2-hydroxyethyl methacrylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro: 

Gene mutation in bacteria:

Salmonella typhimurium TA 1535, TA 1537, TA 98, and TA 100, and E. coli WP2 uvrA, with and without metabolic activation (OECD 471) (MHW Japan, 1997): negative

Gene mutation in mammalian cells:

HGPRT test in CHO cells (OECD 476) by read across with HPMA: negative

Thus, while HEMA does not have a complete evaluation for the endpoint of mutation in mammalian cells in culture [results available only in absence of S9], it is concluded that the available results for HPMA for this endpoint may be read-across to HEMA given the close structural similarities of the two materials and similar behavior in other genotoxicity assays.

Cytogenicity in mammalian cells:
Chromosomal aberration test in CHL cells, with and without metabolic activation (OECD 473), (MHW Japan, 1997): positive

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

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 / 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:

yes

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

Statistics:

yes

Species / strain:

other: Chinese hamster lung (CHL/IU) cells

Metabolic activation:

with and without

Genotoxicity:

positive

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:

valid

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)

Result table see under attachments

Conclusions:

Interpretation of results:
positive

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.

 

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The scenario for this endpoint (gene mutation in mammalian cells) is covered by the RAAF scenario 2 for which the read-across hypothesis is based on different compounds with qualitatively similar properties.

2-Hydroxyethyl methacrylate (HEMA) and Hydroxypropyl methacrylate (HPMA) have very similar chemical structures. The read-across hypothesis for this endpoint is based on the hypothesis that source and target substances have qualitatively similar toxicological properties because they share structural similarities with common functional groups: both substances are esters of methacrylic acid (MMA) and the respective alcohols ethylene glycol (EG) and propylene glycol (PG).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Read across to cover endpoint requirements is performed only from studies with pure substance. The purity of the source substance Hydroxypropyl methacrylate (CAS 27813-02-1) in the study was reported to be 98.96 %, the purity of the target substance 2-Hydroxyethyl methacrylate (868-77-9) is ca. 98 %. Typical impurity of both substances is methacrylic acid (CAS 79-41-4) in the range of 0.1 to 1%, typically 0.1 %

3. ANALOGUE APPROACH JUSTIFICATION

HEMA was evaluated for its ability to cause forward mutation at the hprt locus in Chinese hamster lung fibroblast V79 cells in culture (Schweikl, 1998). This study was assigned a Klimisch rating of 2, reliable with restriction. Results with metabolic activation were not presented in the publication. Ethylmethanesulfonate (200 microgram/ml) was used as a positive control. Concentrations of HEMA up to 5mM did not increase the mutant frequency in this assay; plating efficiencies were 84-113% of control.

Because of the restrictions in the reliability of the study available for HEMA as described above, a fully reliable GLP study conducted with the structurally very similar HPMA was chosen as the key study.

HPMA was evaluated in the in vitro Chinese hamster ovary cell/hypoxanthineguanine- phosphoribosyl transferase (CHO/HGPRT) forward gene mutation assay (Dow, 2010). The genotoxic potential of the test material was assessed in the absence and presence of an externally supplied metabolic activation (S9) system. The concentrations ranged from 45.1 to 1442 µg/ml in the absence and presence of S9. The highest concentration was based on the assay system limit of 10 mM. The adequacy of the experimental conditions for detection of induced mutation was confirmed by employing positive control chemicals, ethyl methanesulfonate for assay in the absence of S9 and 20- methylcholanthrene for assay in the presence of S9. Solvent control cultures were treated with the solvent used to dissolve the test material (i.e. distilled water). The results of the in vitro CHO/HGPRT forward gene mutation assay with hydroxypropyl methacrylate indicate that under the conditions of this study, the test article was non-mutagenic when evaluated in the absence or presence of an externally supplied metabolic activation (S9) system.

Although this read-across is mainly based on the structural similarity of the source and target substances, the approach is further substantiated by a similar mode of action and by supporting toxicological data on other genetic toxicity endpoints.

Mode-of-action analysis:
The only plausible toxicological mode of action of both substances is the weak electrophilic reactivity of the methacrylate ester double bond which is shared by HEMA and HPMA.

Supporting toxicological data available for source and target substances:
Both, source and target substance are negative in the ability to induce gene mutations in bacterial cells in culture. No genotoxic effect was observed in various strains of bacteria (S. typhimurium TA 97a, 98, 100, 102, 1535, 1537, 1538; E. Coli WP2 uvrA) exposed to HEMA with and without metabolic activation (Hatano Research Institiute, 1997). No genotoxic effect was observed in different strains of bacteria (S. typhimurium TA 98, 100, 1535, 1537, 1538; E. Coli WP2 uvrA) exposed to HPMA with and without metabolic activation (Hatano Research Institute , 1996).

Furthermore, supporting the absence of the ability to induce gene mutations in mammalian cells, Arossi et al (2009) reported that HEMA was not active in a test of mutagenicity in Drosophila melanogaster in vivo. The Somatic Mutation and Recombination Test (SMART) detects genotoxicity expressed as homologous mitotic recombination, point and chromosomal mutation. SMART detects the loss of heterozygosity of marker genes expressed phenotypically on the fly’s wings. This fruit fly has an extensive genetic homology to mammalians, which makes it a suitable model organism for genotoxic investigations. HEMA had no statistically significant effect on total spot frequencies – suggesting no genotoxic action in the SMART assay.

While HEMA as well as HPMA cause chromosomal aberrations in mammalian cells in culture, probably at least partially via an oxidative mode of action, both substances are not clastogenic in vivo.
Because of the similarity of the chemical reactivities and supporting toxicological data for other mutagenicity endpoints, the endpoint specific “scientific assessment” of the read across is thus “acceptable with high confidence”.

4. DATA MATRIX
See: Attached document

Reason / purpose for cross-reference:

read-across source

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

negative


The results of the in vitro CHO/HGPRT forward gene mutation assay with the structurally similar substance hydroxypropyl methacrylate indicate that under the conditions of this study, the test article was non-mutagenic when evaluated in the absence or presence of an externally supplied metabolic activation (S9) system.

Executive summary:

Gene mutation in mammalian cells of 2-hydroxyethyl methacrylate was predicted by read across in an analogue approach with the structurally related substance substance 2-hydroxypropyl methacrylate.

Hydroxypropyl methacrylate (2-methyl-2-propenoic acid monoester with 1,2- propanediol) was evaluated in the in vitro Chinese hamster ovary cell/hypoxanthineguanine- phosphoribosyl transferase (CHO/HGPRT) forward gene mutation assay. The genotoxic potential of the test material was assessed in the absence and presence of an externally supplied metabolic activation (S9) system. The concentrations ranged from 45.1 to 1442 µg/ml in the absence and presence of S9. The highest concentration was based on the assay system limit of 10 mM. The adequacy of the experimental conditions for detection of induced mutation was confirmed by employing positive control chemicals, ethyl methanesulfonate for assay in the absence of S9 and 20- methylcholanthrene for assay in the presence of S9. Solvent control cultures were treated with the solvent used to dissolve the test material (i.e. distilled water). The results of the in vitro CHO/HGPRT forward gene mutation assay with hydroxypropyl methacrylate indicate that under the conditions of this study, the test article was non-mutagenic when evaluated in the absence or presence of an externally supplied metabolic activation (S9) system.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

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: Only study summary is written English.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

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

Target gene:

his, trp

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 mix

Test concentrations with justification for top dose:

0, 313, 625, 1250, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: no data provided

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other:  -S9 mix, 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide (TA100, TA98), Sodium azide (TA1535) and 9-Aminoacridine (TA1537) +S9 mix, 2-Aminoanthracene (four strains) S9: Rat liver, induced with phenobarbital and 5,6-benzoflavone

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation
NUMBER OF REPLICATIONS: 2
Plates/test: 3

Rationale for test conditions:

The dosage of 2-hydroxyethyl methacrylic acid ester was determined within the range that a ratio 1:3 within a range of 50 - 5000 g per plate has been set. No antibacterial reaction was observed in any of the bacterial strains used, regardless of whether with or without the addition of S9 mix. Accordingly, in the main study, we set the maximum amount with or without S9 mix was 5000 g per plate.

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Toxicity was not observed up to 5000 µg/plate in four strains with or without S9-mix.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Toxicity was not observed up to 5000 µg/plate in four strains with or without S9-mix.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Toxicity was not observed up to 5000 µg/plate in four strains with or without S9-mix.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Toxicity was not observed up to 5000 µg/plate in four strains with or without S9-mix.

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Toxicity was not observed up to 5000 µg/plate in four strains with or without S9-mix.

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The results show that the number of mutated mutated colonies did not exceed twice the value in the control groups with twice the value in the control groups with /neutral/ medium.
From this we concluded that back mutation due to 2-hydroxyethyl methacrylic acid ester is not evident from the present investigations (negative).

Conclusions:

negative
In conclusion, it can be stated that during the decribed mutagenicity test and under the experimental
conditions reported, the test article did not induce gene mutations by base pair changes or frame s
hifts in the genome of the strains tested.
Therefore, methacrylic acid has to be judged as non mutagenic up to 5000 μg/plate in the presence and absence of mammalian metabolic activation according to the Ames test results.

Executive summary:

In a reverse gene mutation assay in bacteria (Ames test), strains TA1535, TA1537, TA98, TA100 of Salmonella typhimurium ,and E. coli WP2 uvr A were exposed to 2- hydroxyethyl methacrylate ( 97.6 %) at concentrations of 0, 313, 625, 1250, 2500 and 5000 µg/plate in the presence and absence of mammalian metabolic activation S9 -mix. In two independent experiments 2-hydroxyethyl methacrylate was investigated for its potential to induce gene mutations according to the preincubation test. No toxic effects occurred in the test groups with and without metabolic activation in both independent experiments. 2-Hydroxyethyl methacrylate did not induce mutations in the Salmonella typhimurium  strains TA98, TA100, TA1535, TA 1537 and E coli uvr A.

 Appropriate reference mutagens were used as positive controls. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In vivo:

Cytogenicity test in vivo:

Micronucleus assay in rat bone marrow (OECD 474): negative

Supporting information:

Somatic Mutation and Recombinogenic Test (SMART) in Drosophila melanogaster: negative

 

 

Link to relevant study records

Reference

Reference 1

Endpoint:

genetic toxicity in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Documentation sufficient for assessment

Principles of method if other than guideline:

Somatic Mutation and Recombinogenic Test (SMART) to detect mitotic recombination and a diverse set of mutational events.

GLP compliance:

not specified

Remarks:

information not provided in publication

Type of assay:

somatic mutation and recombination test in Drosophila

Species:

Drosophila melanogaster

Strain:

other: marker heterozygous (mwh ⁄flr3 ) and balancer-heterozygous (mwh ⁄TM3) genotypes

Sex:

male/female

Details on test animals or test system and environmental conditions:

The monomer genetic toxicities were accessed using the Standard Cross version of the wing SMART test: flr3 ⁄TM3, BdS females crossed with mwh ⁄mwh males. Eggs from this cross were collected for 8 hr on culture bottle containing a solid agar base (3% w⁄ v) enriched with a layer of live fermenting
baker’s yeast supplemented with sucrose. Three days later the larvae were transferred to vials containing 1.5 g of Drosophila Instant Medium (Carolina Biological Supply, Burlington, NC, USA) rehydrated with 5 ml of the test solutions.

Vehicle:

- Vehicle(s)/solvent(s) used: water

Details on exposure:

The monomer genetic toxicities were accessed using the Standard Cross version of the wing SMART test: flr3 ⁄TM3, BdS females crossed with mwh ⁄mwh males. Eggs from this cross were collected for 8 hr on culture bottle containing a solid agar base (3% w⁄ v) enriched with a layer of live fermenting
baker’s yeast supplemented with sucrose. Three days later the larvae were transferred to vials containing 1.5 g of Drosophila Instant Medium (Carolina Biological Supply, Burlington, NC, USA) rehydrated with 5 ml of the test solutions. Negative solvent controls were always included. The treated individuals remained in the vials until the emergence of the surviving adult flies.

Duration of treatment / exposure:

Flies were collected after eclosion

Frequency of treatment:

Eggs from the cross were collected for 8 hr. Three days later the larvae were transferred to vials containing 1.5 g of Drosophila Instant Medium rehydrated with 5 ml of the test solutions. Flies were collected after eclosion.

Remarks:

Doses / Concentrations:
HEMA diluted in distilled water – 0.675%, 1.25%, 1.875% and 2.5%
Basis:

No. of animals per sex per dose:

30 total

Control animals:

yes

Positive control(s):

ethyl methanesulfonate

Tissues and cell types examined:

presence of cell clones showing malformed wing hairs
number of spots as well as their type and size

Details of tissue and slide preparation:

After eclosion, the flies were collected from the treatment vials and stored in 70% ethanol. Subsequently, the wings were mounted on slides and scored under 400 X· magnification for the presence of cell clones showing malformed wing hairs. The number of spots as well as their type and size were recorded. In test larvae, two genotype configurations are possible: trans-heterozygous for the recessive wing cell markers [mwh and flr3 (mwh +⁄+ flr3)], and balancer- heterozygous (mwh ⁄TM3). Induced loss of heterozygosity on marker-heterozygous flies leads to two types of mutant clones: (i) single spots, either mwh and flr3, which can be produced by somatic point mutation, chromosome aberration as well as mitotic recombination and (ii) twin spots, consisting of both mwh and flr3 sub clones, which are originated exclusively from mitotic recombination. On balancer-heterozygous flies, mwh spots should reflect somatic point mutation and chromosome aberration, as mitotic recombination – involving the TM3 chromosome and its structurally normal homologue – is a lethal event.

Evaluation criteria:

We considered the treatment as positive if the frequency of mutant clones in the treated series was at least m (multiplication factor) times higher than that in the control series.

Statistics:

The conditional binomial test of Kastenbaun and Bowman [1970] was applied to assess differences between the frequencies of each spot type in treated and concurrent NC flies. The multiple decision procedure described by Frei and Wrgler [1988, 1995] was used to judge the overall response of an agent as positive, weakly positive, negative or inconclusive. We considered the treatment as positive if the frequency of mutant clones in the treated series was at least m (multiplication factor) times higher than that in the control series. As small single spots and total spots have a comparatively high spontaneous frequency, m was fixed as 2 (testing for a doubling of the spontaneous frequency). For large single spots and twin spots, which have a low spontaneous frequency, m = 5 was used. The recombinagenic action of the drugs was calculated comparing the standard frequency of clones per 105 cells obtained from mwh ⁄ flr3 and mwh ⁄TM3 genotypes. For an unbiased comparison of this frequency, only mwh clones in mwh single spots and in twin spots were used.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

HEMA did not have a significant effect on total spot frequencies in marker-heterozygous (mwh ⁄ flr3) flies analysed, suggesting that HEMA does not act as a genotoxin in the SMART assay.

Conclusions:

Interpretation of results : negative
HEMA did not have a significant effect on total spot frequencies in marker-heterozygous (mwh ⁄ flr3) flies analysed, suggesting that HEMA does not act as a genotoxin in the SMART assay.

Executive summary:

The present in vivo study investigated the genotoxicity of hydroxyethyl methacrylate (HEMA). The Somatic Mutation and Recombination Test (SMART) in Drosophila melanogaster was applied to analyse their genotoxicity expressed as homologous mitotic recombination, point and chromosomal mutation. SMART detects the loss of heterozygosity of marker genes expressed phenotypically on the fly’s wings. This fruit fly has an extensive genetic homology to mammalians, which makes it a suitable model organism for genotoxic investigations. HEMA had no statistically significant effect on total spot frequencies – suggesting no genotoxic action in the SMART assay. The clinical significance of these observations has to be interpreted for data obtained in other bioassays.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25.10.2000 - 27.02.2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well conducted study, carried out by the Mitsubishi Chemical Safety Institute Ltd. (Japan). Guideline study, GLP

Reason / purpose for cross-reference:

reference to other study

Remarks:

supporting information regarding bone marrow exposure

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Specific details on test material used for the study:

- Supplier: Mitsubishi Rayon Ltd.
- Lot. No. : 0714001
- Analytical purity: 99.7%
- Storage condition of test material: cold, dark storage

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

[Crj: CD(SD)IGS, SPF] obtained from Charles River Japan Inc., on November 22, 2000.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan, Inc.
- Age at study initiation: 7 weeks old
- Weight at study initiation: 259 to 282 g
- Housing: polycarbonate cages; 5 per cage
- Diet (e.g. ad libitum): pellets; ad libitum
- Water (e.g. ad libitum): flitered tap water; ad libitum
- Acclimation period: six days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.2 to 23.2 deg C
- Humidity (%): 47.2 to 60 %
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark

IN-LIFE DATES: From: 2000-11-28 To: 2000-12-03

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: water
- Dosing volume: 10 mL/kg

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was dissolved in water for injection. Then, this solution was diluted with the same solvent. Dosing solutions were administered to rats immediately after preparation.

Duration of treatment / exposure:

2 days

Frequency of treatment:

Twice at 24 hour interval

Post exposure period:

Animals were sacrificed 24 hours after final administration

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5/group

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: intraperitoneal
- Doses / concentrations: dissolved in water for injection at 1 mg/mL just before use.

Tissues and cell types examined:

The femurs were dissected out and the bone  marrow cells were collected.
The animals were weighted just before administration and before  preparation of specimen. The animals were observed once or more per day of administration and  preparation of specimen.

Details of tissue and slide preparation:

The animals in each group were sacrificed 24-hours after the final administration.

Each rat was sacrificed by exsanguination from  the abdominal aorta under anesthethesia with Ravonala (Tanabe Seiyaku  Co.,Ltd. lot no. 07004) and the femurs were dissected out. The bone  marrow cells were collected with PBS(-) (Nissui Pharmaceutical Co.,  Ltd.). The cells were centrifuged at 200 rpm for 5 min and were taken the  supernatant. It wasresuspended in 10 % buffered formalin (Muto Pure  Chemicals,Co., Ltd.) and was centrifuged at 1000 rpm for 5 min. After  centrifugation, the cells were washed twice. Then, the cells were  resusupended in a small amount of 10 % buffered formalin, and was  stocked. The bone marrow suspension was stained with acridine orange, and  was spread on a clean slide glass.

Slides were examined under blind condition and scored under a fluorescent  microscope with B-2 excitation filter. One thousand erythrocytes were  scored from each slide in order to determine the ratio of polychromatic  erythrocytes (PCE's) to the total erythrocytes [PCE's and normochromatic  erythrocytes (NCE's)]. PCEs were further scored up to 2000 cells, the  number of micronucleated PCE's (MNPCE's) in a slide were examined (2  area, Total 2000 cells). PCE's and NCE's were identified according to the  method of Hayashi et al 1.  

Evaluation criteria:

Only when a test substance induced a significant increase in the total number of dose-dependent MNPCEs, the test substance was considered positive in this assay.

Statistics:

For the analysis of the percentage of PCEs, Student's t-test were applied. For the incidence of MNPCEs, tables of Kastenbaum and Bowman were applied.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

There were no significant differences in the incidence of MNPCE's between any treatment group and the negative control group. There were significant increases in the incidences of PCE's between 1000 mg/kg group and the negative control group (p < 0.05).  Clinical signs were not observed with all of test substance groups.  According to Durner (2009), regarding the toxicokinetics and distribution of 2-hydroxyethyl methacrylate in mice, HEMA was found in bone after gastric administration (0.2% and 0.1%, 5 and 24 hours after administration, respectively). Therefore, it is expected that HEMA reaches the bone marrow of rodents after oral administration.

The negative control incidences of MNPCE's among tests was within the range of our laboratory  background data and positive control ones showed remarkable increase.  These findings indicated that the test was conducted appropriately.  This chemical does not induce micronuclei under the test conditions employed.

Results of micronucleus test

				MNPCE
Treatment group	Dosage (mg/kg) *times	Animal number	Number of PECs scored	Number (total)	Incidence (%) (Mean +/- SD)	PCE/(PCE+NCE) (%) (Mean +/- SD)
Negative control (water for injection)	0 * 2^1	1	2000	4	0.20	51.4
		2	2000	5	0.25	54.2
		3	2000	9	0.45	51.1
		4	2000	2	0.10	53.8
		5	2000	2	0.10	50.4
				(22)	(0.22+/-0.14)	(52.2± 1. 71)
Test substance (HEMA)	500 * 2^1	6	2000	2	0.10	55.6
		7	2000	3	0.15	51.8
		8	2000	7	0.35	56.8
		9	2000	3	0.15	53.0
		10	2000	3	0.15	54.3
				(18)	(0.18+/-0.10)	(54.3±1.99)
	1000 * 2^1	11	2000	7	0.35	54.4
		12	2000	5	0.25	55.9
		13	2000	5	0.25	61.7
		14	2000	7	0.35	56.0
		15	2000	2	0.10	54.1
				(26)	(0.26+/-0.10)	(56.4±3.07*3)
	2000 * 2^1	16	2000	8	0.40	53.8
		17	2000	5	0.25	54.1
		18	2000	1	0.05	49.4
		19	2000	3	0.15	60.8
		20	2000	3	0.15	57.8
				(20)	(0.20+/-0.13)	(55.2±4.33)
Positive Control (CP)	10 * 1^2	21	2000	52	2.60	51.3
		22	2000	51	2.55	50.6
		23	2000	51	2.55	51.3
		24	2000	70	3.50	51.8
		25	2000	58	2.90	53.7
				(282^**3)	(2.82±0.41)	(51.7±1.18)

PCE : polycbromatic erytbrocytes, MNPCE : micronucleated PCE,

NCE : normochromatic erytbrocytes, CP : cyclophosphamide

1) twice administered by oral gavage at 24 hours interval

2) once administered by intraperitoneal injection

3) significantly different from the negative control (* p<0.05, ** p<0.01)

Conclusions:

Interpretation of results : negative
Under the conditions of this study, the test substance did not induce micronuclei.

Executive summary:

A bone marrow micronucleus study in seven week old, Sprague Dawley rats was conducted to assess the mutagenic potential of the test substance. The test substance was administered twice at 24 -hour interval by oral gavage at three doses of 500, 1000 and 2000 mg/kg to groups of 5 male tats. Twenty four hours after the final administration bone marrow samples were prepared, and were examined for incidence of micronucleated polychromatic erythrocytes. The test substance did not induce significant increases in the micronucleated polychromatic erythrocytes in any treated groups. Cyclophosphamide, used as positive treatment, did cause an increase in micronucleated PCEs. 

According to Durner (2009), regarding the toxicokinetics and distribution of 2-hydroxyethyl methacrylate in mice, HEMA was found in bone after gastric administration (0.2% and 0.1%, 5 and 24 hours after administration, respectively). Therefore, it is expected that HEMA reaches the bone marrow of rodents after oral administration. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro studies:

 

Bacterial assays

No genotoxic effect was observed in various strains of bacteria (S. typhimurium TA 97a, 98, 100, 102, 1535, 1537, 1538; E. Coli WP2 uvrA) exposed to HEMA with and without metabolic activation. The study conducted by the Japan Ministry of Health and Welfare (Hatano Research Institute, 1997) was chosen as the key study because it was the study with the highest reliability and the most complete documentation. HEMA was not toxic and not mutagenic to Salmonella typhimurium tester strains (TA 1535, 1537, 1538, 98 and 100) or to E. Coli WP2 uvrA at levels up to and including 5000 microgram/plate in the presence or absence of activating enzymes. Rat liver S9 induced with Phenobarbital and with Benzoflavone was the activation system. Appropriate positive controls for each test strain in the presence or absence of S9 were included in the study protocol. Schweikl et al (1994; 1998) also reported that HEMA was not mutagenic at levels up to 25 mg/plate in the presence or absence of rat liver enzymes for Salmonella test strains (TA 97a, 98, 100, 102, 104). Cytotoxicity was not evaluated in these studies. Two additional studies confirmed the absence of a genotoxic effect in various strains of bacteria exposed to HEMA (Waegemaekers, 1984; Heil, 1996). In one individual study, an occasional increase in the number of revertants over the control level was observed with S. typhimurium strain TA 100 with metabolic activation in an Ames assay performed with HEMA (no further specification). This result could not be scientifically assessed due to insufficient level of details available (Carpanini, BPChemicals, 1981).

 

Mammalian cell assays

• Gene mutations

Possible gene mutation with the structurally similar substance HPMA was evaluated in an in vitro Chinese hamster ovary cell/hypoxanthine-guanine-phosphoribosyl transferase (CHO/HGPRT) gene mutation assay (Dow, 2010). The genotoxic potential of the test material was assessed in the absence nd presence of metabolic activation (S9) system. The concentrations ranged from 45.1 to 1442 μg/ml in the absence and presence of S9. Low to moderate cytotoxicity was found: in the presence of S9, RCS (relative cell survival) values ranged from 74.7 to 113.8% and without metabolic activation between 52.9 and 88.4%. HPMA was non-mutagenic in this study either with or without metabolic activation. This study was chosen as the key study because it is reliable without restrictions and performed under GLP.

A supporting study on HEMA is also available but in this assay, HEMA was evaluated only in the absence of metabolic activationHEMA was evaluated for its ability to cause forward mutation at the hprt locus in Chinese hamster lung fibroblast V79 cells in culture (Schweikl, 1998). This study was assigned a Klimisch rating of 2, reliable with restriction. Cells were exposed for 24 hours without metabolic activation or for 4 hours with S9. In the absence of metabolic activation, concentrations of HEMA of 2.5 and 5 mM did not increase the mutant frequency; plating efficiencies were 84-113% of control. Ethylmethanesulfonate (200 microgram/ml) was used as a positive control.

 

Results with metabolic activation were not presented in the publication. However, it is not expected that HEMA induces gene mutations in the presence of metabolic activation in mammalian cells. As demonstrated by several studies, HEMA does not induce mutations in bacterial cells and according to Johannsen review (2008) on the mutagenicity of acrylates and methacrylates, these substances (with few exceptions) are non mutagenic in point mutation tests. Therefore, the results appeared consistent within each of several types of tests across the functional spectrum of acrylates and methacrylates, with no apparent differences in response related to a specific structure.

Johannsen FR (2008) Mutagenicity assessment of acrylate and methacrylate compounds and implications for regulatory toxicology requirements, Regulatory Toxicology and Pharmacology 50 322–335 

The detailed justification for read-across is summarised in the respective source RSS.

 

 

• Chromosomal aberrations

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.

 

 

Lee et al (2006) reported that HEMA could increase micronuclei in vitro in V79-4 cells in culture. Nacetylcysteine (NAc) was effective at preventing induction of micronuclei and the authors considered the role of active oxygen in micronuclei induction. About 2×105V79-4 cells were seeded onto microscopic glass slides in 4ml DMEM and allowed to grow for 12 h. Then, HEMA was added, and the cultures were incubated for 24 h. In a previous study, 24h of the exposure time was proved to be enough for generation of micronuclei by the monomer. Micronuclei were analyzed microscopically in three parallel cultures (slides) of 1000 cells/slide per concentration of resin monomer. Micronuclei were identified as DNA containing structures; their area was less than one-third of that of the main nucleus. Only mononucleated cells containing less than five micronuclei were scored; cells in mitosis and those exhibiting apoptotic nuclear fragmentations were not counted. Ethylmethane sulfonate (EMS) was used as a positive control.

Concentrations of HEMA that increased micronuclei incidence were 3-5 mM and occurred at cell survival that exceeded 60%. NAc (10mM) inhibited the induction of micronuclei by more than 50% at 4-5mM HEMA. NAc was also reported to block HEMA-induced DNA fragmentation and apoptosis in RPC-C2A cells in culture.

 

 

Schweikl et al (2007) also reported the induction of micronuclei in V79 cells in culture exposed to 6.0 mM concentrations of HEMA; NAc inhibited this response.

 

 

• DNA damage

Pawlowska et al (2010) reported that HEMA was able to damage DNA in lymphocytes in culture using the Comet assay system. HEMA at concentrations up to 10 mM did not affect the viability of the cells during a 1 h exposure. However, HEMA induced concentration dependent DNA damage in lymphocytes, as assessed by alkaline and pH 12.1 versions of the comet assay. The increase was over 100% for the highest HEMA concentration (10mM, p< 0.01). No changes in the percent tail DNA in the pH 12.1 and neutral versions of this test were observed, which indicates that the chemical did not introduce DNAstrand breaks in lymphocytes. The inability of HEMA to induce DNA double-strand breaks was confirmed by pulsed-field gel. The results obtained indicated that HEMA induced mainly alkali-labile sites in DNA.

 

Enhanced migration of DNA was also observed in an alkaline Comet assay performed with HEMA (no further specification) at concentrations > 10-6 M, with cell vitality at 84% (Kleinsasser, 2004). This study was performed in human lymphocytes exposed to HEMA for 1 hour without metabolic activation with concentrations ranged from 10-8 to 2.5 x 10-2 M. The DNA migration was assessed using the Olive Tail Moment (OTM) method (relative amount of DNA in the tail of the comet x median migration distance). Positive control was hydrogen peroxide.

 

Urcan et al (2010) reported that high concentrations (0.01 – 100 mM) of HEMA applied to human gingival fibroblast cells in vitro caused double strand DNA breaks. This is a non-guideline study. The relevance of these effects is uncertain given the very high concentrations used.

 

Durner et al (2011) reported that one hour as well as about 24 h after incubation of human gingival fibroblasts with concentrations up to 10 mM HEMA no significant difference in DNA strand breaks was found compared to controls.

 

 

Szczepanska et al (2012) compared the cytotoxic and genotoxic effects induced by HEMA and MAA as inducers of oxidative damage in human gingival fibroblasts in vitro, with MAA studied as the metabolic intermediate of HEMA. Both MAA and HEMA at 5 mM concentration induced DNA damage as measured by modified comet assays in human gingival fibroblasts following 6 h incubations. Hydrogen peroxide was the positive control. The MAA and HEMA induced oxidative damage adducts did not persist longer than 120 min. As assessed in these assays, HEMA produced more oxidative DNA damage than did MAA.

 

Both MAA and HEMA induced single strand breaks in the gingival fibroblasts as indicated by increased DNA migration in the alkaline comet assay beginning at the lowest concentration tested (<1.0 mM) and increasing in a dose dependent manner. There was no difference in efficiency of induction by these two agents. Both MAA and HEMA also induced double strand breaks in the gingival fibroblasts as indicated by increased DNA migration in the neutral comet assay, again beginning at 1.0 mM and increasing with dose. Although HEMA had a greater effect in this regard than did MAA, the difference was not significant. The ability of both MAA and HEMA to induce double strand breaks in the gingival fibroblasts was confirmed by increases in gH2AX (determined by immunofluorescence) production in the cells at exposures to 10 mM of either agent. However, in this regard, HEMA was significantly more efficient than was MAA on a molar concentration basis. Both MAA and HEMA significantly increased rates of apoptosis beginning at 0.5 mM concentrations and increasing in a dose dependent manner. There was no difference in efficiency of the two agents in this regard. Both MAA and HEMA perturbed cell cycle kinetics, decreasing S-phase cells and increasing G0/G1 cells at 0.5 mM concentration and reversing this, with an increase in S phase and decrease in G0/G1 phase cells at 1.0 mM concentrations. There was no difference in the degree of cell cycle perturbations induced by the two agents.

 

 

Neither MAA nor HEMA had an effect in vitro on naked plasmid DNA (Pawlowska et al., 2010; Szczepanska et al., 2012) which demonstrated the requirement for metabolism to produce genotoxicity.

Ginzkey et al (2015) investigated the genotoxic potential of HEMA in distinctly lower concentrations than known to cause cytotoxic damage. HEMA at the concentrations of 10 μM (0.4 ± 0.2) and100 μM (0.5 ± 0.2) did not show any significant difference, where as a concentration of 1 mM was able to increase DNA migration (1.5 ± 0.8) in the alkaline comet assay.

In additions a significant dose-dependent increase in the frequency of chromosome aberrations and sister chromatid exchange rates could be demonstrated in all tested concentrations.

 

 

In vivo studies:

HEMA has been evaluated for the ability to cause chromosomal aberrations in vivo in the micronucleus assay in rats (Mitsubishi Chemical Safety Institute, 2001). This study was assigned a Klimisch rating of 1, reliable without restriction. HEMA was administered by oral gavage twice per day to groups of 5 rats at doses of 500, 1000 and 2000 mg/kg. Doses were selected based on an oral LD50 observed to be 5050 mg/kg. Animals were observed for clinical symptoms of intoxication. Cyclophosphamide (10 mg/kg once) was used as a positive control. Twenty-four hours following the final dosing, bone marrow samples were prepared and examined for incidence of micronucleated polychromatic erythrocytes (PCE). One thousand erythrocytes were calculated to evaluate the ratio of PCEs to erythrocytes; two-thousand PCEs were scored for micronuclei.

In this study, no animals died and there were no clinical symptoms of intoxication even though HEMA was administered at 40% of the LD50. In There was no increase in the number of micronucleated PCEs at any HEMA dose level. Cyclophosphamide treatment did cause an increase in micronucleated PCEs to an extent consistent with laboratory historical controls.

According to Durner (2009), regarding the toxicokinetics and distribution of 2-hydroxyethyl methacrylate in mice, HEMA was found in bone after gastric administration (0.2% and 0.1%, 5 and 24 hours after administration, respectively). Therefore, it is expected that HEMA reaches the bone marrow of rodents after oral administration. 

 

Arossi et al (2009) reported that HEMA was not active in a test of mutagenicity in Drosophila melanogaster in vivo. The Somatic Mutation and Recombination Test (SMART) detects genotoxicity expressed as homologous mitotic recombination, point and chromosomal mutation. SMART detects the loss of heterozygosity of marker genes expressed phenotypically on the fly’s wings. This fruit fly has an extensive genetic homology to mammalians, which makes it a suitable model organism for genotoxic investigations. HEMA has no statistically significant effect on total spot frequencies – suggesting no genotoxic action in the SMART assay.

Justification for classification or non-classification

HEMA has been evaluated for genotoxic potential in bacterial cells in culture and was found not to be mutagenic in this assay system. Further, HPMA was found not to be mutagenic in mammalian cells in culture [CHO HGPRT assay] and HEMA was found not to be mutagenic in mammalian cells in culture [CHL V79 cell assay], albeit HEMA was evaluated only in the absence of S9. Both HEMA and HPMA were reported to cause chromosomal aberrations in mammalian cells in culture, but an in vivo micronucleus study with HEMA indicated that this material was not clastogenic in vivo.

Thus, while HEMA does not have a complete evaluation for the endpoint of mutation in mammalian cells in culture [results available only in absence of S9], it is concluded that the available results for HPMA for this endpoint may be read-across to HEMA given the close structural similarities of the two materials and similar behavior in other genotoxicity assays.

In conclusion, HEMA is not mutagenic in bacterial and mammalian cells in culture. Further, while HEMA may cause chromosomal aberrations in mammalian cells in culture, it is not clastogenic in vivo.

HEMA has not to be classified for genetic toxicity acc. CLP criteria.