2-dimethylaminoethyl methacrylate

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

concentration-driven

Effects on fertility

Description of key information

There is only one study with MADAME available for assessment on toxicity to reproduction. A combined repeated dose and reproductive toxicity study by the oral route (MHW Japan, 1998) was identified as the key study because it was conducted according to OECD guideline 422 in compliance with GLP. The substance was administered at 0/ 40/ 200 and 1,000 mg/kg bw/d by gavage. The NOAEL for systemic and reproductive effects was considered to be 200 mg/kg bw/d.

The methacrylic metabolite donor substance, Methyl methacrylate, was analysed in an OECD 416 study in rats. The substance was administered by gavage at 0/ 50/ 150 and 400 mg/kg bw/d based on th results of a dose range finder study. The NOAEL for systemic effects was considered to be 50 mg/kg bw/d. For reproductive effects, a NOAEL of 400 mg/kg bw/d was derived.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

not specified

Deviations:

yes

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

TEST MATERIAL:
- Name of test material: 2-(dimethylamino) ethyl methacrylate
- Supplier: Sanyo-Kasei Co.
- Purity: 99.9%

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Japan Charles River Co. Ltd.
- Age at study initiation: 9 weeks
- Weight at study initiation: 325-360 g (males); 191-242 g (females)
- Housing:
- Diet (e.g. ad libitum): CRF-1, Oriental Yeast Co., Ltd. (ad libitum)
- Water (e.g. ad libitum): UV-irradiated tap water filtered through 5 µm filter (ad libitum)
- Acclimation period: 6 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-2°C
- Humidity (%): 55+/-15 %
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Oral administration was conducted according to the guidelines. The administration periods were 14 days before mating for both sexes, then during the mating period, and for males, a total of 43 days until the day before autopsy, and 41-52 days for females after mating, pregnancy and delivery and up to the 3rd day of lactation, where it was orally administered once daily in the morning using a gastric tube. Unmated and nulliparous females were administered until the day before necropsy (41 days).

The test substance was dissolved in corn oil (Junsei Chemical Co., Ltd .: lot number 5K2189, 6K2104) for each dose. Although water-soluble, it was confirmed that the test substance reacts with water, so corn oil was used as the vehicle. The administration solution was prepared once a week, refrigerated until use, and used within 8 days of preparation.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: sperm found in vaginal plug or vaginal smear specimen referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: not specified
- After successful mating each pregnant female was caged individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in the administration solution was confirmed under room temperature storage conditions for 5 hours and refrigerated storage conditions for 8 days in the range of 2 to 200 mg / ml before the start of administration. In addition, the administration solution of each dose group was analyzed at the time of initial preparation, and it was confirmed that the concentration was within ± 10% of the set concentration (please see attached material).

Duration of treatment / exposure:

Males: 43 days (starting 14 days before mating)
Females: from 14 days before mating to day 3 of lactation (41 -52 days)

Frequency of treatment:

once daily

Dose / conc.:

40 mg/kg bw/day (actual dose received)

Dose / conc.:

200 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:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: As the LD50 value of > 2000 mg/kg was known, a preliminary test to decide the highest dose level at 30, 100, 300, and 1000 mg/kg/day for 14 days was conducted. At 1000 mg/kg/day, decrease of body weight in males and suppression of body weight increase in females were observed. Then the highest dose level for the test was set at 1000 mg/kg/day.
- Premating exposure period: 14 days
- Duration of test: 41 -52 days
- Post-exposure period: 1 day

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT:
- Time schedule for examinations: 3,7,14 days after the start of administration and once week thereafter; Mated females weighed at 0, 7, 14, 20 days of gestation and 0,4 days of lactation

FOOD CONSUMPTION AND COMPOUND INTAKE
We measured the animals’ weight including the tare using the above balance at administration start date and at 3, 7, 14 days thereafter for both males and females, for males once a week except during the mating period, mated females on 0, 7, 14, 20 days gestation and 0, 4 days of lactation, thereby calculating the average daily food intake per animal.

OPHTHALMOSCOPIC EXAMINATION: Not examined

HAEMATOLOGY:
- Time schedule for collection of blood: on the day of necropsy (from all surviving males; females were not examined)
- Parameters checked:
(1) Erythrocyte count
(2) Hemoglobin concentration
(3) Hematocrit level
(4) Mean red blood cell volume (MCV)
(5) Mean red blood cell hemoglobin (MCH)
(6) Mean red blood cell hemoglobin concentration (MCHC)
(7) Reticulocyte count
(8) Platelet count
(9) Leukocyte count
(10) Leukocyte percentage

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: on the day of necropsy (from all surviving males; females were not examined)
- Animals fasted: Not specified
- Parameters checked
(1) ASAT (GOT)
(2) ALAT (GPT)
(3) LDH
(4) γ GT
(5) ALP
(6) Total bilirubin
(7) Urea nitrogen
(8) Creatinine
(9) Glucose
(10) Total cholesterol
(11) Triglyceride
(12) Total protein
(13) Albumin
(14) A/G ratio
(15) Calcium
(16) Inorganic phosphorus
(17) Sodium
(18) Potassium
(19) Chlorine

URINALYSIS:
- Time schedule for collection of urine: 6 males from each group, two days before necropsy (females were not examined)
- Metabolism cages used for collection of urine: Yes
- Parameters checked:
(1) pH
(2) Protein
(3) Glucose
(4) Ketones
(5) Bilirubin
(6) Occult blood
(7) Urobilinogen
(8) Urine amount
(9) Specific gravity
(10) Urine sediment

NEUROBEHAVIOURAL EXAMINATION: Not examined

IMMUNOLOGY: Not examined

Oestrous cyclicity (parental animals):

not specified

Sperm parameters (parental animals):

not specified
Parameters examined in [all/P/F1/F2] male parental generations:
[testis weight, epididymis weight, daily sperm production, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology, other:]

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No, on Day 4 of lactation, all pups were euthanised and autopsied.

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

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death wasdetermined for pups born or found dead

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were necropsied after euthanasia by cutting and exsanguination of the abdominal aorta, under anesthesia by intraperitoneal administration of sodium thiopental
- Maternal animals: All surviving animals were necropsied after euthanasia by cutting and exsanguination of the abdominal aorta, under anesthesia by intraperitoneal administration of sodium thiopental

GROSS NECROPSY
- not specified

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs and tissues of all animals were collected, fixed with 10% neutral phosphate buffered formalin solution, and stored. Exceptionally, the eyeballs and Harder glands of animals other than dead animals were fixed with Davidson's solution, and the testes and epididymis were fixed with Buan's solution.
Brain, pituitary gland, eyeball / Harder gland, salivary gland (lower jaw / sublingual), thymus, lymph node (lower jaw / mesentery), trachea, lung, esophagus, stomach, intestinal tract (duodenum, intestinum jejunum, ileum, cecum, colon) , Ileum), thyroid / epithelial body, heart, liver, pancreas, spleen, kidney, adrenal gland, bladder, prostatic abdominal lobe, testis, epididymis, seminal vesicle, ovary, uterus, vagina, bone marrow (thymus / femoral bone), sciatic bone nerve, spinal gland, thymus, and any sites exhibiting macroscopic abnormality
For histopathological examination, hematoxylin and eosin-stained specimens were prepared and microscopically examined for the following organs, tissues, and grossly abnormal sites of all animals in all males and females in the control group and 1000 mg / kg group. In addition, dead animals and mother animals in which all newborns died were examined in the same manner.
Brain, pituitary gland, hypothymus, thymus, trachea, lung, stomach, intestinal tract, heart, liver, spleen, kidney, adrenal gland, pancreas, bladder, bone (sternum / femur), bone marrow (sternum / femur), lymph node (lower jaw / mesentery), sciatic nerve, bone marrow, testis, epididymis, prostatic abdominal lobe, sperm, ovary, uterus, vagina, mammary gland (female)
As a result of the examination, changes that seemed to be caused by the test substance were found in the brain, spinal cord, stomach and thymus, therefore the brain, spinal cord, stomach and thymus of males and females in the 40 and 200 mg / kg groups were also examined. In addition, the prostate and seminal vesicles of the male 40 and 200 mg / kg groups were also examined.

Postmortem examinations (offspring):

On Day 4 of lactation, the external apperance including the oral cavita of all surviving infants was examined, and then they were euthanised and autopsied in the same manner as the parent animals.

Statistics:

The metric data were tested for homoscedasticity by the Bartlett method, with one-way ANOVA for equal variances and Kruskal-Wallis test for unequal variances. If there was a significant difference between the groups, Dunnett's method or Dunnett's multiple comparison was performed. However, the following items marked with * were performed from the Kruskal-Wallis test. Of the counted data, the findings of urinalysis and histopathological examination underwent a×b χ2 testing, and if a significant difference was found, a comparison between the control group and each test substance administration group was performed by Armitage χ2 testing. Other count data were tested by Fisher's exact test. The significance level was set to 5% or less. For the data on newborns, the mean value calculated for each mother animal was used as the statistical unit. The weight of non-pregnant animals after mating confirmation, food intake and organ weight, and organ weight of unmated females and mother animals without survivors were excluded from the evaluation. In addition, one male patient (No. 00401) in the 1000 mg / kg group had his upper incisor damaged by an accident 25 days after the start of administration, and subsequently showed poor weight gain, so data other than reproductive function tests were excluded from the aggregation. The target items for statistical analysis are as follows.
Multiple comparison test: Weight, weight gain, feeding amount, hematology test, blood biochemical test, urinalysis (urine volume, specific gravity), organ weight, number of days required for mating *, number of estrus periods missed until mating was established *, gestation period *, number of corpora luteum, number of implantations, rate of implantation *, delivery rate *, number of babies born, birth rate *, 4-day survival rate of newborns *
χ2 test and Armitage χ2 test: Urinalysis (pH, protein, glucose, ketone bodies, bilirubin, occult blood, urobilinogen, sediment), histopathological findings
Fish

Reproductive indices:

Mating rate, Conception rate, Gestation period, Birth rate, implantation rate, delivery rate

Offspring viability indices:

Birth rate, 4-day survival rate of newborns.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the 1000 mg / kg group, convulsions and Straub tail were observed in 4 and 3 cases from 36 days after the start of administration in males and 31 days in females, respectively, and clonic convulsions or irregular respiration were observed in some animals. These symptoms were developed by stimuli such as retention at the time of administration, persisted for several minutes, and then disappeared. In addition, salivation immediately after administration was observed in both males and females from 7 days after the start of administration. Some animals developed the conditions before administration, and by the end of administration, it was observed in almost all males and females.
In the 200 mg / kg group, similar salivation was observed in 4 females 14 days after the start of administration.
Loose stools, red tears, and hair loss were occasionally observed in each group, but they did not tend to occur frequently in the 1000 mg / kg group. It was therefore judged that there was no association with the test substance.

Mortality:

mortality observed, treatment-related

Description (incidence):

In the 1000 mg / kg group, convulsions and Straub tail were observed in 4 and 3 cases from 36 days after the start of administration in males and 31 days in females, respectively, and clonic convulsions or irregular respiration were observed in some animals. These symptoms were developed by stimuli such as retention at the time of administration, persisted for several minutes, and then disappeared. In addition, salivation immediately after administration was observed in both males and females from 7 days after the start of administration. Some animals developed the conditions before administration, and by the end of administration, it was observed in almost all males and females.
In the 200 mg / kg group, similar salivation was observed in 4 females 14 days after the start of administration.
Loose stools, red tears, and hair loss were occasionally observed in each group, but they did not tend to occur frequently in the 1000 mg / kg group. It was therefore judged that there was no association with the test substance.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Weight gain in the females in the 1000 mg / kg group showed significantly low values on the 7th and 14th days of gestation, and then remained low until the 4th day of lactation. Although no significant difference was observed in males, the body weight after mating tended to be low.
The weight gain of males in the 200 mg / kg group showed a significantly high value 41 days after the start of administration, but there was no increasing tendency in the 1000 mg / kg group, so it was judged to be coincidental change.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Females in the 1000 mg / kg group showed significantly higher values 7 and 14 days after the start of administration, but significantly lower values on the 4th day of lactation.
In males, no significant changes were observed between the control group and the test substance-administered group.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In the 200 mg / kg and above group, there were significantly low hemoglobin and hematocrit levels and a significantly high reticulocyte ratio; in the 1000 mg / kg group the number of red blood cells, mean corpuscular volume, and mean corpuscular hemorrhage showed significantly lower values. In the 1000 mg / kg group there was a significantly high white blood cell count, and by leukocyte percentage the number of lymphocytes was significantly low while the number of lobulated nuclei was significantly increased in terms of real numbers. The number of monocytes in the same group also showed a significantly high value, but no significant difference was observed in the percentage, and the value was within the range of the background data (3 to 9%, 1991-1996).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In the 1000 mg / kg group, urea nitrogen showed a significantly higher value and sodium showed a significantly lower value. No significant change was observed in other test items.

Endocrine findings:

not examined

Urinalysis findings:

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Changes due to the test substance were observed in the male and female brain, spinal cord, anterior stomach and female thymus. In the brain, degeneration of nerve fibers in the rubrospinal tract region of the pons was observed symmetrically in 3 males and 4 females in the 1000 mg / kg group. By spinal cord, degeneration of nerve fibers in the dorsal spinocericular tract was observed symmetrically in 8 males and 6 females in the 1000 mg / kg group, and a significant difference in the frequency of occurrence was observed between males and females. As for the anterior stomach, mucosal epithelial hyperplasia was observed in the 1000 mg / kg group in all males and females, including those who died (excluding those who died 2 days after the start of administration with marked postmortem changes); in many cases, edema and inflammatory cell infiltration were associated with the substructure of the further enlarged mucosa, and a significant difference was also observed in the frequency of occurrence. In female thymus, atrophy was observed in 2,0,2 and 6 patients in the control, 40, 200 and 1000 mg / kg groups, respectively, and although no significant difference was observed, it occurred frequently in the 1000 mg / kg group. Of these, 5 of 6 in the 1000 mg / kg group were dead or mothers with all newborns dead.
In the 1000 mg / kg group among the female deaths, lung edema was observed in all cases, white pulp atrophy of the spleen was observed in 2 cases, and lymph node atrophy of the lower jaw and mesenteric lymph nodes was observed in 1 case.
Bleeding was observed in the uterus and thymus in the dead cases in the control group, and it was considered that the patient died due to weakening due to hemorrhaging in the uterus.
Various other changes were observed in each group including the control group, but since was no tendency for it to occur frequently in the 1000 mg / kg group, these was judged to be coincidental findings.

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

Observing delivery, one case in the control group (50102) started labor on the 23rd day of gestation. Only one newborn was delivered and she died the next day without completing delivery. Further, one case in the 200 mg / kg group (50301) delivered on the 24th day of pregnancy and did not show maternal behavior such as breastfeeding, baby licking, placental feeding, and collection behavior. All her newborns died by the first day of feeding. Other maternal animals showed normal delivery on the 22nd or 23rd day of gestation in each group. There were no significant differences between the control group and the test substance-administered group in terms of gestation period, birth rate, number of corpora luteum, number of implantations, implantation rate, and delivery rate.
Observing lactation, all neonatal deaths were observed in one litter in the 200 mg / kg group, which showed delayed delivery, and in three litters (50407, 50408, 50412) in the 1000 mg / kg group. Maternal behavior was observed in these maternal animals, but most of the newborns were poorly fed and all died by the second day of lactation. In addition, many unlactated newborns were observed on the day of delivery in the other litter (50406) in the 1000 mg / kg group, and most of them died. No abnormalities were found in the nursing status of other maternal animals.
Although no significant difference was observed in the 1000 mg / kg group, the number of newborns on the 4th day of lactation and the 4-day survival rate of the newborns were low. This change was caused by the frequent occurrence of neonatal deaths in the four litters where the mother animal showed abnormal nursing. Little neonatal death was observed in the other litter of the group. There were no significant difference between the control group and the test substance-administered groups in the number of babies born, the number of live births, the sex ratio and the birth rate.

The compound had no effects on reproductive parameteres such as the mating index, the fertility index, numbers of corpora Iutea or implantations, the implantation index, the delivery index, the gestation index, gestation length or parturition. Three dams of the 1000 mg/kg group, however, lost all their pups in the lactation period.
Significant adverse effects were observed in animals of the 1000 mg/kg/day group, especially in females. These adverse effects observed in females were as follows:
- 3 females out of 12 died.
- By the observation, late onset of twitching, chronic convulsion, suppression of body weight gain and a decrease in food consumption in lactation period were observed.
- By the histopathological examination, the degeneration of nerve fibers in the brain and the spinal cord, and the hyperplasia of the mucosa in gastric tract, the oedema and inflammatory ceII infiltration in the forestomach, and the atrophy of the thymus were revealed. Also the increases in the weight of the kidney and the adrenals without histopathological changes were observed.

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see remarks

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Treatment related:

yes

Dose response relationship:

not specified

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Although no significant difference was observed in the 1000 mg / kg group, the number of newborns on the 4th day of lactation and the 4-day survival rate of the newborns were low. This change was caused by the frequent occurrence of neonatal deaths in the four litters where the mother animal showed abnormal nursing. Little neonatal death was observed in the other litter of the group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the 1000 mg / kg group, both males and females showed significantly lower body weight on day 0 of lactation. In the group of 200 mg / kg or less, no significant change was observed between the control group and the test substance administration group in both males and females. In the 1000 mg / kg group, excluding the litter where all newborns died and the dead infants frequently occurred, the average body weight on day 0 of lactation was 7.1 g for males and 6.5 g for females, and the average weight gain was 4.0 g for males and 3.8 g for females, which were almost the same as those in the control group.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Subcutaneous hemorrhage was observed in one control group case on the day of birth. Other than that, there was no change in the general condition and no abnormalities on the external appearance were observed except for the frequent occurrence of unlactated newborns in the 1000 mg / kg group.

Histopathological findings:

no effects observed

Description (incidence and severity):

No abnormalities were found in any of the live animals.
In addition, thymic cervical residue was observed in only 1 dead animal in the control group (50108) and 2 dead animals in the 1000 mg / kg group (50406,50410). It was judged that the change was not caused by the substance.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

On examination of neonates, the 1000 mg/kg dose was associated with a decrease in body weight and a low viability index. There were no significant differences in the number of offspring or live offspring, the sex ratio or the live birth index. No abnormalities ascribable to the compound were found for external features, clinical signs or necropsy findings for the offspring.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: body weight of neonates and viability index

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

not specified

Reproductive parameters:

 

Dose (mg/kg)	0	40	200	1000
No. of pairs examined	12	12	12	10
No of pairs with successful mating	12	12	11	10
Mating index (%)	100.0	100.0	91.7	100.0
No. of pregnant females	12	12	11	9
Fertility index (%)	100.0	100.0	100.0	90.0
Pairing days until mating (mean ± SD)	2.5 ± 1.0	3.1 ± 1.0	3.9 ± 3.0	2.8 ± 1.0
No. of estrous stages without mating (mean ± SD)	0.0 ± 0.0	0.0 ± 0.0	0.1 ± 0.3	0.0 ± 0.0

 

Mating index (%) = (No. of pairs with successful mating / No. of pais examined) x 100

Fertility index (%) = (No. of prgnant animals / No. of pairs with successful mating) x 100

 

_

Developmental parameters:

 

Dose (mg/kg)	0	40	200	1000
No. of females examined	12	12	11	8
Live birth index (%)	98.03 ± 4.52	100.00 ± 0.00	93.18 ± 22.61	89.06 ± 12.64
No. of live pups on day 0 (mean)	1.6	1.2	5.1	3.7
No. of live pups on day 4 (mean)	1.5	1.2	3.7	4.2
Body weight of pups on day 0 (g)
Male (mean ± SD)	7.2 ± 0.4	6.6 ± 0.4	6.9 ± 0.7	6.4 ± 1.1*
Female (mean ± SD)	6.8 ± 0.6	6.3 ± 0.5	6.5 ± 0.7	6.0 ± 0.9*
Body weight of pups on day 4 (g)
Male (mean ± SD)	11.1 ± 1.1	10.4 ± 0.9	10.8 ± 2.0	10.2 ± 2.5
Female (mean ± SD)	10.7 ± 1.3	9.8 ± 1.0	10.4 ± 2.0	9.8 ± 1.8

 

* = Significantly different from control; p<0.05

Conclusions:

Under the conditions of this study, the NOAEL for the reproductive/developmental toxicity is considered to be 200 mg/kg/day. A lower body weight gain and a lower viability index were observed in the pups from females of the 1000 mg/kg/day group.

Executive summary:

The study was performed according to OECD TG 422 in compliance with GLP.

SD (Crj: CD) rats received gavage doses of 0 (vehicle; corn oil), 40, 200 and 1000 mg/kg/day, for males starting from 14 days before mating for 43 days and in females from 14 days before mating to day 3 of lactation. The female animals were sacrificed on day 4 of lactation.

There were no effects on the reproductive parameters such as the mating index, the fertility index, the number of corpora lutea or implantations, the implantation index, the delivery index, the gestation index and the gestation length or the parturition. Three females in the 1000 mg/kg/day group, however, lost all of their pups during the lactation period. Females in the 1000 mg/kg/day group showed the following adverse effects in the repeated oral dose test: death of 3 animals out of 12, late onset of twitching, chronic convulsion, the suppression of body weight gain, degeneration of nerve fibers in the brain and spinal cord, hyperplasia of the mucosa in die gastric tract, oedema and inflammatory cell infiltration in the forestomach, atrophy of the thymus, increase in the weight of the kidneys and the adrenals without histopathological changes.

The pups from the females in the 1000 mg/kg/day group showed lower body weights, and the viability index of the pups was decreased due to maternal nursery activity. By external inspection, no abnomalities were found.

Conclusion: Under the conditions of this study, the NOAEL for the reproductive/developmental toxicity is considered to be 200 mg/kg/day. The NOAEL for the parental toxicity is considered to be 200 mg/kg bw/d.

Reference 2

Endpoint:

two-generation reproductive toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Read across to the methacrylic metabolite donor substance

REPORTING FORMAT FOR THE ANALOGUE APPROACH
see chapter "Toxicokinetics, metabolism and distribution"

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
see chapter "Toxicokinetics, metabolism and distribution"

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see chapter "Toxicokinetics, metabolism and distribution"

3. ANALOGUE APPROACH JUSTIFICATION
see chapter "Toxicokinetics, metabolism and distribution"

Reason / purpose for cross-reference:

read-across source

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)

Remarks:

Dose selection rationale: In a Dose range finding study according to OECD 421 with male and female Wistar rats, the terminal body weight in F0 females dosed with 450 mg/kg bw/d was significantly decreased by 10% vs. Ctrl animals (p <= 0.01) and also the food consumption of females in the F0 and the F1 generation (last, during gestation) was significantly reduced by 86-91%. Dosing with 50 and 150 mg/kg bw/d was not related to observed effects. Based on these findings, the highest dose for the main study was set to 400 mg/kg bw/d.

No. of animals per sex per dose:

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

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: In a Dose range finding study according to OECD 421 with male and female Wistar rats, the terminal body weight in F0 females dosed with 450 mg/kg bw/d was significantly decreased by 10% vs. Ctrl animals (p <= 0.01) and also the food consumption of females in the F0 and the F1 generation (last, during gestation) was significantly reduced by 86-91%. Dosing with 50 and 150 mg/kg bw/d was not related to observed effects. Based on these findings, the highest dose for the main study was set to 400 mg/kg bw/d.

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) for general, 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.

Effect on fertility: via oral route

Endpoint conclusion:

no study available (further information necessary)

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

There is only one study with MADAME available for assessment. A combined repeated dose and reproductive toxicity study by the oral route (MHW Japan, 1998) was identified as the key study because it was conducted according to OECD guideline 422 in compliance with GLP.

 

SD (Crj:CD) rats received gavage doses of 0 (vehicle; corn oil), 40, 200 and 1,000 mg/kg/day, males for 43 days starting from 14 days before mating and females for 41 to 52 days from 14 days before mating to day 3 of lactation. The female animals were sacrificed on day 4 of lactation.

There were no effects on the reproductive parameters such as the mating index, the fertility index, the number of corpora lutea or implantations, the implantation index, the delivery index, the gestation index and the gestation length or the parturition. Three females in the 1000 mg/kg/day group, however, lost all of their pups during the lactation period.

Females in the 1000 mg/kg/day group showed many adverse effects in the repeated oral dose test such as death of 3 animals out of 12, late onset of twitching, chronic convulsion, the suppression of body weight gain, degeneration of nerve fibers in the brain and spinal cord, hyperplasia of the mucosa in the gastric tract, oedema and inflammatory cell infiltration in the forestomach, atrophy of the thymus, increase in the weight of the kidneys and the adrenals without histopathological changes.

The pups from the females in the 1000 mg/kg/day showed lower body weights and a lower viability index due to maternal nursery activity. By external inspection of the pups, no abnormalities were found.

 The NOAEL for the reproductive/developmental toxicity is considered to be 200 mg/kg/day.

In addition, a two-generation reproduction toxicity study in rats according to OECD TG 416 in compliance with GLP with the structural analogue methyl methacrylate (CAS 80-62-6) was used to assess the reproduction toxicity of the test substance (REACH Methacrylate Task Force, 2009). In this study, 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. Control parental animals were dosed daily with the vehicle (1% CMC 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. 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).

The NOAEL for general, systemic toxicity was 50 mg/kg bw/day for the P and F1 parental rats, based on adverse 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 was 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 400 mg/kg bw/day, the highest dose tested.

Short description of key information:
In a well-conducted OECD combined repeated dose and reproductive/developmental toxicity screening test (OECD TG 422) there was no sign of reproductive toxicity up to 1000 mg/kg/day (high dose group) for males. Three females of the high dose group, however, lost all of their pups in the lactation period. The NOAEL of the test substance for reproductive/developmental toxicity after oral gavage in rats is considered to be 200 mg/kg/day for both parents and offspring. Supportingly, in a two-generation reproduction toxicity study in rats with the structural analogue methyl methacrylate (CAS 80-62-6) no adverse findings on the fertility and reproductive performance were evident at any of the tested doses and the NOAEL fertility and reproductive performance for the P and F1 parental rats was 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 400 mg/kg bw/day, the highest dose tested.

Effects on developmental toxicity

Description of key information

In a recently performed prenatal developmental toxicity study according to OECD 414 MADAME

was administered to female rats dosed dissolved in corn oil by gavage at dose levels of 0, 100, 300 and 600 mg/kg bw/day from days 6 through 19 of gestation. The NOAEL in this study was found to be 600 mg/kg bw/day for maternal and developmental toxicity.
Developmental effects in a well-conducted OECD combined repeated dose and reproductive/developmental toxicity screening test (OECD TG 422) were that the pups born from the females in the high dose group showed a lower body weight although no external abnormalities were observed. The NOAEL of this chemical for reproductive/developmental toxicity after oral gavage in rats is considered to be 200 mg/kg/day for both parents and offspring.

With respect to animal welfare legislation, new animal testing has to be considered as the last resort. Based on the toxicokinetic data provided in IUCLID section 7.1. showing the rapid ester cleavage by ubiquitous carboxylesterases to methacrylic acid and 2 -dimethylaminoethanol, we are convinced that the Scenario 1 of the RAAF is the most appropriate in order to fulfil the REACH information requirements.

In a prenatal developmental toxicity study in rabbits with the read across donor substance methyl methacrylate (CAS 80-62-6), no adverse fetal findings of toxicological relevance were evident at any of the tested doses and the NOAEL for prenatal developmental toxicity was determined to be 450 mg/kg bw/d with a NOAEL for maternal toxicity of 50 mg/kg/day.
The alcoholic metabolite, 2-dimethylamino ethanol (CAS 108-01-1) was tested in a study similiar to OECD 414 at 0/ 300 and 600 mg/kg bw/d via gavage. No NOAEL for maternal or developmental toxicity could be identified due to the corrosive effect of the substance. The OECD 414 in rabbits is ongoing.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16-03-2014 - 12-05-2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 22 January 2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): Dimethyl aminoethyl methacrylate
- Supplier: Evonik Röhm GmbH, D-64293 Darmstadt, Germany
- Substance type: organic
- Physical state at room temperature: liquid
- Storage condition of test material: 4°C, light protected

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Italy S.p.A., Calco (Lecco), Italy
- Age at study initiation: 9 weeks old (females), 11 weeks old (males)
- Weight at study initiation:females 197 to 236 g, males at least 322 g)
- Fasting period before study: no data
- Housing: during pre-pairing period and after mating animals were housed no more than 5 of one sex to a cage in polisulphone cages (59.5 x 38 x 20 cm); during mating period, the rats were housed 1 male to 1 female in clear polycarbonate cages (42.5 x 26.6 x 18.5 cm)
- Diet (e.g. ad libitum): ad libitum (4 RF 21, Mucedola S.r.1., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy
- Water (e.g. ad libitum): ad libitum
- Acclimation period: approximately 4 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ±2°C
- Relative humidity: 55 ± 15%
- Air changes (per hr): 15 to 20 cycles/hour
- Photoperiod (hrs dark / hrs light): artificial light 12 h/12 h

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): The required amount of Dimethylaminoethyl methacrylate was dissolved in the vehicle
(corn oil). The formulations were prepared daily (concentrations of 20, 60 and 120 mg/mL) and the concentrations were calculated and
expressed in terms of test item as supplied.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Final results for all levels were within the acceptability limits stated in RTC SOPs for concentration (85-115%) and homogeneity (CV < 10 %).
In this study a 24 hour stability at room temperature was verified in the range from 20 to 100 mg/mL.
According to RTC SOPs, suspensions are considered to be stable if concentration after the defined period of storage is still acceptable (85-115% for concentration and CV < 10%) for homogeneity).
Samples of the formulations perpared on week 1 and last week, were analysed to check the homogeneity and concentration. The received results of the analyses were within the above mentioned acceptability limits stated in RTC SOP's for suspensions.

Details on mating procedure:

Females were paired one to one in the home cage of the male and left overnight. Vaginal smears were taken daily in the morning from the day after pairing until a positive identification of mating was made. The presence of sperm in the vaginal smear or by the presence of a copulation plug, was considered as Day0 of gestation.

Duration of treatment / exposure:

6 - 19 day post coitum inclusive

Frequency of treatment:

once a day

Duration of test:

14 d (dams were euthanized on gestation day 20)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

600 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

24 mated female rats per group exposed.

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS: No data
- Time schedule:

DETAILED CLINICAL OBSERVATIONS: Yes (starting from allocation until sacrifice)
- Time schedule: once a day; Morbidity and mortality: at least twice a day including weekends and public holidays

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighted on GD0, 3, 6, 9, 12, 15, 18 and 20 p.c.starting from Day0 post coitum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption: The quantity of food consumed by each female was measured on Days 3, 6, 9, 12, 15, 18 and 20 p.c.starting from Day 0 post coitum. 
- Food consumtion was not reported for cage no. 3 on Day 20 post coitum, since its value was unreliable.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations:

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20 post coitum

Ovaries and uterine content:

The ovaries and uterus of the females were examined to determine:
gravid uterine weight (will not be obtained from animals found dead or killed during the study), number of corpora lutea, number of implantation sites, number, sex and weight of all live foetuses, number and sex of dead fetuses (foetuses at term without spontaneous movements and breathing), number of intra-uterine deaths and gross evaluation of placentae.
A gross evaluation of placentae was also undertaken.

Fetal examinations:

- External examinations: Yes: all live foetuses
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: in all groups
- Head examinations: No data

Statistics:

Continuous data (for instance body weight, food consumption, …) amongst group means were assessed by Dunnett's test or a modified t test, depending on the homogeneity of data.
Statistical analysis of non-continous data are carried out by means of the Kruskal-Wallis test and intergroup differences between the control and treated groups assessed by a non-parametric version of the Williams test. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.

Details on maternal toxic effects:

Maternal toxic effects:no effects

Dose descriptor:

NOAEL

Effect level:

600 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Dose descriptor:

NOAEL

Effect level:

600 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

not specified

Developmental effects observed:

no

Mortality and fate of females

 

One high dose female (animal no. 97130185) died during the study, on gestation Day 10. A total of 4 females were found not pregnant at necropsy: 1 each in the control, low, mid- and high dose groups. One mid-dose female had unilateral implantation and another mid-dose female had unilateral total resorption.

The number of females with live foetuses on gestation Day 20 was 23 in each of the control, low and mid-dose groups and 22 in the high dose group.

 

Clinical signs

 

Scabs and hairloss were the most frequent signs recorded in control and treated females. These signs were not considered of toxicological significance.

 

Body weight and body weight gain

 

No differences in body weight were noted between control and treated groups.

At body weight gain, a statistically significant decrease of approximately 27% was noted on Day 6 post coitum in females receiving 100 mg/kg/day and a statistically significant increase of approximately 33 % was noted in the same group on Day 18 post coitum, when compared to controls. These changes were considered of no toxicological relevance.

 

Food consumption

 

No differences in food consumption were detected between control and treated groups.

Terminal body weight, uterus weight and absolute weight gain

 

No significant differences in terminal body weight, gravid uterus weight and absolute weight gain were observed in treated groups, when compared to the control group.

Litter data and sex ratios

 

Litter data and sex ratios were not affected by treatment.

 

Macroscopic examination of females

                                                                

No cause of death was identified in the animal which died during the study, due to the significant presence of organs and tissues cannibalized. No treatment-related changes were noted at macroscopic observation performed at the final sacrifice. The minor changes observed are suggested to be incidental, having a comparable incidence in control and treated groups, and/or characteristically seen in untreated Sprague Dawley SD rats of the same age.

 

External examination of foetuses

 

Multiple malformations (such as cor bilocular and ectopic cordis of the heart; malpositioned lungs with lobes agenesia; thymus agenesia) and anomalies were detected in one foetus of the low dose group examined externally and internally. One foetus was found dead in the high dose group. A total of 8 small foetuses ( <2.7 g) were detected in control and treated groups, without dose-relationship. All these findings were considered incidental and not dose-related.

 

Skeletal examination of foetuses

 

Malformations were detected in two foetuses of the low dose group (no ossification of pubis and ischium in one foetus and cleaved sternum in the other) and in one foetus of the mid-dose group (no ossification of pubis). No malformations were observed in the control and high dose groups. In addition, most of the anomalies and variants recorded at skeletal examination were noted both in control and treated groups, with a similar incidence. Considering the low incidence and the absence of dose-relation, all the findings were considered incidental.

 

Visceral examination of foetuses

 

No findings that could be considered treatment-related were noted at visceral examination of the foetuses in any group.

Conclusions:

In a developmental toxicity study according to OECD 414 Dimethylaminoethyl methacrylate (purity: 99.79 %) was administered to female rats dosed dissolved in corn oil by gavage at dose levels of 0, 100, 300 and 600 mg/kg bw/day from days 6 through 19 of gestation. The NOAEL in this study was found to be 600 mg/kg bw/day for maternal and developmental toxicity.

Executive summary:

In a developmental toxicity study according to OECD 414 Dimethylaminoethyl methacrylate (purity: 99.79 %) was administered to female rats (Sprague-Dawley, (SD)) by oral gavage at dose levels of 0, 100, 300 and 600 mg/kg bw/day from days 6 through 19 of gestation.

Neither clinical signs nor signs of reaction to treatment were noted in treated females that could be related to treatment. No significant differences were noted in body weight, food consumption, gravid uterus weight, litter data and macroscopic observation of treated females, when compared to controls. Due to the absence of dose-relation, the findings detected at the external, visceral and skeletal examination of foetuses from all groups were considered incidental.

 

The maternal and developmental NOAEL was therefore 600 mg/kg bw/day.

Dimethylaminoethyl methacrylate formulation analyses were 85% to 115% of the nominal concentration (homogeneity (CV < 10%)) and were within the acceptable range.

The developmental toxicity study in the rat is classified acceptable and satisfies the guideline requirement for a developmental toxicity study (OPPTS 870.3700; OECD 414) in rats.

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 2

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Read across to the methacrylic metabolite donor substance

REPORTING FORMAT FOR THE ANALOGUE APPROACH
see chapter "Toxicokinetics, metabolism and distribution"

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
see chapter "Toxicokinetics, metabolism and distribution"

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see chapter "Toxicokinetics, metabolism and distribution"

3. ANALOGUE APPROACH JUSTIFICATION
see chapter "Toxicokinetics, metabolism and distribution"

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rabbit

Strain:

Himalayan

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: 16-21 weeks
- Weight at study initiation: 2187-2917 g
- Fasting period before study: no
- Housing: the rabbits were housed singly in type 12.2395.C stainless steel wire mesh cages supplied by Draht-Bremer GmbH, Marktheidenfeld, Germany (floor area about 3,000 cm²)
- Diet: pelleted “Kliba maintenance diet for rabbits & guinea pigs, GLP”, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: tap water ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12 (6.00 p.m. to 6.00 a.m. dark, 6.00 a.m. to 6.00 p.m. light)

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The aqueous test substance preparations were prepared at the beginning of the administration period and thereafter at maximum intervals of 7 days, which took into account the analytical results of the stability verification. For the test substance preparation, an specific amount of test substance was weighed depending on the dose group, into a graduated flask (conical Erlenmeyer flasks with groundin stopper), topped up (shortly under the marking) with 1% Carboxymethylcellulose solution in drinking water and a few drops Cremophor EL and one drop of 32% hydrochloric acid. Afterwards the preparation was filled up with 1% Carboxymethylcellulose suspension in drinking water. The flask was sealed and the preparation was intensely mixed with a magnetic stirrer. During administration, the preparations were kept homogeneous with a magnetic stirrer and the vessels were kept closed between the withdrawals of the preparations.

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 twice during the study period (at the beginning and towards the end) for verification of the concentrations. Samples were analyzed by GC with external calibration.

Details on mating procedure:

After an acclimatization period of at least 5 days, the female rabbits were fertilized by means of artificial insemination. This implied that 0.2 mL of a synthetic hormone which releases LH and FSH from the anterior pituitary lobe (Receptal) were injected intramuscularly to the female rabbits about 1 hour before insemination. The ejaculate samples used for the artificial insemination were derived from male Himalayan rabbits of the same breed as the females. Each female was inseminated with the sperm of a defined male donor. The male donors were kept under conditions (air conditioning, diet, water) comparable to those of the females participating in this study. The day of insemination was designated as gestation day (GD) 0 and the following day as GD 1.

Duration of treatment / exposure:

from implantation to one day prior to the expected day of parturition (GD 6-28)

Frequency of treatment:

daily

Duration of test:

until GD 29

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

actual dose: 41 mg/kg bw/d

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

actual dose: 132 mg/kg bw/d

Dose / conc.:

450 mg/kg bw/day (nominal)

Remarks:

actual dose: 406 mg/kg bw/d

No. of animals per sex per dose:

25 inseminated females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The dose volume was 10 mL/kg body weight. The calculation of the volume administered was based on the most recent individual body weight.

Maternal examinations:

CLINICAL EXAMINATIONS
- Mortality: Mortality was checked in the females twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0-29).
- Clinical symptoms: A clinical examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. If such signs occurred, the animals were examined several times daily (GD 0-29).
- Food consumption: The food consumption was determined daily on GD 1–29.
- Body weight data: All animals were weighed on GD 0, 2, 4, 6, 9, 11, 14, 16, 19, 21, 23, 25, 28 and 29. The body weight change of the animals was calculated.
- Corrected (net) body weight gain: The corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6).

TERMINAL EXAMINATIONS OF THE DOES
After the does had been sacrificed on GD 29, they were necropsied and assessed by gross pathology in randomized order.

Ovaries and uterine content:

On GD 29, the surviving does were sacrificed in randomized order by an intravenous injection of pentobarbital (Narcoren; dose: 2 mL/animal). After the does had been sacrificed, they were necropsied and assessed by gross pathology in randomized order. The uterus and the ovaries were removed and the following data were recorded:
- Weight of the unopened uterus
- Number of corpora lutea
- Number and distribution of implantation sites classified as:
1) live fetuses
2) dead implantations: a) early resorptions (only decidual or placental tissues visible or according to SALEWSKI (Salewski, 1964) from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single-horn pregnancy); b) late resorptions (embryonic or fetal tissue in addition to placental tissue visible); c) dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened).
After the weight of the uterus had been determined, all subsequent evaluations of the does and the gestational parameters were conducted by technicians unaware of treatment group in order to minimize bias. For this purpose the animal numbers were encoded.
Furthermore, calculations of conception rate and pre- and postimplantation losses were carried out:
The conception rate (in %) was calculated according to the following formula: (number of pregnant animals)/(number of fertilized animals) x 100.
The preimplantation loss (in %) was calculated based on each individual pregnant animal with scheduled sacrifice according to the following formula: (number of corpora lutea – number of implantations)/(number of corpora lutea) x 100.
The postimplantation loss (in %) was calculated based on each individual pregnant animal with scheduled sacrifice from the following formula: (number of implantations – number of live fetuses)/(number of implantations) x 100.

Fetal examinations:

All fetal analyses were conducted by technicians unaware of the treatment group, in order to minimize bias.
- Examination of the fetuses after dissection from the uterus: Each fetus was weighed and examined macroscopically for any external findings.
Furthermore, the viability of the fetuses and the condition of the placentae, the umbilical cords, the fetal membranes, and fluids were examined. Individual placental weights were recorded. Thereafter, the fetuses were sacrificed by a subcutaneous injection of phenobarbital (Narcoren; 0.2 mL/fetus).
- Soft tissue examination of the fetuses: After the fetuses had been sacrificed, the abdomen and the thorax were opened in order to examine the organs in situ before they were removed. The heart and the kidneys were sectioned in order to evaluate the internal structure. The sex of the fetuses was determined by examination of the gonads in situ. After these examinations, the heads of approximately one half of the fetuses per litter and the heads of those fetuses, which revealed severe findings during the external examination (e.g. anophthalmia, microphthalmia or hydrocephalus) were severed from the trunk. These heads were fixed in BOUIN's solution and were, after fixation, processed and evaluated according to WILSON's method (Wilson and Warkany, 1965). About 10 transverse sections were prepared per head. After the examination these heads were discarded. All fetuses (partly without heads) were skinned and fixed in ethyl alcohol. After fixation for approx. 1-5 days, the fetuses were removed from the fixative for awhile. With a scalpel, a transversal incision was made into the frontal / parietal bone in the heads of the intact fetuses. The two halves of the calvarium were then cauteously bent outward and the brain was thoroughly examined. Subsequently, the fetuses were placed back into the fixative for further fixation.
- Skeletal examination of the fetuses: After fixation in ethyl alcohol the skeletons were stained according to a modified method of KIMMEL and TRAMMELL (Kimmel, C.A., and Trammell, C., 1981). Thereafter, the stained skeleton of each fetus was examined. After the examination the stained skeletons were retained individually.

Statistics:

- DUNNETT-test (two-sided) for food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight.
- FISHER'S EXACT test (one-sided) for female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings.
- WILCOXON-test (one-sided) for proportions of fetuses with malformations, variations and/or unclassified observations in each litter.

Details on maternal toxic effects:

Details on maternal toxic effects:
- Mortality: There were no test substance-related or spontaneous mortalities in any group.
- Clinical symptoms: No test substance-related clinical signs or any disturbances of the general behavior were observed in any rabbit during the entire study period.
- Food consumption: The food consumption in the high-dose females (450 mg/kg bw/d) was distinctly and statistically significantly reduced during a significant part of the treatment period (GD 15-23). During the entire treatment period (GD 6-28) the total average food consumption of the high dose rabbits was about 18% below controls. The food consumption of the mid dose females (150 mg/kg bw/d) was similarly affected in terms of magnitude and course of reduction, however the reduction of food consumption reached statistical significance only on GD 22-24. During the treatment period (GD 6-28) the total average food consumption of the mid-dose rabbits was about 13% below controls. Overall, the food consumption of the low-dose does (50 mg/kg bw/d) did not show test substance-related impairments. The reduced food consumption at the 150 and 450 mg/kg bw/d levels is considered to be related to the treatment.
- Body weight data: The mean body weights of the low-, mid- and high-dose rabbits (50; 150 and 450 mg/kg bw/d) were not significantly different from the concurrent control throughout the course of the study. The average body weight gain of the mid- and high-dose rabbits was statistically significantly reduced by about 27% and 31% during the treatment period. A significant reduction of mean body weight gain was also noted for the the high-dose rabbits on GD 19-21.
- Corrected (net) body weight gain: Mean carcass weights and the corrected body weight gain (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6) were comparable among all groups.
- Uterus weight: The mean gravid uterus weights of test groups 1, 2, and 3 (50; 150 or 450 mg/kg bw/d) did not show statistically significant differences in comparison to the control group.
- Necropsy findings: At necropsy, only spontaneous findings were seen in single females of every test group. No test substance-related findings were observed in the does.
- Reproduction data of does: The conception rate reached 96% in test groups 1 and 3 (50 and 450 mg/kg bw/d) and 100% in test groups 0 and 2 (0 and 150 mg/kg bw/d). Importantly, a sufficient number of pregnant females was available for the purpose of the study, as 24-25 pregnant rabbits per group had implantation sites in the uterus, at terminal sacrifice. There were no test substance-related and/or biologically relevant differences between the control and all dosed groups in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation losses, the number of resorptions and viable fetuses. Gestational parameters were within the normal range for animals of this strain and age.

Dose descriptor:

NOAEL

Effect level:

450 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks on result:

other: no adverse effects observed; actual dose 406 mg/kg/d

Dose descriptor:

NOEL

Remarks:

food consumption

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

food consumption and compound intake

Remarks on result:

other: actual dose: 41 mg/kg/d

Abnormalities:

effects observed, treatment-related

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
- Sex distribution of fetuses: The sex distribution of the fetuses in test groups 1-3 (50; 150 and 450 mg/kg bw/d) was comparable to the control fetuses. Observable differences were without biological relevance.
- Weight of placentae: The mean placental weights in test groups 1, 2 and 3 (50; 150 and 450 mg/kg bw/d) were comparable to the controls.
- Weight of fetuses: The mean fetal weights of all treated groups were not influenced by the test substance. Neither female nor male weights showed statistically significant or biologically relevant differences between the test substance-treated groups and the controls.
- Fetal external malformations: One sole external malformation (unilateral microphthalmia) was recorded for two fetuses from 2 litters in the high-dose group (450 mg/kg bw/d). This malformation is present in the historical control data. Thus an association of these individual findings to the treatment is not assumed. The total incidences of external malformations were comparable to the historical control data.
- Fetal external variations: One external variation (paw hyperflexion) occurred in single fetuses of the low- and mid-dose groups and the control. The incidences did not demonstrate a dose-response relationship and were comparable to the historical control data. Thus an association of this finding to the treatment is not assumed.
- Fetal external unclassified observations: Unclassified external observations, such as necrobiotic placentae and discolored amniotic fluid, were recorded for single fetuses of test groups 1 and 2 (50 and 150 mg/kg bw/d). A relation to dosing is not present if normal biological variation is taken into account. Therefore, a test substance induced effect is not assumed.
- Fetal soft tissue malformations: The examination of the soft tissues revealed a variety of malformations in fetuses of all test groups including the controls (0; 50; 150 and 450 mg/kg bw/d). With the exception of a lateral pouch in the tongue of 2 fetuses all individual soft tissue malformations were present in the historical control data at comparable frequencies. No statistically significant differences between the test groups and the control were observed. The total incidences of external malformations were comparable to the historical control data. No malformation pattern was evident. Thus an association of these findings to the treatment is not assumed.
- Fetal soft tissue variations: A number of soft tissue variations, such as absent lung lobe (lobus inferior medialis) and malpositioned carotid branch, was detected in each test group including the controls. Incidences were without a relation to dosing. Neither statistically significant differences between the test groups nor differences to the historical control data were noted.
- Fetal soft tissue unclassified observations: Unclassified soft tissue observations, such as infarct of liver, hemorrhagic thymus or ovary and blood coagulum around urinary bladder, were recorded for some fetuses of test groups 0, 1, 2 and 3 (0; 50; 150 and 450 mg/kg bw/d). A relation to dosing is not present if normal biological variation is taken into account. Therefore, a test substance induced effect is not assumed.
- Fetal skeletal malformations: Malformations of the fetal skeletons were noted in fetuses of test groups 0, 2 and 3 (0; 150 and 450 mg/kg bw/d). Neither statistically significant differences between treated groups and the control were calculated nor a dose-response relationship was observed. All individual skeletal malformations were present in the historical control data at a comparable frequency.
- Fetal skeletal variations: For all test groups, variations in different skeletal structures were detected with or without effects on the corresponding cartilages. The observed skeletal variations were related to various parts of the fetal skeletons and were statistically significant higher in the low- and the
high-dose groups on a fetus per litter basis. Several specific skeletal variations were statistically significant higher than the concurrent control in the dosed groups (on a fetus per litter basis). These findings are delays or minor disturbances of ossification which are reversible or do not considerably affect the integrity of the underlying structures. Such slight changes of the ossification process occur very frequently in gestation day 29 rabbit fetuses of this strain and all observed incidences were within the historical control data. Thus an association of these findings to the treatment is not assumed.
- Fetal skeletal unclassified cartilage observations: Additionally, isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all groups including the control. The observed unclassified cartilage findings did not show a relation to dosing and were comparable to historical control data and, therefore, regarded to be spontaneous in nature.
- Abstract of all classified fetal external, soft tissue and skeletal observations: Various external, soft tissue and skeletal malformations occurred throughout all test groups including the control. All individual malformations are present in the historical control data, with the exception of lateral pouches in the tongue of 2 fetuses. They did neither show a consistent pattern since a number of morphological structures of different ontogenic origin were affected nor a clear dose-response relationship. The overall incidence of malformations was comparable to the historical control data. One external (paw hyperflexion), two soft tissue (absent lobus inferior medialis and malpositioned carotid branch) and a broad range of skeletal variations occurred in all test groups including the controls. All fetal and litter incidences for these variations and the corresponding mean percentages of affected fetuses/litter were not significantly different from the concurrent control and their frequency is comparable to the historical control data. Therefore, they were not considered to be related to the treatment. A spontaneous origin is also assumed for external, soft tissue and unclassified skeletal cartilage observations which were observed in several fetuses of all test groups including controls (0, 50; 150 and 450 mg/kg bw/d). Distribution and type of these findings do not suggest relation to treatment.

Dose descriptor:

NOAEL

Remarks:

prenatal developmental toxicity

Effect level:

450 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no adverse effects observed

Abnormalities:

no effects observed

Developmental effects observed:

no

Table: Occurence of statistically significantly increased fetal skeletal variation (expressed as mean percentage of affected fetuses/litter)

Finding	Group 0 0 mg/kg/d	Group 1 50 mg/kg/d	Group 2 150 mg/kg/d	Group 3 450 mg/kg/d	HCD (range)
Incomplete ossification of parietal; unchanged cartilage	0.0	0.0	1.9*	2.1*	0.4 (0.0 – 2.6)
Incomplete ossification of hyoid; cartilage present	11.2	11.4	19.1	20.4*	9.8 (0.0 – 21.6)
Splitting of skull bone	0.4	3.3*	3.3	2.3	2.9 (0.0 – 7.7)
Incomplete ossification of cervical centrum; unchanged cartilage	2.5	2.2	3.6	7.3*	2.5 (0.0 – 9.3)
Supemumerary 13th rib; cartilage not present	2.5	9.8	6.1	9.9*	6.6 (0.0 – 17.5)
Total fetal skeletal variations	46.3	63.7*	59.3	71.6**	63.5 (46.3 – 81.9)

HCD = Historical control data; * = p ≤ 0.05, ** = p ≤ 0.01 (Wilcoxon-Test [one-sided])

 

 

Table: Total fetal malformations

		Group 0 0 mg/kg/d	Group 1 100 mg/kg/d	Group 2 300 mg/kg/d	Group 3 1000 mg/kg/d
Litter Fetuses	N N	25 171	24 154	25 157	24 158
Fetal incidence	N (%)	4 (2.3%)	2 (1.3%)	6 (3.8%)	9 (5.7%)
Litter incidence	N (%)	4 (16%)	1 (4.2%)	4 (16%)	7 (29%)
Affected fetuses/litter	Mean%	2.3	1.2	3.6	6.2

 

 

Table: Total fetal variations

		Group 0 0 mg/kg/d	Group 1 100 mg/kg/d	Group 2 300 mg/kg/d	Group 3 1000 mg/kg/d
Litter Fetuses	N N	25 171	24 154	25 157	24 158
Fetal incidence	N (%)	106 (62%)	106 (69%)	106 (68%)	122 (77%)
Litter incidence	N (%)	21 (84%)	24 (100%)	24 (96%)	23 (96%)
Affected fetuses/litter	Mean%	59.9	69.8	64.3	74.2

 

Conclusions:

In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 450 mg/kg bw/d and the no observed effect level (NOEL) for maternal toxicity is 50 mg/kg bw/d based on effects on food consumption. The NOAEL for prenatal developmental toxicity is 450 mg/kg bw/d. No adverse fetal findings of toxicological relevance were evident at any dose.

Executive summary:

The study was performed according to OECD TG 414 in compliance with GLP.

Methyl Methacrylate was tested for its prenatal developmental toxicity in Himalayan rabbits. The test substance was administered as an aqueous preparation to 25 inseminated female Himalayan rabbits by stomach tube at doses of 50; 150 and 450 mg/kg body weight/day on gestation days (GD) 6 through GD 28. The control group, consisting of 25 females, was dosed with the vehicle (1% Carboxymethylcellulose CB 30.000 in drinking water and a few drops Cremophor EL and one drop hydrochloric acid [1% CMC]) in parallel. A standard dose volume of 10 mL/kg body weight was used for each test group. At terminal sacrifice on GD 29, 24-25 females per group had implantation sites.

The following test substance-related adverse effects/findings were noted:

Test group 3 (450 mg/kg body weight/day):

-        Reduced food consumption (-18%) and body weight gain (-31%)

-        No test substance-related adverse effects on gestational parameters or fetuses

 

Test group 2 (150 mg/kg body weight/day):

-        Reduced food consumption (-13%) and body weight gain (-27%)

-        No test substance-related adverse effects on gestational parameters or fetuses

 

Test group 1 (50 mg/kg body weight/day):

-        No test substance-related adverse effects on does, gestational parameters or fetuses

In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is nominal 450 mg/kg bw/d (actual 406 mg/kg bw/d), the highest dose tested. The no observed effect level (NOEL) for maternal toxicity is nominal 50 mg/kg bw/d (effective 41 mg/kg bw/d) 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 (actual 132 mg/kg bw/d).

The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is nominal 450 mg/kg bw/d (actual 406 mg/kg bw/d). No adverse fetal findings of toxicological relevance were evident at any dose.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

600 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

GHS classification according to Annex I 1272/2008 CLP (EU GHS):

- No classification required.