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Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 June 2012 to 21 July 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted 22 March 1996.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Benzyl methacrylate
EC Number:
219-674-4
EC Name:
Benzyl methacrylate
Cas Number:
2495-37-6
Molecular formula:
C11H12O2
IUPAC Name:
benzyl methacrylate
Test material form:
liquid
Specific details on test material used for the study:
Identity : Benzyl methacrylate
Alternative names : VISIOMER® BNMA; Benzylmethacrylat
CAS number : 2495-37-6
EINECS number : 219-674-4

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Sprague Dawley [Crl:CD(SD)BR] rats from Charles River Italia S.p.A., Calco (Lecco), Italy
- Age at study initiation: 7 to 8 weeks old and weighing 211-232 g for males and 135-199 g for females, at receipt and approximately 10 weeks at study initiation.
- Weight at study initiation: (P) Males: 335g-338g; Females: 233g-238 g
- Fasting period before study: no
- Housing: 5 sex/cage
- Diet (e.g. ad libitum): ad libitum except for clinical pathology investigation
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +/- 2°C
- Humidity (%): 55% +/- 15%
- Air changes (per hr): approximately 15 to 20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To: 24 May 2012 to 21 July 2012

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was administered orally by gavage and the formulations were prepared daily.

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water): solubility of test item
- Concentration in vehicle: 10, 35 and 100 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg body weight
- Lot/batch no. (if required): ----
- Purity: ----
Details on mating procedure:
Mating was monogamous (one male to one female). A vaginal smear was taken from the day after the start of pairing until positive identification of copulation (sperm identification, vaginal plug in situ or copulation plugs found in the cage tray).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to commencement of treatment, analysis was performed to confirm that the proposed formulation procedure was acceptable (content check and homogeneity). Results of the analyses were within the limits of acceptance.
The stability was found to be 24 hours at room temperature in the concentration range of 10 to 100 mg/mL.
Samples of the formulations prepared on Week 1 and last Week were also analysed to check the concentration and homogeneity. Results of the analyses were within the limits of acceptance.
Chemical analysis was carried out by the Analytical Chemistry Department at RTC according to a validated method (RTC Study No. 90820), in the range from 1 to 200 mg/mL. The software used for this activity was the Empower® Pro build No. 2154
Duration of treatment / exposure:
Males
Animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter through the day before necropsy.

Dose volumes were adjusted once per week for each animal according to the last recorded body weight.

Females
Animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 3 post partum (the day before sacrifice).

Dose volumes were adjusted once per week for each animal according to the last recorded body weight.
During the post coitum period, dose volumes were calculated according to individual body weight on Days 0, 7, 14 and 20 post coitum and on Day 1 post partum. Thereafter individual dose volumes remained constant.
Frequency of treatment:
once a day
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
175 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
3 groups of 10 males and 10 females received the test item at the dose levels of 50, 175 and 500 mg/kg/day. A similarly constituted group of
animals received the vehicle alone (corn oil) and acted as control.
Control animals:
yes, concurrent vehicle
Details on study design:
The oral route was selected as it is a possible route of exposure of the test item in man.
Dose levels of 50, 175 and 500 mg/kg/day were selected by the Sponsor based on information from previous non GLP compliant studies (RTC Study nos.: 90830EXT and 90840EXT).
Positive control:
no

Examinations

Parental animals: Observations and examinations:
Mortality

Throughout the study, all animals were checked early in each working day in the morning and in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.

Clinical signs

Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs were recorded. Observations were performed approximately at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions.

Clinical observations (Functional Observation Battery Tests)

Once before commencement of treatment and once a week thereafter, each animal was given a detailed clinical examination.
Each animal was removed from the home cage and observed in an open arena.
The tests included observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern).
All observations were recorded for individual animals.

Grip strength and sensory reactivity to stimuli

Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group for evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli); an assessment of grip strength was also performed. For males the tests were performed on Day 29 of the study and for females on Day 3 post partum.

Motor activity assessment (MA)

Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group and the motor activity was measured (at least 5 minutes) by an automated activity recording device. Measurements were performed using a computer generated random order. For males the tests were performed on Day 29 of the study and for females on Day 3 post partum.

Body weight

Males were weighed on the day of allocation to treatment groups, on the day of treatment commencement, weekly thereafter and at termination.

Females were weighed on the day of allocation to treatment groups, on the day of treatment commencement, weekly thereafter until pairing and on Days 0, 7, 14 and 20 post coitum. Dams were also weighed on Days 1 and 4 post partum.

Food consumption

The weight of food consumed by each cage of males and females was recorded weekly, where possible, during the pre-mating period following allocation. Individual food consumption for the females was measured on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Day 4 post partum starting from Day 1 post partum.

Clinical pathology investigations

As a part of the sacrificial procedure, samples of blood were withdrawn under isofluorane anaesthesia from the abdominal vena cava from 5 males and 5
females (females with viable litters) randomly selected from each group, under condition of food deprivation.
The blood samples collected were divided into tubes as follows:

EDTA anticoagulant for haematological investigations
Heparin anticoagulant for biochemical tests
Citrate anticoagulant for coagulation tests

The measurements performed on blood samples are listed below:

Haematology

Haematocrit
Haemoglobin
Red blood cell count
Reticulocyte count
Mean red blood cell volume
Mean corpuscular haemoglobin
Mean corpuscular haemoglobin concentration
White blood cell count
Differential leucocyte count
- Neutrophils
- Lymphocytes
- Eosinophils
- Basophils
- Monocytes
- Large unstained cells
Platelets

Coagulation tests

Prothrombin time

Clinical chemistry

Alkaline phosphatase
Alanine aminotransferase
Aspartate aminotransferase
Gamma-glutamyltransferase
Urea
Creatinine
Glucose
Triglycerides
Bile acids
Phosphorus
Total bilirubin
Total cholesterol
Total protein
Albumin
Globulin
A/G Ratio
Sodium
Potassium
Calcium
Chloride







Oestrous cyclicity (parental animals):
Vaginal smears

Vaginal smears were taken daily in the morning starting two weeks before pairing until a positive identification of copulation was made. The vaginal smear data were examined to determine the following:

a) anomalies of the oestrous cycle;
b) pre-coital interval (i.e., the number of nights paired prior to the detection of mating).

Sperm parameters (parental animals):
Parameters examined in [P] male parental generation:
[testis weight, epididymis weight, morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) ]
Litter observations:
Parturition and gestation length

A parturition check was performed from Day 20 to Day 25 post coitum.
Gestation length was calculated as the time between the day of successful mating (Day 0 post coitum) and the day of commencement of birth (i.e. first detected presence of offspring in the cage). The day that offspring were first detected in the cage was considered Day 0 post partum.

Pups identification, weight and observation

As soon as possible, after parturition was considered complete (Days 0 or 1 post partum), all pups (live and dead) were counted, sexed and live pups were
identified.
Live pups were individually weighed on Days 1 and 4 post partum.
Pups dying during the lactation period were weighed before the despatch to necropsy.
Observation was performed once daily for all litters.
Postmortem examinations (parental animals):
Parental animals were killed by exsanguination under isofluorane anaesthesia. All animals were subjected to necropsy.

Parental males:
Males were sacrificed after completion of the mating period after 33 days of treatment.

Parental females:
The females with live pups were killed on Day 4 post partum.

The clinical history of the males and females of the parental generation was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices).
Changes were noted, the requisite organs weighed and the required tissue samples preserved in fixative and processed for histopathological examination.

Females:
All females were examined also for the following:

a) external and internal abnormalities;
b) number of visible implantation sites (pregnant animals);
c) number of corpora lutea (pregnant animals).

Organ weights

From all animals completing the scheduled test period, the organs were dissected free of fat and weighed.
The ratios of organ weight to body weight were calculated for each animal.

Tissues fixed and preserved

Samples of all the tissues were fixed and preserved in 10% neutral buffered formalin (except testes and epididymides which were fixed in modified Davidson's fluid and preserved in 70% ethyl alcohol).
The examination was restricted as detailed below:

a) Tissues from 5 males and 5 females randomly selected (animals evaluated for clinical pathology) in the control and high dose groups killed at term.
b) All abnormalities in all groups











Postmortem examinations (offspring):
Pups were sacrificed by intraperitoneal injection of Sodium Thiopental
All pups found dead in the cage were examined for external and internal abnormalities.
All live pups sacrificed at termination were killed and examined for external abnormalities and sex confirmation by gonadal inspection.
Statistics:
Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by
Dunnett’s test or a modified t test, depending on the homogeneity of data.
The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non-parametric version of the Williams test.
The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.

Reproductive indices:
The following reproductive indices were calculated:

Males
Copulatory Index (%) = no. of animals mated/no. of animals paired x 100
Fertility Index (%)= no. of males which induced pregnancy/no. of animals paired x 100

Females
Copulatory Index (%) = no. of animals mated/no. of animals pairedx 100
Fertility Index (%) = no. of pregnant females/no. of females paired x 100

Males and Females

Pre-coital Interval = Mean number of days between pairing and mating

Sex ratios were calculated at birth and on Day 4 post partum and are presented as the percentage of males per litter.
Offspring viability indices:
Females
Pre-birth loss was calculated as a percentage from the formula:

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

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

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

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

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

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

(no. of corpora lutea - no. of implantations)/ no. of corpora lutea x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

- CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No mortality occurred throughout the study.
All females proved to be pregnant.
The number of females with live pups on Day 4 post partum was 10 both in the control and in all treated groups.

Clinical observations (Functional Observation Battery Tests) - (Table 2)
Neurotoxicity assessment (removal of animals from the home cage and open arena)
Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item.

- BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Means of body weight and body weight gain were, in general, comparable between control and treated groups both in males and females throughout the
study.
No relevant differences in food consumption were observed in treated animals of both sexes during the study.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Oestrus cycle and reproductive parameters (pre-coital intervals, copulatory and fertility indices) were similar in treated and control groups

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Oestrus cycle and reproductive parameters (pre-coital intervals, copulatory and fertility indices) were similar in treated and control groups
Implantation, pre-implantation loss data, pre-birth loss data and gestation length of females: No significant differences were observed in treated and control groups for these parameters. All dams gave birth between Days 21 and 23 post coitum

ORGAN WEIGHTS (PARENTAL ANIMALS)
Terminal body weight was unaffected by treatment in both sexes.
Changes noted in the absolute and relative organ weights were of low magnitude and no histological findings were observed at microscopic examination, therefore they were considered of no toxicological relevance.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No treatment-related changes were noted

HISTOPATHOLOGY (PARENTAL ANIMALS)
No treatment-related changes were noted.
In addition seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and no alterations were noted.

OTHER FINDINGS (PARENTAL ANIMALS)

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Gross pathological findings:
no effects observed

Details on results (F1)

Litter data at birth, on Day 1 and on Day 4 post partum of females and sex ratio of pups: Litter data parameters and sex ratios at birth and on
Day 4 post partum were similar between groups.

Clinical signs of pups: Pre-weaning clinical signs were comparable between treated and control groups.

Necropsy findings in decedent pups and in pups sacrificed on Day 4 post partum: Necropsy findings in decedent pups and in pups sacrificed on
Day 4 post partum did not reveal any treatment-related effect.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result
Critical effects observed:
not specified

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
On the basis of the results obtained in this study, 500 mg/kg/day could be considered the NOAEL (No Observed Adverse Effect Level) for animals of both sexes.
Executive summary:

SUMMARY

 Study design

The toxic effects on rats of both sexes after repeated oral dosing withBenzyl methacrylate, as well as on effects of the test item on male and female reproductive performance, such as gonadal function, conception, parturition and early lactation of the offspring were investigated. The vehicle was corn oil. All doses were administered at a constant volume of 5 mL/kg body weight.

 

Group

Number

Treatment

(mg/kg/day)

Number of animals

1

2

3

4

0

50

175

500

10M+10F

10M+10F

10M+10F

10M+10F

Males were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 33 days. Females were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until Day 3post partum.

The following investigations were performed in all groups: body weight, clinical signs (including neurotoxicity assessment, motor activity and sensory reaction to stimuli), food consumption, oestrous cycle, mating performance, clinical pathology investigations (haematology and clinical chemistry), litter data, macroscopic observations, organ weights and histopathological examination.

Clinical signs and macroscopic observations of pups were also performed. The histopathological examination was performed only on control and high dose groups (five animals/sex/group selected randomly). It included identification of the stages of the spermatogenic cycle in five males.

 Mortality and fate of females

 No mortality occurred throughout the study.

All females proved to be pregnant. The number of females with live pups on Day 4post partumwas 10 both in the control and in all treated groups.

 

Clinical signsand clinical observations (Functional ObservationBatteryTests)

 

No relevant clinical signs were observed throughout the study in all treated animals of both sexes.

Neurotoxicity assessment (removal of animals from the home cage and open arena)

Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item.

 

Body weight and body weight gain

No relevant differences in body weights were recorded in animals of both sexes compared to the control group, throughout the study.

 

Food consumption

 Food consumption was, in general, comparable between control and treated groups.

 

Motor activity and sensory reaction to stimuli

 No relevant differences were noted in all parameters investigated between control and treated groups.

 

 Haematology

  No changes of toxicological significance were recorded.

No significant change was recorded in the coagulation test.

 

 Clinical chemistry

 The alteration (decrease in cholesterol level) noted in males receiving 500 mg/kg/day could not be conclusively attributed to treatment since it was observed in only one sex.

 

  Oestrus cycle, reproductive parameters, pairing combination and mating performance

 Oestrus cycle, pre-coital intervals, copulatory index and fertility index did not show intergroup differences.

 

 Implantation, pre-implantation loss data, pre-birth loss data and gestation length of females

  No significant differences were observed in treated and control groups for these parameters.

All dams gave birth between Days 21 and 23post coitum.

 

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

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

 

Clinical signs of pups

  Pre-weaning clinical signs were comparable between treated and control groups.

 

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

 Necropsy findings in decedent pups and in pups sacrificed on Day 4post partumdid not reveal any treatment-related effect.

 

Terminal body weight and organ weights

 Terminal body weight was unaffected by treatment in both sexes.

Changes noted in the absolute and relative organ weights were of low magnitude and no histological findings were observed at microscopic examination, therefore they were considered of no toxicological relevance.

 

Macroscopic observations

  No treatment-related changes were noted.

 

Microscopic observations

  No treatment-related changes were noted.

In addition seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and no alterations were noted.

 

Conclusions

 On the basis of the results obtained in this study, 500 mg/kg/day could be considered the NOAEL (No Observed Adverse Effect Level) for animals of both sexes.

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.