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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
22.01.2001
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
(August 1988)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

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:
2-[(2-methylprop-2-enoyl)oxy]ethyl 2-methylprop-2-enoate
Details on test material:
- Name of test material (as cited in study report): Ethylene Glycol Dimethacrylate (EGDMA; CAS RN: 97-90-5)

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, L' Arbresle, France
- Age at study initiation: 11 weeks
- Weight at study initiation: mean body weight: 258 g (217 g to 298 g)
- Fasting period before study: no data
- Housing: Animals were housed individually, except during mating, in suspended wire-mesh cages (43.0 x 21.5 x 18.0 cm).
- Diet (e.g. ad libitum): ad libitum SSNIFF R/M-H pelleted maintenance diet
- Water (e.g. ad libitum): ad libitum, filtered tab water
- Acclimation period: 4 or 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ±2°C
- Relative humidity: 50 ± 20%
- Air changes (per hr): ca. 12 cycles/hour filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
The test item was administered as a solution in corn oil.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): Ethylene glycol dimethacrylate dosage formulations were prepared once weekly
- Storage temperature of food: + 4°C, protected from light and humidity

VEHICLE
- Concentration in vehicle: 5, 20 and 100 mg/mL
- Lot/batch no. (if required): 015K0115; Supplier: Sigma (Saint-Quentin-Fallavier, France)
- Purity: commercial grade
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
An aliquot of each dosage formulation was diluted appropriately then analyzed by High Performance Liquid Chromatography with Ultra-Violet
detection at 200 nm. The concentration of the test item was determined from a calibration curve of peak area against concentration of Ethylene Glycol Dimethacrylate (EGDMA; CAS RN 97-90-5) standard solutions (external standard calibration).
The analytical method used was validated according to CIT procedure prior to analysis of the study samples. The validation data (CIT/Study No. 28780 VAL) demonstrated the suitability of the method for analysis of the dosage formulation.

Validation of the analytical method

Specificity
The specificity of the analytical method was demonstrated as follows:
- analysis of a standard solution of Ethylene Glycol Dimethacrylate (EGDMA; CAS RN 97-90-5) in mobile phase,
- analysis of corn oil (vehicle) diluted ten fold in isopropanol.
No relevant interference between the test item peak and corn oil was observed on chromatograms.

Limit of quantification
The limit of quantification of the analytical method was established at 1 μg/mL for a standard solution of Ethylene Glycol Dimethacrylate (EGDMA;
CAS RN 97-90-5). This limit corresponds to a limit of quantification of 0.01 mg/mL for the test item in corn oil.

Linearity
Linearity was checked by analysis of three different sets of seven standard solutions containing 1, 2, 5, 10, 20, 50, and 100 μg/mL of Ethylene Glycol Dimethacrylate (EGDMA; CAS RN 97-90-5) in mobile phase.
Satisfactory linearity was demonstrated in the range of 1 to 100 μg/mL since the coefficients of determination obtained were higher than 0.98.
Details on mating procedure:
Females were mated with males at the breeder's facilities. The day of confirmed mating (detection of a vaginal plug) was considered as gestation day 0 post-coitum (p.c.). 
Duration of treatment / exposure:
6 - 20 day post coitum inclusive
Frequency of treatment:
once a day
Duration of test:
15 d (dams were euthanized on gestation day 21)
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
24 mated female animals per group exposed.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose-levels were selected following the results of a previous range-finding study (CIT study No. 28192 RSR).
- Rationale for animal assignment (if not random): The animals were allocated to the treatment groups, according to a stratified procedure based on body weight recorded on day 2 p.c., to ensure comparatively similar mean body weights among the groups. Body weights of the animals assigned to the study at the start of the treatment period were within 20% of the mean group weight.
- Other: The strain was selected because background development toxicity data exists at CIT company on this rat strain. The test material was given by oral administration (gavage) since the oral route is requested by the regulatory authorities for this type of test item.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS:
- Time schedule: Animals were observed daily for behavioral changes.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day; Morbidity and mortality: at least twice a day including weekends and public holidays, once a day on other days

BODY WEIGHT: Yes
- Time schedule for examinations: Maternal body weights were recorded on GD 2, 6, 9, 12, 15, 18 and 21 p.c. and prior to premature sacrifice (female M28119 (group 4) on GD 15). 

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption: The quantity of food consumed by each female was measured for GD intervals 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 p.c.. 

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations:

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
Ovaries and uterine content:
On GD 21 p.c., all dams were asphyxiated with carbon dioxide, the thoracic and abdominal organs were examined. The weight of the gravid uterus (including the cervix) and liver was recorded for each pregnant female (with at least one live fetus).Subsequently, the liver was discarded.
The ovaries and uterus of the females were examined to determine:
number of corpora lutea, number and distribution of dead and live fetuses, early and late resorptios, uterine scars and implantation sites.
A gross evaluation of placentas was also undertaken.
Fetal examinations:
The live fetuses were sacrificed by a subcutaneous injection of pentobarbital. The following examinations were conducted for all the litters where the female had at least one live fetus.
The fetal external, soft tissue and skeletal findings were described according to the glossary of the International Federation of Teratology Societies
(IFTS) and classified as malformations or variations (Wise, Chahoud):
- malformation refers to permanent structural change that is likely to adversely affect the survival or health,
- variation refers to a change that is unlikely to adversely affect survival or health (this might include a delay in growth or morphogenesis that other
wise followed a normal pattern of development).

Body weight
The body weight of each fetus was recorded.

External examination
Each fetus was subjected to a detailed external examination, which included the observation of all visible structures, surfaces and orifices.

Soft tissue examination
A detailed fresh, soft tissue examination was performed by microdissection on approximately half of the live fetuses in each litter. This included the observation of all the organs and structures of the neck, thorax and abdomen. After the examination, the fetuses were fixed in Harrison’s fluid for
examination of the organs and structures of the head (Wilson, 1965).

Skeletal examination
The remaining live fetuses per litter were eviscerated.
A detailed examination of the skeleton (bone + cartilage) was performed after fixation with ethyl alcohol and staining with alizarin red S and alcian
blue (Peters, 1977). This examination included the observation of all the bone and cartilage structures of the head, spine, rib cage, pelvis and limbs.

Sex of live fetuses
The sex of each fetus was determined at the time of evisceration or at the time of microdissection.
Statistics:
Continuous data (for instance body weight, food consumption, …) were compared by one-way analysis of variance and the Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous).
Incidence data (for instance data on reproductive parameters) were compared by the Fisher exact probability test.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No clinical signs were considered to be treatment-related that were observed in surviving pregnant animals in females given 0, 25, and 100 mg/kg/day. Among the females given 500 mg/kg/day, three pregnant females (3/22) showed clinical signs during the second half of the treatment period.
Mortality:
mortality observed, treatment-related
Description (incidence):
No premature deaths occurred during the study in the control or groups treated at 25 or 100 mg/kg/day groups. At 500 mg/kg/day, one pregnant female (M28119) was prematurely sacrificed on gestation day (GD) 15 due to poor health.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 100 and 25 mg/kg/day, there were no treatment-related effects on body weight and weight gain. Over the treatment period (GD 6-21), the overall body weight gain of the surviving females given 500 mg/kg/day was 12% lower than that of the control mean and statistically significant. The initial effect on body weight coincided with low food consumption values.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 100 and 25 mg/kg/day, there were no treatment-related effects on mean maternal food consumption. Group mean food consumption was lower from GD 6 to 15 for the group given 500 mg/kg/day, when compared to the controls (ranging from -9% to -27%). This difference was more marked at the beginning of the treatment period where the differences were statistically significant during GD 6-9 (p<0.001). There was no effect on food consumption from GD 15 to 21 for this group.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No effects on liver weight, and gravid uterus weight that were considered to be treatment related (all dose-groups).
Histopathological findings: non-neoplastic:
no effects observed

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
mortality

Results (fetuses)

Details on embryotoxic / teratogenic effects:
The mean numbers of corpora lutea, implantation sites and the percentage of pre-implantation loss were similar between treated and control groups.
There were no effects on the group mean numbers of live fetuses, or on the extent of post-implantation loss for any group.
There were no effects of treatment with EGDMA on group mean fetal body weights or on the percentage of male fetuses; values were comparable with controls in all groups.
None of the litter parameters recorded (implantations, live fetuses, percentage of male/female fetuses, fetal body weight) were affected.
There were no indications of any treatment-related malformations and there were no treatment-related variations that were considered to be adverse.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

A total of 0, 1, 1 and 1 females were not pregnant in the 0, 25, 100 or 500 mg/kg/day groups.

Applicant's summary and conclusion

Conclusions:
In a developmental toxicity study according to OECD 414 Ethylene glycol dimethacrylate (purity: 97.5 %) was administered to female rats dose in diet by gavage at dose levels of 0, 25, 100 and 500 mg/kg bw/day from days 6 through 20 of gestation.
Maternal toxicity was observed at 500 mg/kg bw/day as evidenced by, clinical signs of poor health observed in 3/22 surviving pregnant females (and one female sacrificed on GD 15), transient body weight loss and reduction of food consumption. At 100 mg/kg bw/day, body weight gain was transiently reduced with no adverse outcome. There were no maternal effects at 25 mg/kg bw/day. None of the litter parameters recorded (implantations, live fetuses, percentage of male/female fetuses, fetal body weight) were affected. There were no indications of any treatment-related malformations and there were no treatment-related variations that were considered to be adverse.
The maternal NOAEL in this study was found to be 100 mg/kg bw/day and the developmental NOAEL was found to be 500 mg/kg bw/day.
Executive summary:

In a developmental toxicity study according to OECD 414 Ethylene glycol dimethacrylate (purity: 97.5 %) was administered to female rats (Sprague-Dawley, Crl CD® (SD) IGS BR, Caesarian Obtained, barrier sustained-virus antibody free, (COBS-VAF ®)) dose by gavage at dose levels of 0, 25, 100 and 500 mg/kg bw/day from days 6 through 20 of gestation.

 

The administration of 500 mg/kg/day caused evident signs of maternal toxicity (one female was sacrificed on GD 15, clinical signs of poor health were observed in 3/22 surviving pregnent females and there was transient body weight loss and reduction of food consumption). At 100 mg/kg/day, body weight gain was transiently reduced with no adverse outcome. There were no maternal effects at the dose-level of 25 mg/kg/day.

The maternal NOAEL was therefore 100 mg/kg bw/day.

None of the litter parameters recorded (implantations, live fetuses, percentage of male/female fetuses, fetal body weight) were affected.

There were no treatment-related malformations and there were no treatment-related variations that were considered to be adverse. 

The developmental NOAEL was therefore 500 mg/kg bw/day.

Ethylene glycol dimethacrylate formulation analyses were + 2% to + 9% of the nominal concentration and were within the acceptable range.

The developmental toxicity study in the rat is classified acceptable and satisfies the guideline requirement for a developmental toxicity study (OPPTS 870.3700; OECD 414) in rats.

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