Benzyl methacrylate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

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

Test material

Test material information

Constituent 1

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:

A total of 90 Sprague Dawley [Crl:CD(SD)BR] rats (45 males and 45 virgin females), 7 to 8 weeks old and weighing 211-232 g for males and 135-199 g for females, were received from Charles River Italia S.p.A., Calco (Lecco), Italy.
After arrival the weight range for each sex was determined and the animals were temporarily identified within the cage by means of a coloured mark on the tail. A health check was then performed by a veterinarian.
An acclimatisation period of 14 days was allowed before the start of treatment, during which time the health status of the animals was assessed by thorough observations.

Animal room controls were set to maintain temperature and relative humidity at 22°C  2°C and 55%  15% respectively; actual conditions were monitored, recorded and the records retained. No relevant deviations from these ranges were recorded during the study. There were approximately 15 to 20 air changes per hour and the rooms were lit by artificial light for 12 hours each day.

In-life data from: 24 May 2012 to 21 July 2012

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

The test item was administered orally by gavage at a dose volume of 5 mL/kg body weight. Control animals received the vehicle alone at the same dose volume.

The required amount of Benzyl methacrylate was suspended in the vehicle (corn oil) and brought to the final volume appropriate for each concentration (concentrations of 10, 35 and
100 mg/mL) and kept under magnetic stirrer at room temperature prior to use and until the time of dosing of the last animal.
The formulations were prepared daily 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:

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 / concentrations

Doses / concentrations 1

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

175 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

3 groups comprised 10 male and 10 female rats received the test item at the dose levels of 50, 175 and 500 mg/kg/day. A similary constituted group of
animals received the vehicle alone (corn oil) and acted as control.

Control animals:

yes

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

Observations and examinations performed and frequency:

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.

Animals were examined in an open arena for a minimum of three minutes.

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

Sacrifice and pathology:

Parental animals were killed by exsanguination under isofluorane anaesthesia

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

Necropsy

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).


Histopathological examination

After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin.

In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed.

The examination was restricted as detailed below:

a) Tissues specified in section 4.5.6 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

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.

Results and discussion

Results of examinations

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

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

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 4 post partum was 10 both in the control and in all treated groups.

Clinical signs and clinical observations (Functional Observation Battery Tests)

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.

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.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Key result

Critical effects observed:

no

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:

Study design

 

The toxic effects on rats of both sexes after repeated oral dosing with Benzyl 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, clinical pathology investigations (haematology and clinical chemistry), macroscopic observations, organ weights and histopathological examination.

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

 

No mortality occurred throughout the study.

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.

 

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.