Ethylene dimethacrylate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

There are no data available for EGDMA itself.

For the evaluation of EGDMA the data of the primary metabolite, HEMA, and the further metabolites methacrylic acid (and its donor substance methyl methacrylate) and ethylene glycol are used. Read-across to the metabolites is justified by the fact that carboxylesterases are ubiquitous in the body and EGDMA has a half-life of only a few minutes (see 5.1). In the body, HEMA is the first metabolite resulting from the primary ester hydrolysis of EGDMA. Subsequent ester hydrolysis produces methacrylic acid and ethylene glycol. Methyl methacrylate hydrolyses rapidly to methacrylic acid and thus serves as methacrylic acid donor in several test systems investigating systemic effects.

HEMA (primary metabolite)

OECD 422 fertility screening, rat, gavage: parenteral NOAEL 100 mg/kg bw/d, offspring NOAEL 1000 mg/kg bw/d (Nihon Bioresearch, 1997)

MMA (methacrylic metabolite donor substance)

OECD 416 fertility, rat, gavage: parenteral NOAEL 400 mg/kg bw/d/ NOEL 50 mg/kg bw/d based on food consumption), offspring NOAEL 400 mg/kg bw/d (BASF 2009)

EG (alcohol metabolite)

Continuous breeding acc. NTP, mouse, drinking water: NOAEL 2826 mg/kg bw/d, offspring NOAEL 2826 mg/kg bw/d (highest dose; Lamb 1985/ NTP 1984)

Summary

Based on the results of a reproductive toxicity screening study with the primary metabolite HEMA and reliable fertility studies with the alcohol metabolite EG and the methacrylic metabolite donor substance MMA, exposure to EGDMA is not likely to result in reproductive toxicity.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

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 Nippon Bioresearch Inc.Hashima Laboratory (Japan).

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Combined Repeated Dose and Reproductive / Developmental Toxicity Screening Test (Precursor Protocol of GL 422)

Deviations:

yes

Remarks:

older version of method that did not contain the functional observational battery

GLP compliance:

yes

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan
- Age at study initiation: 10 weeks
- Weight at study initiation: male 341~380 g; the female was 232~256 g.
- Housing: suspended, stainless steel cage; 5/cage until breeding, then divided into separate rearing cages.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 day quarantine; 7 day acclimation

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ~ 24 ℃
- Humidity (%): 40 to 70%
- Photoperiod (hrs dark / hrs light): 12 hour light / 12 hour dark

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: dissolved in water

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 14 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): separate rearing cages

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Exposure period: Males, 49 days; Females, from 14 days before mating to day 3 of lactation
Premating exposure period (males): 14 days
Premating exposure period (females): 14 days
Duration of test: Male, 50 days; Females, day 4 of lactation

Frequency of treatment:

Once daily

Remarks:

Doses / Concentrations:
0 (vehicle), 30, 100, 300, 1000 mg/kg/day
Basis:

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

Post-exposure period: Male, 50 days; Females, day 4 of lactation
- Dose selection rationale: based on range-finding
- Rationale for animal assignment (if not random): random

Positive control:

not applicable

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked : general condition and mortality; estrus and abnormal labor conditions in females

BODY WEIGHT: Yes
- Time schedule for examinations: twice per week in males; before mating, twice a week during the mating period, 0, 7 ,14 and 21 days duirng pregnancy, during the feeding period was measured 0 and 4 days in females

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): one week prior to mating, then twice a week; additionally, in females, days 2,9,16 and 21 of pregnancy, four days over the feeding period.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day after treatment
- Anaesthetic used for blood collection: Yes; sodium pentobarbital
- Animals fasted: No data
- How many animals: No data
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day after treatment
- Animals fasted: No data
- How many animals: No data
- Parameters checked in table [No.2] were examined.

URINALYSIS: No

Oestrous cyclicity (parental animals):

Once daily from the dosing start date until successful mating was observed.

Sperm parameters (parental animals):

Parameters examined in P male parental generations:
testis weight, epididymis weight

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead

Postmortem examinations (parental animals):

Terminal kill: Males, day 50; Females, day 4 of lactation

GROSS PATHOLOGY: Yes; Thymus, liver, kidney, testis and epididymis weight in males and ovary in females was measured after removal, adrenal gland, brain, heart and spleen and 10% neutral buffered formalin solution (However, testicular and epididymal fluid Buan) was fixed. Post-mortem examination of feamles who did not give birth to Day 25 of pregnancy. Number of corpora lutea and the number of implantation scars in females.

HISTOPATHOLOGY: Yes; Paraffin-embedded specimens were prepared. Control group and 1000 mg / kg group of heart, liver, spleen, thymus, kidney, testis and epididymis in males ovary in females, adrenal and brain for the Preparation HE staining of tissue was examined histologically. In males, 1000 mg / kg in the kidney was considered to indicate a difference in the number of abnormal animals in the test group compared with the control group; 30, 100 and 300 mg / kg group were similarly examined. In females, 1000 mg / kg differences in the brain was considered to indicate an abnormal number of animals in the test group than the control group and changes in adrenal cases and 30, 100, 300 mg / kg group were similarly examined.

Statistics:

Newborn screening as a unit has an average of one litter.
Weight (the parent animals, babies), food consumption, number of estrus, days mating, pregnancy [Day delivery (feeding 0) - date confirmed mating, the number of implantation scars, the number of birth control mobilize (number of babies stillborn baby + ), the number of newborn, number of children born dead, birth rate [(number of birth control mobilize / number of implantation scars) × 100], rate of production of child [(number of infant feeding 0 days / number of implantation scars) × 100], corpus number, implantation rates [(number of implantation scars / number of corpora lutea) × 100], fertility [(number of infant feeding 0 day / mobilize all of birth control) × 100], feeding baby number four day, feeding 4 day survival rate [(number of infant feeding 4 days / 0 Number of infant feeding day) × 100], unusual occurrence rate [(number of children with abnormal/ number of newborns) × 100], sex ratio (male / female), organ weights ( including the relative weight), results of blood tests, blood biochemistry test results for the mean and standard deviation were calculated for each group.
Significant difference test, Bartlett's test and the homoscedasticity of Law, analysis of variance, Dunnett method. Kruskal-Wallis test.
Copulation rate [(number of established animal mating / number of live animals) × 100], fertility [(number of female fertility / Establishment of animal mating) × 100], the birth rate [(number of female newborns / number of female fertility) × 100] is, χ ^ 2 using the test.
Cochran • Armitage was carried out using a test of dose-response trend test.

Reproductive indices:

Copulation rate [(number of established animal mating / number of live animals) × 100], fertility [(number of female fertility / Establishment of animal mating) × 100], pregnancy [Day delivery (feeding 0) - date confirmed mating, birth rate [(number of birth control mobilize / number of implantation scars) × 100], rate of production of child [(number of infant feeding 0 days / number of implantation scars) × 100], implantation rates [(number of implantation scars / number of corpora lutea) × 100], fertility [(number of infant feeding 0 day / mobilize all of birth control) × 100]

Offspring viability indices:

the birth rate [(number of female newborns / number of female fertility) × 100], unusual occurrence rate [(number of children with abnormal/ number of newborns) × 100], feeding 4 day survival rate [(number of infant feeding 4 days / 0 Number of infant feeding day) × 100]

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

effects observed, treatment-related

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Detail:[Males]
1) General condition: With the survival animals, no death and no moribund  were found for 30, 100 and 300 mg/kg/day groups. At 1000 mg/kg/day, one  death on day 20 of dosing was seen and abnormality wasn't seen except for  salivation until the previous day.  With the dead animals, no abnormality  was found for 30, 100 and 300 mg/kg/day groups.  At 1000 mg/kg/day,  salivation was seen in about 1 to 30-minutes after dosing from day 3. 2) Body weight: No significant difference from control group was seen in  30, 100, and 300 mg/kg/day groups. At 1000 mg/kg/day, the significant low  value was recorded during day 18 to day 25 of dosing andduring day 32 to  day 50 of dosing.
3) Food consumption: At 30 and 300 mg/kg/day, no significant difference  from control was seen. At 100 mg/kg/day, the significant high values were  seen on day 31. but no dose-related changes were obserbed.At 1000  mg/kg/day, the statistically significant low values were recorded on day  13, 31 and during day 38 to day 45. 
4) Hematological examination: No significant difference from control  group was seen for all groups up to 1000 mg/kg/day dose.
5) Blood chemical examination: At 30 and 300 mg/kg/day,the significant  high value in BUN were seen. As the difference was very small, this was  not considered as the adverse effect of HEMA dosing.  At 100 mg/kg/day, a  higher value of BUN but not statistically signifficant difference from  control was recorded. At 1000 mg/kg/day, the significant high values were  recorded in BUN, K, Cl,I-phosphorous and Triglyceride. 
6) Autopsy: No abnormalitywas found for 30 and 100 mg/kg groups. In the  300 mg/kg group, the albedo spot in the kidney of the unilateral in the 1  animal and, the atrophy of the testiculus of the bilaterality and  softening were observed in the 1 animal. In the 1000 mg/kg group, the  dark-red of the thymus gland in the 1 animal and the hypertrophy of the  kidney of bilaterality in the 1 animal were observed. 
7) Weight oforgans: At 30 mg/kg/day, no significant difference from  control group in absolute and relative weight was seen for all organs. At  100 and 300 mg/kg/day, the significant high value was recorded in the  absolute weight of kidneys. At 1000 mg/kg/day, the statistically  significant high values were recorded in the relative weight of liver and  kidneys.
8) Histopathological examination: At 1000 mg/kg/day in the survival  animals, the dilatation of renal tubule in 3 animals in the kidney and  the dilatation of collecting tubules in 2 animals were observed. But, all  these changes were just slight. And the dilatation of renal tubule has a  significant difference but no dose-related changes.  As for the  dilatation of collecting tubules, it has no significant difference but  increase tendency. In the other group, there were hemorrhage of thymus  gland, microgranuloma of the heart, microgranuloma of the liver and  hepatocyte vacuolar degeneration of the centrilobular, renal basophilic  tubules, eosinophilic corpuscle in proximal tubule, cyst, diffusive  mineral deposition and neutrophilic infiltration. But it was judged with  the incidental change, because they were whether it equivalently seems  even in the control group or small number animals. And no abnormality was  observed in spleen, adrenal, testiculus and brain in the control and 1000  mg/kg group. In animal of death of the 1000 mg/kg group, there were  hemorrhage of the thymus gland, edema of the lung, autolysis of adrenal  and lung and thymus gland with the deadanimal of 1000 mg/kg group. As for  those degrees, all were just slight. In the adrenal with the abnormality  in the autopsy, no change which suggested hypertrophy was seen.   
[Females]
1) General condition: With the existence animales, no death and no  moribund were seen for 30, 100 and 300 mg/kg/day groups. At 1000  mg/kg/day, three death on day 6 of dosing, one death on day 12 of dosing  and one death on day 17 of dosing were seen. Salivation, decrease in  locomotor activity, adoption of a prone position, acrimation, soiled fur,  hypothermia, bradypnea were seen at 1000 mg/kg. With the death animals,  no abnormality was found for 30, 100 and300 mg/kg/day groups.  At 1000  mg/kg/day, salivation was seen in about 1 to 30-minutes after dosing from  day 3.
2) Body weight: Before mating period, no significant difference from control  group was seen at 30, 100 and 300 mg/kg/day. At 1000 mg/kg/day, the  significant lower values were recorded on day 4 and 5 of dosing. During  gestation period, no significant difference from control groups was seen  in 30, 300 and 1000 mg/kg/day groups. At 100 mg/kg/day, the significant  high values were recorded on day 21 of gestation,  but no dose-related  changes were observed. During lactation period, no significant difference  from control groups was seen in 300 and 1000 mg/kg/day groups. At 30 and  100 mg/kg/day, the significant high values were recorded on day 4 of  lactation, but no dose-related changes were obserbed.
3) Food consumption:  Before mating period, no significant difference from control group was  seen at 30, 100 and 300 mg/kg/day. At 1000 mg/kg/day, the significant low  value from control group was recorded on day 3, 6 and 13 of dosing. During  gestation period, no significant difference from control groups was seen  in 30 and 300 mg/kg/day groups. At 100 and 1000 mg/kg/day, the  significant high value from control group was recorded on day 16 of  gestation, but no dose-related changes were observed. During lactation  period, no significant difference from control groups was seen.
4) Weight of organs: At 30 mg/kg/day, no significant difference from  control group in absolute and relative weight was seen for all organs. At  100 mg/kg/day,the significant high value was recorded in the absolute  weight of kidneys. At 1000 mg/kg/day, the significant high values were  recorded in the relative and absolute weight of kidneys. 
5) Histopathological examination: Though at 1000 mg/kg/day survival  groups, neutrophilic infiltration (unilateral ) to medulla and papilla  mammae part in the kidney were observed in the 1 animal, the degree was  slight. Though extensive softening of the medulla oblongata in the brain  was observed in the 1 example at 1000 mg/kg group, the degree was slight.   In dead 6 animals of the 1000 mg/kg group, there were the edema in 1  animal in the lung, the atrophy in 1 animal in the thymus gland, the  atrophy in 5 animals and the atrophy of a Malpighian body in 1 animals in  the spleen, the hyperplasia of zona fasciculata in 3 animals and the  autolysis in 1 animal in the the adrenal and the erosion in 1 animal in  the small intestinal mucosa. The degrees of the atrophy in the thymus  gland and the atrophy of a Malpighian body were moderate, but the others  were slight.  All the changes are noted related agonism. No changes which  suggested, though the hypertrophy of the adrenal in 2 animals, dark-red  of the glandular stomach mucosa in 2 animals and dark-red of the  intestinum tenue were observed as abnormal in the autopsy of the 1000  mg/kg group.

There were no effects of the test substance on the estrus frequency, copulation index, number of conceiving days, fertility index, length of gestation, number of corpora lutea or gestation index.

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

There were no effects of the test substance on the number of live pups born, birth index, number of dead pups, number of pups born, delivery index, live birth index, sex ratio, viability index, external anomalies, body weight or necropsy findings.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Reproductive effects observed:

not specified

Conclusions:

An OECD 422 study was conducted with rats by gavage at doses of 0, 30, 100, 300 and 1000 mg/kg. The NOAEL for reproductive/developmental toxicity and is considered to be greater than 1000 mg/kg.

Executive summary:

2-Hydroxyethyl methacrylate was studied for oral toxicity in rats in an OECD combined repeat dose and reproductive/developmental toxicity screening test at doses of 0, 30, 100, 300 and 1000 mg/kg/day.

There were no effects of the test substance on the estrus frequency, copulation index, number of conceiving days, fertility index, length of gestation, number of corpora lutea or gestation index.

There were no effects of the test substance on the number of live pups born, birth index, number of dead pups, number of pups born, delivery index, live birth index, sex ratio, viability index, external anomalies, body weight or necropsy findings.

Therefore, the NOAELs for reproductive/developmental toxicity are considered to be >/=1000 mg/kg/day for reproductin in both males and females and as well as for development of pups.

Reference 2

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-12-06 - 2009-04-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Name of test substance: Methyl Methacrylate
- Batch identification: 012231eda0
- CAS-No.: 80-62-6
- Physical state/appearance: Liquid /colorless, clear
- Purity: 99.9 %
- Homogeneity: Given
- Storage conditions: Refrigerator; avoid temperatures >35°C
- Storage stability: Expiry date: 16 Jan 2009. The stability of the test substance under storage conditions over the test period was guaranteed by the manufacturer, and the manufacturer holds this responsibility.

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: (P) 37 (±1) days at the beginning of treatment; (F1) x wks
- Weight at study initiation: (P) Males: 127.5 - 151.0 g; Females: 110.5 - 145.1 g; (F1) Males: x-x g; Females: x-x g
- Fasting period before study:
- Housing: During the study period, the rats were housed individually in Makrolon type M III cages (Becker & Co., Castrop-Rauxel, Germany), floor area of about 800 cm², with the following exceptions: 1) During overnight matings, male and female mating partners were housed together in Makrolon type M III cages. 2) Pregnant animals and their litters were housed together until PND 21 (end of lactation).
- Diet: ground Kliba maintenance diet mouse/rat “GLP” meal (Provimi Kliba SA, Kaiseraugst, Switzerland) ad libitum
- Water: ad libitum
- Acclimation period: (P) about 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 10 or 15 times
- Photoperiod (hrs dark / hrs light): 12 / 12


IN-LIFE DATES: From: To:

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The aqueous test substance suspensions were prepared at the beginning of the administration period and thereafter at intervals that took into account the analytical results of the stability verification. For the test substance preparation, the specified amount of test substance was weighed into an Erlenmeyer flask, topped up (shortly under the marking) with 1% Carboxymethylcellulose suspension in drinking water and four drops Cremophor EL and one drop of 32% hydrochloric acid. Afterwards the preparation was filled up with 1% Carboxymethylcellulose suspension in drinking water. The Erlenmeyer flask was sealed and the preparation was intensely mixed with a magnetic stirrer. A magnetic stirrer was used to keep the preparations homogeneous during treatment of the animals.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: overnight for a maximum of 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy (= GD 0)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the test substance preparations were sent to the analytical laboratory ten times during the study period (among other things at the beginning and towards the end) for verification of the concentrations. The samples, which were taken for the concentration control analyses at the beginning of the administration period, were also used to verify the homogeneity for the samples of the low and the high concentrations (50 and 400 mg/kg bw/d). Three samples (one from the top, middle and bottom in each case) were taken for each of these concentrations from the beaker with a magnetic stirrer running.

Duration of treatment / exposure:

until one day before sacrifice

Frequency of treatment:

once daily

Details on study schedule:

- F1 parental animals not mated until 75 days after selected from the F1 litters.
- Selection of parents from F1 generation after weaning (PND 21).
- Age at mating of the mated animals in the study: [...] weeks

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

Dose / conc.:

400 mg/kg bw/day (nominal)

Remarks:

In a Dose range finding study according to OECD 421 with male and female Wistar rats, the terminal body weight in F0 females dosed with 450 mg/kg bw/d was significantly decreased by 10% vs. Ctrl animals (p <= 0.01) and also the food consumption of females in the F0 and the F1 generation (last, during gestation) was significantly reduced by 86-91%. Dosing with 50 and 150 mg/kg bw/d was not related to observed effects. Based on these findings, the highest dose for the main study was set to 400 mg/kg bw/d.

No. of animals per sex per dose:

F0 generation parental animals: 25
F1 generation parental animals: 25

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale:
In a Dose range finding study according to OECD 421 with male and female Wistar rats, the terminal body weight in F0 females dosed with 450 mg/kg bw/d was significantly decreased by 10% vs. Ctrl animals (p <= 0.01) and also the food consumption of females in the F0 and the F1 generation (last, during gestation) was significantly reduced by 86-91%. Dosing with 50 and 150 mg/kg bw/d was not related to observed effects. Based on these findings, the highest dose for the main study was set to 400 mg/kg bw/d.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily on working days or once daily (Saturday, Sunday or on public holidays)


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily


BODY WEIGHT: Yes
- Time schedule for examinations: first day of the premating period and then once a week at the same time of the day (in the morning) until sacrifice. The following exceptions are notable for the female parental animals: 1) During each gestation period the F0 and the F1 generation parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20. 2) Females showing no positive evidence of sperm in vaginal smears were weighed once a week during the mating interval (solely for calculation of dose volume). 3) Females with litter were weighed on the day after parturition (PND 1) and on PND 4, 7, 14 and 21. 4) Females without litter were weighed once a week during the lactation phase (solely for calculation of dose volume).



OTHER:
- Food consumption: In general, food consumption was determined once a week for the male and female F0 and F1 parental animals. After the 10th test week, food consumption of the females during pregnancy (animals with evidence of sperm) was determined weekly for GD 0-7, 7-14 and 14-20. During the lactation period (animals with litter) food consumption was determined for PND 1-4, 4-7, 7-14 and 14-21.

Oestrous cyclicity (parental animals):

Estrous cycle length and normality were evaluated daily for all F0 and F1 female parental rats for a minimum of 3 weeks prior to mating. The evaluations were continued throughout the mating period until the female exhibited evidence of mating. Moreover, at the scheduled necropsy a vaginal smear was microscopically examined to determine the stage of the estrous cycle for each F0 and F1 female.

Sperm parameters (parental animals):

Parameters examined in all male parental generations:
testis weight, epididymis weight, cauda epididymis weight, prostate weight, seminal vesicles including coagulation glands weight, sperm head count in testis, sperm head count in cauda epididymis, sperm motility, sperm morphology

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.


PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, clinical symptoms, sexual maturation


GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE AND GROSS NECROPSY
All F0 and F1 parental animals were sacrificed by decapitation under Isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology; special attention was given to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
Weight assessment was carried out on all animals sacrificed at scheduled dates. The following weights were determined: Anesthetized animals, liver, kidneys, adrenal glands, testes, epididymides, cauda epididymis, prostate, seminal vesicles including coagulation glands, ovaries, uterus, spleen, brain, pituitary gland, thyroid glands (with parathyroid glands). The following organs or tissues of the F0 and F1 generation parental animals were fixed in 4% neutral buffered formaldehyde solution or in BOUIN’s solution, respectively: Vagina, cervix uteri, uterus, ovaries (BOUIN), oviducts, left testis (BOUIN), left epididymis (BOUIN), seminal vesicles, coagulation glands, prostate, pituitary gland, adrenal glands, liver, kidneys, spleen, brain, thyroid glands (with parathyroid glands), all gross lesions. After fixation, the organs fixed in BOUIN´s solution were embedded in Paraplast. Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings.

OTHER:
- Differential Ovarian Follicle Count (DOFC) in F1 generation: From both ovaries (”ovary 1” and “ovary 2”) of F1 female animals (control and top dose), five sections were taken from the proximal and the distal part of the ovaries, respectively, at least 100 µm apart from the inner third of the ovary. All ovarian sections were prepared and evaluated. Primordial follicles and growing follicles were counted by light microscope (magnification: 100x) on each of these slides, – according to the definitions given by Plowchalk et al. (PLOWCHALK, D. R., B. J. SMITH, and D. R. MATTISON: Assessment of Toxicity to the Ovary Using Follicle Quantitation and Morphometrics. In: Methods in Toxicology, Vol. 3, Part B: Female Reproductive Toxicology (J. J. HEINDEL and R. E. CHAPIN, Editors), p. 57-68, 1993, Academic Press). To prevent multiple counting on serial slides – especially of the growing follicles – only follicles with an oocyte with visible chromatin on the slide were counted. The number of each type of follicle was recorded individually for ovary 1 and ovary 2 of every animal on any of the slide levels (level 1-10), giving in summary the incidence of each type of the follicles by using EXCEL sheets for the reporting of the results. Finally, the results of all types of follicles were summarized for all animals per group in dose groups 10 and 13. As primordial follicles continuously develop into growing follicles, the assessment of the follicles was extended to the combined incidence of primordial plus growing follicles. In general, the fifth slide of the left and right ovary was evaluated for histological findings. Whenever the diagnosis: ”no abnormalities detected” was used for the ovaries, this implicates all functional statuses of follicles, especially corpora lutea, were present. An attempt was made to correlate gross lesions with histopathological findings.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals were sacrificed at 4 days of age. All F2 offspring were sacrificed after weaning (~ PND 21).
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- All pups were examined externally and eviscerated; their organs were assessed macroscopically.

HISTOPATHOLOGY / ORGAN WEIGHTS
Animals with notable findings or abnormalities were further evaluated on a case-by-case basis, depending on the type of finding noted. After the scheduled sacrifice the brain, spleen and thymus of 1 pup/sex and litter from the F1 and F2 pups were weighed. Normally, the first male and the first female pups/litter were taken for these determinations. For the calculation of the relative organ weights, the pup body weights, determined routinely during the in-life phase on PND 21, were used.

Reproductive indices:

- Male reproduction data: The mating partners, the number of mating days until vaginal sperm could be detected in the female, and the gestational status of the female were noted for F0 and F1 breeding pairs. For the males, mating and fertility indices were calculated for F1 and F2 litters according to the following formulas: Male mating index (%) = (number of males with confirmed mating)/(number of males placed with females) x 100. Male fertility index (%) = (number of males proving their fertility)/(number of males placed with females) x 100.
- Female reproduction and delivery data: The mating partners, the number of mating days until vaginal sperm were detected, and gestational status were recorded for F0 and F1 females. For the females, mating, fertility and gestation indices were calculated for F1 and F2 litters according to the following formulas:
Female mating index (%) = (number of females mated)/(number of females placed with males) x 100.
Female fertility index (%) = (number of females pregnant)/(number of females mated) x 100.
Gestation index (%) = (number of females with live pups on the day of birth)/(number of females pregnant) x 100.
The total number of pups delivered and the number of liveborn and stillborn pups were noted, and the live birth index was calculated for F1 and F2 litters according to the following formula:
Live birth index (%) = (number of liveborn pups at birth)/(total number of pups born) x 100.
The implantations were counted and the postimplantation loss (in %) was calculated according the following formula:
Postimplantation loss (%) = (number of implantations – number of pups delivered)/(number of implantations) x 100.

Offspring viability indices:

The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4, 5-7, 8-14 and 15-21 (lactation period) were determined; however, pups, which died accidentally or had to be sacrificed due to maternal death, were not included in these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation days 4, 7, 14, and 21. Furthermore, viability and lactation indices were calculated according to the following formulas:
Viability index (%) = (number of live pups on day 4 (before culling) after birth)/(number of live pups on the day of birth) x 100.
Lactation index (%) = (number of live pups on day 21 after birth)/(number of live pups on day 4 (after culling) after birth) x 100.

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Test group 03 (400 mg/kg bw/d)
• Statistically significantly decreased food consumption in parental males during premating weeks 5 - 10 (up to 7%)
• Statistically significantly decreased food consumption in parental females during premating weeks 1 - 3 (up to 6%), weeks 5 - 8 (up to 7%) and weeks 9 - 10 (about 6%)
• Statistically significantly decreased food consumption in parental females during gestation days 0 - 7 (about 10%)
• Statistically significantly decreased food consumption in parental females during lactation days 4 - 7 (about 7%)

Test group 02 (150 mg/kg bw/d)
• Statistically significantly decreased food consumption in parental females during premating weeks 1 - 2 (about 5%)
• Statistically significantly decreased food consumption in parental females during gestation days 0 - 7 (about 7%)

Test group 01 (50 mg/kg bw/d)
• no test substance-related adverse effects/findings

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Under the conditions of the present 2-generation reproduction toxicity study the NOAEL (no observed adverse effect level) for general, systemic toxicity is 400 mg/kg bw/d for the F0 parental rats, the highest dose tested.
The NOEL (no observed effect level) is 50 mg/kg bw/d for the F0 parental rats based on effects on food consumption being a consequence of reduced appetite observed at the LOEL (Lowest Observed Effect Level) of 150 mg/kg bw/d in the F0 parental females.
The NOAEL for fertility and reproductive performance for the F0 parental rats is 400 mg/kg bw/d, the highest dose tested.

Dose descriptor:

NOEL

Remarks:

food consumption

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

food consumption and compound intake

Dose descriptor:

LOEL

Remarks:

food consumption

Effect level:

150 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

food consumption and compound intake

Remarks on result:

other: consequence of reduced appetite observed in the F0 parental females

Dose descriptor:

NOAEL

Remarks:

fertility and reproductive performance

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no adverse effects observed

Dose descriptor:

NOAEL

Remarks:

General systemic toxicity

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no adverse effects observed

Critical effects observed:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Test group 03 (400 mg/kg bw/d)
Statistically significantly decreased food consumption in parental males during premating weeks 0 - 1 (about 14%) and weeks 8 - 10 (up to 7%)
• Statistically significantly decreased food consumption in parental females during premating weeks 0 - 1 (about 10%) and weeks 9 - 10 (about 8%)
• Statistically significantly decreased food consumption in parental females during gestation days 0 - 14 (up to 8%)
• Statistically significantly decreased body weights in parental males during weeks 0 - 5 (up to 17%) and weeks 10 - 11 (up to 6%)
• Statistically significantly decreased body weights in parental females during premating weeks 0 - 1 (up to 16%)

Test group 02 (150 mg/kg bw/d)
• no test substance-related adverse effects/findings

Test group 01 (50 mg/kg bw/d)
• no test substance-related adverse effects/findings

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

The NOEL (no observed effect level) is 50 mg/kg bw/d for the F1 parental rats based on effects on food consumption being a consequence of reduced appetite observed at the LOEL (Lowest Observed Effect Level) of 150 mg/kg bw/d in the F0 parental females.
The NOAEL for fertility and reproductive performance for the F1 parental rats is 400 mg/kg bw/d, the highest dose tested.
The NOAEL for developmental toxicity, in the F1 of the test substance is 400 mg/kg bw/d, the highest dose tested.

Dose descriptor:

NOEL

Remarks:

food consumption

Generation:

F1

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

food consumption and compound intake

Remarks on result:

other:

Remarks:

effects on food consumption beeing a consequence of reduced appetite observed at the LOEL of 150 mg/kg bw/d in the F0 parental females

Dose descriptor:

NOAEL

Remarks:

fertility & reproductive performance

Generation:

F1

Effect level:

400 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no adverse effects observed

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

400 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no dverse effects observed

Critical effects observed:

no

Reproductive effects observed:

no

- Analytics:

The various analyses:

• demonstrated the stability of the test substance preparations over a period of 7 days at room temperature
• confirmed the homogeneous distribution of the test substance in the vehicle (1% Carboxymethylcellulose suspension in drinking water and a few drops Cremophor EL and one drop hydrochloric acid)
• showed that the prepared concentrations were close to the expected values ranging between 86.8 and 113.2% of the nominal concentrations

Analytical values (range):

Test group	Nominal Dose (mg/kg bw/d)	Analytical Dose (mg/kg bw/d) [minimum]	Analytical Dose (mg/kg bw/d) [maximum]	% Nominal Dose [minimum]	% Nominal Dose [maximum]
00 / 10	0	0	0
01 / 11	50	43.40	46.61	86.8	93.2
02 / 12	150	132.90	169.80	88.6	113.2
03 / 13	400	359.20	379.90	89.8	95.0

 

Conclusions:

The NOAEL for general, systemic toxicity is 400 mg/kg/d for the parental rats in the highest dose tested.
The NOEL 50 mg/kg bw/day for the P parental rats, based on effects on food consumption observed at the LOAEL of 150 mg/kg bw/day in the P parental females.
The NOAEL for fertility and reproductive performance for the P and F1 parental rats is 400 mg/kg bw/day, the highest dose tested.
The NOAEL for developmental toxicity, in the F1 and F2 progeny, of the test substance is 400 mg/kg bw/day, the highest dose tested.

Executive summary:

The study was performed according to OECD TG 416 in compliance with GLP. Methyl Methacrylate was administered to groups of 25 male and 25 female healthy young Wistar rats (P parental generation) as an aqueous preparation by stomach tube at dosages of 0; 50; 150 and 400 mg/kg body weight/day. At least 73 days after the beginning of treatment, P animals were mated to produce a litter (F1). Mating pairs were from the same dose group and F1 animals selected for breeding were continued in the same dose group as their parents. Groups of 25 males and 25 females, selected from F1 pups to become F1 parental generation, were treated with the test substance at dosages of 0; 50; 150 and 400 mg/kg body weight/day post weaning, and the breeding program was repeated to produce F2 litter. The study was terminated with the terminal sacrifice of the F2 weanlings and F1 adult animals.

Control parental animals were dosed daily with the vehicle (1% Carboxymethylcellulose suspension in drinking water and four drops Cremophor EL and one drop hydrochloric acid).

The mid- and high-dose parental animals (400 mg/kg bw/d) showed clinical signs of systemic toxicity. The only relevant clinical observation was temporary salivation during a short period after dosing, which is considered to be test substance-induced. From the temporary, short appearance immediately after dosing it is likely, that this finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. It is, however, not considered to be an adverse toxicologically relevant finding.

In the mid- and high-dose (150 and 400 mg/kg bw/d) P generation animals, dose-related intermittent reductions of food consumption were noted, either during premating, gestation and lactation phases of this study. Less significant changes were noted for the F1 generation animals where the effects were limited to the high-dose group.

High dose F1 parental males had statistically significant lower body weights during several study segments, which led to a statistically significant reduction of the mean terminal body weight resulting in secondary weight changes of brain.

High dose parental females had statistically significant lower body weights during the first weeks after weaning. This weight decrease during major phases of sexual maturation led to an apparent marginal delay of vaginal patency. This minor delay did, however, not result in any corroborative pathological findings nor did it adversly effect F1 female cyclicity, fertility and reproduction. Thus, an influence of the test substance on female sexual maturation is not assumed.

Pathological examinations revealed no test-substance-related changes in organ weights, gross lesions, changes in differential ovarian follicle counts or microscopic findings, apart from an increase in kidney and liver weights in male and female animals in both generations which is presumably related to the treatment. There was no histopathologic lesion observed, that could explain the weight increase. It is regarded to be an adaptive change, most likely caused by an increase in metabolic activity in the two organs, which does not lead to histopathologic findings. It is not regarded to be an adverse effect.

There were no indications from clinical examinations as well as gross and histopathology, that the administration of methyl methacrylate via the diet adversely affected the fertility or reproductive performance of the P or F1 parental animals up to and including a dose of 400 mg/kg bw/day. Estrous cycle data, mating behavior, conception, gestation, parturition, lactation and weaning as well as sperm parameters, sexual organ weights and gross and histopathological findings of these organs (including differential ovarian follicle counts in the F1 females) were comparable between the rats of all test groups and ranged within the historical control data of the test facility.

All data recorded during gestation and lactation in terms of embryo-/fetal and pup development gave no indications for any developmental toxicity in the F1 and F2 offspring up to a dose level of 400 mg/kg bw/day. Up to this dose level, the test substance did not adversely influence pup viability and pup body weights. Sex ratio and sexual maturation was not directly affected at any dose level, inclusive the high-dose group (400 mg/kg bw/day).

Conclusion:

Under the conditions of the present 2-generation reproduction toxicity study the NOAEL(no observed adverse effect level) forgeneral, systemic toxicityis 400 mg/kg bw/d for the parental rats, the highest dose tested.

The NOEL (no observed effect level) is 50 mg/kg bw/d for the F1 parental rats based on effects on food consumption being a consequence of reduced appetite observed at the LOEL (Lowest Observed Effect Level) of 150 mg/kg bw/d in the F0 parental females.

The NOAEL for fertility and reproductive performance for the F1 parental rats is 400 mg/kg bw/d, the highest dose tested.

 The NOAEL for developmental toxicity, in the F1 of the test substance is 400 mg/kg bw/d, the highest dose tested.

Reference 3

Endpoint:

reproductive toxicity, other

Remarks:

Fertility Assessment by Continuous Breeding (FACB) assay

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

"Fertility Assessment by Continuous Breeding (FACB)" assay.
Well designed study that used adequate numbers of animals and examined reproductive function in two generations. Limitations of the study include examination of reproductive function only in control and high-dose F1 animals, no histopathology in reproductive organs, and no sperm measurements.

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Source of Test material: Ashland Chemical Company
Purity: 99.6 %
water content: 0.243 ± 0.006(0)% (Karl Fischer)
acid content:0.7 ± 0.1(/i) ppm (as acetic acid).
Gas chromatography on a
Tenax-GC column indicated a major peak and two impurities with a combined area totaling 0.30% of the major peak area.

Species:

mouse

Strain:

CD-1

Details on species / strain selection:

COBS Crl: CD-1 (ICR) BR outbred albino mice

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age: CD-1 mice were purchased at 6 weeks of age from Charles River Breeding Laboratories, Inc. (Kingston, NY)
- Housing: Two animals were housed per cage in polycarbonate shoebox type cages with stainless steel wire bar lids. Cages were rotated (relative placement) at least once a week.
- Diet: Purina certified rodent chow animal diet, ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25°C
- Humidity (%): 20 to 70%
- Photoperiod (hrs dark / hrs light): 14-hour light cycle

During 5 week quarantine period, two males and two females were killed and their sera were evaluated against antibodies agaainst 1 l murine viruses: Allsera were negative.
Fcal samples were collected from two animal and examined for endoparaties:Result: negative

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

Ethylene glycol dosing solutions were prepared at 0, 0.25, 0.5, l.0, 2.5, and 5.0% (w/v) in Task 1 (dose finding , 14 day repeated dose study), and 0, 0.25, 0.5, and 1.0% for Task 2 (continous breeding phase). Dosing solutions were prepared fresh once every 2 weeks and stored in distilled water at room temperature under yellow lights. Chemical analyses performed by MRI every 6 weeks indicated that dosage solutions were within 98 to 107% of the intended concentrations.Ethylene glycol dosing solutions were prepared at 0, 0.25, 0.5, l.0, 2.5, and 5.0% (w/v) in Task 1, and 0, 0.25, 0.5, and 1.0% for Task 2. Dosing solutions were prepared fresh once every 2 weeks and stored in distilled water at room temperature under yellow lights.

Details on mating procedure:

11-week-old male and female mice were exposed to the chemical during a 7-day premating period, after which they were randomly paired ( one male: one female) within each dose group and cohabited for 98 days ( 14 weeks) while treatment continued. Newbom litters were evaluated and immediately killed. At the end of the 98-day cohabitation period, males and females were separated to prevent further mating; litters were saved during the following 21-day segregation period. Chemical exposure was continuous during the 98-day cohabitation period and the 21-day segregation period which followed, and throughout the life of the offspring bom during the 21 days. The final litters bom to the control and high-dose pairs during the 21-day segregation period were weaned and evaluated for reproductive performance in Task 4 (Offspring Assessment).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical analyses performed by MRI every 6 weeks indicated that dosage solutions were within 98 to 107 % of the intended concentrations.

Duration of treatment / exposure:

Task 2 (main study): 18 weeks (1 week prior to cohabitation, 14 weeks of cohabitation, and 3 weeks thereafter)

Frequency of treatment:

dosed/undosed water ad libitum

Details on study schedule:

Task 1 (Dose finding study): 14 days treatment
Task 2 (Continuous breeding phase): 1 week + 14 weeks + holding period
In case of negative response: Task 4 (offspring assessment): Control & 1 dose group
In case of positive response: Task 3 (determination of affected sex)and task 4 (offspring assessment): Control & 3 dose groups

Dose / conc.:

410 mg/kg bw/day (actual dose received)

Remarks:

0.25 % ethylene glycol in water

Dose / conc.:

840 mg/kg bw/day (actual dose received)

Remarks:

0.5 % ethylene glycol in water

Dose / conc.:

1 640 mg/kg bw/day (actual dose received)

Remarks:

1 % ethylene glycol in water

No. of animals per sex per dose:

20 per sex per dose group (0.25%, 0.5%, 1.0%)
40 per sex per dose group (control)

Control animals:

yes, concurrent vehicle

Details on study design:

Fertility Assessment by Continuous Breeding (FACB). It consists of four related tasks, not all of which are necessarily performed for a given compound. These tasks include Task 1 - dose finding; Task 2 - cohabitation phase; Task 3 - identification of the affected sex and Task 4 - offspring assessment. This test protocol is designed to provide an alternative to multigeneration studies which produce similar comprehensive reproductive data but in a considerably shorter time and at a lower cost.
Task 1 is conducted to determine suitable doses for the continuous breeding phase. The test chemical is administered for 14 consecutive days and them maximum tolerated dose (MTD) is computed. Task 2 is designed to determine the effect of the MTD and two lower dose levels on fertility and reproduction. In this phase, treatment is continued for 18 weeks (1 week prior to cohabitation, 14 weeks of cohabitation, and 3 weeks thereafter). If the fertility is significantly affected, Task 3 is conducted to determine whether the male, female or both sexes are affected. If the overall response in Task 2 is negative, Task 4 is conducted. It is designed to evaluate reproductive performance in the offspring from the final and generally the fifth litter of the control and high dose groups. If the fertility in the first generation offspring is significantly affected, a Task 3 may be performed using these animals to determine the affected sex. At the conclusion of either Task 3 or 4, experimental animals may be necropsied.
During necropsy the liver, brain, pituitary, female reproductive tract (ovaries, oviduct, uterus, and vagina), testes, epididymis, prostate, and seminal vesicles with coagulating glands are weighed and fixed for histopathology. Based on the overall response during Task 3 or 4, vaginal smears are prepared to check the effect on oestrous cycle and sperm studied in detail to evaluate the effect on sperm density, sperm motility, and sperm head morphology.

Positive control:

no

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS For CLINICAL SIGNS: Yes
- Time schedule: twice dailyed.


BODY WEIGHT: Yes
- Time schedule for examinations: Weekly


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: gravimetically at weekly intervals


- Rough estimation of chemical consumption by multiplying water consumption by chemical concentration/weight of pair

Oestrous cyclicity (parental animals):

no data

Sperm parameters (parental animals):

no data

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

Postmortem examinations (offspring):

The following parameters were examined in [F1 / F2 / F3] offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities
GROSS EXAMINATION OF DEAD PUPS: yes

Statistics:

Yes
Reproductive data were evaluated by the Chi-Square test for homogeneity and/or the Fisher’s Exact test. Pup and litter data were evaluated by Chi-Square approximation to the Kruskal-Wallis test, Fisher’s Exact Test, and the Mann-Whitney U test.

Reproductive indices:

yes

Offspring viability indices:

yes

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not specified

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two females from the 0.5% group and one male and two females from the control group died before the end of the study; at least one of the two deaths from the 0.5% EG group could have been a treatment-
related death since the renal tubules contained a moderate number of oxalate crystals.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

There were slight, but significant adverse effects on fertility in the high-dose group. Exposure to 1% EG in the drinking water was associated with statistically significant decreases in the number of litters per fertile pair, the mean number of live pups per litter, and the mean live pup weight as compared with the controls.

Of the l 00 breeding pairs which began the 14-week cohabitation period, all the pairs had at least one litter and on the average each pair had between four and five litters. There were slight, but significant adverse effects on fertility in the high-dose group. Exposure to 1% EG in the drinking water was associated with statistically significant decreases in the number of litters per fertile pair, the mean number of live pups per litter, and the mean live pup weight as compared with the controls. Neither the 0.25 nor the 0.5% dose groups were significantly affected.

Dose descriptor:

NOEL

Effect level:

840 mg/kg bw/day

Based on:

act. ingr. (dissolved fraction)

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other:

Remarks:

Exposure to ethylene glycol resulted in a small but significant decrease in the number of litters per breeding pair, in the number of live pups per pair and in the live pup weight. A significant number of pups in the 1.0% dose group were born with distinct facial deformities. In the retained litters at this dose, the facial deformities were more obvious with age. These malformed animals also exhibited fused ribs and shortened nasal, parietal, and/or frontal bones of the skull. When pups from the high dose group were raised to adulthood (with continued exposure to ethylene glycol) and mated, they exhibited decreased mating and fertility indices relative to controls handled in the same manner, but there were no effects on litter size, pup weight or sex ratio. These findings that were most likely developmental effects

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not specified

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Mice were necropsied after the mating trial and weights were measured for liver, brain, pituitary, and male and female reproductive organs. [Histopathology findings in reproductive organs were not reported.]

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Although no clinical signs of EG toxicity were reported in the study, unusual facial features were noticed in some of the offspring of the treated mice but not in the controls. The affected offspring generally bad a shorter snout with wide-set eyes compared to the control CD-1 mice. The examination revealed a pattern of skeletal defects in the treated mice affecting the skull, sternebrae, ribs, and vertebrae in both males and females. The defects included shortened frontal, nasal, and parietal bones; one or more pairs of fused ribs; abnormally shaped vertebrae; and twisting of thespine. The untreated mice showed no such defects. It was apparent from low-magnification examination of the histological sections that the size and shape of the bones in treated mice differed from the controls. Bones from treated mice were smaller and bad altered shapes. However, histologic alterations were not evident when bones from treated mice were examined by light microscopy.
Formation of lamellar bone, numbers and activity of osteoblasts and osteoclasts, and cartilage structure were identical in treated and control tissues. Tooth structure was normal in all treated mice. No alterations were seen in nasal turbinates, skeletal muscle, various salivary glands, eyes, or in those portions of the olfactory lobes of the brain which were frequently present in the sections. Deposition of calcium oxalate crystals was not found during careful examination of the microvasculature of bony or adjacent soft tissues.

Histopathological findings:

not specified

Other effects:

not specified

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Description (incidence and severity):

In the F1 generation, 16 control pairs and 11 high-dose pairs produced litters. There were no significant differences in fertility, live litter size, or live pup weight between the control and 1.0% ethylene glycol groups.

A number of F1 animals in the 1,640 mg/kg bw/day group were noted to have unusual facial features. Further examination of the skeleton by staining with Alizarin Red in 4 mice/sex/group in the control and high-dose group revealed a pattern of skeletal defects affecting the skull, sternebrae, ribs, and vertebrae in both sexes of the high-dose group.

Dose descriptor:

NOEL

Generation:

F1

Effect level:

1 640 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Reproductive effects observed:

yes

Lowest effective dose / conc.:

1 640 mg/kg bw/day

Treatment related:

yes

Relation to other toxic effects:

not specified

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

Ethylene glycol is not reprotoxic up to doses of 840 mg/kg/day in CD-1 mice.

Executive summary:

In a continuous breeding study (Lamb et. al. 1994) ethylene glycol (99.6%) was tested for reproductive functions of CD-1 (ICR) BR outbred albino mice (20/dose/sex, 40 control animals) at concentrations of 0.25, 0.5 and 1% w/v (corresponding to 410, 840 and 1640 mg/kgbw/day).

Strengths/Weakness: Lamb et al. is a well designed study that used adequate numbers of animals and examined reproductive function in two generations. Limitations of the study include examination of reproductive function only in control and high-dose F1 animals, no histopathology in reproductive organs, and no sperm measurements.

Utility (Adequacy) for CERHR Evaluation Process: Lamb et al. is useful for demonstrating no effects on reproductive function in mice at doses up to 0.5% (840 mg/kg bw/day). Exposure to 1.0% (1,640 mg/kg bw/day) resulted in a minor effect on fertility (a slight decrease in the number of F1 litters/pair of P0 mice) and findings that were most likely developmental effects (reduced numbers of live F1 pups/litter, decreased F 1 pup weight, and facial and skeletal malformations). No reproductive effects were observed in the high-dose F1 mice.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Species:

rat

Quality of whole database:

The available data are adequate for the assessment. Data from other methacrylates and ethylene glycol, the alcohol metabolite, show an absence of alerts in the relevant dose range.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

There are no data available for EGDMA itself.

For the evaluation of EGDMA the data of the primary metabolite, HEMA, and the further metabolites methacrylic acid (and its donor substance methyl methacrylate) and ethylene glycol are used. In the body, HEMA is the first metabolite resulting from the primary ester hydrolysis of EGDMA. Subsequent ester hydrolysis produces methacrylic acid and ethylene glycol. Methyl methacrylate hydrolyses rapidly to methacrylic acid and thus serves as methacrylic acid donor in several test systems investigating systemic effects.

HEMA (primary metabolite)

HEMA was studied in an OECD 422 combined repeat dose and reproductive/developmental toxicity screening test (Nihon Bioresearch, 1997). Groups of 12 male and 12 female rats were administered by gavage at dose levels of 0, 30, 100, 300, or 1000 mg/kg/day. Male rats were dosed for 49 days and female rats were dosed from 14 days prior to mating through Day 3 of lactation. The NOAEL for parental effects was 100 mg/kg/d. In male rats slight effects were observed in kidneys at 300 mg/kg/d dose and above. In the absence of reproductive effects at the highest dose, the NOAEL for fertility effects is considered as 1000 mg/kg/day.

MMA (methacrylic metabolite donor substance)

Methyl methacrylate (MMA) is the methyl ester of methacrylic acid (MMA) and is rapidly absorbed and metabolised to MAA within the body. It therefore acts as an effective systemic deliver system for MAA avoiding the local high toxicity of MMA due to its acidity. The reference chemical for the methacrylic moiety of the category members, MMA, hasrecently been tested in an OECD TG 416 oral two-generation reproduction toxicity study in rats, in which both, parental and F1 animals were dosed with 0; 50; 150 and 400 mg/kg bw/day (BASF, 2009).

In mid- and high-dose parental animals (150 and 400 mg/kg bw/d) temporary salivation, presumably due to a bad taste of the test substance and associated dose-related intermittent reductions of food consumption were noted. Less significant changes were noted for the F1 generation animals where the effects were limited to the high-dose group and not associated with effects on histopathology or reproductive performance.

The NOAEL for fertility and reproductive performance for the P and F1 parental rats was determined to be 400 mg/kg bw/day, the highest dose tested. The NOAEL for developmental toxicity, in the F1 and F2 progeny, of the test substance was determined to be 400 mg/kg bw/day, the highest dose tested. There were no signs of systemic toxicity other than reduced body weight gain associated with reduced food consumption, presumably due to bad palatability (NOEL 50 mg/kg bw/day for the P and F1 parental rats).

The 2-generation study with MMA provides confidence that the absence of reproductive effects below maternal toxic doses seen at in the screening studies with the three category members, are a reliable indication for an absence of fertility effects below maternal toxic doses in higher tier studies such as a 2 generation study when considering the methacrylic moiety of the category members.

EG (alcohol metabolite)

Ethylene glycol (EG) was tested for reproductive toxicity in rats and mice. The NTP-CERHR Expert Panel (2004) concluded that “there are sufficient data to conclude that ethylene glycol is not a reproductive toxicant in rats exposed orally to 1,000 mg/kg bw/day via diet. The study in mice was essentially negative at doses up to 2,826 mg/kg bw/day via drinking water. The studies available for review included a continuous breeding study in mice (selected for the IUCLID dataset of EGDMA, also for reliability reasons – NTP study), a two-generation study in rats, and sub-chronic toxicity studies in rats.

The Expert Panel concluded that data in mice are sufficient to demonstrate no effect on fertility of male or female mice following oral exposure to up to 2,826 mg/kg bw/day ethylene glycol for approximately 22 weeks.

The Expert Panel concluded that the data are sufficient to demonstrate that ethylene glycol is not a reproductive toxicant in male and female rats following dietary exposure with up to 1,000 mg/kg bw/day for 7 weeks prior to mating in parental rats or from the time of conception through mating in their offspring.

The Expert Panel is confident that these data are useful in judging hazard to humans because the doses tested far exceeded the doses relevant to humans based on knowledge of absorption, distribution, metabolism, and excretion in rats, mice, and humans. It was further noted that the pattern of general toxicity is similar in experimental animal studies and instances of human poisoning.”

Justification for selection of Effect on fertility via oral route
For the evaluation of EGDMA the data of the primary metabolite HEMA are used. In the body, HEMA is the first metabolite resulting from the ester hydrolysis of EGDMA. HEMA was studied in an OECD 422 combined repeat dose and reproductive/developmental toxicity screening test. Up to the highest dose tested (1000 mg/kg/d) there were no reproductive effects - in the presence of parental toxicity.

Compliance to REACh requirements

The screening study requirement is covered with a reliable OECD 422 oral rat study, performed with the the primary metabolite HEMA. The reproduction toxicity requirements are covered with a reliable two generation study in rats with the methacrylic metabolite donor MMA and a continuous breeding study with the alcohol metabolite EG. All mentioned studies are reliable (Reliability 1 or 2) and the read across is done with a high level of confidence (see chapter "Toxicokinetics" and the attached category document).

Effects on developmental toxicity

Description of key information

There are data with the rodent species rat available for EGDMA itself. Developmental toxicity was not observed at any dose up to 500 mg/kg bw/d.

For the evaluation of the developmental toxicity of EGDMA in a non-rodent species, screening data of the primary metabolite, HEMA, and reliable data from the further metabolites methacrylic acid (and its donor substance methyl methacrylate) and the alcohol metabolite ethylene glycol are available.

Read-across to the metabolites is justified by the fact that carboxylesterases are ubiquitous in the body and EGDMA has a half-life of only a few minutes (see 5.1). In the body, HEMA is the first metabolite resulting from the primary ester hydrolysis of EGDMA. Subsequent ester hydrolysis produces methacrylic acid and ethylene glycol. Methyl methacrylate hydrolyses to methacrylic acid rapidly and thus serve as methacrylic acid donor in several test systems investigating systemic effects. In the selected studies, developmental toxicity was not observed at any dose.

EGDMA

OECD 414, developmental, rat, gavage: parenteral NOAEL 100 mg/kg bw/d, developmental NOAEL 500 mg/kg bw/d (CIT 2006)

MMA (methacrylic metabolite donor substance)

OECD 414 developmental, rabbit, gavage: parenteral NOAEL 50 mg/kg bw/d, developmental NOAEL 405 mg/kg bw/d (BASF 2009; study selected from a larger data set for REACh requirement reasons)

EG (alcohol metabolite)

OECD 414 developmental, rabbit, gavage: parenteral NOAEL 1000 mg/kg bw/d, developmental NOAEL 2000 mg/kg bw/d (Tyl 1993; study selected from a larger data set for REACh requirement reasons)

Summary

Based on the results of a developmental toxicity study with the rodent species rat and reliable developmental toxicity studies with the alcohol metabolite EG and the methacrylic metabolite donor substance MMA, exposure to EGDMA is not likely to result in developmental toxicity.

Link to relevant study records

Reference

Reference 1

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

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

Specific details on test material used for the study:

- Name of test material (as cited in study report): Ethylene Glycol Dimethacrylate (EGDMA; CAS RN: 97-90-5)

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

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)

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.

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.

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

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

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.

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

Abnormalities:

not specified

Developmental effects observed:

not specified

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

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.

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.

Reference 2

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Name of test substance: Methyl Methacrylate
Test substance No.: 07/0751-1
Batch identification: 012231eda0
CAS No.: 80-62-6
Purity: 99.9%
Homogeneity: Homogeneous
Expiry date: 16 Jan 2009
The stability of the test substance under storage
conditions over the test period was guaranteed by
the manufacturer, and the manufacturer holds this
responsibility.
Supplier: Sigma Aldrich

Species:

rabbit

Strain:

Himalayan

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 16-21 weeks
- Weight at study initiation: 2187-2917 g
- Fasting period before study: no
- Housing: the rabbits were housed singly in type 12.2395.C stainless steel wire mesh cages supplied by Draht-Bremer GmbH, Marktheidenfeld, Germany (floor area about 3,000 cm²)
- Diet: pelleted “Kliba maintenance diet for rabbits & guinea pigs, GLP”, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: tap water ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12 (6.00 p.m. to 6.00 a.m. dark, 6.00 a.m. to 6.00 p.m. light)

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The aqueous test substance preparations were prepared at the beginning of the administration period and thereafter at maximum intervals of 7 days, which took into account the analytical results of the stability verification. For the test substance preparation, an specific amount of test substance was weighed depending on the dose group, into a graduated flask (conical Erlenmeyer flasks with groundin stopper), topped up (shortly under the marking) with 1% Carboxymethylcellulose solution in drinking water and a few drops Cremophor EL and one drop of 32% hydrochloric acid. Afterwards the preparation was filled up with 1% Carboxymethylcellulose suspension in drinking water. The flask was sealed and the preparation was intensely mixed with a magnetic stirrer. During administration, the preparations were kept homogeneous with a magnetic stirrer and the vessels were kept closed between the withdrawals of the preparations.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the test substance preparations were sent to the analytical laboratory twice during the study period (at the beginning and towards the end) for verification of the concentrations. Samples were analyzed by GC with external calibration.

Details on mating procedure:

After an acclimatization period of at least 5 days, the female rabbits were fertilized by means of artificial insemination. This implied that 0.2 mL of a synthetic hormone which releases LH and FSH from the anterior pituitary lobe (Receptal) were injected intramuscularly to the female rabbits about 1 hour before insemination. The ejaculate samples used for the artificial insemination were derived from male Himalayan rabbits of the same breed as the females. Each female was inseminated with the sperm of a defined male donor. The male donors were kept under conditions (air conditioning, diet, water) comparable to those of the females participating in this study. The day of insemination was designated as gestation day (GD) 0 and the following day as GD 1.

Duration of treatment / exposure:

from implantation to one day prior to the expected day of parturition (GD 6-28)

Frequency of treatment:

daily

Duration of test:

until GD 29

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

actual dose: 41 mg/kg bw/d

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

actual dose: 132 mg/kg bw/d

Dose / conc.:

450 mg/kg bw/day (nominal)

Remarks:

actual dose: 406 mg/kg bw/d

No. of animals per sex per dose:

25 inseminated females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The dose volume was 10 mL/kg body weight. The calculation of the volume administered was based on the most recent individual body weight.

Maternal examinations:

CLINICAL EXAMINATIONS
- Mortality: Mortality was checked in the females twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0-29).
- Clinical symptoms: A clinical examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. If such signs occurred, the animals were examined several times daily (GD 0-29).
- Food consumption: The food consumption was determined daily on GD 1–29.
- Body weight data: All animals were weighed on GD 0, 2, 4, 6, 9, 11, 14, 16, 19, 21, 23, 25, 28 and 29. The body weight change of the animals was calculated.
- Corrected (net) body weight gain: The corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6).

TERMINAL EXAMINATIONS OF THE DOES
After the does had been sacrificed on GD 29, they were necropsied and assessed by gross pathology in randomized order.

Ovaries and uterine content:

On GD 29, the surviving does were sacrificed in randomized order by an intravenous injection of pentobarbital (Narcoren; dose: 2 mL/animal). After the does had been sacrificed, they were necropsied and assessed by gross pathology in randomized order. The uterus and the ovaries were removed and the following data were recorded:
- Weight of the unopened uterus
- Number of corpora lutea
- Number and distribution of implantation sites classified as:
1) live fetuses
2) dead implantations: a) early resorptions (only decidual or placental tissues visible or according to SALEWSKI (Salewski, 1964) from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single-horn pregnancy); b) late resorptions (embryonic or fetal tissue in addition to placental tissue visible); c) dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened).
After the weight of the uterus had been determined, all subsequent evaluations of the does and the gestational parameters were conducted by technicians unaware of treatment group in order to minimize bias. For this purpose the animal numbers were encoded.
Furthermore, calculations of conception rate and pre- and postimplantation losses were carried out:
The conception rate (in %) was calculated according to the following formula: (number of pregnant animals)/(number of fertilized animals) x 100.
The preimplantation loss (in %) was calculated based on each individual pregnant animal with scheduled sacrifice according to the following formula: (number of corpora lutea – number of implantations)/(number of corpora lutea) x 100.
The postimplantation loss (in %) was calculated based on each individual pregnant animal with scheduled sacrifice from the following formula: (number of implantations – number of live fetuses)/(number of implantations) x 100.

Fetal examinations:

All fetal analyses were conducted by technicians unaware of the treatment group, in order to minimize bias.
- Examination of the fetuses after dissection from the uterus: Each fetus was weighed and examined macroscopically for any external findings.
Furthermore, the viability of the fetuses and the condition of the placentae, the umbilical cords, the fetal membranes, and fluids were examined. Individual placental weights were recorded. Thereafter, the fetuses were sacrificed by a subcutaneous injection of phenobarbital (Narcoren; 0.2 mL/fetus).
- Soft tissue examination of the fetuses: After the fetuses had been sacrificed, the abdomen and the thorax were opened in order to examine the organs in situ before they were removed. The heart and the kidneys were sectioned in order to evaluate the internal structure. The sex of the fetuses was determined by examination of the gonads in situ. After these examinations, the heads of approximately one half of the fetuses per litter and the heads of those fetuses, which revealed severe findings during the external examination (e.g. anophthalmia, microphthalmia or hydrocephalus) were severed from the trunk. These heads were fixed in BOUIN's solution and were, after fixation, processed and evaluated according to WILSON's method (Wilson and Warkany, 1965). About 10 transverse sections were prepared per head. After the examination these heads were discarded. All fetuses (partly without heads) were skinned and fixed in ethyl alcohol. After fixation for approx. 1-5 days, the fetuses were removed from the fixative for awhile. With a scalpel, a transversal incision was made into the frontal / parietal bone in the heads of the intact fetuses. The two halves of the calvarium were then cauteously bent outward and the brain was thoroughly examined. Subsequently, the fetuses were placed back into the fixative for further fixation.
- Skeletal examination of the fetuses: After fixation in ethyl alcohol the skeletons were stained according to a modified method of KIMMEL and TRAMMELL (Kimmel, C.A., and Trammell, C., 1981). Thereafter, the stained skeleton of each fetus was examined. After the examination the stained skeletons were retained individually.

Statistics:

- DUNNETT-test (two-sided) for food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight.
- FISHER'S EXACT test (one-sided) for female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings.
- WILCOXON-test (one-sided) for proportions of fetuses with malformations, variations and/or unclassified observations in each litter.

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no test substance-related or spontaneous mortalities in any group.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weights of the low-, mid- and high-dose rabbits (50; 150 and 450 mg/kg bw/d) were not significantly different from the concurrent control throughout the course of the study. The average body weight gain of the mid- and high-dose rabbits was statistically significantly reduced by about 27% and 31% during the treatment period. A significant reduction of mean body weight gain was also noted for the the high-dose rabbits on GD 19-21.

Corrected (net) body weight gain: Mean carcass weights and the corrected body weight gain (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6) were comparable among all groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The food consumption in the high-dose females (450 mg/kg bw/d) was distinctly and statistically significantly reduced during a significant part of the treatment period (GD 15-23). During the entire treatment period (GD 6-28) the total average food consumption of the high dose rabbits was about 18% below controls. The food consumption of the mid dose females (150 mg/kg bw/d) was similarly affected in terms of magnitude and course of reduction, however the reduction of food consumption reached statistical significance only on GD 22-24. During the treatment period (GD 6-28) the total average food consumption of the mid-dose rabbits was about 13% below controls. Overall, the food consumption of the low-dose does (50 mg/kg bw/d) did not show test substance-related impairments. The reduced food consumption at the 150 and 450 mg/kg bw/d levels is considered to be related to the treatment.

Food efficiency:

not specified

Ophthalmological findings:

not specified

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The mean gravid uterus weights of all test groups did not show staatistically significant differences in comparison to control.

Gross pathological findings:

no effects observed

Description (incidence and severity):

At necropsy, only spontaneous findings were seen in single females of every test group. No test substance-related findings were observed in the doses.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The conception rate reached 96% in test groups 1 and 3 (50 and 450 mg/kg bw/d) and 100% in test groups 0 and 2 (0 and 150 mg/kg bw/d). Importantly, a sufficient number of pregnant females was available for the purpose of the study, as 24-25 pregnant rabbits per group had implantation sites in the uterus, at terminal sacrifice. There were no test substance-related and/or biologically relevant differences between the control and all dosed groups in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation losses, the number of resorptions and viable fetuses. Gestational parameters were within the normal range for animals of this strain and age.

Dose descriptor:

NOAEL

Effect level:

450 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks on result:

other: no adverse effects observed; actual dose 406 mg/kg/d

Dose descriptor:

NOEL

Remarks:

food consumption

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

food consumption and compound intake

Remarks on result:

other: actual dose: 41 mg/kg/d

Abnormalities:

effects observed, treatment-related

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean fetal weights of all treated groups were not influenced by the test substance. Neither female nor male weights showed statistically significant or biologically relevant differences between the test substance-treated groups and the controls.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution of the fetuses in test groups 1-3 (50; 150 and 450 mg/kg bw/d) was comparable to the control fetuses. Observable differences were without biological relevance.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

One sole external malformation (unilateral microphthalmia) was recorded for two fetuses from 2 litters in the high-dose group (450 mg/kg bw/d). This malformation is present in the historical control data. Thus an association of these individual findings to the treatment is not assumed. The total incidences of external malformations were comparable to the historical control data.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Malformations of the fetal skeletons were noted in fetuses of test groups 0, 2 and 3 (0; 150 and 450 mg/kg bw/d). Neither statistically significant differences between treated groups and the control were calculated nor a dose-response relationship was observed. All individual skeletal malformations were present in the historical control data at a comparable frequency.
- Fetal skeletal variations: For all test groups, variations in different skeletal structures were detected with or without effects on the corresponding cartilages. The observed skeletal variations were related to various parts of the fetal skeletons and were statistically significant higher in the low- and the high-dose groups on a fetus per litter basis. Several specific skeletal variations were statistically significant higher than the concurrent control in the dosed groups (on a fetus per litter basis). These findings are delays or minor disturbances of ossification which are reversible or do not considerably affect the integrity of the underlying structures. Such slight changes of the ossification process occur very frequently in gestation day 29 rabbit fetuses of this strain and all observed incidences were within the historical control data. Thus an association of these findings to the treatment is not assumed.
- Fetal skeletal unclassified cartilage observations: Additionally, isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all groups including the control. The observed unclassified cartilage findings did not show a relation to dosing and were comparable to historical control data and, therefore, regarded to be spontaneous in nature.

Visceral malformations:

no effects observed

Description (incidence and severity):

- Fetal soft tissue malformations: The examination of the soft tissues revealed a variety of malformations in fetuses of all test groups including the controls (0; 50; 150 and 450 mg/kg bw/d). With the exception of a lateral pouch in the tongue of 2 fetuses all individual soft tissue malformations were present in the historical control data at comparable frequencies. No statistically significant differences between the test groups and the control were observed. The total incidences of external malformations were comparable to the historical control data. No malformation pattern was evident. Thus an association of these findings to the treatment is not assumed.
- Fetal soft tissue variations: A number of soft tissue variations, such as absent lung lobe (lobus inferior medialis) and malpositioned carotid branch, was detected in each test group including the controls. Incidences were without a relation to dosing. Neither statistically significant differences between the test groups nor differences to the historical control data were noted.
- Fetal soft tissue unclassified observations: Unclassified soft tissue observations, such as infarct of liver, hemorrhagic thymus or ovary and blood coagulum around urinary bladder, were recorded for some fetuses of test groups 0, 1, 2 and 3 (0; 50; 150 and 450 mg/kg bw/d). A relation to dosing is not present if normal biological variation is taken into account. Therefore, a test substance induced effect is not assumed.

Details on embryotoxic / teratogenic effects:

- Abstract of all classified fetal external, soft tissue and skeletal observations: Various external, soft tissue and skeletal malformations occurred throughout all test groups including the control. All individual malformations are present in the historical control data, with the exception of lateral pouches in the tongue of 2 fetuses. They did neither show a consistent pattern since a number of morphological structures of different ontogenic origin were affected nor a clear dose-response relationship. The overall incidence of malformations was comparable to the historical control data. One external (paw hyperflexion), two soft tissue (absent lobus inferior medialis and malpositioned carotid branch) and a broad range of skeletal variations occurred in all test groups including the controls. All fetal and litter incidences for these variations and the corresponding mean percentages of affected fetuses/litter were not significantly different from the concurrent control and their frequency is comparable to the historical control data. Therefore, they were not considered to be related to the treatment. A spontaneous origin is also assumed for external, soft tissue and unclassified skeletal cartilage observations which were observed in several fetuses of all test groups including controls (0, 50; 150 and 450 mg/kg bw/d). Distribution and type of these findings do not suggest relation to treatment.

Key result

Dose descriptor:

NOAEL

Remarks:

prenatal developmental toxicity

Effect level:

450 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no adverse effects observed

Abnormalities:

no effects observed

Developmental effects observed:

no

Table: Occurence of statistically significantly increased fetal skeletal variation (expressed as mean percentage of affected fetuses/litter)

Finding	Group 0 0 mg/kg/d	Group 1 50 mg/kg/d	Group 2 150 mg/kg/d	Group 3 450 mg/kg/d	HCD (range)
Incomplete ossification of parietal; unchanged cartilage	0.0	0.0	1.9*	2.1*	0.4 (0.0 – 2.6)
Incomplete ossification of hyoid; cartilage present	11.2	11.4	19.1	20.4*	9.8 (0.0 – 21.6)
Splitting of skull bone	0.4	3.3*	3.3	2.3	2.9 (0.0 – 7.7)
Incomplete ossification of cervical centrum; unchanged cartilage	2.5	2.2	3.6	7.3*	2.5 (0.0 – 9.3)
Supemumerary 13th rib; cartilage not present	2.5	9.8	6.1	9.9*	6.6 (0.0 – 17.5)
Total fetal skeletal variations	46.3	63.7*	59.3	71.6**	63.5 (46.3 – 81.9)

HCD = Historical control data; * = p ≤ 0.05, ** = p ≤ 0.01 (Wilcoxon-Test [one-sided])

 

 

Table: Total fetal malformations

		Group 0 0 mg/kg/d	Group 1 100 mg/kg/d	Group 2 300 mg/kg/d	Group 3 1000 mg/kg/d
Litter Fetuses	N N	25 171	24 154	25 157	24 158
Fetal incidence	N (%)	4 (2.3%)	2 (1.3%)	6 (3.8%)	9 (5.7%)
Litter incidence	N (%)	4 (16%)	1 (4.2%)	4 (16%)	7 (29%)
Affected fetuses/litter	Mean%	2.3	1.2	3.6	6.2

 

 

Table: Total fetal variations

		Group 0 0 mg/kg/d	Group 1 100 mg/kg/d	Group 2 300 mg/kg/d	Group 3 1000 mg/kg/d
Litter Fetuses	N N	25 171	24 154	25 157	24 158
Fetal incidence	N (%)	106 (62%)	106 (69%)	106 (68%)	122 (77%)
Litter incidence	N (%)	21 (84%)	24 (100%)	24 (96%)	23 (96%)
Affected fetuses/litter	Mean%	59.9	69.8	64.3	74.2

 

Conclusions:

In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 450 mg/kg bw/d and the no observed effect level (NOEL) for maternal toxicity is 50 mg/kg bw/d based on effects on food consumption. The NOAEL for prenatal developmental toxicity is 450 mg/kg bw/d. No adverse fetal findings of toxicological relevance were evident at any dose.

Executive summary:

The study was performed according to OECD TG 414 in compliance with GLP.

Methyl Methacrylate was tested for its prenatal developmental toxicity in Himalayan rabbits. The test substance was administered as an aqueous preparation to 25 inseminated female Himalayan rabbits by stomach tube at doses of 50; 150 and 450 mg/kg body weight/day on gestation days (GD) 6 through GD 28. The control group, consisting of 25 females, was dosed with the vehicle (1% Carboxymethylcellulose CB 30.000 in drinking water and a few drops Cremophor EL and one drop hydrochloric acid [1% CMC]) in parallel. A standard dose volume of 10 mL/kg body weight was used for each test group. At terminal sacrifice on GD 29, 24-25 females per group had implantation sites.

The following test substance-related adverse effects/findings were noted:

Test group 3 (450 mg/kg body weight/day):

-        Reduced food consumption (-18%) and body weight gain (-31%)

-        No test substance-related adverse effects on gestational parameters or fetuses

 

Test group 2 (150 mg/kg body weight/day):

-        Reduced food consumption (-13%) and body weight gain (-27%)

-        No test substance-related adverse effects on gestational parameters or fetuses

 

Test group 1 (50 mg/kg body weight/day):

-        No test substance-related adverse effects on does, gestational parameters or fetuses

In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is nominal 450 mg/kg bw/d (actual 406 mg/kg bw/d), the highest dose tested. The no observed effect level (NOEL) for maternal toxicity is nominal 50 mg/kg bw/d (effective 41 mg/kg bw/d) based on effects on food consumption being a consequence of reduced appetite observed at the LOEL (Lowest Observed Effect Level) of 150 mg/kg bw/d (actual 132 mg/kg bw/d).

The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is nominal 450 mg/kg bw/d (actual 406 mg/kg bw/d). No adverse fetal findings of toxicological relevance were evident at any dose.

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

98% purity

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

5-month-old

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

destilled water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dosing solutions were verified to be within 10% of theoretical concentrations.

Details on mating procedure:

artificially inseminated

Duration of treatment / exposure:

gd 6–19

Frequency of treatment:

daily

Duration of test:

gd 30

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

0 mg/kg bw/day (nominal)

No. of animals per sex per dose:

23-24

Control animals:

yes

Details on study design:

- Dose selection rationale not presented

Maternal examinations:

At scheduled necropsy on gd 30, maternal liver and kidneywere recorded. Kidneys were examined histologically in 10–17 dams/group.
At necropsy, 20–22 dams and litters/group were evaluated in the control and 3 lowest dose groups; 9 does and litters were evaluated in the highest dose group due to a high mortality rate.

Ovaries and uterine content:

At scheduled necropsy on gd 30, gravid uterus weights were recorded. Ovarian corpora lutea were counted and uterine implantation sites were recorded.

Fetal examinations:

All live fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and variations. Viscera were examined according to the Staples method and the skeleton was stained with Alcian Blue/Alizarin Red S. Heads from half the fetuses were fixed in Bouin’s solution and examined.

Statistics:

The litters were considered the experimental unit. Data were analyzed with the General Linear Trend Models procedures for ANOVA, Bartlett’s test for homogeneity of variance, Williams’ and Dunnett’s multiple comparison tests, and/or Fisher’s Exact Probability Test.

Clinical signs:

not specified

Mortality:

mortality observed, treatment-related

Description (incidence):

At 2,000 mg/kg bw/day, 42% of the does died.

Body weight and weight changes:

no effects observed

Water consumption and compound intake (if drinking water study):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Kidney weights were slightly increased in the 2,000 mg/kg bw/day group (not statistically significant).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Necropsy revealed renal toxicity including tubule dilatation and degeneration, epithelial necrosis, and intraluminal oxalate crystal deposition.

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

At 2,000 mg/kg bw/day, one doe aborted.

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): At 2,000 mg/kg bw/day, three does delivered early.

Other effects:

effects observed, treatment-related

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

gross pathology

mortality

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Dose descriptor:

NOAEL

Effect level:

2 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: no adverse effects observed

Developmental effects observed:

no

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Additional information

EGDMA

In a study performed according to OECD 414 administration of EGDMA to pregnant female Sprague-Dawley rats from gestation days 6 to 20 inclusive, at 25, 100 and 500 mg/kg/day caused evident signs of maternal toxicity at 500 mg/kg/day (one female was sacrificed on GD 15, clinical signs of poor health were observed in 3/22 surviving pregnant 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. 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 No Observed Adverse Effect Level (NOAEL) for maternal toxicity was 100 mg/kg/day and the NOAEL for developmental toxicity was 500 mg/kg/day.

For the evaluation of the developmental toxicity of EGDMA especially in a non-rodent species, screening data of the primary metabolite, HEMA, and reliable data from the further metabolites methacrylic acid (and its donor substance methyl methacrylate) and the alcohol metabolite ethylene glycol are available. In the body, HEMA is the first metabolite resulting from the primary ester hydrolysis of EGDMA. Subseque MMA (methacrylic metabolite donor substance)nt ester hydrolysis produces methacrylic acid and ethylene glycol. Methyl methacrylate hydrolyses to methacrylic acid rapidly and thus serve as methacrylic acid donor in several test systems investigating systemic effects. In the selected rabbit studies, developmental toxicity was not observed at any dose.

MAA (methacrylic metabolite; including donor substance MMA)

Methacrylic acid (MAA), the common metabolite for all esters, was tested in a developmental toxicity study in groups of 19-25 pregnant female rats (whole-body inhalation exposure for 6 hr/day, during days 6 to 20 of gestation), at 0, 50, 100, 200, and 300 ppm (0, 179, 358, 716 and 1076 mg/m³) and produced no embryo- or fetal lethality, nor fetal malformations after exposure with MAA at any concentration, despite overt maternal toxicity (decreased body weight and feed consumption) at 300 ppm (1076 mg/m³). The NOAEL for developmental toxicity was considered 300 ppm (1076 mg/m³) MAA (Saillenfait et al., 1999).

 

In a developmental toxicity study according to OECD 414 MMA (methyl methacrylate) was administered by inhalation exposure to 99, 304, 1178, and 2028 ppm (412, 1285, 4900, 8436 mg/m³; Solomon et al., 1993). No relevant maternal, treatment-related effects, except reduced body weight, were noted at any concentration tested. No embryo or foetal toxicity was evident and no increase in the incidence in the malformations or variations was noted at exposure levels up to and including 2028 ppm. In this study the NOAEC for developmental toxicity was 2028 ppm (8436 mg/m³).

 

In addition, another study with MMA has been performed, an oral OECD 414 study in rabbits at 50, 150 , and 405 mg/kg/d. The no observed adverse effect level (NOAEL) for prenatal developmental toxicity was 450 mg/kg bw/d. No adverse foetal findings of toxicological relevance were evident at any dose, even in the presence of maternal toxicity (BASF, 2009). MMA is not a selective teratogen.

 

EG (alcohol metabolite)

The developmental toxicity of ethylene glycol has been assessed by several animal studies. From those, oral studies are considered most relevant for the primary metabolite of HEMA and have thus been generally selected for the IUCLID dataset of EGDMA; a dermal study is added for supportive reasons. In brief, developmental effects in animal studies have been shown to be species specific and influenced by the dosing regime. These effects have to be interpreted in the light of the the toxicokinetics of the substance (NTP-CERHR 2004): “Ethylene glycol metabolism yields a variety of products with glycolic acid and oxalic acid of principle toxicological interest. There are several studies that have attempted to characterize the oxidation of glycolic acid to glyox­ylate, a saturable process that leads to accumulation of glycolic acid. This is reflected most clearly in the non-linear increase in glycolic acid levels in blood and urine as the dose of ethylene glycol is in­creased. This saturation occurs beginning at doses as low as 150 mg/kg bw in rodents with the mouse enzyme system appearing to be somewhat more readily saturable than the rat system(Neeper-Bradley 1995 vs. Neeper-Bradley 1990, see Table below: Summary of maternal toxic and developmental NOAELs in studies with ethylene glycol with respect to animal species and dosing regime of selected studies taken from NTP-CERHR monograph (2004)).It is evident that glycolic acid metabolism is saturated at the bolus doses in rats required to produce developmental toxicity (1,000 mg/kg bw).” The role of saturation kinetics in developmental toxicity is also suggested by a lack of toxicity after ethylene glycol continuous dosing (e.g., feeding, dermal) when compared to bolus dosing (gavage; Neeper-Bradley 1990 vs. Maronpot 1983, seeTable1: Summary of key physico-chemical properties). The lower dose rates in these continuous dosing studies “leads to a glycolic acid formation rate that apparently does not exceed saturation”. The NTP-CERHR expert panel stated that the continuous dosing is more relevant for human exposure than bolus dosing.

 

Table: Summary of maternal toxic and developmental NOAELs in studies with ethylene glycol with respect to animal species and dosing regime of selected studies taken from NTP-CERHR monograph (2004)

Study	Animal species	Dosing regime	NOAEL maternal	NOAEL developmental
Neeper-Bradley 1995	mouse	oral gavage (bolus)	≥ 1500 mg/kg/d	150 mg/kg/d
Neeper-Bradley 1990	Rat	oral gavage (bolus)	1000 mg/kg/d	500 mg/kg/d
Maronpot 1983	Rat	oral feed (continuous)	≥ 1000 mg/kg/d	≥ 1000 mg/kg/d
Tyl 1993	Rabbit	oral gavage (bolus)	1000 mg/kg/d	2000 mg/kg/d
Tyl 1995	Mouse	dermal (continuous)	≥ 3550 mg/kg/d	≥ 3550 mg/kg/d

 

In addition, developmental toxicity was not observed in the non-rodent species rabbit orally exposed to ethylene glycol at doses associated with severe maternal toxicity even with bolus dosing (Tyl 1993). As stated by NTP-CERHR (2004), “rabbits demonstrated no developmental toxicity following gavage exposure to doses as high as 2,000 mg/kg bw/day on gd 6–19, as noted by a lack of malformations, prenatal deaths, or decrease in fetal weights. Severe maternal toxicity was observed at 2,000 mg/kg bw/day as evidenced by maternal deaths, increased early delivery, and lesions as well as oxalate crystals in the kidneys. Maternal and fetal NOAELs were identified as 1,000 and 2,000 mg/kg bw/ day, respectively. Thus, the data were sufficient to demonstrate a lack of developmental toxicity in rabbits following oral gavage throughout organogenesis at doses ≤ 2,000 mg/kg bw/day.”

The absence of developmental effects of ethylene glycol after dermal application is shown in the sensitive species mouse by Tyl (1995).

With respect to human relevance, NTP-CERHR Expert Panel noted the doses tested in animal studies “far exceeded the doses relevant to humans based on knowledge of absorption, distribution, metabolism, and excretion in rats, mice, and humans”; and this is believed especially true for ethylene glycol as alcohol metabolite of EGDMA. In addition, the Expert Panel recognized that “the rat and mouse models are possibly more sensitive than humans because of the dependence of these species on the inverted yolk sac placenta, which is not present in humans.”

 

Overall, there is no concern of developmental potential for EGDMA with respect to the ethylene glycol metabolite when considering relevant exposure routes and quantities.

Summary

Based on the results of a developmental toxicity study with the rodent species rat and reliable developmental toxicity studies with the alcohol metabolite EG and the methacrylic metabolite donor substance MMA in the non-rodent species rabit, exposure to EGDMA is not likely to result in developmental toxicity.

Justification for selection of Effect on developmental toxicity: via oral route:
The EGDMA study is a valid, guideline study for developmental toxicity, in which maternal toxicity but, up to the highest dose tested, no developmental toxicity was observed. The studies with the metabolites are also reliable studies covering a wide range of animal species and dose regimes.

Compliance to REACh requirements

The development toxicity requirement for one species is covered with reliable oral rat study, while the second species (rabbit) requirement is covered with reliable studies with the methacrylic metabolite donor MMA and the alcohol metabolite EG. All mentioned studies are reliable (Reliability 1 or 2) and the read across is done with a high level of confidence (see chapter "Toxicokinetics" and the attached category document).

Justification for classification or non-classification

In the absence of toxicity to reproduction (fertility and developmental effects) of either EGDMA or its proven metabolites, no classification is required.