Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The results of in vitro mutagenicity evaluations of EGDMA showed inconsistent findings. Positive finding were observed in studies that had inherent weakness in the design.

Link to relevant study records

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
S. typhimurium TA102 or E. coli WP2 uvrA not tested
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
S. typhimurium TA102 or E. coli WP2 uvrA not tested
Principles of method if other than guideline:
Method: Ames test according to Ames et al., Mutat. Res. 31, 347-364 (1975)
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat and hamster liver S9
Test concentrations with justification for top dose:
100, 333, 1.000, 3.333, and 10.000 ug/plate
Vehicle / solvent:
DMSO
Details on test system and experimental conditions:
Methode: Ames test
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Conclusions:
Interpretation of results (migrated information):
negative

In this Salmonella typhimurium reverse mutation assays EGDMA did not increase the reversion rates in tester strains TA 98, TA100, TA1535 and TA1537 (test concentrations: 100-10,000 µg/plate with and without metabolic activation).
In conclusion, it can be stated that during the decribed mutagenicity test and under the experimental conditions reported, Ethyleneglycol dimethacrylate did not induce gene mutations by base pair changes or frameshifts in the genome of the strains tested.
Therefore, the test substance has to be judged as nonmutagenic up to 10000 µg/plate in the presence and absence of mammalian metabolic activation according to the Ames test results (study conducted similar to OECD 471).
Executive summary:

In a reverse gene mutation assay (similar to OECD 471) in bacteria (Ames test), strains TA1535, TA1537, TA98, TA100 of Salmonella typhimurium were exposed to Ethyleneglycol dimethacrylate (86%) at concentrations of 100 to 10000 µg/plate in the presence and absence of mammalian metabolic activation S9 -mixIn conclusion, it can be stated that during the decribed mutagenicity test and under the experimental conditions reported, Ethyleneglycol dimethacrylate did not induce gene mutations by base pair changes or frameshifts in the genome of the strains tested. Therefore, the test substance has to be judged as nonmutagenic up to 10000 µg/plate in the presence and absence of mammalian metabolic activation according to the Ames test results.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
TA102 or E. coli WP2 uvrA not tested
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
TA102 or E. coli WP2 uvrA not tested
Principles of method if other than guideline:
Method: Ames test according to Ames et al., Mutat. Res. 31: 347-364 (1975)
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium, other: TA98, TA100, TA1535, TA1537, TA1538
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital- and Aroclor-induced rat S9-mix
Test concentrations with justification for top dose:
40, 160, 625, 2500 µg/plate
Details on test system and experimental conditions:
Method: Ames test, plate incorporation method
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535, TA1537, TA1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Conclusions:
Interpretation of results (migrated information):
negative

In this Salmonella typhimurium reverse mutation assays EGDMA did not increase the reversion rates in tester strains TA 98, TA100, TA1535, TA1537 and TA1538 (test concentrations: 40 -2,500 µg/plate with and without metabolic activation).
In conclusion, it can be stated that during the decribed mutagenicity test and under the experimental conditions reported, Ethyleneglycol dimethacrylate did not induce gene mutations by base pair changes or frameshifts in the genome of the strains tested.
Therefore, the test substance has to be judged as nonmutagenic up to 2500 µg/plate in the presence and absence of mammalian metabolic activation according to the Ames test results (study conducted similar to OECD 471).
Executive summary:

In this Salmonella typhimurium reverse mutation assays EGDMA did not increase the reversion rates in tester strains TA 98, TA100, TA1535, TA1537 and TA1538 (test concentrations: 40 -2,500 µg/plate with and without metabolic activation).

In conclusion, it can be stated that during the decribed mutagenicity test and under the experimental conditions reported, Ethyleneglycol dimethacrylate did not induce gene mutations by base pair changes or frameshifts in the genome of the strains tested.

Therefore, the test substance has to be judged as nonmutagenic up to 2500 µg/plate in the presence and absence of mammalian metabolic activation according to the Ames test results (study conducted similar to OECD 471).

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
TA102 or E. coli WP2 uvrA not tested
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
TA102 or E. coli WP2 uvrA not tested
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Test concentrations with justification for top dose:
0.001, 0.01, 0.1, 1.0, and 5.0 µl/plate
Details on test system and experimental conditions:
Ames test
Species / strain:
S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Conclusions:
Interpretation of results (migrated information):
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 shifts in the genome of the strains tested.
Therefore, the test substance has to be judged as nonmutagenic up to 5.0 µL/plate in the presence and absence of mammalian metabolic activation according to the Ames test results.
Executive summary:

In a reverse gene mutation assay (similar to OECD 471) in bacteria (Ames test), strains TA1535, TA1537, TA98, TA100 of Salmonella typhimurium were exposed to Ethyleneglycol dimethacrylate at concentrations of 0.001 to 5 µl/plate in the presence and absence of mammalian metabolic activation S9 -mix

In conclusion, it can be stated that during the decribed mutagenicity test and under the experimental conditions reported, Ethyleneglycol dimethacrylate did not induce gene mutations by base pair changes or frameshifts in the genome of the strains tested.Therefore, the test substance has to be judged as nonmutagenic up to 5 µl/plate in the presence and absence of mammalian metabolic activation according to the Ames test results.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
The mouse lymphoma assay evaluates the ability of a test substance to induce forward mutation in the L5178Y (TK+/-) mouse lymphoma cell line as determined by cell growth in medium containing the anti-metabolite trifluorothymidine (TFT).

GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
TK locus of L5178Y mouse lymphoma
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
AROCLOR 1254 induced rat liver microsomal fraction S9 mix
Test concentrations with justification for top dose:
Experiment i and II (+/- S9-mix): 78, 156, 313, and 625 mg/l
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO, Dimethylsulphoxide
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties.
Positive controls:
yes
Positive control substance:
other: N-nitrosodimethylamine (NDMA), with metabolic activation, final dose 600 µg/mL
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Migrated to IUCLID6: without metabolic activation, final dose 750 µg/mL
Untreated negative controls:
yes
Remarks:
Dimethylsulphoxide (DMSO)
Negative solvent / vehicle controls:
yes
Remarks:
Dimethylsulphoxide (DMSO)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION

- Exposure duration: 4 hours
- Expression time (cells in growth medium): 72 hours
- Selection agent: Trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2
Evaluation criteria:
Acceptability of the Assay
The gene mutation assay is considered acceptable if it meets the following criteria:
Cell growth and maintenance
- post-expession plating efficiencies of 50% or greater should be achieved for the negative control viability plates

Spontaneous control data
- spontaneous mutant frequency with and without metabolic activation S9-mix should be within the range of 0.8 - 6.0 x 10E4 mutants per survivor (based on historical data)
- must be reproducible

Positive control data
- positive control substances tested must give unequivocal positive responses
- failure of the positive control does not itself invalidate the data for the test substance that gives a positive effect if all the other criteria are satisfied
- should be reproducible in a separate experiment

Criteria for positive response
- statistically significant dose-related increase in mutant frequency is required, but not only at dose levels showing excessive toxicity (ie, less than 10% survival)
- absolute increase in mutant number must be above the negative control values
- must be reproducible in a separate experiment

Criteria for a negative response
- no reproducible statistically significant dose-related increase in mutant frequency
- reproducible statistically significant increasees in mutant frequencyy are only seen at levels of high toxicity (less than 10% survival), or when such increases are not accompanied by an increase in absolute numbers of mutants over negative control values.
Statistics:
Interpretation of data are supported by statistical analysis where considered necessary. It monitors both for significance of individual doses and trend of dose response.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
in forward gene mutations in mammalian cells
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: other: L5178Y TK+/-
Remarks:
Migrated from field 'Test system'.

Summary of data Experiment I

Dose µg/mL EGDMA Experiment I (- S9) Survival % Experiment I (- S9) Mutant frequency (x10E-4) Experiment I (+ S9) Survival %  Experiment I (+ S9) Mutant frequency (x10E-4)
 625  6  4.2  75  2.9
 313  83  2.8  105  2.5
 156  81  2.1  93  2.7
 78  97  3.7  107  3.5
Negative control DMSO (10 µl/mL)  100  2.8  100  3.1
Positive control EMS (750 µg/mL)  45  21.9  N.D.  N.D.
Positive control NDMA (600 µg/mL)  N.D.  N.D.  30  27.9

N.D.: Not determined

Summary of data Experiment II

Dose µg/mL EGDMA Experiment II (- S9) Survival % Experiment II (- S9) Mutant frequency (x10E-4) Experiment II (+ S9) Survival %  Experiment II (+ S9) Mutant frequency (x10E-4)
 625  15  2.4  98  2.7
 313  70  2.2  99  3.3
 156  80  2.6  115  3.1
 78  86  3.3  128  2.4
Negative control DMSO (10 µl/mL)  100  2.6  100  1.9
Positive control EMS (750 µg/mL)  62  17.8  N.D.  N.D.
Positive control NDMA (600 µg/mL)  N.D.  N.D.  68  15.1

N.D.: Not determined

Conclusions:
Interpretation of results (migrated information):
negative in vitro

In conclusion it can be stated that under the experimental conditions reported the test item Ethylene glycol dimethacrylate is non-mutagenic in mouse lymphoma assay to L5178Y cells in the presence and absence of mammalian metabolic activation system (S9) when tested to concentrations up to 625 µg/mL (+/- S9-Mix) limited by solubility in the treatment medium.
Executive summary:

In a mammalian cell gene mutation assay mouse lymphoma test with L5178Y TK+/- cells cultured in vitro were exposed to Ethylene glycol dimethacrylate (purity: 98 % a.i. (w/w)) at concentrations of  78, 156, 313, and 625 µg/mL in the presence and absence of mammalian metabolic activation S9 -mix (Aroclor 1254 induced rat liver homogenate). 

Mutant frequencies were assessed by cell growth in the presence of trifluorothymidine after 72 hour expression time.

The assay was performed in two independent experiments both in the presence and absence of S9 up to the dose level 625 µg/mL limited by solubility of Ethylene glycol dimethacrylate in the treatment medium. In both experiments 625 µg/mL produced dose-related cytotoxicity.

Although limited by solubility, the doses tested reduced cell survival to minima of 6 % and 75 % in the absence and
presence of S9 respectively at the maximum dose, indicating biological activity.

There were no reproducible increases in mutant frequency in the presence or absence of S9.

 

The positive controls did induce the appropriate response. 

There was no evidence of induced mutant colonies over background.

 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data. 

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

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
Qualifier:
according to
Guideline:
other: OECD 473 (1983)
Deviations:
yes
Remarks:
the stability of the test substance has not been reported; the stability and achieved concentration of the test substance in the vehicle used were not determined by analysis
Principles of method if other than guideline:
Method: according to OECD Guide-line 473 (1983) and UKEMS Recommended Procedure for Basic Mutagenicity Tests (Kirkland, 1990)
GLP compliance:
yes
Type of assay:
other: cultured human lymphocytes
Species / strain / cell type:
other: human lymphocytes
Metabolic activation:
with and without
Metabolic activation system:
AROCLOR 1254 induced rat liver microsomal fraction S9 mix
Test concentrations with justification for top dose:
Range-finding cytotoxicity test:
8, 40, 200, 1000 and 5000 µg/mL
Main cytogenetic tests:
-S9-mix: 25, 100, 200, 400, 600, and 800 µg/mL
+S9-mix: 100, 500, 1000, 2000, 3500 and 5000 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO, Dimethylsulphoxide
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties.
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Dosing solutions prepared in sterile double deionised water. Migrated to IUCLID6: Final concentration: 1.0 µg/mL
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Dosing solutions prepared in sterile double deionised water. Migrated to IUCLID6: Final concentration: 50 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period:
- Exposure duration: 72 and 96 hours
- Expression time (cells in growth medium): 3 hours
- Fixation time (start of exposure up to fixation or harvest of cells): at 72 or 96 hours

SPINDLE INHIBITOR (cytogenetic assays): colcemid (0.4 µg/mL)
STAIN (for cytogenetic assays): Giemsa and mounted in DPX

NUMBER OF REPLICATIONS:2

NUMBER OF CELLS EVALUATED: 100 cells per culture were evaluated (examination of cells with abberations); 1000 lymphocytes per culture (examination of mitose index)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
Statistics:
Evaluation was performed only for cells carrying aberrations exclusive gaps. The cells for each of the treatment groups were compared to the solvent control values using the Fisher's Exact Test (one sided).
Species / strain:
lymphocytes: human lymphocytes
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
at 600 µg/mL
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1000 and 5000 µg/mL in the absence of S9 or at 5000 µg/mL in the presence of S9-mix.
Vehicle controls validity:
valid
Positive controls validity:
valid

Results:
Increases in the percentage of aberrant cells were observed at 100, 400, and 600 mg/l in male donor cultures at 72 h sampling time and in female donor cultures at 600 mg/l harvested after 72 h; no significant increases were observed
at 96 h sampling time.

Conclusions:
Interpretation of results (migrated information):
positive in vitro in the absence of S9-mix

In the Chromosomal aberration test with human lymphocytes according to OECD 473 in the absence of mammalian activation S9-mix structural chromosomal aberrations were observed. Therefore, Ethyleneglycol dimethacrylate is considered to be clastogenic (in vitro in the absence of S9-mix) at 600 µg/mL under the experimental conditions reported.
Executive summary:

In a cytogenetic assay (Chromosome aberration test with human lymhocytes from two donors (1 f, 1 m) according to OECD 473) human lymphocytes were exposed to Ethylene glycol dimethacrylate (purity: 98 % w/w, solvent: DMSO) at concentrations in the range of 25 to 5000 µg/mL

in the presence and absence of mammalian metabolic activation S9 -mix (Aroclor 1254 induced rat liver homogenate). 

 

The assay was performed in two independent experiments using Ethylene glycol dimethacrylate (EGDMA) concentrations of 25, 100, 200, 400, 600 and 800 µg/mL in the presence of S9 -mix and concentrations of 100, 500, 1000, 2000 , 3500 and 5000 µg/mL in the absence of S9 -mix. Dose related reductions in mitotic activity were observed in cultures from both donors in the presence and absence of S9 -mix at the 72 hour sampling time.

Cultures treated with higher concentrations of EGDMA (600 µg/mL -S9 -mix and 1000 µg/mL +S9 -mix) in the main cytogenetic tests were considered not to be suitable for chromosomal aberration analysis due to lack of metaphases as a result of toxicity or due to cytotoxic effects on the chromosome structure.

Cultures from both donors exposed with EGDMA at concentrations of 100, 400 and 600 µg/mL in the absence of S9 -mix and 100, 500 and 1000 µg/mL in the presence of S9 -mix were choosen for chromosomal aberration analysis at 72 hours sampling time. Additionally, cultures from the female donor treated at 400 µg/mL in the absence of S9 -mix and 1000 µg/mL in the presence of S9 -mix were selected for analysis at 96 hour sampling time.

No statistically or biologically significant increases in percentage of aberrant cells, compared to the solvent control values, were seen at any of the EGDMA concentrations tested in either donor, in the presence of S9 -mix at the 72 hour sampling time or in the presence or absence of S9 -mix at 96 hour sampling time. Statistically significant increases of chromosomal aberrations were seen in cultures from both donors treated with EGDMA at 600 µg/mL in the absence of S9 -mix and examined at 72 hours sampling time.

No significant increases were observed at the 96-hours.

Positive controls (mitomycin C and cyclophosphamide) induced the appropriate response confirming the sensitivity of the test system. 

Therefore EGDMA showed under the experimental conditions reported reproducibly induced chromosomal damage in human peripheral blood lymphocytes in vitro in the absence of auxiliary mammalian metabolic activation S9 -mix.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 473 for in vitro cytogenetic mutagenicity data. 

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

Genetic toxicity in vivo

Description of key information

The two in vivo mutagenicity tests with EGDMA the mouse micronucleus test and the unscheduled DNA synthesis assay in male rats were negative. Therefore, it is concluded that EDGMA is unlikely to be mutagenic in vivo.

Link to relevant study records

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Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
(1983)
Principles of method if other than guideline:
Pre-Experiment for Toxicity
A preliminary study on acute toxicity was performed with 1250 and 2000 mg/kg with groups of 2 male animals. 2/2 animals were dead after application of 2000 mg/kg EGDMA. The male animals exposed to 1250 mg/kg EGDMA survived the treatment. Therefore a further group of 3 males and 5 females were than dosed with Ethylene glycol dimethacrylate at a dose level of 1250 mg/kg. Result all animals survived the exposure.
The animals were treated orally with a single dose 1250 mg/kg bw of the test item and examined for acute toxic symptoms (four day observation period). Based on clinical signs and lethalities 1250 mg/kg Ethylene glycol dimethacrylate was selected as the maximum tolerated dose for the males and females.
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Margate, UK
- Number of animals. 54; 27 males and 27 females
- Age at study initiation: 5-13 weeks
- Weight at study initiation: 25.5 to 39.6 g males and 20.4 to 32.7 g females
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: 5 per cage
- Diet:Porton combined diet, ad libitum
- Water: filtered tap water, ad libitum
- Acclimation period: yes, but no data how long


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-21°C
- Humidity (%): 40-70 %
- Air changes (per hr): approximately 25 air changes
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs artifical light
Route of administration:
oral: unspecified
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: The vehicle was chosen to its relative non-toxicity for animals. All animals received a single standard
volume of 10 mL/kg (0.1 mL/10g) body weight orally.
- Concentration of test material in vehicle: 1250 mg/kg MTD
- Supplier: Central Dispensary, CTL
- Lot/batch no.: CTL reference number: Y00790/004
- Purity: no data
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

The test material was prepared in corn oil. A further solution of Cyclophosphamide was prepared in sterilised physiological saline. All animals received a single standard volume of 10 ml/kg body weight orally.

DIET PREPARATION
- Rate of preparation of diet (frequency): no data
- Mixing appropriate amounts with (Type of food): no data
- Storage temperature of food: no data
Duration of treatment / exposure:
single dose
Frequency of treatment:
once
Post exposure period:
24h and 48 hours
Dose / conc.:
1 250 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
CPA; cyclophosphamide
- Supplier: Sigma Chemical Company Ltd, Poole, UK
- Purity: commercial grade
- Dissolved in: sterilised physiological saline
- Route of administration: orally, once
- Doses / concentrations: 65 mg/kg bw
- Volume administered: 10 mL/kg bw
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or
the highest dose that can be formulated and administered reproducibly.
The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 72
hours.
The volume to be administered should be compatible with physiological space available.

TREATMENT AND SAMPLING TIMES:

Treatment:
At the beginning of the treatment the animals (including the controls) were weighed and the individual volume to be administered was adjusted to the animals body weight. The animals received the test item, the vehicle or the positive control substance once. Ten animals, five males and five females, were treated per dose group and sampling time.
Sampling of bone marrow was done 24 and 48 hours after treatment, respectively.


DETAILS OF SLIDE PREPARATION:

Preparation of the Animals:
The animals were sacrificed by asphyxiation in a rising concentration of carbon dioxide followed by cervical dislocation 24 and 48 hours after application of the test material. The femora were removed and stripped clean of muscle. The iliac end of the femur was removed and a fine paint brush rinsed in saline and wetted with a solution of albumin (6% w/v in physiological saline). Then this was dipped into the marrow canal and two smears were painted on a microscope slide. This was repeated to give 4 smears per slide. The smear was air-dried and then stained with polychrome methylene blue and eosin using an Ames Hema-Tek staining machine (Hema-Tek, Miles Laboratory Incorporated, Berkshire, UK).

METHOD OF ANALYSIS:
Analysis of Cells:
Evaluation of the slides was performed using x10 or x12.5 eye pieces and a 100x oil immersion objective lens for each animal. At least 1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 1000 erythrocytes. The analysis was performed with coded slides.
Ten animals (5 males, 5 females) per test group were evaluated as described. The remaining animal of each test group was evaluated in case an
animal had died in its test group spontaneously or due to gavage error.
Evaluation criteria:
A test article is classified as mutagenic if it induces either a statistically significant dose-related increase in the number of micronucleated
polychromatic erythrocytes or a reproducible statistically significant positive response for at least one of the test points.
A test article producing neither a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes nor a
statistically significant and reproducible positive response at anyone of the test points is considered nonmutagenic in this system.

However, both biological and statistical significance should be considered together.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Positive controls validity:
valid

Table 1

Mean incidence of micronucleated polychromatic erythrocytes/1000 polychromatic erythrocytes ± standard deviation (SD) at two sampling times:

group       compound       dose       mean incidence of       mean incidence of

MPE/1000 PE ± SD        MPE/1000 PE ± SD

mean animal data        mean animal data

males                   females

24h       48h           24h       48h

---------------------------------------------------------------------------------

11       vehicle control  10mL/kg    0.6 ±0.6 0.6 ±0.6       0.8 ±0.8 0.6 ±0.6

(corn oil)

12       Cycolphosphamide 65mg/kg    20.6 ±4.5**             14.0 ±6.9**

13       EGDMA            1250mg/kg  0.6 ±0.6 0.8 ±0.5(4)    1.2 ±1.1 0.2 ±0.5

---------------------------------------------------------------------------------

PE= polychromatic erythrocytes

MPE= micronucleated polychromatic erythrocytes

SD= standard deviation

** statistically significant increase in micronucleated polychromatic erythrocytes at p<0.01 in the Student's 't' test (one-side) on transformed data.

All means based on 5 animals except where indicated in parentheses.

Table 2

Mean percentage of polychromatic erythrocytes ± standard deviation (SD) at two sampling times:

group      compound      dose      mean % of polychromatic  mean % polychromatic                                   erythrocytes ± SD       erythrocytes ± SD                                    mean animal data       mean animal data

males                   females

24h       48h           24h       48h

---------------------------------------------------------------------------------

11       vehicle control 10mL/kg    54.1 ±17.0 49.6 ±10.3    52.4 ±8.3 48.0 ±13.8

(corn oil)

12      Cycolphosphamide 65mg/kg    40.4 ±4.9*              47.6 ±10.2

13      EGDMA            1250mg/kg  51.7 ±15.5 39.0 ±8.8(4)  49.1 ±7.7 54.6 ±8.8

---------------------------------------------------------------------------------

SD= standard deviation

* statistically significant decrease in the percentage of polychromatic erythrocytes at p<0.05 in the Student's 't' test (one-sided).

All means based on 5 animals except where indicated in parentheses.

Conclusions:
Interpretation of results (migrated information): negative
During the study decribed and under the experimental conditions reported, Ethylene glycol dimethacrylate did not induce micronuclei. This was determined by the micronucleus test with bone marrow cells of the mouse (OECD guideline 474). Therefore, Ethylene glycol dimethacrylate is considered to be non-mutagenic in this micronucleus assay.
Executive summary:

In a CD-1 mouse bone marrow micronucleus assay, 5 animals/male/female/dose were treated orally with Ethylene glycol dimethacrylate (98.8%, stabilizied) at a single dose of 1250 mg/kg bw. The test article was suspended in corn oil. This suspending agent was used as negative control. The volume administered orally was 10 ml/kg b.w.. 24 h and 48 hours after a single application of the test article the bone marrow cells were collected for micronuclei analysis. Ten animals (5 males and 5 females) per test group were evaluated for the occurrence of micronuclei. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei.

To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE.

The following dose level of the test article was investigated:

24 h and 48 h preparation interval: 1250 mg/kg b.w..

In a pre-experiment this dose level was estimated to be the maximum attainable dose. After treatment with the test article the ratio between PCEs and NCEs was not affected as compared to the corresponding negative control thus indicating no cytotoxic effects.

In comparison with the corresponding negative control there was no enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article.

An appropriate reference mutagen was used as positive control which showed a distinct increase of induced micronucleus frequency. 

This study is classified as accceptable. This study satisfies the requirement for Test Guideline OECD 474 for in vivo cytogenetic mutagenicity data.

Therefore, Ethylene glycol dimethacrylate is considered to be non-mutagenic in this micronucleus assay.

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods
Remarks:
ASTM
Principles of method if other than guideline:
Method: according to Butterworth et al., Mutat. Res. 189: 123-133 (1987) described as an ASTM guideline.
GLP compliance:
yes
Type of assay:
unscheduled DNA synthesis
Species:
rat
Strain:
other: Alderley Park
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: ICI Barriered Animal Breeding Unit, Alderley Park, Macclesfield
- Number of animals: 36 males
- Age at study initiation: approximately 6-8 weeks
- Weight at study initiation: 190 to 302 g males
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: stainless steel cages, wire mesh floored, suspended cages (ICI specification R2); 5 animales per cage
- Diet:Porton combined diet (supplied by Special Diets Services Ltd, ad libitum
- Water: drinking water, ad libitum
- Acclimation period: not considered appropriate


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2°C
- Humidity (%): 40-60 %
- Photoperiod (hrs dark / hrs light): 12 hrs light, 12 hrs dark
- Air changes (per hr): approximately 25 air changes
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: dried corn oil
- Justification for choice of solvent/vehicle: The vehicle was chosen to its relative non-toxicity for animals. The animals received a single standard
volume of 10 mL/kg (0.1 mL/10g) body weight orally.
- Concentration of test material in vehicle: 1250 or 2000 mg/kg
- Supplier: no data
- Lot/batch no.: CTL reference number: Y00990/005
- Purity: no data
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

The test material was prepared in dried corn oil. Concentrations prepared for UDS assay were 125 and 200 mg/mL. EGDMA suspensions were used within a day of preparation.

DIET PREPARATION
- Rate of preparation of diet (frequency): no data
- Mixing appropriate amounts with (Type of food): no data
- Storage temperature of food: no data
Duration of treatment / exposure:
single dose
Frequency of treatment:
once
Post exposure period:
2 or 16 hours post-treatment
Dose / conc.:
1 250 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
No of animals for the whole study: 36 male rats (inclusive positive and negative control animals); 8 groups with 2-3 animals per dose group (2 experiments dose group)
Control animals:
yes, concurrent vehicle
Positive control(s):
2-Acetylaminofluorene (2-AAF)
- Supplier: no data
- Purity: no data but commercial grade assumed
- Route of administration: orally, once
- Treatment time: 16 hours
- Doses / concentrations: 2.5 mg/mL bw suspension in 0.5 % w/v Hydroxypropylmethyl cellulose in 0.1% aqueous PS80 (HPMC)

N-Nitrosodimethylamine (NDMA)
- Supplier: no data
- Purity: no data but commercial grade assumed
- Dissolved in: dissolved at 1 mg/mL in deionised water
- Route of administration: orally, once
- Treatment time: 2 hours
- Doses / concentrations:1mg/mL mg/kg bw
Evaluation criteria:
Negative Controls: Cytoplasmic counts of less than 40 are considered acceptable. Mean net nuclear grain counts for the negative controls should be less than zero (Ashby et al 1987).
Positive Controls: Mean net grain counts for the positive control in each experiment should have a value of 5 or greater, with at least 20% of the cells in repair. A cell in repair is one with a net nuclear grain count of at least 5.

Criteria for a Negative (ie non-genotoxic) Response: A negative response is obtained where the mean net nuclear grain count of test compound treated animals is less than 0, and the percentage of cells in repair is less than 20.

Criteria for a Positive (ie genotoxic) Response: In this laboratory no negative control animal has given a mean net nuclear grain count of greater than zero. In Butterworth et al (1987) a survey of four laboratories sampling over 200 animals gave no negative control treatment with an [N-C] value of
greater than zero. It appears, therefore, that a mean animal [N-C] value of greater than zero represents a biologically significant departure from
normal. Furthermore, at this point the radiolabelling of the nucleus exceeds that of the cytoplasm, so there is confidence that areal net synthesis of nuclear DNA has occurred. The occurrence of a mean net nuclear grain count of zero or greater in a treated animal is therefore taken as indicative of a UDS response.
A compound is assigned as an unequivocal genotoxic agent in this assay if such a response is reproduced in animals treated concurrently and in an
independent repeat experiment.
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 20, 200 and 2000 mg/kg bw as a solution in dried corn oil
- Exposure application: single dose (10 ml/kg bw)
- Observation: 4 days
In the range finding study for UDS a preliminary oral toxicity study was carried out. No animals died after application of Ethylene glycol dimethacrylate at the highest test concentration of 2000 mg/kg (test duration 96 hours). All groups were observed for four days and no signs of toxicity were observed.

Results:
Over the period of the main UDS experiments Ethylene glycol dimethacrylate exposed animals showed no signs of acute toxic effects.

Cytotoxicity: Hepatocytes of the Ethylene glycol dimethacrylate treated animals showed no signs of excessive cytotoxicity. Therfore both dose levels were assessed for UDS.

Ethylene glycol dimethacrylate caused no significant increases over control in mean net nuclear grain count, nor in percentage of cells in repair, at either dose level (1250 or 2000 mg/kg) or time point (2 or 16 hours). Hepatocytes from

Ethylene glycol dimethacrylate treated animals had mean net nuclear grain

values of less than zero.

Therefore the test substance did not induce DNA repair in rat liver hepatocytes.

Ethylene glycol dimethacrylate: Assessment for the induction of UDS synthesis in rat hepatocytes in vivo

Summary data

Dose       No. of        Mean        Mean        Mean (N-C)       Mean %

(mg/kg) Animals      N ± SD      C ± SD      ± SD              Cells in

Repair (a)

------------------------------------------------------------------------------- 16 Hours

Corn oil

lOml/kg)     2        5.1        6.9       -1.8                 1

EGDME(1250)   5        5.4 ± 0.9   8.0 ± 1.7 -2.6 ± 0.8           1

EGDME(2000)  5        4.8 ± 1.1 7.3±1.5   -2.5 ± 0.5           1

2-AAF (25)   2        21.6        9.2       +12.3               84

2 Hours

Corn oil

(10ml/kg)    2        5.2        8.1       -2.9                 1

EGDME(1250)  5        5.0 ± 1.5   8.0 ± 1.7 -3.0 ± 0.6           1

EGDME(2000) 5         6.6 ± 3.7 9.2 ± 3.2 -2.5 ± 1.3           4

NDMA (10)    2        26.2        8.4      +17.9               84

--------------------------------------------------------------------------------

N ± SD = mean nuclear grain count ± standard deviation

C ± SD = mean cytoplasmic grain count ± standard deviation

(N-C) ± SD = mean net nuclear grain count ± standard deviation

(a) Cells in repair have net nuclear grain counts >=5.

Note: Values shown are the means of individual cell observations.

Conclusions:
Interpretation of results (migrated information): negative
Under the above mentioned test conditions the test substance Ethylene glycol dimethacrylate did not induce DNA repair in rat liver in vivo, when tested up to a limit dose of 2000 mg/kg. The test substance did not induce unscheduled DNA synthesis (UDS) in rat hepatocytes in vivo.
Executive summary:

In an unscheduled DNA synthesis assay, primary rat hepatocyte cultures were exposed to Ethylene glycol dimethacrylate (98.8%, solvent used: corn oil) at concentrations of 0, 1250 or 2000 mg/kg (sampling time: 2 and 16 hours after administration of the test substance). 

 

The positive controls the carcinogens 2 -Acetylaminofluorene or N-Nitrosodimethrylamine induced the appropriate response. 

There was no evidence of a positive response that unscheduled DNA synthesis, as determined by radioactive tracer procedures [nuclear silver grain counts] was induced.

 

This study is classified as acceptable.  This study satisfies the requirement for unscheduled DNA synthesis genotoxic mutagenicity data. 

Therefore under the above mentioned test conditions the test substance Ethylene glycol dimethacrylate was not mutagenic. It did not induce DNA repair in rat liver in vivo, when tested up to a limit dose of 2000 mg/kg.

Additional information

Assessment by endpoint

a) Gene Mutation Tests

While bacterial mutagenicity tend to be negative as in the case of methacrylic acid and other methacrylate esters, mammalian gene mutation data throughout the category of multifunctional methacrylates, including EGDMA, indicate the presence of high-toxicity potential for mutagenicity in vitro. At the same time, this activity has also been found in chromosome aberration tests in vitro. Particularly the mouse lymphoma assay is known to be sensitive also to clastogenic substances, and therefore positive findings in this test are not always a clear indication of the induction of gene mutations.

The two UDS tests in EGDMA and TMPTMA, both investigating a relevant target tissue (hepatocytes) indicate the absence of mutagenicity, and gene mutations in particular, in vivo.

b) Chromosome Aberration Tests

Three in-vitro tests with EGDMA, 1,4-BDDMA and TMPTMA indicate the presence of a weak clastogenic potential in vitro. That is consistent with findings with other groups of methacrylates, e.g. lower alkyl methacrylates. The corresponding in vivo tests, three micronucleus tests in mouse bone marrow and the two UDS tests are all negative, which indicates the absence of mutagenicity in vivo. This likely reflects that it is the parent ester that is responsible for the clastogenicity in vitro and that these chemicals are rapidly metabolised in vivo to the corresponding acid and alcohol, which are not mutagenic

c) Aneugenicity

Data on aneugenicity have been presented in three of the in vitro tests on chromosome damage as well as in the three micronucleus tests in vivo and the one in vitro. The only test that indicated a slight increase above background for the induction of aneuploidy was the chromosome aberration test in CHO cells in vitro with 1,4-BDDMA. With the absence of a corresponding effect in the micronucleus test in vivo there is no indication that EGDMA or the other multifunctional methacrylates are aneugenic.

Overall, EGDMA is not regarded to be mutagenic.


Endpoint Conclusion: No adverse effect observed (negative)

Compliance to REACh requirements

The in vitro mutagenicity test requirements are all covered with an Ames test with 4 tester strains, a chromosome aberration test, and a Mouse Lymphoma assay. The in vivo mutagenicity test requirement is covered by an Unscheduled DNA Synthesis test and an in vivo Micronucleus test. All mentioned studies are reliable (Reliability 1 or 2) and were performed with the substance itself. The test requirement for a fifth tester strain (E. coli/ S. typh. TA 102) in the Ames test is covered by consistent negative results of other category members and other methacrylate esters (category read across, assessed as acceptable with high confidence).

Justification for classification or non-classification

The overall data do confirm the non-mutagenicity of the substance. Therefore, no classification is required.