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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
Study period:
12 June 2012 to 04 August 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: compliance to GLP and testing guideline, coherence between data, results and conclusions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 422
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Name: Ethyltriglycol methacrylate

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
A total of 90 Sprague Dawley [Crl:CD(SD)BR] rats (45 males and 45 virgin females), 7 to 8 weeks old and weighing 218-230 g for males and 195-
200 g for females, were received from Charles River Italia S.p.A., Calco (Lecco), Italy.
After arrival the weight range for each sex was determined and the animals were temporarily identified within the cage by means of a coloured mark on the tail. A health check was then performed by a veterinarian.
An acclimatisation period of 14 days was allowed before the start of treatment, during which time the health status of the animals was assessed by
thorough observations.

The animals were housed in a limited access rodent facility. Animal room controls were set to maintain temperature and relative humidity at 22°C
+/- 2°C and 55% +/- 15% respectively; actual conditions were monitored, recorded and the records retained. No relevant deviations from these
ranges were recorded during the study. There were approximately 15 to 20 air changes per hour and the rooms were lit by artificial light for 12 hrs
each day.

In-life phase: 07 June 2012 to 04 August 2012

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
The test item was administered orally by gavage at a dose volume of 5 mL/kg body weight. Control animals received the vehicle alone at the
same dose volume.

The required amount of Ethyl triglycol methacrylate was suspended in the vehicle (corn oil) and brought to the final volume appropriate for each
concentration (concentrations of 20, 60 and 200 mg/mL) and kept under magnetic stirrer at room temperature prior to use and until the time of
dosing of the last animal.
The formulations were prepared daily and the 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:
Prior to commencement of treatment, analysis was performed to confirm that the proposed formulation procedure was acceptable (content check
and homogeneity). Results of the analyses were within the limits of acceptance.
The stability was found to be 24 hours at room temperature in the concentration range of 20 to 200 mg/mL.
Samples of the formulations prepared on Week 1 and last Week were also analysed to check the concentration and homogeneity. Results of the
analyses were within the limits of acceptance.
Chemical analysis was carried out by the Analytical Chemistry Department at RTC according to a validated method (RTC Study No. 90900), in the
range from 1 to 200 mg/mL. The software used for this activity was the Empower® Pro build No. 2154
Details on mating procedure:
Mating was monogamous (one male to one female). A vaginal smear was taken from the day after the start of pairing until positive identification of
copulation (sperm identification, vaginal plug in situ or copulation plugs found in the cage tray).
The female was paired with the same male until positive identification occurred or 14 days had elapsed.
Duration of treatment / exposure:
Males
Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter through the day before necropsy (Day 32 of the study).

Dose volumes were adjusted once per week for each animal according to the last recorded body weight.

Females
Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 3 post partum (the day before sacrifice).

Dose volumes were adjusted once per week for each animal according to the last recorded body weight.
During the post coitum period, dose volumes were calculated according to individual body weight on Days 0, 7, 14 and 20 post coitum and on Day 1 post partum. Thereafter individual dose volumes remained constant.
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
Dose levels had been selected in consultation with the Sponsor based on information from a previous non GLP Compliant study (2 week preliminary oral toxicity study in rats)

Examinations

Maternal examinations:
yes
Ovaries and uterine content:
yes
Fetal examinations:
yes
Statistics:
Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated
groups were assessed by the non-parametric version of the Williams test.
The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.

Results and discussion

Results: maternal animals

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
On the basis of the results obtained in this study with Ethytriglycol methacrylate, the NOAEL (No Observed Adverse Effect Level) for
developmental toxicity was found to be 300 mg/kg/day for males and females.
Executive summary:

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test according to OECD Guideline 422 (22 March 1996) Ethyltriglycol methacrylate was administered to 10 Hsd: Sprague Dawley SD rats/sex/dose orally by gavage at dose levels of 0 (control), 100, 300 and 1000 mg/kg bw/d. The treatment schedule included 2 weeks before pairing, during pairing, post coitum and post partum periods up to day 3 post partum. Animals were administered for approximately 5 and 8 weeks for males and females, respectively.

One male receiving 100 mg/kg/day of Ethyltriglycol methacrylate was found dead on Day 14 of the premating phase. The animal was found cannibalised on the day of death.

At macroscopic observation the found dead male showed: dark and/or red cervical lymph nodes, thymus, and thyroids; swollen lobe of the liver; dark lungs and incomplete collapse; thin stomach wall and a single abnormal area in the limiting ridge.

Microscopically the cervical lymph nodes, liver, lungs, thymus and thyroids were congested.

No cause of death could be identified in this animal.

One female receiving 1000 mg/kg/day was sacrificed for humane reasons with its litter on Day 0post partum. The female showed prolapse of the uterus and pale aspect as clinical signs.

Macroscopically, this animal showed: abnormal content (dark red) in the thoracic cavity, stomach, ileum and jejunum; pale liver and kidneys. The uterus was dark red, oedematous, with presence of prolapse.

Microscopically, changes were seen only in the uterus, consisting of presence of decidual reaction, associated with necrosis of the endometrial epithelium and presence of abnormal (i.e., plant-like material) material within the lumen.

The factors contributing to the illness status of this female were related to uterine necrosis associated with presence of plant-like material within the lumen. These changes were considered to be incidental and of no treatment related.

A total of 8 females were found not pregnant: 3 females in the mid-dose group (300 mg/kg/day) and 5 females in the high dose group (1000 mg/kg/day).

The number of females with live pups on Day 4post partum was: 10 in each of the control and low dose groups, 7 in the mid-dose group and 4 in the high dose group.

No relevant clinical signs were observed throughout the study in all treated animals of both sexes.

Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item.

No relevant differences in body weights were recorded in animals of both sexes compared to the control group, throughout the study.

Food consumption was, in general, comparable between control and treated groups.

Motor activity and sensory reaction to stimuli

No relevant differences were noted in all parameters investigated between control and treated groups.

Haematology

No changes of toxicological significance were recorded in animals sacrificed at the end of the study.

No change was recorded in the coagulation test.

No changes in clinical chemistry parameters were observed in treated animals when compared to the control group.

Urinalysis

No changes were observed between control and treated males.

Oestrus cycle, pre-coital intervals and copulatory index in males and females were similar in treated and control groups.

No copulation plugs were found in the cage tray of 9 out of 10 females receiving 1000 mg/kg/day. In addition low sperm was found in the vaginal smear of these females.

Fertility index of males and females receiving the dose levels ≥ 300 mg/kg/day was decreased compared to the control group.

Implantation, pre-implantation loss data, pre-birth loss data and gestation length of females

No significant differences were observed in treated and control groups for these parameters.

All pregnant females gave birth between Days 22 and 23post coitum.

Litter data parameters and sex ratios at birth and on Day 4post partum were similar between groups.

Clinical signs of pups were comparable between treated and control groups.

Necropsy findings in unscheduled dead pups and in pups sacrificed on Day 4post partumdid not reveal any treatment-related effect.

Terminal body weight was unaffected by treatment in both sexes.

No relevant differences were recorded in the absolute and relative organ weights of treated animals.

Macroscopic observations

No treatment-related changes were noted.

Microscopic observations

No treatment-related changes were noted.

In addition seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and no alterations were noted between control males and those receiving 1000 mg/kg/day.

Conclusions:

On the basis of the results obtained in this study, the NOAEL (No Observed Adverse Effect Level) for general toxicity was considered to be 1000 mg/kg/day for males and females.

The NOAEL for developmental toxicity was found to be 1000 mg/kg/day for males and females.