Registration Dossier

Diss Factsheets

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-12-06 - 2009-04-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Version / remarks:
22nd January 2001
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Methyl methacrylate
EC Number:
201-297-1
EC Name:
Methyl methacrylate
Cas Number:
80-62-6
Molecular formula:
C5H8O2
IUPAC Name:
methyl methacrylate
Test material form:
liquid
Specific details on test material used for the study:
- Name of test substance: Methyl Methacrylate
- Batch identification: 012231eda0
- CAS-No.: 80-62-6
- Physical state/appearance: Liquid /colorless, clear
- Purity: 99.9 %
- Homogeneity: Given
- Storage conditions: Refrigerator; avoid temperatures >35°C
- Storage stability: Expiry date: 16 Jan 2009. The stability of the test substance under storage conditions over the test period was guaranteed by the manufacturer, and the manufacturer holds this responsibility.

Test animals

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:

Administration / exposure

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
Doses / concentrationsopen allclose all
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:
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 n
ot 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.

Examinations

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):
Immediately after necropsy and organ weight determination, the right testis and cauda epididymis wer
e taken from F0 and F1 males of all dose groups.
Parameters examined in all male parental generations:
testis weight, epididymis weight, cauda epididymis weight, prostate weight, seminal vesicles includingcoagulation glands weight, sperm head count in testis, sperm head count in cauda epididymis, sperm motility, sperm morphology Sperm motility and sperm head count (cauda epididymis and testis) were evalutated for the control and highest dose group.
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.
Statistics:
Simultaneous comparison of all dose groups with the control group using Dunnett-test (2-sided) for t
he hypothesis of equal means:
- food consumption (parental animals)
- body weight and body weight change (parental animals and pups; for the pup weights, the litter
means were used)
- oestrus cycle duration
- number of mating days
- duration of gestation
- number of implantation sites
- postimplantation loss and % postimplantation loss
- number of pups delivered per litter
- duration of sexual maturation (days to vaginal opening or preputial separation)
Pairwise comparison of each dose group with the control group using Fisher's exact test for the hypo
thesis of equal proportions:
- male and female mating indices
- male and female fertility indices
- gestation index
- females with stillborn pups
- live birth index
- pups stillborn, died, cannibalised, sacrificed moribund
- viability index
- lactation index
- number of litter with affected pups at necropsy
- sexual maturation (vaginal opening and preputial separation)
- males with certain amount of abnormal sperm (cutoff value: 0.9-quantile of control groups)
Pairwise comparison of each dose group with the control group using the Wilcoxon-test (1-sided) for
the hypothesis of equal medians:
- Proportion of affected pups per litter with necropsy observations
Pairwise comparison of each dose group with the control group using the Wilcoxon-test (1-sided) for
the hypothesis of equal medians:
-Total spermatids/ g testis, total sperm/g cauda epididymis
Pairwise comparison of each dose group with the control group using the Wilcoxon-test (1-sided) with
Bonferoni-Holm-Adjustement for the hypothesis of equal medians:
- Sperm motility(%)
Non parametric one-way analysis using Kruskal-Wallis-test (2-sided). If resulting p-value was <=
than 0.05, a pairwise comparison of each dose group with the control group was performed using
Wilcoxon-test (2-sided) for the equal medians:
-pup organ weights (absolute and relative)
Reproductive indices:
Male reproduction data: The mating partners, the number of mating days until vaginal sperm could b
e 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 vagina
l 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 p
regnant) 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 fol
lowing formula:
Postimplantation loss (%) = (number of implantations – number of pups delivered)/(number of im
plantations) 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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
Clincial signs of males and females except gestation and lactation period:
A male and 7 females of the high dose group showed transient salivation during major parts of the treatment period. Salivation persisted in the respective animals only for some minutes after daily
gavage (i.e. upto 15 minutes). This symptom is initially observed during study week 5.
The temporary salivation is considered to be test item-related. Form the temporary, short appearance immediately after dosing, it is likely that this finding was induced by the bad taste of the test item or lo
cal affection of the upper digestivve tract. It was clearly considered to be not an adverse toxicological finding. There were no clinical signs in low and mid-dose animals.
Clinical signs of females in females during gestation of F1 litters:
NIne out of 25 high dose females showed transient salivation during major parts of the gestation period (GD 0-16 and GD 20). There were no clinical signs in low and mid-dose females.
Clinical signs of females and offspring during lactation of F1 litters:
Seven out of 24 high dose females showed transient salivation during major parts of the lactation period (OND 0-8; PND 10; PND 14-16 and PND 18-21). The F0 animals and the low and mid-dose
females showed no clinical signs.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no test item-related mortalities in any of the male and female parental animals in any of the groups. During study week 11, one high dose female was found dead, after showing hypo
thermia, salivation and labored respiration. Pathological examination revealed pleuritis and pericarditis as the cause of death, most likely secondary to a gavage error.
During lactation period, two high dose females were found dead. There were no macroscopic or histopathologic findings that could explain the death of these animals.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weights and body weight change of F0 parental males and females in the treated groups were comparable to the control group throughout the entire study. The statistically significantly increa
sed or decreased body weight change in the low-dose females during week 0-1 and weeks 4-5, in the mid-dose females during study week 1-2 and weeks 4-6 and in high dose females during weeks 4-6
was considered as spontaneous in nature.
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 examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
All findings were either single observations, or were similar in distribution pattern and severity in control rats compared to treatment groups. All of them were considered to be incidental and/or spo
ntaneous in origin and without any relation to treatment. Male animals and their female mating partners did not show any histopathologic findings that could explain their infertility. One male animal
revealed oligospermia and debris within the tubules of the testes and the epididymides. This could have caused the infertility in this mating pair. The female mating partner did not show any histopathologic finding that could explain the infertility.
Information on descendents: see in section mortality
Histopathological findings: neoplastic:
not examined
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Oestrus cycle data, generated during the last three weeks prior to mating for the F1 litter, revealed regular cycles in the females of all test groups. The mean oestrus cycle duration in the different
groups was comparable: 4.0 (control), 4.5 (low-dose), 5.7 (mid-dose) and 5.3 (high-dose).
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
No treatment-related effects were noted for the sperm measures. The number of homogenisation resistant testicular spermatids of cauda epididymal sperm, the percentages of abnormal and normal
sperm and sperm motility data were comparable between the groups.
Reproductive performance:
no effects observed
Description (incidence and severity):
For all F0 males, copulation was confirmed. Fertility was proven for most of the F0 males. 3 control , 3 low-dose, 3 mid-dose and 1 high-dose male did not generate F1 pups. The male fertility index ra
nged from 88-96% without showing any relation to the dosing. The infertile male rats did not show histopathological findings that could explain infertility. One male of the low-dose had an oligosperma
and debris within the tubules of the testes.
The female mating index calculated after mating period of F1 litter was 100% in all groups. The mean duration until sperm was detected varied from 2.3 and 2.6 days without any relation to dosing. All sperm positive rats delivered pups or had implants in utero (except 3 control 3 low-dose, 3 mid-dose and 1 high-dose female which did not become pregnant). The fertility index varied between 88 and 96%.
There were no corroborative histopathological findings in the sexual organs of the non-pregnant females. The mean duration of gestation varied between 22.0 and 22.2 days without any relation to dosing.
Implanation was not affected by treatment since the number of implanation sites was comparable to control. There were no indications for test item-related intrauterine embryo-/fetolethality since po
stimplantation loss did not show any statistically significant differences between the groups. The mean number of F1 pups delivered per dam remained unaffected. The rate of liveborn pups was also
not affected by treatment with the test item, as indicated by live birth index of 99 (control, mid- and high-dose) and 100% (low-dose). Moreover, the number of stillborn pups was comparable between the groups.

Details on results (P0)

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.

Effect levels (P0)

open allclose all
Dose descriptor:
NOEL
Remarks:
P0 systemic
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
food consumption and compound intake
Key result
Dose descriptor:
NOAEL
Remarks:
P0 systemic
Effect level:
400 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
no effects observed
Description (incidence and severity):
24 out of 25 males and 12 out of 25 females of the high dose F1 parental animals showed transient salivation during major parts of the treatment period. Salivation persisted in the respective animals only
for some minutes after daily gavage (i.e. upto 15 minutes). The salivation persisted in the respective animals only for some minutes after daily gavage dosing (i.e. up to 15 minutes). This symptom was
initially observed during study week 6 in males and towards the end of the study in females.
The temporary salivation is considered to be test item-related. Form the temporary, short appearance immediately after dosing, it is likely that this finding was induced by the bad taste of the test item or
local affection of the upper digestivve tract. It was clearly considered to be not an adverse toxicological finding. Other clinical findings like labored respiration, microphthalmia, poor general state,
palpable mass in the neck and throat region in some low-and mid-dose individuals were considered as spontaneous in nature and not as test item-related.
Clinical signs of females in females during gestation of F2 litters:
Five out of 25 high dose females showed transient salivation during major parts of the gestation period (GD 4, 6-7, 9 and 11-12). There were no clinical signs in low and mid-dose females.
Clinical signs of females and offspring during lactation of F2 litters:
There were no clinical findings in F1 females during the lacation period for the F2 litters at all dose levels.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were not deaths observed in the F1 parental dose groups.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
HIgh dose F1 parental males had statistically significantly lower body weights during week 0-5 (up to 17%) and 10-11 (up to 6%). Mean body weight gain of these parental males was comparable to
control group throughout the entire study.
Mean body weights and body weight gain of F1 parental males in the low and mid dose were comparable to control throughout the entire study.
HIgh dose F1 parental females had statistically significantly lower body weights during premating week 0-1 (up to 16%). High dose females' body weights were comparable to control group during
remaining premating, entire gestation and lacation period. Mean body weight gain of these parental females was comparable to control animals.
Mean body weights and body weight gain of F1 parental males in the low and mid dose were comparable to control throughout the entire study. The statistically significantly increased body weight gain of
high dose females during premating weeks 1-2 and premating weeks 0-10 was considered as spontaneous in nature.
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%) a nd 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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Absolute organ weights
Group male animals female animals
50 150 400 50 150 400
_________________________________________________________________________________________
Body weight -1% -5% -7%**
Liver +3% +9%** +10%**
Kidney +7%** +12%** +12%**
Brain 0% -2% -3%*
Cauda epididymis +7%* +7%* +6%*
*:p<=0.05 **:p<=0.01
All other mean absolute weight parameters did not show any significant differences compared to control.
The increase of absolute liver and kidney weights of females were considered as treatment-related.
The increase of cauda epididymis weight in all treated males is considered incidental and not treatment-related as there was no histopathologic correlate observed. The decrease of brain weights in highdose males is regarded to be a consequence of the reduced terminal body weight.
Relative organ weights
Group male animals female animals
50 150 400 50 150 400
_________________________________________________________________________________________
Liver -1% +3% +5%* +3%* +9%** +13%**
Kidney 0% +5%* +10%** +6%** +12%** +15%**
Epididymides +5%* +7%* +7%**
Cauda epididymides +8%* +13%** +14%**
*:p<=0.05 **:p<=0.01

All other parameters did not show significant differences in comparison to control.
The increase relative liver and kidney weights of females of all treated groups was regarded as treatment-related. The increase in liver weight in high-dose males and of kindey in mid- and high-dose males were considered to be treatment-related. The increase of epididymides in all treated males were thought to be reflective to the lower body weight. In addition, there was no histopathologic correlate corresponding to the weight decrease in the high-dose group.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All gross lesions observed in test animals occured singularly. They are considered to be spontaneous lesions in origin and are not related to treatment.
The females that were not pregnant and the mating partners did not show relevant gross lesions.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
All findings were single observations either or were similarly in distribution pattern and severity in control rats compared to treatment groups. All of them are considered to be incidental and/ or sp
ontaneous in origin and without any relation to treatment.
The non-pregant females and the male mating partners did not show any histopathologic lesion that could explan the infertility.
Descendents: In the F1 generation, all animals survived until the end of the study.
Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Oestrus cylce data, generated during the last 3 weeks prior to mating for the F2 litter, revealed regular cycles in the females of all groups. The mean oestrus cycle duration was similar: 4.1 (control), 4.2 (lo
w-dose), 4.3 (mid-dose) and 4.1 days (high-dose).
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
No test item-related effects were noted for the different sperm measures examined at or after the sacrifice of the parental F1 males. The number of homogenisation resistend testicular spermatids or ca
udal epididymal sperm, the percentages of abnormal and normal sperm and sperm motility data were comparable to control animals
Reproductive performance:
no effects observed
Description (incidence and severity):
For all F1 males, copulation was confirmed. The male mating index is 100%. Fertility was proven for most of the F1 males within the scheduled mating interval for F2 litter. One control, one low-dose and
one high-dose male did not generate F2 pups. The male fertility index ranges from 96-100 % without any relation to dosing. This reflects the normal range of biological variation inherent in the strain of rats used for the study. All respective values are within the range of historical control data of the test laboratory. The apparently infertile males did not show histopathological findings that could explain infertility.
The female mating index calculated after the mating period for F2 litter was 100% in all dose group s. The mean duration until sperm was detected (GD 0) varied between 2.2 and 2.8 days without any
relation to dosing. All sperm positive rats delivered pups or had implants in utero, except one control, one low-dose and one high-dose female rat.
The female fertility index ranges from 96 (control, low and high-dose) to 100 (mid-dose) % without any relation to dosing. This reflects the normal range of biological variation inherent in the strain of
rats used for the study. All respective values are within the range of historical control data of the test laboratory. The apparently non pregnant females did not show histopathological findings.
The mean duration of gestation values varied from 22.0 and 22.2 days without any relation to dosing.
The gestation index was 100% in all dose groups.
Implanation was not affected by treatment since the number of implanation sites was comparable to control. There were no indications for test item-related intrauterine embryo-/fetolethality since pos
timplantation loss did not show any statistically significant differences between the groups. The mean number of F2 pups delivered per dam remained unaffected. The rate of liveborn pups was also
not affected by treatment with the test item, as indicated by live birth index of 99% in all dose groups.
Moreover, the number of stillborn pups was comparable between the groups.

Effect levels (P1)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
P1/ F1 parental systemic
Effect level:
400 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
NOEL
Remarks:
P1/ F1 parental systemic
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Target system / organ toxicity (P1)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
The F1 pups did not display any treatment-related clinical signs until weaning. One pup of the control group displayed a domed head on PND 14-16 and was cannibalised on PND 16.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
The viability index indication pup mortality during early lactation (OND 0-4) varied between 100 (low dose) and 99% (other dose groups). The lacation index indicating pup mortality on PND 4-21 varied
between 100 (low-dose), 99 (control), 97 (mid-dose) and 92%**(high-dose). The statistically significant decreased lactation index in the high-dose group was caused by the non test item-related death
of 2 dams on PND 13 and 18, the litters of which had to be sacrified. These litters losses were considered as spontaneous in nature.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test item-related influence on F1 pup body weight was noted in all treated groups. Mean body weight gain of low and mid-dose pups was comparable to control group. A temporary decrease of mean
body weight gain was noted for the high dose pups on PND 4-7. Considering the absence of significant differences in weights themselves, these decreases were regarded as incidential and non treatment-related.
Food consumption and compound intake (if feeding study):
not examined
Description (incidence and severity):
The food consumption of the parental F1 animals is reported above.


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
Description (incidence and severity):
The first day when vaginal opening was observed was PND 28, the last was PND 42. The mean age at vaginal patency was 31.6, 33.0, 33.2* and 33.9* days in the control, low-, mid- and high-dose,
respectively. The mean body weight on the day, when vaginal patency was noted, amounted to 95.5, 101.4, 101.5 and 98.1 g in the control, low-, mid- and high-dose, respectively. This minimal apparent
increase in the high-dose group is considered secondary to the lower female body weight during major phase of sexual maturation.
The first day of preputial separation was observed was PND 39, the last was PND 49. The mean number of days to reach the criterion in the control and 50, 150 and 400 mg/kg bw/d test group was 41.6, 41.4, 41.6 and 42.5 days, indication that male sexual maturation was not influenced by the test item. The mean body weight, when preputial separation was recorded, amounted to 171.1, 168.9, 166.9 and 162.2*g. All ages and corresponding body weight values were either below or at the lower end of the historical control range. The significantly low high-dose body weight corresponds to the slightly, but not-significantly, later high-dose male sexual maturation.
Anogenital distance (AGD):
not specified
Nipple retention in male pups:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The absolute organ weight of the brain were statistically significant changed in comparison to the control.
absolute brain weight (male +female): 99%( low-dose); 97% (mid-dose); 97% * (high-dose)
absolute brain weight (male): 99% (low-dose); 95%*(mid-dose); 96%*(high-dose)
These marginal differences in absolute brain weights reflect normal biological variation in this strain of rats as they are comparable to historical control data. Relative brain weights remained unchanged.
These findings are neither adverse nor toxicologially relevant.
Gross pathological findings:
no effects observed
Description (incidence and severity):
A few F1 pups showed spontaneous findings in gross necropsy (e.g. situs inversus, supernumerary lung lobe, small kidney, enlarged kidney). There findings occured without any relation to dosing and/
or can be found in the historical control data at comparable or even higher incidences. Therefore, the se findings were not considered to be associated to the test item.
Histopathological findings:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not specified

Details on results (F1)

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.

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
fertility
Generation:
F1
Effect level:
400 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
400 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Results: F2 generation

General toxicity (F2)

Clinical signs:
no effects observed
Description (incidence and severity):
The F2 pups did not show any treatment-related clinical signs until weaning. Female pup of one control dam had multiple malformed left hindlimb and for one female pup of a high-dose dam a lateral posi
tion on one occasion during lactation (OND 19) was recorded.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
The viability index indicating pup mortality during early lactation (OND 0-4) varied between 98 (lowdose) and 99% (other dose groups). The lactation index indicating pup mortality on OND 4-21 varied
between 98 (low-and mid-dose), 99 (high-dose) and 100% (control). The statistically significantly in creased number of cannibalised pups in the low-dose was considered as spontaneous in nature.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test item-related influence of F2 pup body weight was noted in all dose groups. Temporary decreases of mean body weight change were noted in F2 male and/or female pups of high-dose
group on PND 1-4 and PND 4-7 as well as in low-dose group on PND 14-21. Considering the absence of significant differences in the weights themselves, these decreases were regardrd as incidental and not treatment-related.
Food consumption and compound intake (if feeding study):
not examined
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:
not examined
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no changes in absolute or relative organ weights noted in any of the dose groups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Single F2 pups showed some spontaneous findings at gross necropsy such as incisors sloped, hemorrhagic thymus, diaphragmatic hernia, empty stomach, dilated renal pelvis, dilated ureter,
small testis, haematoma and multiple malformed hindlimb. Every pup necropsy finding occured without any relation to dosing and/ or can be found in the historical control data at comparable or even higher incidences.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
The sex distribution and sex ratios of live F2 pups on day of birth and PND 21 were comparable to control group.

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Effect levels (F2)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
fertility
Generation:
F2
Effect level:
400 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F2
Effect level:
400 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects found
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F2)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

- 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

 

Applicant's summary and conclusion

Conclusions:
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 P0 and F1 parental rats, the highest dose tested. The respective NOEL (no observed effect level) is 50 mg/kg bw/d for the P0 parental rats and 150 mg/kg bw/d for the F1 parental rats based on effects on food consumption (and on body weight, in case of F1) being a consequence of reduced appetite in the next higher doses.

The NOAEL for fertility and reproductive performance for the P0 and F1 parental rats is 400 mg/kg bw/d, the highest dose tested.

The NOAEL for developmental toxicity, in the F1 and F2 progeny, of the test substance is 400 mg/kg bw/d, 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 (P0 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, P0 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 (= P1 in the current IUCLID nomenclature), 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 P0 and F1 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) P0 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 parental 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 F1 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 P0 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 toxicity is 400 mg/kg bw/d for the P0 and F1 parental rats, the highest dose tested. The respective NOEL (no observed effect level) is 50 mg/kg bw/d for the P0 parental rats and 150 mg/kg bw/d for the F1 parental rats based on effects on food consumption (and on body weight, in case of F1) being a consequence of reduced appetite in the next higher doses.


The NOAEL for fertility and reproductive performance for the P0 and F1 parental rats is 400 mg/kg bw/d, the highest dose tested.


The NOAEL for developmental toxicity, in the F1 and F2 progeny, of the test substance is 400 mg/kg bw/d, the highest dose tested.