 µg/mL and 1980 µg/mL, induced approximately 67% and 54% mitotic inhibition (reduction in mitotic index) in the absence and presence of S-9 respectively.

The proportion of cells with structural aberrations in negative control cultures fell within historical solvent control ranges. Untreated cultures were not analysed. Cells receiving the positive controls were sampled 20 hours after the start of treatment; both compounds induced statistically significant increases in the proportion of cells with structural aberrations.

Treatment of cultures with 2-ethylhexyl methacrylate in the absence of S-9 resulted in frequencies of cells with aberrations which were similar to and not significantly different from those seen in concurrent negative control ranges. Numbers of cells with aberrations in all treated cultures fell within the historical negative control (normal) range at both sampling times.

Treatment of cultures with 2-ethylhexyl methacrylate in the presence of S-9 resulted in frequencies of cells with aberrations which were significantly higher than those in concurrent controls. Numbers of aberrant cells, however, exceeded the normal range in only a single replicate at the highest dose level at each sampling time. Insofar as increases at this concentration were very small and not reproduced in both replicates the observation was not considered biologically significant.

Any other information on results incl. tables

2-Ethylhexyl methacrylate: summary of the numbers and types of structural aberrations observed without S9

20+0 hours

Treatment (µg/mL)	Rep	Cells *	g	Chr del	Chr exch	Ctd del	Ctd exch	Other	Abs +g	Abs -g
Solvent	A	100	0	0	0	0	0	1	1	1
	B	100	0	0	0	0	0	0	0	0
	Total	200	0	0	0	0	0	1	1	1
41.94	A	100	1	0	0	0	0	0	1	0
	B	100	1	1	0	5	0	0	7	6
	Total	200	2	1	0	5	0	0	8	6
52.43	A	100	0	1	0	0	0	1	2	2
	B	100	2	1	0	0	0	0	3	1
	Total	200	2	2	0	0	0	1	5	3
65.54	A	80	0	0	0	1	0	0	1	1
	B	80	0	0	0	2	0	0	2	2
	Total	160	0	0	0	3	0	0	3	3
NQO, 2.5	A	25	2	3	0	3	11	3	22	20
	B	25	0	1	0	1	3	2	7	7
	Total	50	2	4	0	4	14	5	29	27

* = Total cells examined for structural aberrations

44+0 hours

Treatment (µg/mL)	Rep	Cells *	g	Chr del	Chr exch	Ctd del	Ctd exch	Other	Abs +g	Abs -g
Solvent	A	100	0	1	0	0	0	0	1	1
	B	100	0	0	0	2	0	0	2	2
	Total	200	0	1	0	2	0	0	3	3
41.94	A	100	2	1	0	2	0	0	5	3
	B	100	2	0	0	3	0	0	5	3
	Total	200	4	1	0	5	0	0	10	6

2-Ethylhexyl methacrylate: summary of the numbers and types of numerical aberrations observed without S9

20+0 hours, Donor sex: male

Treatment (µg/mL)	Rep	Cells **	H	E	P	Tot abs	% with num abs
Solvent	A	102	0	0	2	2	2.0
	B	101	0	0	1	1	1.0
	Total	203	0	0	3	3	1.5
41.94	A	101	0	0	1	1	1.0
	B	103	1	0	2	3	2.9
	Total	204	1	0	3	4	2.0
52.43	A	100	0	0	0	0	0
	B	102	0	0	2	2	2.0
	Total	202	0	0	2	2	1.0
65.54	A	80	0	0	0	0	0
	B	81	1	0	0	1	1.2
	Total	161	1	0	0	1	0.6
NQO, 2.5	A	25	0	0	0	0	0
	B	25	0	0	0	0	0
	Total	50	0	0	0	0	0

44+0 hours, Donor sex: male

Treatment (µg/mL)	Rep	Cells **	H	E	P	Tot abs	% with num abs
Solvent	A	102	0	0	2	2	2.0
	B	103	1	0	2	3	2.9
	Total	205	1	0	4	5	2.4
41.94	A	103	0	0	3	3	2.9
	B	101	0	0	1	1	1.0
	Total	204	0	0	4	4	2.0

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative

2-Ethylhexyl methacrylate did not induce chromosome aberrations in cultured human peripheral blood lymphocytes when tested to its limit of toxicity in both the absence and presence of S-9

Executive summary:

In an OECD guideline 473and GLP in vitro cytogenetics assay, 2-Ethylhexyl methacrylate was tested using duplicate human lymphocyte cultures from a male human donor. Treatments covering a broad range of doses, separated by narrow intervals, were performed both in the absence and presence of metabolic activation by a rat liver post-mitochondrial fraction (S-9) from Aroclor 1254 induced animals. The highest dose level used, 1980 µg/mL was equivalent to a 10 mM concentration and exceeded the limit of solubility. Treatment in the absence of S-9 was continuous for 20 or 44 hours (20+0, 44+0). Treatment in the presence of S-9 was for 3 hours only followed by a 17 or 41 hour recovery period (3+17, 3+41). The test article dose levels for chromosome analysis were selected by evaluating the effect of 2-ethylhexyl methacrylate on mitotic index. Following 20+0 hour treatments, -S-9 and 3+17 hour treatments, +S-9, chromosome aberrations were analysed at three consecutive dose levels. The highest concentrations chosen for analysis, 65.54 µg/mL and 1980 µg/mL, induced approximately 67% and 54% mitotic inhibition (reduction in mitotic index) in the absence and presence of S-9 respectively. The effects of single concentrations only, (41.94 µg/mL, without and 1980 µg/mL with S-9) were investigated at the delayed (44+0, 3+41) sampling time.

Appropriate negative (solvent) control cultures and untreated cultures were included in the test system under each treatment condition. The proportion of cells with structural aberrations in negative control cultures fell within historical solvent control ranges. Untreated cultures were not analysed. 4-Nitroquinoline 1-oxide and cyclophosphamide were employed as positive control chemicals in the absence and presence of liver S-9 respectively. Cells receiving these were sampled 20 hours after the start of treatment; both compounds induced statistically significant increases in the proportion of cells with structural aberrations.

Treatment of cultures with 2-ethylhexyl methacrylate in the absence of S-9 resulted in frequencies of cells with aberrations which were similar to and not significantly different from those seen in concurrent negative control ranges. Numbers of cells with aberrations in all treated cultures fell within the historical negative control (normal) range at both sampling times.

Treatment of cultures with 2-ethylhexyl methacrylate in the presence of S-9 resulted in frequencies of cells with aberrations which were significantly higher than those in concurrent controls. Numbers of aberrant cells, however, exceeded the normal range in only a single replicate at the highest dose level at each sampling time. Insofar as increases at this concentration were very small and not reproduced in both replicates the observation was not considered biologically significant.

It is concluded that 2-Ethylhexyl methacrylate did not induce chromosome aberrations in cultured human peripheral blood lymphocytes when tested to its limit of toxicity in both the absence and presence of S-9.