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Genetic toxicity: in vitro

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in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted study, carried out by the Hatano research Institute, Food and Drug Safety Center (Japan), Guideline study, GLP; german translation available, only study summary is written English.

Data source

Referenceopen allclose all

Reference Type:
study report
Report date:
Reference Type:
Relevance of chemical structure and cytotoxicity to the induction of chromosome aberrations based on the testing results of 98 high production volume industrial chemicals.
Kusakabe H, Yamakage K, Wakuri S, Sasaki K, Nakagawa Y, Watanabe M, Hayashi M, Sofuni T, Ono H, Tanaka N
Bibliographic source:
Mutation Research 517 (1-2): 187-198

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
according to guideline
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
GLP compliance:
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
2-hydroxyethyl methacrylate
EC Number:
EC Name:
2-hydroxyethyl methacrylate
Cas Number:
Molecular formula:
2-hydroxyethyl methacrylate
Test material form:
Specific details on test material used for the study:
Supplier: Nippon Shokubai
Batch No. 5P05LA
Purity: 97.6 %
Ethylenglycol dimethacrylate: 0.2 - 0.3 %
Diethylenglycol monomethacrylate: 2.0 -2.5%
Stabilzer: 50 ppm Hydrochinon monomethylether


Species / strain
Species / strain / cell type:
other: Chinese hamster lung (CHL/IU) cells
Cytokinesis block (if used):
The cytotoxic effect of the test substance on CHL/IU cells was determined using a monolayer cell density meter (Monocellater TM, manufactured by Olympus Kogaku Kogyo (AG)) by determining the growth rate for each group.
The cell growth was compared control group with /neutral/ medium. It was found that with both continuous and short-term medication, the cytotoxic did not exceed 50% at all concentrations used.
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix
Test concentrations with justification for top dose:
-S9 mix (continuous treatment): 0, 0.16, 0.33, 0.65, 1.3 mg/ml; -S9-mix and +S9-mix(short-term treatment): 0, 0.33, 0.65 and 1.3 mg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: solubility tests
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
Positive control substance:
other: -S9 mix, Mitomycin C; +S9 mix, Cyclophosphamide S-9: Rat liver, induced with phenobarbital and 5,6-benzoflavone
Details on test system and experimental conditions:
Test duration: continous treatment: 24-hours                
short-term treatment: 6-hours
Plates/test: 2
Evaluation criteria:
A substance is considered clastogenic if: - any dose level shows
a statistically signicant increase in aberration-bearing
cells - the increase is over historical controls - the increase is present in both replicates

Results and discussion

Test results
Species / strain:
other: Chinese hamster lung (CHL/IU) cells
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
other: Toxicity was not observed up to 0.65 mg/ml in continuous and short-term treatment with or without S9-mix.
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
Additional information on results:
Structural chromosomal aberrations (including gap) were induced under the following conditions: 
24 h continuous treatment (0.65 and 1.3 mg/ml: mid and high concentrations, 10.0 and 70.6 %, respectively); 
48 h continuous treatment (0.16 - 0.65 mg/ml: all concentrations, 6.0 - 84.0 %);
short-term treatment with an exogenous metabolic activation system (1.3 mg/ml: high concentration, 13.0 %). 
Polyploidy was induced under the following conditions: the 48 h continuous treatment (0.65 mg/ml, 3.25 %); 
short-term treatment with an exogenous metabolic activation system (0.65 mg/ml: mid concentration, 1.25 %); 
short-term treatment without the metabolic activation system (0.33 and 1.3 mg/ml: low and high concentrations, 0.88 and 6.13 %, respectively). 
However, a trend test showed no dose-dependency for the  polyploidy with short-term treatment and the metabolic activation system.
Lowest concentration producing cytogenetic effects in vitro:
Without metabolic activation (continuous treatment): 0.16 mg/ml (clastogenicity)                           
0.65 mg/ml (polyploidy)
Without metabolic activation (short-term treatment):  0.33 mg/ml (polyploidy)
With metabolic activation (short-term treatment): 1.3 mg/ml (clastogenicity)      
0.65 mg/ml (polyploidy)

Any other information on results incl. tables

Result table see under attachments

Applicant's summary and conclusion

Interpretation of results:

Structural chromosomal aberrations (including gap) were induced under the conditions of the study.
Executive summary:

HEMA has been evaluated for its ability to induce chromosomal aberrations in mammalian cells in culture (MHW 1997). Kusakabe et al. (2002) evaluated the clastogenic potential of HEMA along with a large number of other substances in Chinese hamster lung cells in culture, exposed to concentrations up to 1.3 mg/ml. HEMA was reported to induce structural chromosome aberrations following 6-hour exposure of cells but only in the presence of S9 at 1.3 mg/ml. Continuous exposure of cells for 24 or 48 hours without S9 also caused an elevated incidence of chromosome aberrations (from 0.16 mg/ml for the 48-hour exposure and from 0.65 mg/ml for the 24-hour exposure). Polyploidy was reported after both short-term treatment and 48-hour continuous treatment exposures. However, no dose-dependency was observed for polyploidy in the short-term treatment with metabolic activation. These effects were found at exposure levels without cytotoxicity or at concentrations which caused <50% cell death (no toxicity up to 0.65 mg/ml). For careful interpretation of these results two publications by Fujita et al (2016) should be considered. If the cytotoxicity index relative cell count (RCC) is replaced with a new index, RICC or RPD (relative increase in cell count/relative population doubling), the result was identified as being possibly false positive.