Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
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:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Clear yellowish liquid (supplied by the Sponsor)
Details on test material:
Identity : Ethyl triglycol methacrylate
Alternative names : VISIOMER® ETMA; Ethyltriglycol methacrylate
CAS number : 39670-09-2
EINECS number : 254-588-0
Batch number : 1201180308
Date of expiry : 27 August 2012
Appearance : Clear yellowish liquid (supplied by the Sponsor)
Appearance at first use: Clear liquid
Storage conditions : Room temperature, protected from light

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
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 hours each day.

In-life data from: 07 June 2012 to 04 August 2012

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral 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
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 a daily
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg/day
Basis:

No. of animals per sex per dose:
The test item was administered orally, by gavage. The oral route was selected as it is a possible route of exposure of the test item in man.

3 groups comprised 10 males and 10 females rats received the test item at dose level of 100, 300 and 1000 mg/kg/day .
Each group comprised 10 male and 10 female rats.
Control animals:
yes
Details on study design:
Dose levels of 100, 300 and 1000 mg/kg/day were selected by the Sponsor based on information from a previous non GLP compliant study
(RTC Study no.: 90910EXT).

Examinations

Observations and examinations performed and frequency:
Mortality

Throughout the study, all animals were checked early in each working day 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.
Severely debilitated animals were observed carefully. One animal judged to be in extremis was killed. A complete necropsy was performed in all cases.

Clinical signs

Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs were recorded.
Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions.

Clinical observations (Functional Observation Battery Tests)

Once before commencement of treatment and 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).
All observations were recorded for individual animals.

Animals were examined in an open arena for a minimum of three minutes.

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); an assessment of grip strength was also performed. For malesthe tests were performed on Day 28 of the study and for females on Day 3 post partum, with the exception of one high dose female (no. 90920067) for which the tests were performed on Day 25 post coitum.

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 (at least 5 minutes) by an automated activity recording device. Measurements were performed using a computer generated random order. For males the tests were performed on Day 28 of the study and for females on Day 3 post partum, with the exception of one high dose female (no.
90920067) for which the tests were performed on Day 25 post coitum.

Body weight

Males were weighed on the day of allocation to treatment groups, on the day of treatment commencement, weekly thereafter and at termination.

Females were weighed on the day of allocation to treatment groups, on the day of treatment commencement, weekly thereafter until pairing and on Days 0, 7, 14 and 20 post coitum. Dams were also weighed on Days 1 and 4 post partum.

Food consumption

The weight of food consumed by each cage of males and females was recorded weekly, where possible, during the pre-mating period following allocation.
Individual food consumption for the females was measured on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Day 4 post partum starting from Day 1 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 (all females with viable litters with the exception of one high dose female) randomly selected from each group, under condition of food deprivation (male and females) and water deprivation (only males).
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 and urine 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

Coagulation tests
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 of the clinical pathology investigations, individual overnight urine samples were also collected from the same animals underthe 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



Sacrifice and pathology:
One parental animal killed for humane reasons (no. 90920061) and those that had completed the scheduled test period were killed by exsanguination
under isofluorane anaesthesia.

Parental males:
The males were killed after the mating of all females, after 32 days of treatment period.

Parental females:
The females with live pups were killed on Day 4 post partum.
The females which did not give birth were sacrificed 26/27 days after positive identification of mating.

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 (excluding animals sacrificed for humane reasons or found dead) and the required tissue samples
preserved in fixative and processed for histopathological examination.

Females:
All females were examined also for the following:

a) external and internal abnormalities;
b) number of visible implantation sites (pregnant animals);
c) number of corpora lutea (pregnant animals).

Organ weights

From all animals completing the scheduled test period, the organs were dissected free of fat and weighed.
The ratios of organ weight to body weight were calculated for each animal.

Tissues fixed and preserved

Samples of all the tissues were fixed and preserved in 10% neutral buffered formalin (except eyes, optic nerves and Harderian glands, testes and
epididymides which were fixed in modified Davidson's fluid and preserved in 70% ethyl alcohol).

Histopathological examination

The tissues required for histopathological examination are listed in Annex 1. After dehydration and embedding in paraffin wax, sections of the
tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin.
In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed.

The examination was restricted as detailed below:

a) Tissues specified in sAnnex 1 from 5 males and 5 females randomly selected (animals evaluated for clinical pathology) in the control and high
dose groups killed at term.
b) Tissues specified in Annex 1 from all animals killed or dying during the treatment period.
c) All abnormalities in all groups.
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 of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Description (incidence and severity):
only males
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
Mortality

One male receiving 100 mg/kg/day of Ethyl triglycol 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 0 post 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.

Clinical signs and clinical observations (Functional Observation Battery Tests)

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

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

Body weight and body weight gain

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

Food consumption

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.

Clinical chemistry

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.

Terminal body weight and organ weights

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.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: 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.

Target system / organ toxicity

Critical 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 general toxicity was considered to be 1000 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 Ethyl triglycol methacrylatewas 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 0 post 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.

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 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.

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.