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Toxicological information

Genetic toxicity: in vivo

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Administrative data

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

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1992
Report date:
1992
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report date:
1992

Materials and methods

Test guideline
Qualifier:
according to guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethylene dimethacrylate
EC Number:
202-617-2
EC Name:
Ethylene dimethacrylate
Cas Number:
97-90-5
Molecular formula:
C10H14O4
IUPAC Name:
ethane-1,2-diyl bis(2-methylacrylate)

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system 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

Administration / exposure

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
Doses / concentrations
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

Examinations

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.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.