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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
2-ethylhexyl methacrylate
EC Number:
211-708-6
EC Name:
2-ethylhexyl methacrylate
Cas Number:
688-84-6
Molecular formula:
C12H22O2
IUPAC Name:
2-ethylhexyl methacrylate
Test material form:
liquid
Specific details on test material used for the study:
Batch #: 2027013127
Purity: 99.1%

Test animals

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

Administration / exposure

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
Doses / concentrationsopen allclose all
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.

Examinations

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.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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

Maternal developmental toxicity

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

Effect levels (maternal animals)

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

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

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.

Effect levels (fetuses)

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.

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.