Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
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
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
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 phase: 07 June 2012 to 04 August 2012
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.
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.
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
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 the dose levels 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).
Parental animals: Observations and examinations:
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 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.

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.
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 [all/P/F1/F2] male parental generations:
testis weight, epididymis weight,. 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.
Litter observations:
A parturition check was performed from Day 20 to Day 25 post coitum.
Gestation length was calculated as the time between the day of successful mating (Day 0 post coitum) and the day of commencement of birth
(i.e. first detected presence of offspring in the cage). The day that offspring were first detected in the cage was considered Day 0 post partum.

As soon as possible, after parturition was considered complete (Days 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 or sacrificed for humane reasons during the lactation period were weighed before the despatch to necropsy.
Observation was performed once daily for all litters.
Postmortem examinations (parental animals):
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.

Necropsy

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).

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


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 listed 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 section 4.5.6. 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 Annex 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 section 4.5.6 from all animals killed or dying during the treatment period.
c) All abnormalities in all groups.
Postmortem examinations (offspring):
Pups killed for humane reasons or those that had completed the scheduled test period (Day 4 post partum) were euthanised by intraperitoneal
injection of Sodium Thiopenthal.

Pups
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 killed and examined for external abnormalities and sex confirmation by gonadal inspection.
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.
Reproductive indices:
Group mean values were calculated for all parameters. Data from non-pregnant females and from dams without live young on Day 4 post partum were
excluded from group mean calculations.

The following reproductive indices were calculated:

Males

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

Fertility Index (%) = no. of males which induced pregnancy/no. of animals pairedx 100

Females

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

Fertility Index (%) = no. of pregnant females/no. of females paired x 100

Males and Females

Pre-coital Interval = Mean number of days between pairing and mating
Sex ratios were calculated at birth and on Day 4 post partum and were presented as the percentage of males per litter.
Offspring viability indices:
Females

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

(No. of visible implantations-total litter size at birth)/No. of visible implantations x 100

Pup loss at birth was calculated as a percentage from the formula:

(Total litter size-live litter size)/Total litter size x 100

Cumulative pup loss on Day 4 post partum was calculated as a percentage from the formula:

(Total litter size at birth-live litter size at Day 4 post partum)/Total litter size at birth x 100

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

(no. of corpora lutea - no. of implantations)/no. of corpora lutea x 100
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
spermatogenic cycle
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
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
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 lumenThese 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.

Oestrus cycle, reproductive parameters, pairing combination and mating performance

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.


No significant differences were observed in treated and control groups for these parameters.
All pregnant females gave birth between Days 22 and 23 post coitum.


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.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: general toxicity
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: fertility (reproductive toxicity)
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Gross pathological findings:
no effects observed
Clinical signs of pups

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

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 23 post coitum.

Litter data at birth, on Day 1 and on Day 4 post partum of females and sex ratio of pups

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



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

Necropsy findings in unscheduled dead pups and in pups sacrificed on Day 4 post partum did not reveal any treatment-related effect.
Reproductive effects observed:
not specified

Oestrus cycle – Before pairing - Group summary data

  

 -----------------------------------------------------------------------------------------------------------------------------------

           Group 1              Group 2             Group 3             Group 4

 Animal   Oestrus    Animal   Oestrus   Animal   Oestrus   Animal   Oestrus

 Number   Cycles     Number   Cycles    Number   Cycles    Number   Cycles

 -----------------------------------------------------------------------------------------------------------------------------------

  90920001    4      90920021    4      90920041    4      90920061    4

  90920003    4      90920023    4      90920043    3      90920063    4

  90920005    4      90920025    3      90920045    4      90920065    4

  90920007    4      90920027    4      90920047    4      90920067    3

  90920009    4      90920029    4      90920049    4      90920069    4

  90920011    3      90920031    2      90920051    4      90920071    4

  90920013    4      90920033    4      90920053    4      90920073    3

  90920015    4      90920035    2      90920055    4      90920075    4

  90920017    3      90920037    3      90920057    3      90920077    4

  90920019    4      90920039    4      90920059    4      90920079    3

 

    Means  3.8                 3.4                 3.8                 3.7

 -----------------------------------------------------------------------------------------------------------------------------------

 Note: The number of oestrus cycles is based on the number of non sequential days the dams were in oestrus.

Reproductive parameters of males - Summary

   

 -----------------------------------------------------------------------------------------------------------------------------------

                              Group       1          2         3          4

 -----------------------------------------------------------------------------------------------------------------------------------

 

   Copulatory Index%                    100.0     100.0     100.0      100.0

 

 -----------------------------------------------------------------------------------------------------------------------------------

 

   Fertility Index%                     100.0     100.0      70.0       50.0

 

 -----------------------------------------------------------------------------------------------------------------------------------

Reproductive parameters of females – Summary

  

 -----------------------------------------------------------------------------------------------------------------------------------

                              Group       1          2         3          4

 -----------------------------------------------------------------------------------------------------------------------------------

 

   Copulatory Index%                    100.0     100.0      100.0      100.0

 

 -----------------------------------------------------------------------------------------------------------------------------------

 

   Fertility Index%                     100.0     100.0       70.0       50.0

 

 -----------------------------------------------------------------------------------------------------------------------------------
Conclusions:
Conclusions
On the basis of the results obtained in this study with Ethytriglycol methacrylate, the NOAEL (No Observed Adverse Effect Level) for
reproductive 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 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 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 23 post 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 reproductive toxicity was found to be 300 mg/kg/day for males and females.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Neither the methacrylate moiety nor the higher glycol ethers appear to affect fertility This is indicating that the effect on fertility at the highest dose in the ET3EGMA study is an isolated finding.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

ET3EGMA was studied in an OECD 422 combined repeat dose and reproductive/developmental toxicity screening test (RTC, 2013). Groups of 10 male and 10 female rats were administered by gavage at dose levels of 0, 100, 300, or 1000 mg/kg/day. Male rats were dosed for 29 days and female rats were dosed from 14 days prior to mating through Day 3 of lactation. On the basis of the results obtained in this study, there were no significant signs of toxicity up to 1000 mg/kg/day for males and females. No copulation plugs were found in the cage tray of 9 out of 10 females receiving 1000 mg/kg/day and 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. The NOAEL for effects on fertility was found to be 300 mg/kg/day males and females.

Methacrylates and the vinyl group do not cause effects on fertility as illustrated by MMA, for which a 2 generation study is available and more than ten other negative 422 screening studies with a wide variety of methacrylate esters. Furthermore, higher glycol ethers, for example ethoxydiethyleneglycol, a lower ethoxylated analogue of ethoxytriethylene glycol, do not appear to affect fertility either indicating that the effect on fertility at the highest dose in the ET3EGMA study is an isolated finding.


Short description of key information:
OECD 422 reproductive toxicity screening study: Reproductive NOAEL 300 mg/kg, no apparent systemic toxicity up to 1000 mg/kg

Justification for selection of Effect on fertility via oral route:
Available reproductive toxicity screening study, reliability 1, GLP

Effects on developmental toxicity

Description of key information
OECD 422 reproductive toxicity screening study: Developmental and systemic NOAEL 1000 mg/kg
Developmental toxicity studies with primary metabolites:
Methacrylic acid: Saillenfait et al. (1999); Developmental study in rats; No effects on development up to 300 ppm (1232 mg/m³), equivalent to a body burden of 409 mg/kg/d
Ethoxytriethylene glycol: Leber et al. 1990; Developmental toxicity study at 250 and 1000 mg/kg/d, Chernoff-Kavlock protocol; no developmental effects
Link to relevant study records
Reference
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
Qualifier:
according to
Guideline:
other: OECD Guideline 422
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
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
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
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)
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.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Abnormalities:
not specified
Developmental effects observed:
not specified
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.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Screening level data available from OECD 422 study, supported by developmental toxicity data from primary metabolites methacrylic acid and ethoxytriethylene glycol, both indicating an absence of developmental toxicity
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

ET3EGMA was studied in an OECD 422 combined repeat dose and reproductive/developmental toxicity screening test (RTC, 2013). Groups of 10 male and 10 female rats were administered by gavage at dose levels of 0, 100, 300, or 1000 mg/kg/day. Male rats were dosed for 29 days and female rats were dosed from 14 days prior to mating through Day 3 of lactation. On the basis of the results obtained in this study, there were no significant signs if toxicity up to 1000 mg/kg/day for males and females. While there were fertility-related effects at 1000 mg/kg/day there were no effects on developmental parameters compared to controls. The NOAEL for developmental effects was found to be 1000 mg/kg/day.

In terms of the primary metabolites, methacrylic acid (MAA), the common metabolite for all the esters, also was tested 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).

 

From the alcohol side chain there are no alerts regarding specific developmental toxicity for the other members of the H&EMA category, the simple glycols (ethylene and propylene glycol; ATSDR 2010; ATSDR 1997 and 2008) and the complex glycol ethers (diethylene glycol butyl ether, DEGBE and trietheylene glycol monomethyl ether, TEGEE; Leber et al. 1990, ECETOC, 2005).


Justification for selection of Effect on developmental toxicity: via oral route:
Available reproductive toxicity screening study, reliability 1, GLP

Justification for classification or non-classification

Overall, in consideration of all data available for ET3EGMA and its methabolites, there is no convincing evidence of reproductive toxicity and a general absence of developmental effects.

In summary, classification of ethoxytrithylene glycol methacrylate for reproductive toxicity is not required.