Reaction mass of 2-hydroxy-1,3-propanediyl bismethacrylate and 101525-90-0

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No data are available for GDMA. However, data from the metabolites (HPMA as analogous substance of the primary metabolites plus MMA and glycerol as further metabolites/ metabolite donor substances) allow the evaluation of reproduction toxicity hazard of GDMA.

Read-across to the metabolites is justified by the fact that carboxylesterases are ubiquitous in the body and half-lifes of the other category substances are only a few minutes (see category document, chapter 5.1). In the body, methyl methacrylate hydrolyses rapidly to methacrylic acid and thus serves as methacrylic acid donor in several test systems investigating systemic effects.

Following data were considered for read across:

OECD 422 repeated dose/ reproduction screening study, rat, gavage (GLP): parenteral

NOAEL = 300 mg/kg bw/d; NOAELreproduction = 1000 mg/kg bw/d; read-across from the analogous substance of the primary metabolites, HPMA (Nihon Bioresearch 1996)

OECD 416 fertility, rat, gavage (GLP): parenteral NOAEL 400 mg/kg bw/d/ NOEL 50 mg/kg bw/d based on food consumption), offspring NOAEL 400 mg/kg bw/d (BASF 2009); read-across from the metabolite donor substance MMA

Pre-guideline 2 gen study, rat, dw: parenteral and offspring NOAEL 2000 mg/kg bw/d (Wegener 1953); read-across from the alcohol metabolite Glycerol (endogeneous metabolite in mammals)

Summary

Based on the results of a reliable screening study with the primary metabolite HPMA and two gen fertility studies with the methacrylic metabolite donor substance MMA and the alcohol metabolite glycerol, exposure to GDMA is not likely to result in reproductive toxicity.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study, GLP, japanese study

Qualifier:

according to guideline

Guideline:

OECD Combined Repeated Dose and Reproductive / Developmental Toxicity Screening Test (Precursor Protocol of GL 422)

Deviations:

yes

Remarks:

, An old version of OECD 422 (not containing functional observation battery test) had been conducted.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan
- Age at study initiation: 8 weeks
- Weight at study initiation: male 315 ~ 359 g; females 210 ~ 243 g,
- Fasting period before study: yes
- Housing: During the quarantine: suspended using a stainless steel cage type 1 with 5 per cage; Breeding: divided into separate rearing cages. Moved to a separate plastic cage, having had a natural birth .
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: five-day quarantine period and then set up a six-day acclimation period

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 deg C
- Humidity (%): 40-70%
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12 hour light / 12 hour dark

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Concentration of samples was prepared by dissolving the required water for injection. The concentrations of preparation is protectedc from light at room temperature for 7 days to ensure that there were no stability issues. Preparation of 0.6% solution concentration is below the threshold level of determination, because we could not confirm the stability during the preparation for the 6% solution diluted with water for injection prepared in concentration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): water
- Concentration in vehicle: 0.6, 0.8, 2.6 and 20%
- Amount of vehicle (if gavage): 5 ml/kg

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 14 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): moved to put a separate plastic cage, having had a natural birth and feeding.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

No data

Duration of treatment / exposure:

Exposure period: 49 days

Frequency of treatment:

daily

Details on study schedule:

No data

Remarks:

Doses / Concentrations:
0 (vehicle), 30, 100, 300 and 1000 mg/kg/day
Basis:
nominal in water

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: 2-week preliminary study
- Rationale for animal assignment (if not random): random by weight

Positive control:

not applicable

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day
- Cage side observations: general condition

DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: twice a week

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): 2 times per week
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood:day after treatment
- Anaesthetic used for blood collection: Yes (identity) : sodium pentobarbital
- Animals fasted: No data
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day after treatment
- Animals fasted: No data
- Parameters checked in table [No.2] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: sexual cycle until confirmation; status of delivery

Oestrous cyclicity (parental animals):

Sexual cycle, the daily dose from the start date until the date confirmed

Litter observations:

observed at birth
Mobilize the number of preterm birth and sex, number of stillborn children, the presence of abnormalities observed and the number of newborn外表.
(2) observation of the newborn
Newborns, the presence of daily survival and mortality in a general state who observed times.
(3) measurement of body weight
Weight, feeding 0 days (date of birth) and measured four days.

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes; Males: Thymus, liver, kidney, testis and epididymis weight was measured after removal, adrenal gland, brain, heart and spleen and 10% neutral buffered formalin solution (However, testicular and epididymal fluid Buan) was fixed; Females: counting the number of corpora lutea and the number of implantation scars. Liver, kidney, ovary and thymus weight was measured after removal, adrenal gland, brain, heart and spleen with a fixed 10% neutral buffered formalin solution.
HISTOPATHOLOGY: Yes; Control group and 1000 mg / kg group of heart, liver, spleen, thymus, kidney, testis, epididymis, ovary, adrenal and brain for the Preparation HE staining of tissue was examined histologically.

Postmortem examinations (offspring):

Autopsy performed after four days of feeding by exsanguination from the abdominal aorta under ether anesthesia.

Statistics:

In either test, significant risk factors were less than 5%.
1) multiple comparison test
Weight (the parent animals, babies), food consumption, number of estrus, days mating, pregnancy [Day delivery (feeding 0) - date confirmed mating, the number of implantation scars, the number of birth control mobilize (number of babies stillborn baby + ), the number of newborn, number of children born dead, birth rate [(number of birth control mobilize / number of implantation scars) × 100], rate of production of child [(number of infant feeding 0 days / number of implantation scars) × 100], corpus number, implantation rates [(number of implantation scars / number of corpora lutea) × 100], fertility [(number of infant feeding 0 day / mobilize all of birth control) × 100], feeding baby number four day, feeding 4 day survival rate [(number of newborn feeding 4 days / 0 Number of infant feeding day) × 100], unusual occurrence rate [(number of children with abnormal/ number of newborns) × 100], sex ratio (male / female), organ weights ( including the relative weight), results of blood tests, blood biochemistry test results for the mean and standard deviation were calculated for each group. Significant difference test, Bartlett's test and the homoscedasticity of Law, Law-way layout analysis of variance if the variance 1) and, if significant Dunnett method 2) or Scheff Method 3) were using. However, if the variance was not approved, the analysis method using centrally located position (Kruskal-Wallis test of 4)) and a significant if you use the ranking method or Dunnett Scheff Method was used.
2) χ ^ 2 test
Copulation rate [(number of established animal mating / number of live animals) × 100], fertility [(number of female fertility / Establishment of animal mating) × 100], the birth rate [(number of female newborns / number of female fertility) × 100] is, χ ^ 2 using the test.

Reproductive indices:

Estrus frequency, copulation index, number of days to conception, fertility index, length of gestation, number of corpora lutea or gestation index.

Offspring viability indices:

Number of live pups born, birth index, number of dead pups, number of pups born, delivery index, live birth index, sex ratio, viability index

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Haematological findings:

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

For the males, salivation, decrease in locomotor activity and ptosis were found in the 1000 mg/kg group, and 2 animals of the group died. Decrease in hematocrit, tendencies for decrease in RBC and hemoglobin, and increase in the relative liver weights were also found in the 1000 mg/kg group.
For the females, salivation, decrease in locomotor activity and ptosis were found in the 1000 mg/kg group, and 1 animal died.

There were no effects of the test substance on the estrus frequency, copulation index, number of days to conception, fertility index, length of gestation, number of corpora lutea or gestation index.

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

haematology

organ weights and organ / body weight ratios

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

There were no effects of the test substance on the number of live pups born, birth index, number of dead pups, number of pups born, delivery index, live birth index, sex ratio, viability index, external anomalies, body weight or necropsy

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: lack of overall effects

Reproductive effects observed:

not specified

Conclusions:

In an OECD 422 study in rats, the NOAEL for reproduction toxicity was determined to be 1000 mg/kg based on body weights.

Executive summary:

The combined repeated dose toxicity study with the reproductive/developmental toxicity screening test [MHW Japan, 1996] was conducted by OECD TG 422 in compliance with GLP. SD (Crj: CD) rats (12 animals/dose/sex) were administrated with this substance by gavage at doses of 0 (vehicle; water for injection), 30, 100, 300, and 1,000 mg/kg bw/day (12 animal/dose/sex). Males were dosed for total of 49 days beginning 14 days before mating, and females were dosed for total of 41 to 48 days starting from 14 days before mating to day 4 of lactation throughout the mating and pregnancy period.

No adverse effects were observed in reproductive parameters such as estrous cyclicity, copulation index, fertility index, number of females with live pups, length of gestation, number of implantation sites, gestation index. Further, no adverse effects were found in developmental parameters such as number of live pups born, sex ratio, birth index, number of dead pups on day 0 of lactation, number of pups born, delivery index, live birth index, number of live pups on day 4 of lactation, viability index, number of external anomalies, body weight of pups, and autopsy findings of neonates. External examination of offspring revealed no morphological abnormality. In conclusion, the NOAELs for the reproductive/developmental toxicity is considered to be 1000 mg/kg bw/day.

Reference 2

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: (P) 37 (±1) days at the beginning of treatment; (F1) x wks
- Weight at study initiation: (P) Males: 127.5 - 151.0 g; Females: 110.5 - 145.1 g; (F1) Males: x-x g; Females: x-x g
- Fasting period before study:
- Housing: During the study period, the rats were housed individually in Makrolon type M III cages (Becker & Co., Castrop-Rauxel, Germany), floor area of about 800 cm², with the following exceptions: 1) During overnight matings, male and female mating partners were housed together in Makrolon type M III cages. 2) Pregnant animals and their litters were housed together until PND 21 (end of lactation).
- Diet: ground Kliba maintenance diet mouse/rat “GLP” meal (Provimi Kliba SA, Kaiseraugst, Switzerland) ad libitum
- Water: ad libitum
- Acclimation period: (P) about 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 10 or 15 times
- Photoperiod (hrs dark / hrs light): 12 / 12


IN-LIFE DATES: From: To:

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The aqueous test substance suspensions were prepared at the beginning of the administration period and thereafter at intervals that took into account the analytical results of the stability verification. For the test substance preparation, the specified amount of test substance was weighed into an Erlenmeyer flask, topped up (shortly under the marking) with 1% Carboxymethylcellulose suspension in drinking water and four drops Cremophor EL and one drop of 32% hydrochloric acid. Afterwards the preparation was filled up with 1% Carboxymethylcellulose suspension in drinking water. The Erlenmeyer flask was sealed and the preparation was intensely mixed with a magnetic stirrer. A magnetic stirrer was used to keep the preparations homogeneous during treatment of the animals.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: overnight for a maximum of 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy (= GD 0)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the test substance preparations were sent to the analytical laboratory ten times during the study period (among other things at the beginning and towards the end) for verification of the concentrations. The samples, which were taken for the concentration control analyses at the beginning of the administration period, were also used to verify the homogeneity for the samples of the low and the high concentrations (50 and 400 mg/kg bw/d). Three samples (one from the top, middle and bottom in each case) were taken for each of these concentrations from the beaker with a magnetic stirrer running.

Duration of treatment / exposure:

until one day before sacrifice

Frequency of treatment:

once daily

Details on study schedule:

- F1 parental animals not mated until 75 days after selected from the F1 litters.
- Selection of parents from F1 generation after weaning (PND 21).
- Age at mating of the mated animals in the study: [...] weeks

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

Dose / conc.:

400 mg/kg bw/day (nominal)

No. of animals per sex per dose:

F0 generation parental animals: 25
F1 generation parental animals: 25

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily on working days or once daily (Saturday, Sunday or on public holidays)


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily


BODY WEIGHT: Yes
- Time schedule for examinations: first day of the premating period and then once a week at the same time of the day (in the morning) until sacrifice. The following exceptions are notable for the female parental animals: 1) During each gestation period the F0 and the F1 generation parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20. 2) Females showing no positive evidence of sperm in vaginal smears were weighed once a week during the mating interval (solely for calculation of dose volume). 3) Females with litter were weighed on the day after parturition (PND 1) and on PND 4, 7, 14 and 21. 4) Females without litter were weighed once a week during the lactation phase (solely for calculation of dose volume).



OTHER:
- Food consumption: In general, food consumption was determined once a week for the male and female F0 and F1 parental animals. After the 10th test week, food consumption of the females during pregnancy (animals with evidence of sperm) was determined weekly for GD 0-7, 7-14 and 14-20. During the lactation period (animals with litter) food consumption was determined for PND 1-4, 4-7, 7-14 and 14-21.

Oestrous cyclicity (parental animals):

Estrous cycle length and normality were evaluated daily for all F0 and F1 female parental rats for a minimum of 3 weeks prior to mating. The evaluations were continued throughout the mating period until the female exhibited evidence of mating. Moreover, at the scheduled necropsy a vaginal smear was microscopically examined to determine the stage of the estrous cycle for each F0 and F1 female.

Sperm parameters (parental animals):

Parameters examined in all male parental generations:
testis weight, epididymis weight, cauda epididymis weight, prostate weight, seminal vesicles including coagulation glands weight, sperm head count in testis, sperm head count in cauda epididymis, sperm motility, sperm morphology

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.


PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, clinical symptoms, sexual maturation


GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE AND GROSS NECROPSY
All F0 and F1 parental animals were sacrificed by decapitation under Isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology; special attention was given to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
Weight assessment was carried out on all animals sacrificed at scheduled dates. The following weights were determined: Anesthetized animals, liver, kidneys, adrenal glands, testes, epididymides, cauda epididymis, prostate, seminal vesicles including coagulation glands, ovaries, uterus, spleen, brain, pituitary gland, thyroid glands (with parathyroid glands). The following organs or tissues of the F0 and F1 generation parental animals were fixed in 4% neutral buffered formaldehyde solution or in BOUIN’s solution, respectively: Vagina, cervix uteri, uterus, ovaries (BOUIN), oviducts, left testis (BOUIN), left epididymis (BOUIN), seminal vesicles, coagulation glands, prostate, pituitary gland, adrenal glands, liver, kidneys, spleen, brain, thyroid glands (with parathyroid glands), all gross lesions. After fixation, the organs fixed in BOUIN´s solution were embedded in Paraplast. Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings.

OTHER:
- Differential Ovarian Follicle Count (DOFC) in F1 generation: From both ovaries (”ovary 1” and “ovary 2”) of F1 female animals (control and top dose), five sections were taken from the proximal and the distal part of the ovaries, respectively, at least 100 µm apart from the inner third of the ovary. All ovarian sections were prepared and evaluated. Primordial follicles and growing follicles were counted by light microscope (magnification: 100x) on each of these slides, – according to the definitions given by Plowchalk et al. (PLOWCHALK, D. R., B. J. SMITH, and D. R. MATTISON: Assessment of Toxicity to the Ovary Using Follicle Quantitation and Morphometrics. In: Methods in Toxicology, Vol. 3, Part B: Female Reproductive Toxicology (J. J. HEINDEL and R. E. CHAPIN, Editors), p. 57-68, 1993, Academic Press). To prevent multiple counting on serial slides – especially of the growing follicles – only follicles with an oocyte with visible chromatin on the slide were counted. The number of each type of follicle was recorded individually for ovary 1 and ovary 2 of every animal on any of the slide levels (level 1-10), giving in summary the incidence of each type of the follicles by using EXCEL sheets for the reporting of the results. Finally, the results of all types of follicles were summarized for all animals per group in dose groups 10 and 13. As primordial follicles continuously develop into growing follicles, the assessment of the follicles was extended to the combined incidence of primordial plus growing follicles. In general, the fifth slide of the left and right ovary was evaluated for histological findings. Whenever the diagnosis: ”no abnormalities detected” was used for the ovaries, this implicates all functional statuses of follicles, especially corpora lutea, were present. An attempt was made to correlate gross lesions with histopathological findings.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals were sacrificed at 4 days of age. All F2 offspring were sacrificed after weaning (~ PND 21).
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- All pups were examined externally and eviscerated; their organs were assessed macroscopically.

HISTOPATHOLOGY / ORGAN WEIGHTS
Animals with notable findings or abnormalities were further evaluated on a case-by-case basis, depending on the type of finding noted. After the scheduled sacrifice the brain, spleen and thymus of 1 pup/sex and litter from the F1 and F2 pups were weighed. Normally, the first male and the first female pups/litter were taken for these determinations. For the calculation of the relative organ weights, the pup body weights, determined routinely during the in-life phase on PND 21, were used.

Reproductive indices:

- Male reproduction data: The mating partners, the number of mating days until vaginal sperm could be detected in the female, and the gestational status of the female were noted for F0 and F1 breeding pairs. For the males, mating and fertility indices were calculated for F1 and F2 litters according to the following formulas: Male mating index (%) = (number of males with confirmed mating)/(number of males placed with females) x 100. Male fertility index (%) = (number of males proving their fertility)/(number of males placed with females) x 100.
- Female reproduction and delivery data: The mating partners, the number of mating days until vaginal sperm were detected, and gestational status were recorded for F0 and F1 females. For the females, mating, fertility and gestation indices were calculated for F1 and F2 litters according to the following formulas:
Female mating index (%) = (number of females mated)/(number of females placed with males) x 100.
Female fertility index (%) = (number of females pregnant)/(number of females mated) x 100.
Gestation index (%) = (number of females with live pups on the day of birth)/(number of females pregnant) x 100.
The total number of pups delivered and the number of liveborn and stillborn pups were noted, and the live birth index was calculated for F1 and F2 litters according to the following formula:
Live birth index (%) = (number of liveborn pups at birth)/(total number of pups born) x 100.
The implantations were counted and the postimplantation loss (in %) was calculated according the following formula:
Postimplantation loss (%) = (number of implantations – number of pups delivered)/(number of implantations) x 100.

Offspring viability indices:

The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4, 5-7, 8-14 and 15-21 (lactation period) were determined; however, pups, which died accidentally or had to be sacrificed due to maternal death, were not included in these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation days 4, 7, 14, and 21. Furthermore, viability and lactation indices were calculated according to the following formulas:
Viability index (%) = (number of live pups on day 4 (before culling) after birth)/(number of live pups on the day of birth) x 100.
Lactation index (%) = (number of live pups on day 21 after birth)/(number of live pups on day 4 (after culling) after birth) x 100.

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Test group 03 (400 mg/kg bw/d)
• Statistically significantly decreased food consumption in parental males during premating weeks 5 - 10 (up to 7%)
• Statistically significantly decreased food consumption in parental females during premating weeks 1 - 3 (up to 6%), weeks 5 - 8 (up to 7%) and weeks 9 - 10 (about 6%)
• Statistically significantly decreased food consumption in parental females during gestation days 0 - 7 (about 10%)
• Statistically significantly decreased food consumption in parental females during lactation days 4 - 7 (about 7%)

Test group 02 (150 mg/kg bw/d)
• Statistically significantly decreased food consumption in parental females during premating weeks 1 - 2 (about 5%)
• Statistically significantly decreased food consumption in parental females during gestation days 0 - 7 (about 7%)

Test group 01 (50 mg/kg bw/d)
• no test substance-related adverse effects/findings

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Under the conditions of the present 2-generation reproduction toxicity study the NOAEL (no observed adverse effect level) for general, systemic toxicity is 400 mg/kg bw/d for the F0 parental rats, the highest dose tested.
The NOEL (no observed effect level) is 50 mg/kg bw/d for the F0 parental rats based on effects on food consumption being a consequence of reduced appetite observed at the LOEL (Lowest Observed Effect Level) of 150 mg/kg bw/d in the F0 parental females.
The NOAEL for fertility and reproductive performance for the F0 parental rats is 400 mg/kg bw/d, the highest dose tested.

Dose descriptor:

NOEL

Remarks:

food consumption

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

food consumption and compound intake

Dose descriptor:

LOEL

Remarks:

food consumption

Effect level:

150 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

food consumption and compound intake

Remarks on result:

other: consequence of reduced appetite observed in the F0 parental females

Dose descriptor:

NOAEL

Remarks:

fertility and reproductive performance

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no adverse effects observed

Dose descriptor:

NOAEL

Remarks:

General systemic toxicity

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no adverse effects observed

Critical effects observed:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Test group 03 (400 mg/kg bw/d)
Statistically significantly decreased food consumption in parental males during premating weeks 0 - 1 (about 14%) and weeks 8 - 10 (up to 7%)
• Statistically significantly decreased food consumption in parental females during premating weeks 0 - 1 (about 10%) and weeks 9 - 10 (about 8%)
• Statistically significantly decreased food consumption in parental females during gestation days 0 - 14 (up to 8%)
• Statistically significantly decreased body weights in parental males during weeks 0 - 5 (up to 17%) and weeks 10 - 11 (up to 6%)
• Statistically significantly decreased body weights in parental females during premating weeks 0 - 1 (up to 16%)

Test group 02 (150 mg/kg bw/d)
• no test substance-related adverse effects/findings

Test group 01 (50 mg/kg bw/d)
• no test substance-related adverse effects/findings

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

The NOEL (no observed effect level) is 50 mg/kg bw/d for the F1 parental rats based on effects on food consumption being a consequence of reduced appetite observed at the LOEL (Lowest Observed Effect Level) of 150 mg/kg bw/d in the F0 parental females.
The NOAEL for fertility and reproductive performance for the F1 parental rats is 400 mg/kg bw/d, the highest dose tested.
The NOAEL for developmental toxicity, in the F1 of the test substance is 400 mg/kg bw/d, the highest dose tested.

Dose descriptor:

NOEL

Remarks:

food consumption

Generation:

F1

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

food consumption and compound intake

Remarks on result:

other:

Remarks:

effects on food consumption beeing a consequence of reduced appetite observed at the LOEL of 150 mg/kg bw/d in the F0 parental females

Dose descriptor:

NOAEL

Remarks:

fertility & reproductive performance

Generation:

F1

Effect level:

400 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no adverse effects observed

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

400 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no dverse effects observed

Reproductive effects observed:

no

- Analytics:

The various analyses:

• demonstrated the stability of the test substance preparations over a period of 7 days at room temperature
• confirmed the homogeneous distribution of the test substance in the vehicle (1% Carboxymethylcellulose suspension in drinking water and a few drops Cremophor EL and one drop hydrochloric acid)
• showed that the prepared concentrations were close to the expected values ranging between 86.8 and 113.2% of the nominal concentrations

Analytical values (range):

Test group	Nominal Dose (mg/kg bw/d)	Analytical Dose (mg/kg bw/d) [minimum]	Analytical Dose (mg/kg bw/d) [maximum]	% Nominal Dose [minimum]	% Nominal Dose [maximum]
00 / 10	0	0	0
01 / 11	50	43.40	46.61	86.8	93.2
02 / 12	150	132.90	169.80	88.6	113.2
03 / 13	400	359.20	379.90	89.8	95.0

 

Conclusions:

The NOAEL for general, systemic toxicity is 400 mg/kg/d for the parental rats in the highest dose tested.
The NOEL 50 mg/kg bw/day for the P parental rats, based on effects on food consumption observed at the LOAEL of 150 mg/kg bw/day in the P parental females.
The NOAEL for fertility and reproductive performance for the P and F1 parental rats is 400 mg/kg bw/day, the highest dose tested.
The NOAEL for developmental toxicity, in the F1 and F2 progeny, of the test substance is 400 mg/kg bw/day, the highest dose tested.

Executive summary:

The study was performed according to OECD TG 416 in compliance with GLP. Methyl Methacrylate was administered to groups of 25 male and 25 female healthy young Wistar rats (P parental generation) as an aqueous preparation by stomach tube at dosages of 0; 50; 150 and 400 mg/kg body weight/day. At least 73 days after the beginning of treatment, P animals were mated to produce a litter (F1). Mating pairs were from the same dose group and F1 animals selected for breeding were continued in the same dose group as their parents. Groups of 25 males and 25 females, selected from F1 pups to become F1 parental generation, were treated with the test substance at dosages of 0; 50; 150 and 400 mg/kg body weight/day post weaning, and the breeding program was repeated to produce F2 litter. The study was terminated with the terminal sacrifice of the F2 weanlings and F1 adult animals.

Control parental animals were dosed daily with the vehicle (1% Carboxymethylcellulose suspension in drinking water and four drops Cremophor EL and one drop hydrochloric acid).

The mid- and high-dose parental animals (400 mg/kg bw/d) showed clinical signs of systemic toxicity. The only relevant clinical observation was temporary salivation during a short period after dosing, which is considered to be test substance-induced. From the temporary, short appearance immediately after dosing it is likely, that this finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. It is, however, not considered to be an adverse toxicologically relevant finding.

In the mid- and high-dose (150 and 400 mg/kg bw/d) P generation animals, dose-related intermittent reductions of food consumption were noted, either during premating, gestation and lactation phases of this study. Less significant changes were noted for the F1 generation animals where the effects were limited to the high-dose group.

High dose F1 parental males had statistically significant lower body weights during several study segments, which led to a statistically significant reduction of the mean terminal body weight resulting in secondary weight changes of brain.

High dose parental females had statistically significant lower body weights during the first weeks after weaning. This weight decrease during major phases of sexual maturation led to an apparent marginal delay of vaginal patency. This minor delay did, however, not result in any corroborative pathological findings nor did it adversly effect F1 female cyclicity, fertility and reproduction. Thus, an influence of the test substance on female sexual maturation is not assumed.

Pathological examinations revealed no test-substance-related changes in organ weights, gross lesions, changes in differential ovarian follicle counts or microscopic findings, apart from an increase in kidney and liver weights in male and female animals in both generations which is presumably related to the treatment. There was no histopathologic lesion observed, that could explain the weight increase. It is regarded to be an adaptive change, most likely caused by an increase in metabolic activity in the two organs, which does not lead to histopathologic findings. It is not regarded to be an adverse effect.

There were no indications from clinical examinations as well as gross and histopathology, that the administration of methyl methacrylate via the diet adversely affected the fertility or reproductive performance of the P or F1 parental animals up to and including a dose of 400 mg/kg bw/day. Estrous cycle data, mating behavior, conception, gestation, parturition, lactation and weaning as well as sperm parameters, sexual organ weights and gross and histopathological findings of these organs (including differential ovarian follicle counts in the F1 females) were comparable between the rats of all test groups and ranged within the historical control data of the test facility.

All data recorded during gestation and lactation in terms of embryo-/fetal and pup development gave no indications for any developmental toxicity in the F1 and F2 offspring up to a dose level of 400 mg/kg bw/day. Up to this dose level, the test substance did not adversely influence pup viability and pup body weights. Sex ratio and sexual maturation was not directly affected at any dose level, inclusive the high-dose group (400 mg/kg bw/day).

Conclusion:

Under the conditions of the present 2-generation reproduction toxicity study the NOAEL(no observed adverse effect level) forgeneral, systemic toxicityis 400 mg/kg bw/d for the parental rats, the highest dose tested.

The NOEL (no observed effect level) is 50 mg/kg bw/d for the F1 parental rats based on effects on food consumption being a consequence of reduced appetite observed at the LOEL (Lowest Observed Effect Level) of 150 mg/kg bw/d in the F0 parental females.

The NOAEL for fertility and reproductive performance for the F1 parental rats is 400 mg/kg bw/d, the highest dose tested.

 The NOAEL for developmental toxicity, in the F1 of the test substance is 400 mg/kg bw/d, the highest dose tested.

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

Reference 3

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: no guideline available

Principles of method if other than guideline:

Test material was administered in drinking water of male and female rats through 2 generations.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

Number of animals: 10/sex/treatment for Parent and F1.No additional information available.

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on exposure:

ADMINISTRATION / EXPOSURE- Test durations: until F2-generation was 100 days of age- Premating period: 8 weeks- Exposure period: 12 weeks (until weaning of F1)- Route of administration: oral (gavage, dose volume 10 mL/kg)- Doses: 20% solution in water, ~2000 mg/kg bw

Details on mating procedure:

not indicated (starting when females were between 170 and 215 g)

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No additional information available.

Duration of treatment / exposure:

8-12 weeks (starting before mating and continuing, in females, until weaning).- Test durations: until F2-generation was 100 days of age- Premating period: 8 weeks- Exposure period: 12 weeks (until weaning of F1)

Frequency of treatment:

Daily

Details on study schedule:

Test durations: until F2-generation was 100 days of age- Premating period: 8 weeks- Exposure period: 12 weeks (until weaning of F1)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10/sex/treatment for Parent and F1

Control animals:

yes, concurrent vehicle

Details on study design:

No additional information available.

Positive control:

No data.

Parental animals: Observations and examinations:

Clinical observations: frequency not indicated- Estrous cycle: in F1 and F2 between 60 and 100 days- Body weight: in F1 and F2 during day 15 and 60 at 2-dayintervals

Oestrous cyclicity (parental animals):

Estrous cycle: in F1 and F2 between 60 and 100 days

Sperm parameters (parental animals):

Sperm examination: not performed

Litter observations:

Litter size and survival were monitored

Postmortem examinations (parental animals):

No additional information available.

Postmortem examinations (offspring):

ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC): - Organ weights F1 and F2: pituitary, thyroid, adrenals,ovaries, testicles- Histopathology F1 and F2: endocrine system organs on 26animals

Statistics:

No additional information available.

Reproductive indices:

No additional information available.

Offspring viability indices:

No additional information available.

Clinical signs:

not specified

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: sperm measures:

not specified

Reproductive performance:

no effects observed

No effects were found on the reproductive efficiency of the parents.-Onset of oestrus cycle and weight and microscopy of the endocrine organs were comparable to control values for both F1 and F2 animals.-In the parent generation all 10 females became pregnant (litter size 9.0, controls 8.1) and in the F1 9/10 females became pregnant (litter size 8.7, controls 8.1).

Dose descriptor:

NOAEC

Effect level:

> 2 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no effects observed

Critical effects observed:

no

Clinical signs:

not specified

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Description (incidence and severity):

males not examined

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

no effects observed

There was no effect on the growth, fertility and reproductive performance of the F1 generation, and no histological changes occurred in the tissues of both the F1 and F2 generation.

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

> 2 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Remarks on result:

other: no effects observed

Critical effects observed:

no

Reproductive effects observed:

no

No effects were found on the reproductive efficiency of the parents, nor on the growth, fertility and reproductive performance of the untreated F1 generation, and no histological changes occurred in the tissues of both the F1 and F2 generation.
Onset of oestrus cycle and weight and microscopy of the endocrine organs were comparable to control values for both F1 and F2 animals.
In the parent generation all 10 females became pregnant (litter size 9.0, controls 8.1) and in the F1 9/10 females became pregnant (litter size 8.7, controls 8.1).

Conclusions:

Glycerin was administered by oral gavage to groups of male and female rats through two generations. There was no effect noted on growth, fertility and reproductive performance through two generations.

Executive summary:

Glycerin was administered by oral gavage to groups of male and female rats through two generations. There was no effect noted on growth, fertility and reproductive performance through two generations at a dose level of ~2000 mg/kg/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

No study is available for GDMA. Reliable, relevant and adequate studies are however available on reproductive toxicity conducted with the analogous substance of the primary metabolites, Hydropropyl Methacrylate (HPMA, screening), and the secondary metabolites MAA/MMA and Glycerol as follows. Like other investigated substances from this category, GDMA is expected to be rapidly hydrolysed by carboxylesterases in two subsequent steps to Methacrylic Acid (MAA) and the respective alcohol Glycerol, with HPMA as analogue of the primary metabolites after the first hydrolysis step. The read across is justified in detail in the attached category document.

 

HPMA (analogous substance of the primary metabolites of GDMA)

As a part of the Japanese HPV program HPMA has been evaluated for reproduction toxicity in Sprague-Dawley rats in the OECD 422 combined repeat-dose developmental/ reproductive toxicity screening test in 1996.

Here, male rats (12/group) were given daily gavage doses of 0 (vehicle), 30, 100, 300 or 1000 mg/kg for 50 days including pre-mating, mating and post-mating intervals. Females (12/group) were administered the same doses for two weeks prior to mating, during mating and gestation up until day 4 of lactation (study duration of approximately 54 days depending upon time to conception). Animals were observed for clinical symptoms of intoxication daily and food consumption and body weight were monitored throughout the study. Blood samples were taken for hematological and clinical chemistry analysis at study termination. Thymus, liver kidney, testes, epididymes, and ovaries were weighed. In addition to these tissues, adrenal gland, brain, heart and spleen were fixed in 10% neutral buffered formalin solution for subsequent staining and histopathological evaluation.

Reproductive observations included observations which permit characterization of male and female fertility and fecundity. These include: number of live births and post implantation loss; number of pups with grossly visible abnormalities, number of runts; number of implantations, corpora lutea, litter size and litter weights. Copulation, fertility, implantation, gestation, live birth, delivery, and viability indices were calculated from the data. Pup sex, body weights and viability at birth through day 4 of lactation were determined; pups were autopsied at this time.

 There were no effects of HPMA on any fertility related reproductive index including: estrus frequency, copulation index, number of days to conception, fertility index, length of gestation, number of corpora lutea and gestation index. Further, there were no effects on the number of live pups born, birth index, number of dead pups, number of pups born, delivery index, live birth index.. No histological changes in male and female reproductive organs were described.

The NOAEL for reproductive/developmental toxicity of HPMA was 1000 mg/kg/d, the highest dose tested.Two male and one female of 12 exposed animals died on study at the 1000 mg/kg dose level. Clinical symptoms of intoxication in parenteral animals observed at 1000 mg/kg included: salivation, decrease in locomotor activity and ptosis for both sexes. Liver weight was increased in males only at 1000 mg/kg/d, with minimal hepatocyte vacuolation as a histopathological correlate. Also in males, a decrease in RBCs was observed at 1000 mg/kg only. The parenteral NOAEL for these effects was considered to be 300 mg/kg for both males and females.

 

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.

Glycerol (metabolite of GDMA; endogeneous metabolite in mammals)

Male and female rats (10/treatment) were dosed daily with glycerol by gavage (20% solution in water; approx. 2 g/kg bw/d) during 8 weeks before mating until weaning of the F1 generation in a two generation study (in total 12 weeks). Females received glycerol throughout pregnancy or until weaning of the F1 generation (5 each). When the F1 generation was ~100 days of age, pups were killed except for 10/sex. These animals were used to produce the F2-generation. The study was terminated when F2-generation was 100 days of age. OECD (2002) stated that the study did not fully matched current OECD Guidelines and evaluated it as valid with restrictions. No effects were found on the reproductive efficiency of the parents, nor on the growth, fertility, reproductive performance of the untreated F1 generation, and no histological changes occurred in the tissues of both the F1 and F2 generation. Although the data were considered as limited, a NOAEL of 2000 mg/kg bw was identified (Wegener 1953).

Summary

Based on the results of a reliable screening study with HPMA as analogous substance of the primary metabolites and two gen fertility studies with the methacrylic metabolite donor substance MMA and the alcohol metabolite glycerol, exposure to GDMA is not likely to result in reproductive toxicity.

Compliance to REACh requirements

The screening study requirement is covered with a reliable OECD 422 oral rat study, performed with HPMA, the analogous substance of the primary metabolites of GDMA. The read across is done with a medium level of confidence as a rapid hydrolysis is expected but not confirmed experimentally (see above table and chapters1.1and5.1.1).

Effects on developmental toxicity

Description of key information

No data are available for GDMA. However, screening data from the analogous substance of the primary metabolites, H

PMA allow the initial evaluation of developmental toxicity hazard of GDMA.

Read-across to the metabolites is justified by the fact that carboxylesterases are ubiquitous in the body and half-lifes of the other category substances are only a few minutes (see category document, chapter 5.1). In the body, methyl methacrylate hydrolyses rapidly to methacrylic acid and thus serves as methacrylic acid donor in several test systems investigating systemic effects.

Following data were considered for read across:

OECD 422 repeated dose/ reproduction screening study, rat, gavage (GLP): parenteralNOAEL = 300 mg/kg bw/d;

reproduction

NOAEL = 1000 mg/kg bw/d; read-across from the analogous substance of the primary metabolites HPMA (Nihon Bioresearch 1996)

Summary

Based on the results of a reliable screening study with the analogous substance of the primary metabolites, HPMA, exposure to GDMA is not likely to result in developmental toxicity.

Supportive data from both the methacrylic and alcohol metabolites are available in the category document.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study, GLP, japanese study, only abstract english

Qualifier:

according to guideline

Guideline:

other: OECD guideline 422

Deviations:

yes

Remarks:

, An old version of OECD 422 (not containing functional observation battery test) had been conducted.)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan
- Age at study initiation: 8 weeks
- Weight at study initiation: male 315 ~ 359 g; females 210 ~ 243 g,
- Fasting period before study: yes
- Housing: During the quarantine: suspended using a stainless steel cage type 1 with 5 per cage; Breeding: divided into separate rearing cages. Moved to a separate plastic cage, having had a natural birth .
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: five-day quarantine period and then set up a six-day acclimation period

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 deg C
- Humidity (%): 40-70%
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12 hour light / 12 hour dark

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Concentration of samples was prepared by dissolving the required water for injection. The concentrations of preparation is protectedc from light at room temperature for 7 days to ensure that there were no stability issues. Preparation of 0.6% solution concentration is below the threshold level of determination, because we could not confirm the stability during the preparation for the 6% solution diluted with water for injection prepared in concentration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): water
- Concentration in vehicle: 0.6, 0.8, 2.6 and 20%
- Amount of vehicle (if gavage): 5 ml/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

No data

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 14 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): moved to put a separate plastic cage, having had a natural birth and feeding.

Duration of treatment / exposure:

49 days

Frequency of treatment:

daily

Duration of test:

Exposure period: 49 days

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: 2-week preliminary study
- Rationale for animal assignment (if not random): random by weight

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day
- Cage side observations: general condition

DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: twice a week

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): 2 times per week
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood:day after treatment
- Anaesthetic used for blood collection: Yes (identity) : sodium pentobarbital
- Animals fasted: No data
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day after treatment
- Animals fasted: No data
- Parameters checked in table [No.2] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: sexual cycle until confirmation; status of delivery

GROSS PATHOLOGY: Yes; Males: Thymus, liver, kidney, testis and epididymis weight was measured after removal, adrenal gland, brain, heart and spleen and 10% neutral buffered formalin solution (However, testicular and epididymal fluid Buan) was fixed; Females: counting the number of corpora lutea and the number of implantation scars. Liver, kidney, ovary and thymus weight was measured after removal, adrenal gland, brain, heart and spleen with a fixed 10% neutral buffered formalin solution.

HISTOPATHOLOGY: Yes; Control group and 1000 mg / kg group of heart, liver, spleen, thymus, kidney, testis, epididymis, ovary, adrenal and brain for the Preparation HE staining of tissue was examined histologically.

Fetal examinations:

observed at birth
Mobilize the number of preterm birth and sex, number of stillborn children, the presence of abnormalities observed and the number of newborn.
(2) observation of the newborn
Newborns, the presence of daily survival and mortality in a general state who observed times.
(3) measurement of body weight
Weight, feeding 0 days (date of birth) and measured four days.

Autopsy performed after four days of feeding by exsanguination from the abdominal aorta under ether anesthesia.

Statistics:

In either test, significant risk factors were less than 5%.
1) multiple comparison test
Weight (the parent animals, babies), food consumption, number of estrus, days mating, pregnancy [Day delivery (feeding 0) - date confirmed mating, the number of implantation scars, the number of birth control mobilize (number of babies stillborn baby + ), the number of newborn, number of children born dead, birth rate [(number of birth control mobilize / number of implantation scars) × 100], rate of production of child [(number of infant feeding 0 days / number of implantation scars) × 100], corpus number, implantation rates [(number of implantation scars / number of corpora lutea) × 100], fertility [(number of infant feeding 0 day / mobilize all of birth control) × 100], feeding baby number four day, feeding 4 day survival rate [(number of newborn feeding 4 days / 0 Number of infant feeding day) × 100], unusual occurrence rate [(number of children with abnormal/ number of newborns) × 100], sex ratio (male / female), organ weights ( including the relative weight), results of blood tests, blood biochemistry test results for the mean and standard deviation were calculated for each group. Significant difference test, Bartlett's test and the homoscedasticity of Law, Law-way layout analysis of variance if the variance 1) and, if significant Dunnett method 2) or Scheff Method 3) were using. However, if the variance was not approved, the analysis method using centrally located position (Kruskal-Wallis test of 4)) and a significant if you use the ranking method or Dunnett Scheff Method was used.
2) χ ^ 2 test
Copulation rate [(number of established animal mating / number of live animals) × 100], fertility [(number of female fertility / Establishment of animal mating) × 100], the birth rate [(number of female newborns / number of female fertility) × 100] is, χ ^ 2 using the test.

Indices:

Reproductive indices
Estrus frequency, copulation index, number of days to conception, fertility index, length of gestation, number of corpora lutea or gestation index.

Offspring viability indices
Number of live pups born, birth index, number of dead pups, number of pups born, delivery index, live birth index, sex ratio, viability index

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
There were no effects of the test substance on the estrus frequency, copulation index, number of days to conception, fertility index, length of gestation, number of corpora lutea or gestation index.

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

clinical signs

mortality

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no effects of the test substance on the number of live pups born, birth index, number of dead pups, number of pups born, delivery index, live birth index, sex ratio, viability index, external anomalies, body weight or necropsy findings.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

In an OECD 422 study in rats, no effects in developmental toxicity related endpoints were seen up to 1000 mg/kg bw/d.

Executive summary:

The combined repeated dose toxicity study with the reproductive/developmental toxicity screening test [MHW Japan, 1996] was conducted by OECD TG 422 in compliance with GLP. SD (Crj: CD) rats (12 animals/dose/sex) were administrated with this substance by gavage at doses of 0 (vehicle; water for injection), 30, 100, 300, and 1,000 mg/kg bw/day (12 animal/dose/sex). Males were dosed for total of 49 days beginning 14 days before mating, and females were dosed for total of 41 to 48 days starting from 14 days before mating to day 4 of lactation throughout the mating and pregnancy period.

No adverse effects were observed in reproductive parameters such as estrous cyclicity, copulation index, fertility index, number of females with live pups, length of gestation, number of implantation sites, gestation index. Further, no adverse effects were found in developmental parameters such as number of live pups born, sex ratio, birth index, number of dead pups on day 0 of lactation, number of pups born, delivery index, live birth index, number of live pups on day 4 of lactation, viability index, number of external anomalies, body weight of pups, and autopsy findings of neonates. External examination of offspring revealed no morphological abnormality. In conclusion, no effects in developmental toxicity related endpoints were seen up to 1000 mg/kg bw/d.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

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

No study is available for GDMA. A reliable, relevant and adequate study is however available on screening level on developmental toxicity conducted with the primary metabolite Hydropropyl Methacrylate(HPMA). Like other investigated substances from this category, GDMA is expected to be rapidly hydrolysed by carboxylesterases in two subsequent steps to Methacrylic Acid (MAA) and the respective alcohol Glycerol, with HPMA as primary metabolite after the first hydrolysis step. The read across is justified in detail in the attached category document. Supportive data, namely on the methacrylic metabolites, are available and currently displayed in the category document but not in the CSR for REACh requirement reasons.

 

HPMA (analogous substance of the primary metabolites of GDMA)

For the evaluation of GDMA the data of HPMA as analogous substance of the primary metabolites are used. In the body, HPMA is closely comparable to the first metabolites resulting from the ester hydrolysis of GDMA. In the study for HPMA (Furuhashi, 1996), male rats (12/group) were given daily gavage doses of 0 (vehicle), 30, 100, 300 or 1000 mg/kg for 50 days including pre-mating, mating and post-mating intervals. Females (12/group) were administered the same doses for two weeks prior to mating, during mating and gestation up until day 4 of lactation (study duration of approximately 54 days depending upon time to conception). Animals were observed for clinical symptoms of intoxication daily and food consumption and body weight were monitored throughout the study. Blood samples were taken for hematological and clinical chemistry analysis at study termination. Thymus, liver kidney, testes, epididymes, and ovaries were weighed. In addition to these tissues,adrenal gland, brain, heart and spleen were fixed in 10% neutral buffered formalin solution for subsequent staining and histopathological evaluation.

Reproductive observations included observations which permit characterization of male and female fertility and fecundity. These include:number of live births and post implantation loss; number of pups with grossly visible abnormalities, number of runts; number of implantations,corpora lutea, litter size and litter weights. Copulation, fertility, implantation, gestation, live birth, delivery, and viability indices were calculated from the data. Pup sex, body weights and viability at birth through day 4 of lactation were determined; pups were autopsied at this time.

There were no effects of HPMA on any reproductive index. This includes reproductive parameters related to developmental toxicity including: number of live pups born, birth index, number of dead pups, number of pups born, delivery index, live birth index..

The NOAELs for reproductive/developmental toxicity of either HEMA or HPMA were 1000 mg/kg/d, the highest dose tested.Two male and one female of 12 exposed animals died on study at the 1000 mg/kg dose level. Clinical symptoms of intoxication in parenteral animals observed at 1000 mg/kg included: salivation, decrease in locomotor activity and ptosis for both sexes. Liver weight was increased in males only at 1000 mg/kg/d, with minimal hepatocyte vacuolation as a histopathological correlate. Also in males, a decrease in RBCs was observed at 1000 mg/kg only. The parenteral NOAEL for these effects was considered to be 300 mg/kg for both males and females.

Summary

Based on the results of a reliable screening study with HPMA as analogous substance of the primary metabolites, exposure to GDMA is not likely to result in developmental toxicity.

Supportive data from both the methacrylic and alcohol metabolites are available in the category document.