Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test with Triethyleneglycol dimethacrylate (TREGDMA); oral (gavage); rat (Hsd: Sprague Dawley SD, m/f (OECD guideline 422, GLP): NOAEL = 1000 mg/kg bw/d

 

TREGDMA will rapidly be hydrolyzed by unspecific carboxyl esterases in the liver into methacrylic acid and Triethylene glycol (see chapter Toxikokinetics and the Category document, respectively). Therefore data from the hydrolyses products methyl methacrylate (the metabolite donor substance for methacrylic acid) and Triethylene glycol can be used to assess the reproductive toxicity of TREGDMA.

 

Methyl methacrylate

No reproductive effects were observed in a 2-generation oral gavage study with rats acc. OECD 416 (BASF 2009) up to 400 mg/kg bw/day with Methyl methacrylate. NOAEL for fertility: 400 mg/kg bw/day.

 

Triethylene glycol

No reproductive effects were observed in a 2-generation oral drinking water study with rats acc. National Toxicolgy Program's RACB protocol (Bossert et al 1992) up to 3% or 6780 mg/kg bw/day with Triethylene glycol. NOAEL for fertility: 6780 mg/kg bw/day.

Based on available screening data on TREGDMA and two generation studies of its metabolites TREG and the metabolite donor substance Methyl methacrylate, TREGDMA is not considered to show reproductive effects.

Link to relevant study records
Reference
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
Qualifier:
according to
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
Specific details on test material used for the study:
Name: Triethyleneglycol dimethacrylate
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals 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
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
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.
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.
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: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
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.
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
Critical effects observed:
no
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
Necropsy findings in pups did not reveal any treatment-related effect.
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
Critical effects observed:
no
Reproductive effects observed:
not specified

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.

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). 

 

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

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 urin analysis 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 4 post partum did 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 the NOAEL for this study. No relevant toxic effects were seen in parental animals 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). 

 

Metabolite data

TREGDMA will rapidly be hydrolyzed by unspecific carboxyl esterases in the liver into methacrylic acid and Triethylene glycol, TREG (see chapter Toxikokinetics and the Category document, respectively). Therefore data from the hydrolyses products methacrylic acid, methyl methacrylate (the metabolite donor substance for methacrylic acid) and Triethylene glycol can be used to assess the reproductive toxicity of TREGDMA.

 

Triethylene glycol (alcohol metabolite of TREGDMA)

A two generation study on mice is available, conducted under the National Toxicolgy Program's RACB protocol (NTP, 1989) with the alcohol triethylene glycol, which is a metabolite in the first step after ester hydrolysis. TEG was tested on Swiss CD1 -Mice in concentrations of 0, 0.3, 1.5 and 3 % (= 0, 590, 3300 and 6780 mg/kg bw/day) in drinking water. Triethylene glycol was noted to produce developmental effects in the first generation as slightly but significantly reduced pup body weight at birth at 1.5 and 3 % TEG in drinking water. In overall view the substance was not considered to be a reproductive toxicant in either generation of mice when administered in drinking water at concentrations of up to 3%. NOAELs were determined to be 6780 mg/kg bw/d both fertility and overall effects.

 

Methyl methacrylate MMA (donor substance for the common primary metabolite MAA)

Methyl methacrylate (MMA) is the methyl ester of methacrylic acid (MMA) and is rapidly absorbed and metabolised to MAA within the body. It therefore acts as an effective systemic deliver system for MAA avoiding the local high toxicity of MMA due to its acidity. The reference chemical for the methacrylic moiety of the category members, MMA, hasrecently been tested in an OECD TG 416 oral two-generation reproduction toxicity study in rats, in which both, parental and F1 animals were dosed with 0; 50; 150 and 400 mg/kg bw/day (BASF, 2009a).

In mid- and high-dose parental animals (150 and 400 mg/kg bw/d) temporary salivation, presumably due to a bad taste of the test substance and associated dose-related intermittent reductions of food consumption were noted. Less significant changes were noted for the F1 generation animals where the effects were limited to the high-dose group and not associated with effects on histopathology or reproductive performance.

The NOAEL for fertility and reproductive performance for the P and F1 parental rats was determined to be 400 mg/kg bw/day, the highest dose tested. The NOAEL for developmental toxicity, in the F1 and F2 progeny, of the test substance was determined to be 400 mg/kg bw/day, the highest dose tested. There were no signs of systemic toxicity other than reduced body weight gain associated with reduced food consumption, presumably due to bad palatability (NOEL 50 mg/kg bw/day for the P and F1 parental rats).

The 2-generation study with MMA provides confidence that the absence of reproductive effects below maternal toxic doses seen at in the screening studies with the three category members, are a reliable indication for an absence of fertility effects below maternal toxic doses in higher tier studies such as a 2 generation study when considering the methacrylic moiety of the category members.

 

Summary

In summary based on available screening data on TREGDMA and two generation studies of its metabolites TREG and the metabolite donor substance Methyl methacrylate, TREGDMA is not considered to show reproductive effects.

 

 

Compliance to REACh requirements

The screening study requirement is covered with a reliable OECD 422 oral rat study, performed with the substance itself. The development toxicity requirements for two species are covered with reliable oral or inhalation studies with the methacrylic metabolite MAA, its donor MMA and the alcohol metabolite TREG. The reproduction toxicity requirements are covered with reliable two generation studies either in rats with the methacrylic metabolite donor MMA or in mice with the alcohol metabolite TREG. All mentioned studies are reliable (Reliability 1 or 2) and the read across is done with a high level of confidence.

Effects on developmental toxicity

Description of key information

Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test with Triethyleneglycol dimethacrylate (TREGDMA); oral (gavage); rat (Hsd: Sprague Dawley SD, m/f (OECD guideline 422, GLP): NOAELmaternal/developmental = 1000 mg/kg bw/d

 

TREGDMA will rapidly be hydrolyzed by unspecific carboxyl esterases in the liver into methacrylic acid and Triethylene glycol, TREG (see chapter Toxikokinetics and the Category document, respectively). Therefore data from the hydrolyses products methacrylic acid, methyl methacrylate (the metabolite donor substance for methacrylic acid) and Triethylene glycol can be used to assess the developmental toxicity of TREGDMA.

 

Methacrylic acid

Methacrylic acid (MAA) was tested in 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).

 

Methyl methacrylate

In a developmental toxicity study according to OECD 414 with Crl:CDBR rats, MMA was administered by inhalation exposure to 99, 304, 1178, and 2028 ppm (412, 1285, 4900, 8436 mg/m³; Rohm and Haas, 1991). Treatment related maternal effects on body weight and food consumption were noted at all exposure levels, consequently the LOEC for maternal toxicity is 99 ppm. In this study the NOAEC for developmental toxicity was 2028 ppm (8436 mg/m³) due to the absence of adverse developmental effects.

In addition, an oral OECD 414 study was performed with rabbits at 50, 150, and 405 mg/kg/d. The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 405 mg/kg bw/d due to the absence of adverse developmental effects, even in the presence of maternal toxicity (body weight and food consumption effects, NOEL 50 mg/kg/d; BASF, 2009). MMA is not a selective teratogen.

 

Triethylene glycol

In a reliable study comparable to OECD 414, CD rats were dosed daily by gavage with undiluted TEG over gestational days (gd) 5–15 at 0.0 (water control), 1126, 5630 or 11 260 mg kg−1day−1with rats. The NOEL for TEG given by gavage over the period of organogenesis was 1126 mg kg−1day−1in the rat for maternal toxicity due to reduced body weight and increased water consumption. The NOEL was 5630 mg kg−1day−1 for developmental toxicology due to reduced fetal body weight and delayed ossification.

In the same study, CD-1 mice were dosed daily by gavage with 0.0 (water control), 563, 5630 or 11260 mg undiluted TEG kg−1 day−1. The NOEL for TEG given by gavage over the period of organogenesis was 5630 mg kg−1 day−1 in the mouse for maternal toxicity due to clinical signs and increased relative kidney weight, and 563 mg kg−1 day−1 (mouse) for developmental toxicology due to due to reduced fetal body weight and delayed ossification. (Ballantyne & Snellings 2005).

For both species, the authors concluded that “no biologically significant embryotoxicity or teratogenicity was observed at any dosage.

 

Based on the available data on Triethylene glycol dimethacrylate or its metabolites Triethylene glycol and Methacrylic acid plus Methyl Methacrylate in rodents and non-rodents, Triethylene glycol dimethacrylate is not considered to cause prenatal developmental toxicity.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
13 April 2012-16 June 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD 422 screening study
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Italy S.p.A., Calco (Lecco), Italy

- Age at study initiation: (P) Males/females: approximately 8 - 9 wks
- Weight at study initiation: (P) Males: 196.5 - 204.7 g; Females: 166.1 - 189.3 g

- Fasting period before study:
- Housing: Pre mating period: no more than 5 per cage in clear polycarbonate cages measuring 59X39X20 cm with a stainless steel
mesh lid and floor (Techniplast - Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray will hold absorbent material which will be
inspected daily and changed at least three times a week.
During mating period: 1 male to one female per cage in clear polycarbonate cages measuring 36X19X24 cm with a stainless steel
mesh lid and floor (Techniplast - Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray will hold absorbent material which will be
inspected daily.
Pregnant females: will be transferred to individual cages after mating: solid bottomed, breeding cages (Techniplast - Gazzada S.a.r.l.,
Buguggiate, Varese), for the gestation period, birth and lactation.
Suitable nesting material will be provided and will be changed as necessary.
- Diet: ad libitum, commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4,
20019, Settimo Milanese (MI), Italy)
- Water: ad libitum, supplied via water bottles

- Acclimation period: aproximately 14 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ±2 °C
- Humidity (%): 55 ±15 %
- Air changes (per hr): 15 - 20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark/ 12 hrs light
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
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Once during week 1 and week 6 of treatment, samples of prepared formulations were analysed for verification of concentration.
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.
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.
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 or the day before sacrifice.
Dose volumes were adjusted once per week for each animal according to the last recorded body weight. During the gestation 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.
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
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 (RTC Study no.: 90790EXT).
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 were assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
Statistical analysis of histopathological findings was carried out by means of the nonparametric Kolmogorov-Smirnov test if n was more than 5. 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 nonparametric version of the Williams test. The criteria for statistical significance were p<0.05 and p<0.01.
The mean value, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.
Details on maternal toxic effects:
Maternal toxic effects:no effects
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks on result:
other: No adverse effects observed
Abnormalities:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no adverse effects observed
Abnormalities:
no effects observed
Developmental effects observed:
no
Conclusions:
Triethyleneglycol dimethacrylate did not show developmental toxicity via oral gavage to rats at doses as high as 1000 mg/kg bw/day in the decribed reproductive/developmental toxicity screening study according to OECD 422.
Executive summary:

Triethyleneglycol dimethacrylate did not show developmental toxicity via oral gavage to rats at doses as high as 1000 mg/kg bw/day in the decribed reproductive/developmental toxicity screening study according to OECD 422 (for more details please also see IUCLID chapter 7.5.1 and 7.8.1). There were no signs of neonatal toxicity or external malformations at doses of 1000 mg/kg bw/day.

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
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Quality of whole database:
OECD guideline 422 study, no deviations, GLP
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

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 (see endpoint summary “Toxicity for reproduction”). 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). 

 

 

Metabolite data

TREGDMA will rapidly be hydrolyzed by unspecific carboxyl esterases in the liver into methacrylic acid and Triethylene glycol, TREG (see chapter Toxikokinetics and the Category document, respectively). Therefore data from the hydrolyses products methacrylic acid, methyl methacrylate (the metabolite donor substance for methacrylic acid) and Triethylene glycol can be used to assess the developmental toxicity of TREGDMA.

 

Triethylene glycol (alcohol metabolite of TREGDMA)

In a reliable study comparable to OECD 414, timed-pregnant CD rats and CD-1 mice were dosed daily by gavage with undiluted TREG over gestational days (gd) 5–15 at 0.0 (water control), 1126, 5630 or 11 260 mg kg−1 day−1 with rats and 0.0, 563, 5630 or 11 260 mg kg−1 day−1 with mice. They were examined daily, and gestational body weights and food and water consumption measured throughout gestation. At necropsy on gd 21 (rats) or gd 18 (mice) dams were

examined for body, gravid uterine, liver and kidney weights, and implantation sites. Maternal kidneys were examined histologically. Fetuses were weighed, sex determined, and examined for external, soft tissue and skeletal variations and malformations. Rat dams had reduced body weights, body weight gains, and food consumption, and increased water consumption and relative kidney weights at 11 260 mg kg−1 day−1. They also had reduced body weight and increased water consumption at 5630 mg kg−1 day−1. Mice had clinical signs and increased relative kidney weight at 11 260 mg kg−1 day−1. Renal histology was normal in both species. Neither species had treatment-related effects on corpora lutea or implantations. Fetal body weights were reduced at 11 260 mg kg−1 day−1 (both species) and 5630 mg kg−1 day−1 (mice). In rat fetuses there was a pattern of delayed ossification in the thoracic region at 11 260 mg kg−1 day−1. Mouse fetuses had delayed ossification in the frontal and supraoccipital bones, cervical region, hindlimb proximal phalanges and reduced caudal segments at 11 260 mg kg−1 day−1, and in the skull bones at 5630 mg kg−1 day−1. These patterns of delayed ossification are consistent with reduced fetal body weights. No biologically significant embryotoxicity or teratogenicity was observed at any dosage in either species. The NOEL for TEG given by gavage over the period of organogenesis was 1126 mg kg−1 day−1 in the rat and 5630 mg kg−1 day−1 in the mouse for maternal toxicity, and 5630 mg kg−1 day−1 (rat) and 563 mg kg−1 day−1 (mouse) for developmental toxicology (Ballantyne & Snellings 2005).

The lowest NOEL in this study, i.e. 563 mg TREG/kg bw/d for fetotoxic effects in the mouse, corresponds to 3.75 mM TREG/ kg bw/d (with a molar weight of 150 g/mol for TREG). Regarding TREDGMA with a molar weight of 286 g/mol, this dosage would not be reached in a modern guideline study with a limit dose of 1000 mg/kg bw/d as this dose corresponds to 3.49 mM TREGDMA/ kg bw/d. Thus, the hazard derived from the alcohol moiety is considered as provenly safe tested.

 

Methacrylic acid, MAA (common primary metabolite)

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).

 

Methyl methacrylate MMA (donor substance for the common primary metabolite MAA)

In a developmental toxicity study according to OECD 414 with Crl:CDBR rats, MMA was administered by inhalation exposure to 99, 304, 1178, and 2028 ppm (412, 1285, 4900, 8436 mg/m³; Rohm and Haas, 1991).Treatment related maternal effects on body weight and food consumption were noted at all exposure levels, consequently the LOEC for maternal toxicity is 99 ppm. No embryo of foetal toxicity was evident and no increase in the incidence in the malformations or variations was noted at exposure levels up to and including 2028 ppm. In this study the NOAEC for developmental toxicity was 2028 ppm (8436 mg/m³).

In addition, another study with MMA has been performed, an oral OECD 414 study in rabbits at 50, 150 , and 405 mg/kg/d. The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 450 mg/kg bw/d. No adverse foetal findings of toxicological relevance were evident at any dose, even in the presence of maternal toxicity (body weight and food consumption effects at 150 and 405 mg/kg/d; BASF, 2009). MMA is not a selective teratogen.

 

Summary

In summary based on available screening data on TREGDMA and developmental studies of its metabolites TREG and MAA (including its metabolite donor substance MMA), TREGDMA is not considered to show developmental effects.

Justification for classification or non-classification

Based on the available data, TREGDMA does not need to be classified for toxicity to reproduction, developmental toxicity and teratogenicity according to the criteria given in regulation (EC) 1272/2008. Thus, no labelling is required.