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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

OECD 422 (screening study), oral, rat: NOAEL fertility 300 mg/kg bw/d (decreases in the number of corpora lutea and the number of implantation sites)

Metabolite data

MMA (donor substance of the methacrylic moiety), 2 gen, oral gavage, rat NOEL P/F1 generation 50 mg/kg bw/d (decreased food consumption); NOAEL fertility 400 mg/kg bw/d (no adverse effects observed)

Based on these data, 2-ethylhexyl methacrylate is not expected to be a reproductive toxicant.

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Combined Repeated Dose and Reproductive / Developmental Toxicity Screening Test (Precursor Protocol of GL 422)
Deviations:
no
GLP compliance:
yes
Limit test:
no
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 Co. (Hino Breeding Center)
- Age at study initiation: 10 weeks old for males and females
- Weight at receipt: 351~384g for males and 212~237 for females
- Fasting period before study: no
- Housing: Stainless steel cage systems (W: 240 x D: 380 x H: 200mm) were used during quarantine and acclimatization, and 5 rats were housed in each cage. After dividing into groups, a rack of five stainless steel cages (W: 755 x D: 210 x H: 170mm) were used for individual housing. Mating was conducted in stainless steel cage systems. Also, on the 18th day of gestation, pregnant animals were moved to plastic cages (W: 310 x D: 360 x H: 175mm) in which autoclaved floors (Sunflake, Charles River Japan Co.) had been inserted to facilitate natural delivery and lactation
- Diet (ad libitum): CRF-1, Oriental Yeast Co.
- Water (ad libitum): tap water
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 44-59
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The amount of the test substance needed at each concentration was estimated, and the substance administered was produced in the required concentration by diluting with corn oil

VEHICLE
- Justification for use and choice of vehicle (if other than water): solubility
- Concentration in vehicle: 6, 20 or 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal plug or sperm in vagina referred to as day 0
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The 6, 20 and 200 mg/ml prepared solutions were confirmed to have no problems with stability for 7 days under cool and dark conditions after preparation, and for four hours under dark conditions at room temperature.
The concentration of the test substance in the administration substance at each concentration used on the administration starting date and the administration ending date for males was measured by HPLC. The results of the test substance concentration were 99.5~110% of the displayed concentration, and as they were within the range of acceptable concentration (within ±10% of the displayed concentration).
Duration of treatment / exposure:
Males: 14 days before mating and 35 days after, for a total of 49 days
Females: 14 days before mating and a total of 41~47 days during mating, gestation and day 4 of lactation to the day before necropsy
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
0(vehicle), 30, 100, 300, 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
Post-exposure period: 1 day
- Dose selection rationale:
The dose was determined by the results of the preliminary testing (administration doses: 0, 125, 250, 500 and 1000 mg/kg, 5 in each group) with oral administration using male rats for a period of two weeks. Salivation was noted immediately after administration in the groups greater than 125 mg/kg, and low body weight values were noted in the 1000 mg/kg group but there were no confirmed fatalities in any of the groups. Therefore, the dose for this study was set with a maximum dose of 1000 mg/kg in conjunction with the dose limits found in the OECD guidelines, and included ratio of approximately 3, with doses of 300, 100 and 30 mg/kg. Furthermore, a group administered the same quantities of a medium (corn oil) as the test substance was considered the control group.
- Rationale for animal assignment (if not random):
To divide into groups, the computer was used to separate by weights and then random sampling was performed to make each group uniform in terms of average weight and distribution for the administration starting date. Animals remaining after allocation into groups were euthanized under ether anesthesia on the administration starting date and discarded
Positive control:
not required
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations:
Males: twice weekly (Days 1, 4, 8, 11, 15, 18, 22, 25, 29, 32, 36, 39, 43, 46 of administration and the necropsy day)
Female: 14 days before mating started and two times each week during mating season (Measurement days: Days 1, 4, 8, 11, 15 and 18 of administration), and measured on day 0, 7, 14 and 21 during gestation, and day 0 and 4 during lactation.

FOOD CONSUMPTION : Yes
Males: twice weekly (Days 3, 6, 10, 13, 24, 27, 31, 34, 38, 41, 45 and 48 of administration)
Female: twice weekly (14 days before mating, on day 2, 9, 16 and 21 during gestation as well as on day 4 of lactation)
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: Yes

WATER CONSUMPTION: No

Oestrous cyclicity (parental animals):
Estrous cycles were observed daily from the date administration started to the date mating was confirmed. If heat was observed continuing for two consecutive days, it was calculated as one occurrence.
Sperm parameters (parental animals):
Parameters examined in all P male parental generations: testis weight, epididymis weight
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- Observations at Birth
Observations were made on the total number and gender of pups at birth, number of stillbirths, number of live births and presence of external abnormalities. Stillbirths were fixed in 20% neutral buffered formalin and stored.
- Observations of the Pups
Observations were made daily on the general status and fatalities during their lifetimes. After necropsy, stillbirths were fixed in 20% neutral buffered formalin and stored (excluding those with dramatic changes after death).
- Body Weight
Body weights were measured on day 0 of lactation (date of birth) and day 4.

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):
GROSS PATHOLOGY: Yes

ORGAN WEIGHTS: Yes
Ovaries, testes and epididymides.

HISTOPATHOLOGY: Yes
H-E stained tissue specimens were produced from the ovaries, uterus, vagina, testes, epididymides, seminal vesicle, prostate, pituitary gland and histopathological examinations performed. During the necropsy for one case (No. 308) in the 300 mg/kg group, an abnormality was confirmed so the epididymides were also examined. section 7.5.1
Postmortem examinations (offspring):
SACRIFICE
After completing the observations on day 4 of lactation, the pups were euthanized under ether anesthesia after blood was collected from the abdominal aorta, and necropsy was performed. Pups with abnormalities confirmed during necropsy were fixed in 20% neutral buffered formalin and stored.
Statistics:
The mean values and standard deviations were calculated for each group for body weight (parent animals, pups), food consumption, number of mating seasons, number of conceiving days, gestation term (delivery day (day 0 of lactation) – day fertilization confirmed), number of implantation scars, total number of births (live births + stillbirths), number of live births, number of stillbirths, number of corpus luteum, reproductive indices, offsping viability indices, gender ratio (males/females), absolute and relative weights of organs results. Next, equal distribution examination was conducted using the Bartlett method, and if there was equal distribution, a distribution analysis was performed using analysis of variance, and if significant, the Dunnett method was performed. On the other hand, if equal distribution was not confirmed, analysis of variance was conducted using a ranking (Kruskal-Wallis test), and if significant, the ranking was used and a Dunnett type of examination method was performed.
The copulation rate, fertilization and delivery rate was conducted using χ2 calibration.
In the histopathological examination, in addition to the suggestion that there was a toxicological impact in the group with the maximum dose, the findings on the organs and tissues from the other groups that were subject to examination were conducted by using the Dunnett method with the ranking given above to compare the groups with the control group. If a significant difference with the control group was confirmed, the dose response was studied using the Cochran Armitage test for trend.
Reproductive indices:
- Reproductive indices:
Delivery rate [(overall birth rate/number of implantation scars) x 100], pup birth rate [(number of pups at day 0 of lactation/number of implantation scars) x 100], implantation rate [(number of implantation scars/number of corpus luteum) x 100], birth rate [(number of pups at day 0 of lactation/total number of births) x 100], rate of external abnormalities [(number of pups with external abnormalities/number of new pups) x 100]
- Copulation rate [(number of animals mating/number of animals cohabitating) x 100],
- Fertilization rate [(number of fertilized females/number of animals mating) x 100]
- Delivery rate [(number of females delivering pups/number of fertilized females) x 100
Offspring viability indices:
- number of pups at day 4 of lactation
- survival rate at day 4 of lactation [(number of live pups at day 4 of lactation/number of new pups at day 0 of lactation) x 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:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined
Reproductive function: oestrous cycle:
effects observed, treatment-related
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
- Clinical signs:
MALES: There were no confirmed deaths or near-deaths in any of the groups.
In the observations on the overall condition, nothing abnormal was noted in any of the animals in the control group during the observation period. In the 30 mg/kg group, there were 1~3 cases where salivation was noted on day 9, 10, 12, 13 and 24 of administration. In the 100 mg/kg group, there were 1~3 cases where salivation was noted on day 5, 9, 10, 12~18, 23~28, 30, 33 and 43 of administration. In the 300 mg/kg group, there were 1~4 cases where salivation was noted on day 4~28, 33, 35, 39, 44~46, 48 and 49 of administration. In the 1000 mg/kg group, there was 1 case of soiled fur on days 16~23 of administration, and there were 4~all cases where salivation was noted on days 2~49 of administration. Salivation was seen for approximately 3~30 minutes after administration in all groups, and no changes were seen in the length of time that salivation continued, even during administration.
FEMALES: Neither dead nor moribund animal was observed in the control group and the 30, 100 and 300 mg/kg group. In the 1000 mg/kg group, one animal died on Day 15 of administration. In the dead animal, salivation and abnormal gait were observed from Days 2 and 8 of administration to the day before death, respectively. In clinical observation in the surviving animals, there was no abnormality in any animal in the control group throughout the observation period. In the 30 mg/kg group, there were 1~2 cases where salivation was noted on day 10, 12~14 of administration. In the 100 mg/kg group, there was 1 case where salivation was noted on day 12 and 13 of gestation. In the 300 mg/kg group, there were 1~3 cases where salivation was noted on day 10, 12~15 and 17 of administration, day 0, 3, 4, 9, 12~14 and 17 of gestation. In the 1000 mg/kg group, there was 1 case of soiled fur on days 16~19 of administration and day 0~3 of gestation, and there were 1~11 cases where salivation was noted on days 2, 4~19 of administration, 0~23 days of gestation, and day 0 and 1 of lactation. Salivation was seen for a period of approximately 3~30 minutes after administration in all groups.

- Body weight:
MALES: The body weight of animals in the 30, 100 and 300 mg/kg groups showed almost similar changes to those in the control group, and no significant difference was observed on any determination day. In the 1000 mg/kg group, significantly low body weights were observed from Day 18 to Day 50 of administration compared with the control group.
FEMALES: The body weight of animals in the 30, 100 and 300 mg/kg groups showed almost similar changes to those in the control group before mating, and no significant difference was observed on any day of determination. In the 1000 mg/kg group, significantly low body weights were observed from Day 4 to Day 15 of administration compared with the control group. In the dead animal in the 1000 mg/kg group, decreased body weight was observed before death. During the gestation period, the body weights in the 30, 100 and 300 mg/kg groups showed almost similar changes to the control group, and no significant difference was observed on any determination day. In the 1000 mg/kg group, significantly low body weights were observed on Days 14 and 21 of gestation compared with the control group. During the lactation period, the body weights in the 30, 100 and 300 mg/kg groups showed almost similar changes to the control group, and no significant difference was observed on any day of determination. In the 1000 mg/kg group, significantly low body weight was observed on Days 0 and 4 of lactation compared with the control group.

- Food consumption:
MALES: Food consumption in the 30 mg /kg group was almost similar to as that in the control group, and no significant difference was observed on any determination day. In the 100 mg /kg group, significantly high food consumption was observed compared with the control group on Day 3 of administration, but it was not a dose-dependent change. Food consumption in the 300 mg /kg group was almost similar to that in the control group, and no significant difference was observed on any day of determination. In the 1000 mg/kg group, significantly low food consumption was observed on Days 6, 10, 31 and 34 of administration compared with the control group.
FEMALES: Food consumption in the 30, 100 and 300 mg/kg groups showed almost the same changes as that in the control group before mating, and no significant difference was observed on any determination day. In the 1000 mg/kg group, significantly low food consumption was observed compared with the control group on Days 3 and 6 of administration. In the dead animals in the 1000 mg/kg group, a marked decrease in food consumption was observed before death. During the gestation period, food consumption in each group showed almost the same change as that in the control group, and no significant difference was observed on any day of determination. During the lactation period, food consumption in the 30, 100 and 300 mg/kg groups showed no significant difference compared with the control group. In the 1000 mg/kg group, no significant difference was observed compared with the control group, but food consumption tended to be low on Day 4 of lactation.

- Organ Weight:
MALES: there were no significant differences in any of the absolute and relative testis or epidydimis organ weights compared with the control group.
FEMALES: there were no significant differences in the absolute and relative ovary weight compared with the control group.

- Histopathology:
MALES: in the 1000 mg/kg group, hemiatrophy of the seminiferous tubule of the testis, unilateral spermatic granuloma of the epididymis, decreased sperms and intraluminal cell debris were observed in 1/12 animals. Since they were the changes sometimes observed in the control group and observed in one animals, they were judged as incidental changes. There were no abnormalities in the pituitary gland, prostate gland and seminal vesicles in the control group and the 1000 mg/kg group.
FEMALES (Table 7): there were no abnormalities in the ovaries, uterus, vagina, pituitary gland and mammary gland in the control group and the 1000 mg/kg group.

- Impact on Reproduction/Development of Parent Animals (P)
1) Number of Estrous Cycles
There were no significant differences in the number of estrous cycles during the administration period (14 days) prior to mating between the control group and the 30, 100 and 300 mg/kg groups. In the 1000 mg/kg group, there were significantly low values noted in the number of estrous cycles when compared to the control group.
2) Number of Conceiving Days, Copulation Rate, Fertilized Females and Fertilization Rate
Copulation was confirmed in all cases, so the copulation rate for all groups was 100%. There were no significant differences between the control group and any of the administration groups for the number of conceiving days.
There were two unfertilized females in the 30 mg/kg group, one case in the 100 mg/kg group, one case in the 300 mg/kg group and two cases in the 1000 mg/kg group so there were 9~12 fertilized females in each group. However, there was no significant difference in the fertilization rate between the control group and each of the administration groups. There were two cases of fertilized females not resulting in pups noted in the 1000 mg/kg group (No. 451 and 452).
3) Gestation Period and Delivery Status
There were no significant differences in gestation period between the control group and the 30, 100 and 300 mg/kg groups. In the 1000 mg/kg group, the gestation period was significantly longer than that of the control group. There were no abnormalities in the delivery status of any mother animals in the control group, 30, 100, 300 and 1000 mg/kg groups.
4) Number of Corpus Luteum, Number of Implantation Scars and Implantation Rate
There were no significant differences in the number of corpus luteum, number of implantation scars and implantation rate between the control group and the 30, 100 and 300 mg/kg groups. In the 1000 mg/kg group, significantly lower values were noted in the number of corpus luteum and number of implantation scars when compared to the control group.
5) Birth Rate and Lactation Status
The birth rate in the control group, and the 30, 100 and 300 mg/kg groups was 100%. In the 1000 mg/kg group, two mother animals (No. 451 and 452) did not have pups so the birth rate was 77.8%. In the control, 30, 100 and 300 mg/kg groups, nothing abnormal was noted with the lactation status. In the 1000 mg/kg group, there was one case (No. 454) with insufficient mammary gland growth and three cases (No. 453, 454 and 461) where all of the pups perished during lactation.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Sex:
male
Basis for effect level:
other: highest dose tested; no effect on copulation and fertility
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Sex:
female
Basis for effect level:
other: low values of the number of corpora lutea and the number of implantation sites at 1000 mg/kg
Clinical signs:
not examined
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

1) Overall Birth Rate and Delivery Rate
There were no significant differences in overall birth rate and delivery rate between the control group and the 30, 100 and 300 mg/kg groups. In the 1000 mg/kg group, significantly lower values were noted in the overall birth rate when compared to the control group.

2) Birth Rate, Survival Rate, Sex Ratio
In the 30 and 100 mg/kg groups, there were no significant differences for survival rate, number of new pups at day 0 of lactation, number of stillbirths, birth rate and sex ratio when compared to the control group. In the 300 mg/kg group, the number of new pups at day 0 of lactation had significantly lower values when compared to the control group. In the 1000 mg/kg group, the survival rate, number of new pups at day 0 of lactation and birth rate had significantly lower values when compared to the control group and significantly higher values for the number of stillbirths.

3) General Status of Pups, Number of Surviving Pups at Day 4 of Lactation, Survival Rate at Day 4 of Lactation and Observation of External Abnormalities
In the 30, 100 and 300 mg/kg groups, there were no significant differences for the number of new pups at day 4 of lactation, and sex ratio when compared to the control group. In the 1000 mg/kg group, the number of new pups at day 4 of lactation and survival rate at day 4 of lactation had significantly lower values when compared to the control group.
For observation of external abnormalities of pups, nothing abnormal was noted for the surviving pups in any of the groups. For stillbirths, there was one case of a short tail in the 30 mg/kg group, one case of a missing tail in the 1000 mg/kg group but both were deemed accidental.
For the general status of pups, there was one case of a damaged tail in the 30 mg/kg group but it was deemed accidental.

4) Body Weight of Pups
In the 30, 100 and 300 mg/kg groups, there were no significant differences for the average by gender, average stomach and total stomach weight at day 0 and day 4 of lactation when compared to the control group. In the 1000 mg/kg group, significantly low values were noted for the average by gender, average stomach and total stomach weight at day 0 of lactation, and significantly low values for total stomach weight were noted at day 4 of lactation.

5) Necropsy Findings of Pups
In the 30 mg/kg group, there was one case each of bilateral renal pelvis dilatation and damaged tail but both were deemed to be accidental. Otherwise, nothing abnormal was noted in any of the groups.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
body weight and weight gain
organ weights and organ / body weight ratios
Remarks on result:
other:
Remarks:
There are two elements which indicate that the lower number of F1 at day 0 of lactation are biologically not significant: Firstly, the difference is transient and not statistically significant by day 4 of lactation (due to reduced survival of neonates in the control group) and secondly, the observed mean number of neonates (13.4) is within the range of newborn F1 in the controls of other 422 studies in the same laboratory (13.1 – 15.2, n=10).
Critical effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
gastrointestinal tract
Organ:
stomach
Reproductive effects observed:
not specified

(1) Number of estruses, copulation index and fertility index

------------------------------------------------------------
Dose level (mg/kg/day)
      0         30        100         300         1000
No. of females
     12         12         12          12           12
 No. of estrous cases before mating (Mean± SD)
   3.3±0.5    3.3±0.9     3.6±0.5     3.4±0.5      2.4±0.8*
Significantly different from control (*: p < 0.05)
------------------------------------------------------------



(2) Body weight of neonates 
------------------------------------------------------------
Dose level (mg/kg/day)
    0         30        100         300         1000
 Body weight of pups (g) (Mean± SD)
  Male  Day 0
 6.59±0.59  6.80±0.66  6.37±0.47  6.70±0.34   5.62±0.60** (6)
  Male  Day 4
 9.94±1.11 10.73±1.26 10.247±1.01  10.34±0.70   8.83±0.91 (4)
  Female  Day 0
 6.18±0.52  6.43±0.65  6.14±0.42   6.25±0.29   5.37±0.56**(7)
  Female  Day 4
 9.43±1.17 10.15±1.01  9.857±0.99   9.83±0.80   8.40±1.31 (4)
Significantly different from control (**: p < 0.01)
------------------------------------------------------------


Conclusions:
The reproductive and developmental toxicological NOAEL is considered as 1000 mg/kg/day for males since there was no effect on copulation and fertility; 300 mg/kg/day for females since low values of the number of corpora lutea and the number of implantation sites were observed after administration at a dose of 1000 mg/kg.
Executive summary:

In an OECD Guideline 422 and GLP study, 2 -ethylhexyl methacrylate (EHMA) in corn oil was administered by oral gavage to 10 male and 10 female rats at 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. Only one high-dose female rat died during the study. Treatment-related decreases in body weight and food consumption were observed in high dose animals only. Relative to reproductive parameters, treatment-related effects observed primarily at 1000 mg/kg/day included: significantly low number of estrus cycles, prolonged gestation period, decreased number of corpora lutea and implantation sites, and decreased parturition index [77.8%]. Maldevelopment of the mammary gland was observed in one animal, and three dams’ neonates all died during the lactation period in the 1000 mg/kg/day dose group. There was a significantly low number of total offspring in the high dose group compared to controls. In the 300 mg/kg/day dose group, there was a significantly low number of neonates on Day 0 of lactation compared with the control group. At 1000 mg/kg/day, there were significantly low body weights of male and female neonates on Day 0 of lactation compared to controls. However, no gross abnormalities were observed in neonates at any dose level. Based on effects observed in parental females in the 1000 mg/kg/day dose group (i.e. decreases in the number of corpora lutea and the number of implantation sites), the NOAEL for reproductive toxicity is considered to be 300 mg/kg/day EHMA. There are two elements which indicate that the lower number of F1 at day 0 of lactation are biologically not significant: Firstly, the difference is transient and not statistically significant by day 4 of lactation (due to reduced survival of neonates in the control group) and secondly, the observed mean number of neonates (13.4) is within the range of newborn F1 in the controls of other 422 studies in the same laboratory (13.1 – 15.2, n=10).

Endpoint:
two-generation reproductive toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP
Justification for type of information:
Read across from the methacrylic metabolite donor substance.
REPORTING FORMAT FOR THE ANALOGUE APPROACH
see attached category document RE_PM-Lower Alkyl (C1-C8) Methacrylates

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
see attached category document

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see attached category document

3. ANALOGUE APPROACH JUSTIFICATION
see attached category document, chapter 5 (Toxikokinetics) and endpoint specific chapters

4. DATA MATRIX
see attached category document, tables in endpoint specific chapters

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)
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
Remarks:
Doses / Concentrations:
0, 50, 150, 450 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
F0 generation parental animals: 25
F1 generation parental aniumals: 25
Control animals:
yes, concurrent vehicle
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.
Test group 03 (400 mg/kg bw/d):
P PARENTAL ANIMALS:
CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/ PATHOLOGY
- 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%)

F1 PARENTAL ANIMALS:
CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY PATHOLOGY
- 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 17%)

Test group 02 (150 mg/kg bw/d):
P PARENTAL ANIMALS:
CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY PATHOLOGY
- 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%)

F1 PARENTAL ANIMALS:
CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY PATHOLOGY
- no test substance-related adverse effects/findings

Test group 01 (50 mg/kg bw/d):
P PARENTAL ANIMALS:
CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY PATHOLOGY
- no test substance-related adverse effects/findings

F1 PARENTAL ANIMALS:
CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY PATHOLOGY
- no test substance-related adverse effects/findings
Dose descriptor:
NOAEL
Remarks:
general, systemic toxicity
Effect level:
50 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: adverse effects on food consumption observed at the LOAEL of 150 mg/kg bw/day in the F0 parental females
Remarks on result:
other: Generation: P and F1 parental animals
Remarks:
Read across to Methyl methacrylate (CAS: 80-62-6) donor substance of the methacrylic moiety.
Dose descriptor:
NOAEL
Remarks:
fertility and reproductive performance
Effect level:
400 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: highest dose tested
Remarks on result:
other: Generation: P and F1 parental animals
Remarks:
Read across to Methyl methacrylate (CAS: 80-62-6) donor substance of the methacrylic moiety.
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
400 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: highest dose tested
Remarks on result:
other: Generation: F1 and F2 progeny
Remarks:
Read across to Methyl methacrylate (CAS: 80-62-6) donor substance of the methacrylic moiety.
Test group 03 (400 mg/kg bw/d):
F1 PUPS:
CLINICAL EXAMINATIONS/ SEXUAL MATURATION/ PUP ORGAN WEIGHTS/ GROSS FINDINGS
- no test substance-related adverse effects/findings

F2 PUPS:
CLINICAL EXAMINATIONS/ SEXUAL MATURATION/ PUP ORGAN WEIGHTS/ GROSS FINDINGS
- no test substance-related adverse effects/findings

Test group 02 (150 mg/kg bw/d):
F1 PUPS:
CLINICAL EXAMINATIONS/ SEXUAL MATURATION/ PUP ORGAN WEIGHTS/ GROSS FINDINGS
- no test substance-related adverse effects/findings

F2 PUPS:
CLINICAL EXAMINATIONS/ SEXUAL MATURATION/ PUP ORGAN WEIGHTS/ GROSS FINDINGS
- no test substance-related adverse effects/findings

Test group 01 (50 mg/kg bw/d):
F1 PUPS:
CLINICAL EXAMINATIONS/ SEXUAL MATURATION/ PUP ORGAN WEIGHTS/ GROSS FINDINGS
- no test substance-related adverse effects/findings

F2 PUPS:
CLINICAL EXAMINATIONS/ SEXUAL MATURATION/ PUP ORGAN WEIGHTS/ GROSS FINDINGS
- no test substance-related adverse effects/findings
Dose descriptor:
NOEL
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: Read across to Methyl methacrylate (CAS: 80-62-6) donor substance of the methacrylic moiety.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
400 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
Remarks on result:
other: Read across to Methyl methacrylate (CAS: 80-62-6) donor substance of the methacrylic moiety.
Reproductive effects observed:
not specified

- 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:
Read across to Methyl methacrylate (CAS: 80-62-6) donor substance of the methacrylic moiety:

The NOAEL for general, systemic toxicity is 50 mg/kg bw/day for the P and F1 parental rats, based on adverse 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:

Read across to Methyl methacrylate (CAS: 80-62-6) donor substance of the methacrylic moiety:

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: The NOAEL for general, systemic toxicity is 50 mg/kg bw/day for the P and F1 parental rats, based on adverse 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.

Effect on fertility: via oral route
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Additional information

Non-human data of EHMA

In an OECD Guideline 422 and GLP study, 2-ethylhexyl methacrylate (2-EHMA) in corn oil was administered by oral gavage to 10 male and 10 female rats at 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 (Furuhashi et al., 1998). One high-dose female rat died during the study. Treatment-related decreases in body weight and food consumption were observed in high dose animals only. Relative to reproductive parameters, treatment-related effects observed primarily at 1000 mg/kg/day included: significantly low number of oestrus cycles, prolonged gestation period, decreased number of corpora lutea and implantation sites, and decreased parturition index [77.8%]. Based on effects observed in parental females in the 1000 mg/kg/day dose group (i. e. decreases in the number of corpora lutea and the number of implantation sites), the NOAEL for reproductive toxicity (fertility) is considered to be 300 mg/kg/day 2-EHMA.

Metabolite data

Clearance of 2-EHMA as parent ester from the body by hydrolysis is in the order of minutes. For 2-EHMA the half-life is 23.8 minutes and 99.9 % was removed by first-pass metabolism in the liver and hydrolysed to Methacrylic acid (MAA) and 2-Ethylhexanol so the parent ester will not have a significant presence in the body, particularly after oral dosing (see chapter Toxicokinetics or Category document chapter 5). Thus, scenario 3 according to RAAF is considered as most relevant for this endpoint. A reliable 2-generation study from Methyl methacrylate (which is also rapidly metabolized to MAA thereby acting as a donor substance) and reliable information on Ethylhexanol are considered to provide a robust basis for the assessment of reproductive toxicity of 2 -EHMA with a high level of certainty.

Methyl methacrylate (MMA, donor substance of the methacrylic metabolite methacrylic acid)

MMA has been tested in a two-generation reproduction toxicity study in rats (OECD TG 416). Methyl methacrylate has been tested in a reliable two-generation reproduction toxicity study in rats with oral administration (gavage). The study was performed according to OECD TG 416 in compliance with GLP (BASF SE, 2009). In this study, 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 adversely 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 histopathological 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 histopathological 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. Oestrous cycle data, mating behaviour, 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-/foetal 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).

The NOAEL for general, systemic toxicity was determined to be 50 mg/kg bw/day for the P and F1 parental rats, based on adverse 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 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.

The 2-generation study with MMA provides confidence that the absence of effects seen at 300 mg/kg/d in the screening study with 2-EHMA are a reliable indication for an absence of fertility effects at this concentration in a higher tier study such as a 2 generation study. For the purpose of the risk assessment, a NOAEL for reproductive toxicity (fertility) of 300 mg/kg/d is taken forward for 2-EHMA.

2-ethylhexanol (alcohol metabolite)

For 2-ethylhexanol a SCOEL review (2011) concluded that no developmental effects are to be expected at non-irritating concentrations and that embryotoxic, foetotoxic and teratogenic effects would only occur at high doses that were maternally toxic - an observation also made with 2-ethylhexanoic acid (EHA), the main metabolite of EH.

Human data

There are no reliable data available.

Based on these data, 2-ethylhexyl methacrylate is not expected to be a reproductive toxicant.

References

SCOEL, 2011. Recommendation from the Scientific Committee on Occupational Exposure Limits for 2-ethylhexanol. SCOEL/SUM/158, March 2011

Effects on developmental toxicity

Description of key information

OECD 414, oral, rabbit: NOAEL maternal toxicity 100 mg/kg/day; NOAEL developmental toxicity 300 mg/kg/day

Metabolite data

MMA (donor substance of the methacrylic moiety), OECD 414, inhal, rat: NOAEC developmental 2028 ppm (8436 mg/m³).

MMA (donor substance of the methacrylic moiety), OECD 414, oral, rabbit: NOAEL developmental 450 mg/kg bw/d

Based on these data, 2-ethylhexyl methacrylate is not expected to be a developmental toxicant.

Link to relevant study records

Referenceopen allclose all

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
Remarks:
GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
22 January 2001
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
Batch #: 2027013127
Purity: 99.1%
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River France
- Age at study initiation: 15 to 16 weeks
- Weight at study initiation: 2.5 kg body weight, with a range between 2.6 and 4.12 kg (females)
- Housing: animals were housed in couple at delivery and individually from the allocation in polycarbonate/stainless steel cages with perforated NorylTM floor suspended over trays.
- Diet (e.g. ad libitum): Mucedola 2 RB 15, Mucedola S.r.l., Via G. Galilei 4, 20019 SettimoMilanese (MI), Italy, ad lib.
- Water (e.g. ad libitum): drinking water ad lib.
- Acclimation period: 34 days before the start of pairing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19°C ± 2°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): yes, but not defined
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1% Carboxymethylcellulose (medium viscosity) in softened water, 0,014% Kolliphor EL and 0.0035% Hydrochloric Acid (concentrated at 37%).
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of the test item was suspended in the vehicle. The formulations (concentrations of 3, 10 and 30mg/mL) were prepared daily for up to 7 days. Concentrations were calculated and expressed in terms of test item as supplied.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis was performed in a separate study in order to validate the analytical method, the formulation procedure and to verify the stability of the formulations (RTC Study No. A2652).
A 28 hour stability at room temperature and an 8 day stability at +5°C ± 3°C were verified in the range from 3 to 30 mg/mL.
Samples of the formulations prepared during the current study (on the first and the last week of treatment) were analysed to check the homogeneity and concentration. The results of the analyses were within the acceptability limits stated in RTC SOPs for suspensions (85-115% for concentration and CV’s ¡ 10% for homogeneity).
Chemical analysis was carried out by the Analytical Chemistry Department at RTC. The software used for this activity was Empower® 2 Build No. 2154.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1
- Length of cohabitation: Females were introduced to sexually mature males obtained from the same supplier. Each female remained with the male for at least 1 hour after successful mating had been observed.
- Proof of pregnancy: Each female received 50 I.U. of luteinizing hormone in a marginal ear vein upon completion of the mating procedure. The day successful mating was detected was considered Day 0 post coitum (or gestation Day 0).
Duration of treatment / exposure:
All females were be treated once a day from Day 6 through Day 28 post coitum. Dose volumes were calculated according to individual body weights on Days 6, 9, 12, 15, 18, 21, 23 and 26 post coitum.
Frequency of treatment:
daily
Duration of test:
until Day 29 post coitum
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels have been selected in consultation with the Sponsor based on the outcomes from the preliminary maternal toxicity study in rabbits, RTC no. Y0150.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holiday a similar procedure was followed except that the final check was carried out at approximately mid-day. This allowed post mortem examinations to be carried out during the working period of that day.


DETAILED CLINICAL OBSERVATIONS: Yes
- All clinical signs were recorded for individual animals. Each animal was observed at least once daily and any clinical signs recorded starting from allocation until sacrifice.

BODY WEIGHT: Yes
- Each animal was weighed on the day of allocation to treatment group (Day 0 post coitum) and on Days 3, 6, 9, 12, 15, 18, 21, 23, 26 and 29 post coitum.

FOOD CONSUMPTION: Yes
- Food consumption was measured on Days 3, 6, 9, 12, 15, 18, 21, 23, 26 and 29 post coitum starting from Day 0 post coitum.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 29
- Organs examined: stomach (weight & histopathology)
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:
• number, sex and weight of all live foetuses;
• number and sex of dead foetuses (foetuses at term without spontaneous
movements and breathing);
• number of intra-uterine deaths;
• gross evaluation of placentae.
Fetal examinations:
- External examinations: Yes: [all per litter ]
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: Yes: [half of litter ] - External examinations: Yes, all per litter
- Soft tissue examinations: Yes, all per litter
- Skeletal examinations: Yes, all per litter
- Head examinations: Yes: head section: all per litter/ fixed in Bouin's solution for internal structur examination: half of the litter
Statistics:
For continuous variables the significance of the differences amongst group means will be assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
Statistical analysis of non-continuous variables will be carried out by means of the Kruskal-Wallis test and intergroup differences between the control and treated groups assessed by a non-parametric version of the Williams test.
Indices:
Pre-implantation loss; post-implantation loss; total implantation loss; sex ratios of the foetuses; number of foetuses affected with structural deviations and the corresponding litter percentage.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The most recurrent findings were reduced and/or soft faeces observed in all groups but with a higher incidence in the high dose group (13 females affected out of 25).
Red staining and foetuses on the cage tray were also recorded in females which aborted. No other clinical signs were observed in the low dose group.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Abortion towards the end of the study occurred in two females at dose level of 100 mg/kg (gd 26/27) and in one female at dose level of 300 mg/kg/day (gd 28), therefore these females were sacrificed before the scheduled necropsy.
In absence of a clear dose dependent pattern and due to isolated occurrence, the cause of abortion could not be fully attributed to the treatment with the test item.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No differences in body weight were recorded between groups through the entire duration of the study. Mean body weight gain was comparable between the control and treated groups. The lower mean body weight gain which occurred at dose of 300 mg/kg/day was considered to be a transient fluctuation.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 300 mg/kg/day, a statistically significant reduction in food consumption was recorded on Days 21, 23 and 26 post coitum (ca. -20%). This was considered to be due to the treatment with the test item. At 30 and 100 mg/kg/day, no effects were observed in mean food consumption.
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 examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No changes were observed in absolute and relative weight of the stomach of treated females, when compared to the control group.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Unscheduled deaths
Three females aborted and were sacrificed before the scheduled necropsy. One female at 300mg/kg/dayand two females at dose of 100mg/kg/day were sacrificed for humane reasons on Days 28, 27 and 26 of the gestation period, respectively. At post mortem examination, red staining of the urogenital region was noted in the high dose group female, whereas red area of the fundic mucosa of the stomach was observed in the high dose female and in one of the two females of the mid-dose group. No abnormalities were detected in the remaining female of the mid-dose group.

Final sacrifice
A slightly increased incidence of red discoloration of fundic mucosa of the stomach was noted at dose of 300mg/kg/day, when compared to the controls (5 versus 1). One female within the same dose level was noted to have uterus with unilateral implantation. No other abnormalities were recorded.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes were noted in the stomach of the selected high dose females, when compared to the selected control animals.
Histopathological findings: neoplastic:
no effects observed
Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
Abortion towards the end of the study occurred in two females at dose level of 100 mg/kg (gd 26/27) and in one female at dose level of 300 mg/kg/day (gd 28). In absence of a clear dose dependent pattern and due to isolated occurrence, the cause of abortion could not be fully attributed to the treatment with the test item.
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
At 300 mg/kg/day, the number of females with higher incidence of post-implantation loss (> 5%) was slighlty higher compared to the control group (9 versus 4). Although not statistically significant, this effect was likely due to the treatment with the test item. The effect was however not statistically significant, and considering the mean number of vialbe foetuses which was not affected, it is concluded it was not adverse.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
gross pathology
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
The incidence of small foetuses (body weight below 35 g) was similar between groups. No effects were noted in sex ratios and foetal weights.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
At dose level of 300 mg/kg/day, higher incidence of post-implantation loss was observed, when compared to the control group. The effect was however not statistically significant, and considering the mean number of vialbe foetuses which was not affected, it is concluded it was not adverse.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No effects were noted in sex ratios.
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No anomalies were detected in all groups.
Skeletal malformations:
no effects observed
Description (incidence and severity):
During the skeletal examination, absence of articulation point on the pelvic girdle was observed in the control and all test item treated groups. The incidence of this malformation among the groups did not give any indication of a test item related effect. One foetus at dose level of 300 mg/kg/day was noted to have a malrotated hindpaw. Due to the isolated occurrence, this was considered to be incidental.
The incidence of unossified or incomplete ossified bones such as humerus head, trochlea, proximal or distal phalanx, tibia head, hyoid body and sternebrae was similar between the control and treated groups.
No differences occurred in the incidence of bilateral insertion on the 2nd sacral vertebrae, in the number of supernumerary ribs or in the presence of fixure on the skull.
Enlarged ventricles of the brain, slight to moderate, were recorded in all groups but without giving indication of a test item-related effect. No other variants or abnormalities were detected.
Visceral malformations:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Enlarged ventricles of the brain, slight to moderate, were recorded in all groups but without giving indication of a test item-related effect. No other variants or abnormalities were detected.
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: No relevant developmental toxicity was observed at any tested dose.
Abnormalities:
no effects observed
Developmental effects observed:
no
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)

BODY WEIGHT (kg) OF PREGNANT FEMALES – GROUP MEAN DATA
    Day of Phase                    
Group(s)   0! 3 6” 9 12 15 18 21 23 26 29
1 (n) 25 25 25 25 25 25 25 25 25 25 25
  Mean 3.901 4.008 4.053 4.082 4.132 4.184 4.177 4.198 4.216 4.238 4.274
  SD 0.338 0.340 0.328 0.336 0.335 0.332 0.360 0.347 0.351 0.332 0.327
2 (n) 25 25 25 25 25 25 25 25 25 25 25
  Mean 3.993 4.087 4.174 4.222 4.260 4.316 4.336 4.339 4.338 4.372 4.408
  SD 0.340 0.334 0.351 0.341 0.341 0.333 0.317 0.323 0.325 0.322 0.300
3 (n) 25 25 25 25 25 25 25 25 25 24 23
  Mean 3.989 4.082 4.137 4.181 4.234 4.284 4.305 4.327 4.342 4.375 4.370
  SD 0.395 0.378 0.399 0.409 0.413 0.409 0.416 0.421 0.423 0.364 0.295
4 (n) 24 24 24 24 24 24 24 24 24 24 23
  Mean 3.953 4.078 4.124 4.130 4.152 4.184 4.121 4.113 4.143 4.154 4.166
  SD 0.402 0.415 0.400 0.422 0.415 0.404 0.429 0.418 0.427 0.420 0.445

* = mean value of group is significantly different from control at p < 0.05

** = mean value of group is significantly different from control at p < 0.01

Statistical analysis: Dunnett`s test if group variances are homogeneous

Modified t test if group variances are inhomogeneous ($)

TERMINAL BODY WEIGHT, UTERUS WEIGHT AND ABSOLUTE WEIGHT GAIN (g) OF FEMALES WITH LIVE FOETUSES - GROUP MEAN DATA
Group(s)   Terminal body weight (kg) Gravid uterus weight (g) Absolute weight gain (g)
1 Mean 4.27 490.15 -125.88
  SD 0.32 104.77 288.29
  N 25 25 25
2 Mean 4.40 487.73 -81.04
  SD 0.30 101.87 229.15
  N 25 25 25
3 Mean 4.36 454.85 -38.96
  SD 0.30 93.87 171.98
  N 23 23 23
4 Mean 4.17 459.01 -206.62
  SD 0.44 141.66 266.18
  N 23 23 23

# = Body weight at necropsy minus gravid uterine wt., minus body wt. at Day 0 of pregnancy* Statistically significant different from control group value at p < 0.05

LITTER DATA AND SEX RATIOS – GROUP MEAN DATA
    Corpora Lutea Implan-tations Uterine deaths Viable young Implantation loss (%) Litter weight (g) Mean foetal weight (g)
Group(s)   Early Late Total Total male female % male Pre Post Total
1 Mean 9.16 8.92 0.40 0.08 0.48 8.44 3.88 4.56 46.77 2.69 4.28 6.97 330.2 39.81
  SD 2.49 2.58 1.19 0.28 1.36 2.36 1.42 1.80 15.01 6.74 11.85 12.72 85.88 5.50
  N 25 25 25 25 25 25 25 25 25 25 25 25 25 25
2 Mean 8.52 8.28 0.16 0.04 0.20 8.08 4.48 3.60 54.79 3.14 2.39 5.53 328.3 41.04
  SD 1.64 1.84 0.47 0.20 0.65 1.93 1.69 1.19 13.06 6.77 7.93 9.65 70.39 4.07
  N 25 25 25 25 25 25 25 25 25 25 25 25 25 25
3 Mean 7.61 7.39 0.22 0.00 0.22 7.17 3.22 3.96 46.57 2.23 2.13 4.30 306.2 43.41
  SD 2.17 1.99 0.85 0.00 0.85 1.83 1.17 1.64 18.52 5.29 7.91 9.53 72.24 5.26
  N 23 23 23 23 23 23 23 23 23 23 23 23 23 23
4 Mean 8.43 8.39 0.52 0.09 0.61 7.78 4.04 3.74 47.78 0.62 6.45 7.06 292.3 38.64
  SD 2.98 3.01 1.08 0.42 1.12 2.88 2.31 1.39 20.55 2.98 10.25 10.26 107.4 7.11
  N 23 23 23 23 23 23 23 23 23 23 23 23 23 23

* Statistically significant different from control group value at p < 0.05

EXTERNAL AND INTERNAL EXAMINATION IN FOETUSES - GROUP INCIDENCE
        No. Foetuses No. Litters
Group(s) Organ Cat Observation(s) Observed Affected % Observed Affected %
1 Whole foetus   No abnormalities detected 211 167 79.15 - - -
  Whole foetus AN Small 211 44 20.85 25 12 48.00
2 Whole foetus   No abnormalities detected 202 175 86.63 - - -
  Whole foetus AN Small 202 27 13.37 25 13 52.00
3 Whole foetus   No abnormalities detected 165 150 90.91 - - -
  Whole foetus AN Small 165 15 9.09 23 6 26.09
4 Whole foetus   No abnormalities detected 179 120 67.04 - - -
  Whole foetus AN Small 179 59 32.96 23 11 47.83

Each foetus may have more than one finding

Conclusions:
Based on the outcome of this study, the NOAEL (No Observed Adverse Effect Level) for maternal and developmental toxicity of orally administered EHMA to pregnant rabbits was established at 100 mg/kg/day.
Executive summary:

The aim of this study was to investigate the toxicity of EHMA in New Zealand rabbits during pregnancy and embryo-foetal development in a GLP conform study accordingto OECD 414. The test item was administered daily by oral gavage at the following dose levels: 30, 100 and 300mg/kg/day from Day 6 through Day 28 post coitum at constant volume of 10mL/kg body weight. Control animals received the vehicle alone (CMC l% in softened water, 0.014% Kolliphor EL and 0.0035% Hydrochloric Acid 37%).

The following results were obtained:

Maternal toxicity

Abortion towards the end of the study occurred in two females at dose level of 100mg/kg and in one female at dose level of 300mg/kg/day, therefore these females were sacrificed before the scheduled necropsy. Reduced body weight gain was observed in all three females before the abortions. Due to the single occurrences, it could not be clearly established if the abortions were incidental or due to the treatment with the test item. With the exception of one female at 300mg/kg, all the others achieved pregnancy. During the treatment period, soft and/or reduced faeces were noted in all groups but the

number of females affected was slightly higher in the high dose group. Treatment with EHMA at 300mg/kg/day caused a statistically significant reduction in food consumption over the last week of the study. Although the statistical analysis did not reveal any differences, mean absolute weight gain at 300mg/kg/day was lower when compared to the control group. No effects were observed in mean body weight gain and terminal body weight.

At necropsy, a slightly increased incidene of red discolored fundic mucosa of the stomach was observed at dose level of 300mg/kg/day. The histopathological examination of the stomach in the control and high dose groups did not indicate any treatment related changes. The absolute and relative weights of the stomach were similar between the groups.

Developmental toxicity

At dose level of 300 mg/kg/day, higher incidence of post-implantation loss was observed, when compared tot he control group. The number of small foetuses was similar in all groups and no effects were noted in sex ratios and foetal weights. No abnormalities were detected at macroscopic examination of foetuses. The examination of fixed head examination did not indicate any malformations, the incidence of variations or anomalies was similar in all groups. The skeletal examination of foetuses did not reveal any test item related effect.

In conclusion, maternal toxicity was observed at dose level of 300mg/kg/day in terms of reduced food consumption, absolute weight gain, clinical signs and macroscopic findings. Developmental toxicity was also noted at dose level of 300 mg/kg/day, due to a slightly higher number of females with post-implantation loss. The effect was however not statistically significant, and considering the mean number of viable foetuses which was not affected, it is concluded that it was not adverse.

Based on the outcome of this study, the NOAEL (No Observed Adverse Effect Level) for maternal toxicity of orally administered EHMA to pregnant rabbits was established at 100 mg/kg/day and for developmental toxicity at 300 mg/kg/day.

Endpoint:
developmental toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Justification for type of information:
Read across from the methacrylic metabolite donor substance.
REPORTING FORMAT FOR THE ANALOGUE APPROACH
see attached category document RE_PM-Lower Alkyl (C1-C8) Methacrylates

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
see attached category document

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see attached category document

3. ANALOGUE APPROACH JUSTIFICATION
see attached category document, chapter 5 (Toxikokinetics) and endpoint specific chapters

4. DATA MATRIX
see attached category document, tables in endpoint specific chapters
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CDBR
Details on test animals or test system and environmental conditions:
Nulliparous female rats, weighing 183-240 grams upon arrival.
AGE AT TIME OF MATING: 88-95 days. 
ACCLIMATION PRIOR TO MATING: 7 days 
SOURCE: Charles River Breeding Laboratories Inc., Kingston, NY.
Animals were housed individually, except during mating, in suspended stainless-steel cages (7" x 8" x 13.5"). During exposures, females were housed individually in suspended stainless-steel, wire mesh cages (6" x 7" x 11"). Temperature range was 23 ± 2°C and the relative humidity ranged from 40-60% during cohabitation and 63-80% during the exposure and post-exposure periods. Food (Certified Purina Rodent Chow #5002) and filtered tap water were available ad libitum except during exposures. A photoperiod of 12 hrs dark/ 12 hrs light was maintained.
Route of administration:
inhalation
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
The test material exposure concentrations were generated by metering the test material with calibrated Fluid Metering Pumps (Fluid Matering Inc., Oyster Bay, NY) into 500 mL three-necked round bottom flasks (Lab Glass Inc., Vineland, NJ).
Exposures were whole body and were conducted in 2000 L stainless steel, glass and Plexiglas® chambers. Cage positions within the chamber were rotated daily. The temperature and relative humidity within the chambers during exposure were 22-24°C and 55-67%, respective.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the test substance in the chambers was determined by the use of a Miran gas analyzer attached to a strip chart recorder. A probe was placed into the center of the chamber and the chamber atmosphere was drawn into the Miran A1 gas analyzer at a rate of 9.5 L/min. Each chamber was analyzed initially within 40 min. of the end of the t99 to insure that each chamber was within the accepted target range. Subsequently, each chamber was sampled every 120 min. A range of  plus or minus 10% of the target chamber concentration was maintained by making minor adjustments on the generator pump delivery rates whenever necessary.
Details on mating procedure:
Females were mated with males overnight (one male:one female) and the presence of sperm in the vaginal smear was considered  gestation day 0. Mated females were exposed via inhalation to the test material for 6 hrs/day on gestation days 6 through 15 and then sacrificed on day 20.  
Duration of treatment / exposure:
6 - 15 day of gestation
Frequency of treatment:
6 hours/day
Duration of test:
20 d (dams were euthanized on gestation day 20)
Dose / conc.:
99 ppm
Remarks:
corresponding to 412 mg/m3 or 0.41 mg/L
Dose / conc.:
304 ppm
Remarks:
corresponding to1285 mg/m3 or 1.29 mg/L
Dose / conc.:
1 178 ppm
Remarks:
corresponding to 4900 mg/m3 or 4.9 mg/L
Dose / conc.:
2 028 ppm
Remarks:
corresponding to 8436 mg/m3 or 8.44 mg/L
No. of animals per sex per dose:
27 animals per group exposed; 22-25 pregnant  females per exposure group.
Control animals:
yes, sham-exposed
Details on study design:
- Other: The strain was selected because background development toxicity data exists as Rohm and Haas Company on this rat strain. The test material was given by inhalation since the respiratory route is a potential route of human exposure.
Maternal examinations:
Maternal body weights were recorded on GD 0, 6, 8, 10, 13, 16 and 20. Food consumption was measured for GD intervals 0-6, 6-10, 10-16 and 16-20. Animals were observed daily for behavioral changes.
Ovaries and uterine content:
On GD 20, all dams were asphyxiated with carbon dioxide, the thoracic and abdominal cavities were examined and the uterus was removed and weighed, and corpora lutea, implantation sites and resorptions were counted. The number of fetuses per litter was counted and position inside the uterus recorded. The uteri of apparently non pregnant rats were stained with a 10% ammonium sulfide solution to detect very early resorptions. All fetuses were weighed, examined for external alterations and the sex of each fetus was determined. 
Fetal examinations:
One half of the fetuses from each litter were examined for visceral alterations using the Staples' technique. Head alterations were recorded for these fetuses examined for soft tissue alterations using the technique of Barrow and Taylor (1969, J. Morphol., 127: 291-306). The carcasses of all fetuses  were stained with alizarin red S and examined for skeletal alterations. 
Statistics:
For the purpose of statistical evaluation, the litter was considered the experimental unit for fetal parameters. Pregnancy rate, clinical signs, maternal deaths, gross necropsy findings  and liters with total resorptions were statistically analyzed using the  Fisher's exact test. Maternal body weight data and feed consumption values were statistically analyzed using Dunnett's test when the one-way ANOVA was significant. The number of implantations, live fetuses, resorptions, corpora lutea, mean fetal body weight/litter, and incidence of fetal alterations were statistically analyzed using the Mann-Whitney U test. When more than 75% ties occurred, then Fisher's exact test was used in place of the Mann-Whitney U test to detect significant differences between groups.
Details on maternal toxic effects:
Details on maternal toxic effects:
No animals died and no treatment-related clinical signs were noted for the dams in the 99, 304 or 1178 ppm groups. Scant feces was noted in the 2028 ppm group throughout the exposure period (GD 6-15). Treatment-related decreases on maternal body weight and feed consumption were noted at all exposure levels. The decreases in maternal body weight at 99 and 304 ppm were minimal and transient since they occurred only during the first 2 days of exposure and returned to control values by the  next weighing period. The body weight and feed consumption values returned to control values for all groups during the post exposure period  (GD 16-20). At 1178 and 2028 ppm, treatment-related effects included losses in maternal body and/or decreased body weight gain throughout the exposure period (GD 6 - 16) and decreased corrected maternal body weight gain. The gross necropsy evaluations did not indicate any treatment-related effects and there were no treatment-related differences between the control and treated groups in any reproductive parameter.  
Dose descriptor:
LOEC
Remarks:
maternal toxicity
Effect level:
ca. 0.41 mg/L air (analytical)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Remarks on result:
other: Read across to Methyl methacrylate (CAS: 80-62-6) donor substance of the methacrylic moiety.
Dose descriptor:
NOAEC
Remarks:
maternal toxicity
Effect level:
8.44 mg/L air (analytical)
Based on:
test mat.
Remarks on result:
other: no adverse effects observed
Remarks:
Read across to Methyl methacrylate (CAS: 80-62-6) donor substance of the methacrylic moiety.
Abnormalities:
no effects observed
Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
 Fetal body weight was not affected by exposure to MMA vapors. The fetal external, visceral and skeletal examinations did not show any treatment related effects.
Dose descriptor:
NOAEC
Remarks:
fetotoxicity
Effect level:
8.44 mg/L air (analytical)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: corresponding to 2028 ppm; no substance related effects observed
Remarks:
Read across to Methyl methacrylate (CAS: 80-62-6) donor substance of the methacrylic moiety.
Dose descriptor:
NOAEC
Remarks:
teratogenicity
Effect level:
8.44 mg/L air (analytical)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: corresponding to 2028 ppm; no substance related effects observed
Remarks:
Read across to Methyl methacrylate (CAS: 80-62-6) donor substance of the methacrylic moiety.
Abnormalities:
no effects observed
Developmental effects observed:
no

Mean measured concentrations (± SD) within the chambers for the 0, 100, 300, 1200 and 2000 ppm groups were 98.8 (±3.4), 304.4 (±9.1), 1178.1  (±69.1) and 2028.2 (±107.3) ppm, respectively.

Endpoint:
developmental toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Read across from the methacrylic metabolite donor substance.
REPORTING FORMAT FOR THE ANALOGUE APPROACH
see attached category document RE_PM-Lower Alkyl (C1-C8) Methacrylates

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
see attached category document

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see attached category document

3. ANALOGUE APPROACH JUSTIFICATION
see attached category document, chapter 5 (Toxikokinetics) and endpoint specific chapters

4. DATA MATRIX
see attached category document, tables in endpoint specific chapters
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. QA statement)
Limit test:
no
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.
Details on maternal toxic effects:
Details on maternal toxic effects:
- Mortality: There were no test substance-related or spontaneous mortalities in any group.
- Clinical symptoms: No test substance-related clinical signs or any disturbances of the general behavior were observed in any rabbit during the entire study period.
- Food consumption: 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.
- Body weight data: 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.
- Uterus weight: The mean gravid uterus weights of test groups 1, 2, and 3 (50; 150 or 450 mg/kg bw/d) did not show statistically significant differences in comparison to the control group.
- Necropsy findings: At necropsy, only spontaneous findings were seen in single females of every test group. No test substance-related findings were observed in the does.
- Reproduction data of does: 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
Remarks:
Read across to Methyl methacrylate (CAS: 80-62-6) donor substance of the methacrylic moiety.
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
Remarks:
Read across to Methyl methacrylate (CAS: 80-62-6) donor substance of the methacrylic moiety.
Abnormalities:
effects observed, treatment-related
Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
- Sex distribution of fetuses: 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.
- Weight of placentae: The mean placental weights in test groups 1, 2 and 3 (50; 150 and 450 mg/kg bw/d) were comparable to the controls.
- Weight of fetuses: 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.
- Fetal external malformations: 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.
- Fetal external variations: One external variation (paw hyperflexion) occurred in single fetuses of the low- and mid-dose groups and the control. The incidences did not demonstrate a dose-response relationship and were comparable to the historical control data. Thus an association of this finding to the treatment is not assumed.
- Fetal external unclassified observations: Unclassified external observations, such as necrobiotic placentae and discolored amniotic fluid, were recorded for single fetuses of test groups 1 and 2 (50 and 150 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.
- 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.
- Fetal skeletal malformations: 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.
- 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.
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
Remarks:
Read across to Methyl methacrylate (CAS: 80-62-6) donor substance of the methacrylic moiety.
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:
Read across to Methyl methacrylate (CAS: 80-62-6) donor substance of the methacrylic moiety:

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:

Read across to Methyl methacrylate (CAS: 80-62-6) donor substance of the methacrylic moiety:

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.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Non-human data of EHMA

In a GLP study according to OECD 414, the toxicity of EHMA in New Zealand rabbits during pregnancy and embryo-foetal development was investigated. The test item was administered daily by oral gavage at the following dose levels: 30, 100 and 300mg/kg/day from Day 6 through Day 28 post coitum at constant volume of 10mL/kg body weight. Control animals received the vehicle alone (CMC l% in softened water, 0.014% Kolliphor EL and 0.0035% Hydrochloric Acid 37%).

The following results were obtained:

Maternal toxicity

Abortion towards the end of the study occurred in two females at dose level of 100mg/kg and in one female at dose level of 300mg/kg/day, therefore these females were sacrificed before the scheduled necropsy. Reduced body weight gain was observed in all three females before the abortions. Due to the single occurrences, it could not be clearly established if the abortions were incidental or due to the treatment with the test item. With the exception of one female at 300mg/kg, all the others achieved pregnancy. During the treatment period, soft and/or reduced faeces were noted in all groups but the number of females affected was slightly higher in the high dose group. Treatment with EHMA at 300mg/kg/day caused a statistically significant reduction in food consumption over the last week of the study. Although the statistical analysis did not reveal any differences, mean absolute weight gain at 300mg/kg/day was lower when compared to the control group. No effects were observed in mean body weight gain and terminal body weight.

At necropsy, a slightly increased incidene of red discolored fundic mucosa of the stomach was observed at dose level of 300mg/kg/day. The histopathological examination of the stomach in the control and high dose groups did not indicate any treatment related changes. The absolute and relative weights of the stomach were similar between the groups.

Developmental toxicity

At dose level of 300 mg/kg/day, higher incidence of post-implantation loss was observed, when compared tot he control group. The number of small foetuses was similar in all groups and no effects were noted in sex ratios and foetal weights. No abnormalities were detected at macroscopic examination of foetuses. The examination of fixed head examination did not indicate any malformations, the incidence of variations or anomalies was similar in all groups. The skeletal examination of foetuses did not reveal any test item related effect.

In conclusion, maternal toxicity was observed at dose level of 300mg/kg/day in terms of reduced food consumption, absolute weight gain, clinical signs and macroscopic findings. Developmental toxicity was also noted at dose level of 300 mg/kg/day, due to a slightly higher number of females with post-implantation loss. The effect was however not statistically significant, and considering the mean number of viable foetuses which was not affected, it is concluded that it was not adverse.

Based on the outcome of this study, the NOAEL (No Observed Adverse Effect Level) for maternal toxicity of orally administered EHMA to pregnant rabbits was established at 100 mg/kg/day and for developmental toxicity at 300 mg/kg/day.

Metabolite data

Clearance of 2-EHMA as parent ester from the body by hydrolysis is in the order of minutes. For 2-EHMA the half-life is 23.8 minutes and 99.9 % was removed by first-pass metabolism in the liver and hydrolysed to Methacrylic acid (MAA) and 2-Ethylhexanol so the parent ester will not have a significant presence in the body, particularly after oral dosing (see chapter Toxicokinetics or Category document chapter 5). Thus, scenario 3 according to RAAF is considered as most relevant for this endpoint.

For the evaluation of the developmental toxicity of EHMA in a rodent species, reliable data from its metabolites methacrylic acid (and its donor substance methyl methacrylate) and the alcohol metabolite2-ethylhexanol

are considered to provide a robust basis for the assessment of reproductive toxicity of 2 -EHMA with a high level of certainty.

 

MAA (methacrylic metabolite; including donor substance MMA)

OECD SIAR concluded that “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).” (study not included in this dataset)

 

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.

 

EH (alcohol metabolite)

For 2-ethylhexanol (EH) a recent SCOEL review (2011) concluded that based on studies in rats or mice exposed to concentrations of about 850 mg/m3 (160 ppm) or oral doses up to 1300 mg/kg per day that no developmental effects are to be expected at non-irritating concentrations. They added that that effects observed only at higher doses of EH which were toxic to the dams were comparable to those observed with 2-ethylhexanoic acid (EHA), the main metabolite of EH.

 

References

SCOEL, 2011. Recommendation from the Scientific Committee on Occupational Exposure Limits for 2-ethylhexanol. SCOEL/SUM/158, March 2011

 

Justification for classification or non-classification

Based on the available data, 2 -EHMA does not need to be classified for toxicity to reproduction, developmental toxicity and teratogenicity according to the criteria given in regulation (EC) 1272/2008 or the former European directive on classification and labelling 67/548/EEC. Thus, no labelling is required.

Additional information