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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 April 2012-16 June 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted 22 March 1996
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-ethylenedioxydiethyl dimethacrylate
EC Number:
203-652-6
EC Name:
2,2'-ethylenedioxydiethyl dimethacrylate
Cas Number:
109-16-0
Molecular formula:
C14H22O6
IUPAC Name:
2-(2-{2-[(2-methylprop-2-enoyl)oxy]ethoxy}ethoxy)ethyl 2-methylprop-2-enoate
Test material form:
liquid
Specific details on test material used for the study:
Name: Triethyleneglycol dimethacrylate

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source:Hsd: Sprague Dawley SD rats from Harlan Italy s.r.l., San Pietro al Natisone (UD), Italy
- Age at study initiation: 7 to 8 weeks old and weighing 200-204g for males and 178-179g for females.
- Weight at study initiation: (P) Males: 297- 361g; Females:208-255g;
- Fasting period before study:no
- Housing:5sex/cage
- Diet (e.g. ad libitum):ad libitum throughout the study, except during clinical pathology investigations
- Water (e.g. ad libitum):d libitum throughout the study, except during clinical pathology investigations - Males only. Ad libitum for females
- Acclimation period:18 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22+/-2
- Humidity (%):55+/-15
- Air changes (per hr):There were approximately 15 to 20 air changes per hour
- Photoperiod (hrs dark / hrs light):rooms were lit by artificial light for 12 hours each day

IN-LIFE DATES: From: To:05 April 2012 to 16 June 2012

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The dosing formulations were prepared daily


VEHICLE
- Justification for use and choice of vehicle (if other than water):solubility of the test item
- Concentration in vehicle:of 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage):5 mL/kg
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).

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before the start of treatment, analysis was performed to confirm that the proposed formulation procedure was acceptable and that the stability of the formulation was satisfactory. Samples of the formulations prepared during the study were also analysed to check the homogeneity and concentration.
Chemical analysis was carried out by the Analytical Chemistry Department at RTC and the results, were within the limits
Duration of treatment / exposure:
Males were treated for a total of 5 weeks (2 weeks before pairing and 3 weeks during the paring period). Females were treated for approximately 6 weeks (2 weeks before pairing, 3 weeks of gestation until post partum Day 3).
Frequency of treatment:
daily
Details on study schedule:
- Parental animals mated after 2 weeks of treatment
- Age at mating of the parental animals in the study: 10-11 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
The oral route was selected as it is a possible route of exposure of the test item in man.

Dose levels of 100, 300 and 1000 mg/kg/day were selected in consultation with the Sponsor based on information from preliminary studies.

Examinations

Parental animals: Observations and examinations:
Mortality

Throughout the study, all animals were checked early in the morning and in the afternoon. At
weekends and Public Holidays 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.

Clinical signs

All observations were recorded for individual animals.
Examination of individual animals for signs of reaction to treatment was carried out daily
prior to dosing and at suitable intervals after dosing. The number and timing of these daily
observations were reviewed by the Study Director at the end of the first week of treatment.

Clinical observations (Functional Observation Battery Tests)

Once before commencement of treatment and at least once a week thereafter, each animal
was given a detailed clinical examination. Each animal was removed from the home cage
and observed in an open arena. The tests included observation of changes in gait and posture,
reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre
behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection,
pupil size, unusual respiratory pattern).
Grip strength and sensory reactivity to stimuli

Once during the study, towards the end of treatment, 5 males and 5 females were randomly
selected from each group for evaluation of sensory reactivity to stimuli of different
modalities (e.g. auditory, visual and proprioceptive stimuli) and for assessment of grip
strength. Measurements were performed using a computer generated random order.

Motor activity assessment (MA)

Once during the study, towards the end of treatment, 5 males and 5 females were randomly
selected from each group and the motor activity was measured (for approximately 5 minutes)
by an automated activity recording device. Measurements were performed using a computer
generated random order.

Food consumption

The weight of food consumed by each cage of males and females was recorded weekly
during the pre-mating period starting from allocation. Individual food consumption for the
females was measured on gestation Days 7, 14 and 20 starting from Day 0 post coitum and
on Day 4 post partum starting from Day 1 post partum.

Body weight

Males were weighed weekly from allocation to termination.
Females were weighed weekly from allocation to pairing and on gestation Days 0, 7, 14 and
20. Dams were also weighed on Days 1 and 4 post partum.
Clinical pathology investigations

As a part of the sacrificial procedure, samples of blood were withdrawn under isofluorane
anaesthesia from the abdominal vena cava from 5 males and 5 females (females with viable
litters, if possible) randomly selected from each group, under condition of food deprivation.
The blood samples collected were divided into tubes as follows:

EDTA anticoagulant for haematological investigations
Heparin anticoagulant for biochemical tests
Citrate anticoagulant for coagulation tests
The measurements performed on blood samples are listed below:

Haematology

Haematocrit
Haemoglobin
Red blood cell count
Reticulocyte count
Mean red blood cell volume
Mean corpuscular haemoglobin
Mean corpuscular haemoglobin concentration
White blood cell count
Differential leucocyte count - Neutrophils
- Lymphocytes
- Eosinophils
- Basophils
- Monocytes
- Large unstained cells
- Platelets
- Prothrombin time
Clinical chemistry

Alkaline phosphatase
Alanine aminotransferase
Aspartate aminotransferase
Gamma-glutamyltransferase
Urea
Creatinine
Glucose
Triglycerides
Bile acids
Phosphorus
Total bilirubin
Total cholesterol
Total protein
Albumin
Globulin
A/G Ratio
Sodium
Potassium
Calcium
Chloride
Urinalysis (Only males)

At the same time interval as the clinical pathology investigations, individual overnight urine
samples were also collected from the same animals under the same conditions. Before
starting urine collection, water bottles were removed from each cage and each animal
received approximately 10 mL/kg of drinking water by gavage, in order to obtain urine
samples suitable for analysis.

Appearance
Volume
Specific gravity
pH
Protein
Glucose
Ketones
Bilirubin
Urobilinogen
Blood

The sediment, obtained from centrifugation at approximately 3000 rpm for 10 minutes, was
examined microscopically for:

Epithelial cells
Leucocytes
Erythrocytes
Crystals
Spermatozoa and precursors
Other abnormal components
Oestrous cyclicity (parental animals):
Vaginal smears were taken daily in the morning starting two weeks before pairing until a positive identification of copulation was made. The vaginal smear data were examined to determine the following:

a) anomalies of the oestrous cycle;
b) pre-coital interval (i.e., the number of nights paired prior to the detection of mating).

Sperm parameters (parental animals):
Parameters examined in male parental generations:
testis weight, epididymis weight morphologicqal evaluation of the seminiferus epithelium (staging of spermatogenica cycle)
Litter observations:
As soon as possible, after parturition was considered complete (Day 0 or 1 post partum), all pups (live and dead) were counted, sexed and live pups were identified.
Live pups were individually weighed on Days 1 and 4 post partum. Pups dying during the lactation period were also weighed before the despatch to nnecropsy.
Litter observation was performed once daily.
Postmortem examinations (parental animals):
The clinical history of the males and females of the parental generation was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices).
Changes were noted, the requisite organs weighed and the required tissue samples preserved in fixative and processed for histopathological examination.

Females:
All females were examined also for the following:

a) number of visible implantation sites (pregnant animals);
b) number of corpora lutea (pregnant animals).

Uteri of females with no visible implantations were immersed in a 10-20% solution of ammonium sulphide to reveal evidence of implantation.

From all animals completing the scheduled test period, the organs indicated in section 4.5.6 (annex1 to the study protocol) were dissected free of fat and weighed.
The ratios of organ weight to body weight was calculated for each animal.
The tissues required for histopathological examination

Postmortem examinations (offspring):
Pups:
Pups that has completed the scheduled test period (Day 4 post partum) were euthanised by intraperitoneal injection of Thiopenthal.
All pups found dead in the cage or sacrificed for humane reasons were examined for external and internal abnormalities.
All live pups sacrificed at termination were examined for external abnormalities and sex confirmation by gonadal inspection.
Statistics:
Means and standard deviations were calculated as appropriate. For body weight, body weight gain, food consumption, clinical pathology, sensory reaction to stimuli and motor activity, terminal body weight and organ weights (absolute and relative) 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 all the other parameters. The criterion for statistical significance was p<0.05.

Reproductive indices:
The following reproductive indices were calculated:

Males
Copulatory Index (%) = no. of animals mated x 100
no. of animals paired

Fertility Index (%) = no. of males which induced pregnancy x 100
no. of males paired

Females
Copulatory Index (%) = no. of animals mated x 100
no. of animals paired

Fertility Index (%) = no. of pregnant females x 100
no. of females paired
Males and females
Pre- coital interval = Mean number of days between pairing and mating

Offspring viability indices:
Pre-birth loss % = (No. of visible implantations - total litter size at birth) x 100/No. of visible implantations


Pup loss at birth % = (Total litter size at birth - live litter size at birth) x 100/ Total litter size at birth

Cumulative pup loss % at Day 4 post partum = (Total litter size at birth - live litter size at Day 4) x 100/Total litter size at birth

Pre- implantation loss was calculated as a percentage from the formula:

(no. of corpora lutea - no. of implantations) x 100/no. of corpora lutea

Sex ratios were calculated at birth and on Day 4 post partum and presented as the percentage of males per litter.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
body weight gain in high dose males
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
body weight gain in high dose males
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

Although changes in body weight gain of high dose males, bile acids and liver weight of high dose females were observed and since the microscopic examination revealed no lesion in any organs, the dosage of 1000 mg/kg/day is considered the NOAEL for this study.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Remarks on result:
other: no effects observed

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

Necropsy findings in pups did not reveal any treatment-related effect.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Remarks on result:
other: no effects observed

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Fate of females

A total of 10 females were proved not pregnant at necropsy: 4 females in the control group, 2 in the low dose group (including one female which did not mate), 2 in the mid-dose group and 2 in the high dose group. One control female showed unilateral total resorption. All remaining females gave birth and the number of dams with litters per group were: 5 in the control and 8 each in the remaining treated groups.

Clinical signsand clinical observations (Functional Observation Battery Tests)

Clinical signs and functional observation battery tests were unaffected by treatment.

Body weight and body weight gain

Statistically significant reduction in body weight was noted in high dose males compared to controls from Day 15 of treatment to sacrifice.

Food consumption

No effects on food consumption were observed.

Motor activity and sensory reactivity to stimuli

No differences of toxicological significance were seen.

Haematology

No changes of toxicological significance were seen. No changes were recorded for coagulation parameters.

Clinical chemistry

Bile acids showed an increase in almost all treated females.

Urinalysis – males only

No changes were observed.

Oestrus cycle, reproductive parameters, pairing combination and mating performance

No treatment related changes were seen.

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

No treatment-related effects were seen.

Litter data and sex ratio of pups

Litter data and sex ratios were unaffected by treatment.

Clinical signs of pups

Clinical signs were comparable between treated and control groups.

Necropsy findings in decedent pups and in pups sacrificed on Day 4 post partum

No treatment-related effects were seen.

Terminal body weight and organ weights

A slight increase in absolute and relative liver weight was observed high dose females compared to controls.

Macroscopic observations

No relevant changes were detected at post mortem examination in treated animals, when compared with controls.

Microscopic observations

No treatment-related changes were seen in selected organs/tissues evaluated in males or females receiving Triethyleneglycol dimethacrylate nor in the abnormalities detected in all groups at post mortem including the staging in the spermatogenic cycle.

Applicant's summary and conclusion

Conclusions:
No relevant toxic effects were seen in parental animals as well as in pups up to the highest dose group of 1000 mg/kg bw/d. On the basis of the results obtained in the study, the NOAEL for both, general toxicity and reproduction/developmental toxicity was 1000 mg/kg bw/d (males/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) TREGDMA (95.8% a.i.) 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.  

No mortality occurred in the study. No clinical signs of toxicological significance were reported. Statistically significant reduction in body weight was noted in high dose males compared to controls from Day 15 of treatment to sacrifice; body weights of females were unaffected by treatment.

Food consumption was comparable between the control and treated groups.

No differences in motor activity, grip strength and sensory reactivity to stimuli were observed. The differences noted in land foot splay noted in low dose males and females were considered incidental since they were inconsistent between males (increase) and females (reduction) and without any dose correlation.

In haematology and urinanalysis no changes of toxicological significance were seen. The statistically significant decrease of reticulocytes recorded in females dosed with 1000 mg/kg bw/d was considered of no toxicological relevance since no associated alterations of the erythrocytes were observed. No changes in prothrombine time were noted. Bile acids showed a dose-related increase in almost all treated females. No other changes of toxicological significance were observed. Two males of the high dose group showed an increase of urea (mean value 35% above controls). However, due to the low incidence, this finding cannot be conclusively attributed to treatment.

Other statistically significant fluctuations of some biochemical parameters were recorded in treated animals, such as: chloride, calcium, sodium and potassium. Changes were of minimal magnitude, not consistent between sexes and/or not dose-related, therefore considered incidental.

All females mated with the exception of on female of the low dose group. Two females, 1 in the low dose group and 1 in the mid-dose group showed an irregular cycle (oestrus was never observed); the low dose female did not mate, the mid-dose female was found sperm positive after 8 days of paring

but not pregnant at necropsy. These isolated cases were considered incidental. In the control group a total of 5 females were found not pregnant and 1 female had unilateral implantation with total resorption. In addition, 1 female in the low dose group, 2 females in the mid-dose group and 2 females in the high dose group were not pregnant.

Measurements of copulatory index, fertility index Pre-coital interval and the number of copulation plugs did not show differences between treated and control groups. No significant differences were observed in the number of implantation, corpora lutea, total litter size, pre-implantation loss, pre-birth loss and gestation length between control and treated groups.

Litter data and sex ratios were unaffected by treatment. Clinical signs of pups were comparable between groups. Decedent pups were found in all groups without dose relationship. Necropsy findings in decedent pups and in pups sacrificed on Day 4post partumdid not reveal any treatment-related effect.

A slight reduction in terminal body weight was noted in the mid- and high dose males (statistically significant in high dose). Terminal body weight of females was unaffected by treatment. A slight increase in absolute and relative liver weight was observed in high dose females compared to controls. No relevant changes were detected at post mortem examination in treated animals, when compared with controls.

No treatment-related changes were seen in selected organs/tissues evaluated in males or females nor in the abnormalities detected in all groups at post mortem including the staging in the spermatogenic cycle.

Although changes in body weight gain of high dose males, bile acids and liver weight of high dose females were observed and since the microscopic examination revealed no lesion in any organs, the dosage of 1000 mg/kg bw/d is considered to be the NOAEL for this study.

No relevant toxic effects were seen in parental animals as well as in pups up to the highest dose group of 1000 mg/kg bw/d. On the basis of the results obtained in the study, the NOAEL for both, general toxicity and reproduction/developmental toxicity was 1000 mg/kg bw/d (males/females).