Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 August to 22 October 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: compliance to GLP and testing guideline, coherence between data, results and conclusions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014

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 on 22 March 1996
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2-(2-butoxyethoxy)ethyl methacrylate
EC Number:
230-813-8
EC Name:
2-(2-butoxyethoxy)ethyl methacrylate
Cas Number:
7328-22-5
Molecular formula:
C12H22O4
IUPAC Name:
2-(2-butoxyethoxy)ethyl 2-methylprop-2-enoate
Test material form:
other: Clear colourless liquid (supplied by Evonik Industries AG, Darmstadt, Germany)

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
A total of 90 Sprague Dawley [Crl:CD(SD)BR] rats (45 males and 45 virgin females), 6 to 7 weeks old and weighing 190 - 217 g for males and 175 - 189 g for females, were received from Charles River Italia S.p.A., 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 13 days was allowed before the start of treatment, during which time the health status of the animals was assessed by thorough observations.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +/- 2°C
- Humidity (%): 55% +/- 15%
- Air changes (per hr): 15 to 20 air changes per hour
- Photoperiod (hrs dark / hrs light): artificial light for 12 hours each day
No relevant deviations from these ranges were recorded during the study.

In-life phase: August 2013 to 22 October 2013

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on 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 Butyl diglycol methacrylate was suspended in the vehicle (corn oil) and brought to the final volume appropriate for each
concentration (concentrations of 20, 60 and 200 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.
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).
The female was paired with the same male until positive identification occurred or 14 days had elapsed.
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 (concentration
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 20 to 200 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. 96370), in the range from 5 to 200 mg/mL.
Duration of treatment / exposure:
Males
Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter through the day before necropsy
(Day 29 - 30 of treatment).

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 a minimum of 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 gestation 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 daily
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
The test item was administered orally, by gavage. The oral route was selected as it is a possible route of exposure of the test item in man.
3 groups comprised 10 males and 10 females rats received the test item at the dose levels of 100, 300 and 1000 mg/kg/day.
Each group comprised 10 male and 10 female rats.
Control animals:
yes
Details on study design:
Dose levels of 100, 300 and 1000 mg/kg/day were selected by the Sponsor based on information from a previous non GLP compliant study (2 week preliminary study) (RTC Study no.: 96020EXT).

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 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.

All observed parameters are reported in a group incidence table.

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. Measurements were performed using a computer generated random order. For males the tests were performed on day before necropsy 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 (for approximately 5 minutes) by an automated activity recording device. Measurements were performed using a computer generated random order. For males the tests were performed the day before necropsy and for females on Day 3 post partum.

Body weight

Males were weighed weekly from allocation to termination.

Females were weighed weekly from allocation to positive identification of mating 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 during the pre-mating period starting from allocation.
Individual food consumption for the females was measured on gestation 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.



Oestrous cyclicity (parental animals):
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 [all/P/F1/F2] male parental generations:
Testis weight, epididymis weight. 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.
Litter observations:
A parturition check was performed from Day 20 to Day 25 post coitum. Females which did not give birth after 25 days of post coitum period were sacrificed shortly after. 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.

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 killed or 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.
Pups were euthanised by intraperitoneal injection of Thiopenthal

Parental males:
The males were killed after the mating of all females (after 29-30 days of treatment).

Parental females:
The females with live pups were killed on Day 4 post partum.
The females which did not give birth 25 days after positive identification of mating were killed shortly after.

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:

- number of visible implantation sites (pregnant animals);
- number of corpora lutea (pregnant animals).

Uteri of female with no visible implantations were immersed in a 10-20% solution of ammonium sulphide to reveal evidence of implantation.


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 listed were fixed and preserved in 10% neutral buffered formalin (except eyes, optic nerves and Harderian glands, testes and epididymides which were fixed in modified Davidson's fluid and preserved in 70% ethyl alcohol).

Histopathological examination

The tissues required for histopathological examination are listed in Annex 1. 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 Annex 1 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
All pups found dead in the cage were examined for external and internal abnormalities.
All live pups sacrificed at termination were examined for external abnormalities and sex confirmation by gonadal inspection. All pups with
abnormalities were retained in an appropriate fixative.
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.
Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test if n was more than 5. 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 criterion for statistical significance was p<0.05. The mean values,
standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.
Reproductive indices:
Group mean values were calculated for all parameters. Data from non-pregnant females were excluded from group mean calculations.

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 paired x 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 were 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
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
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)

Mortality

No mortality occurred throughout the study. A total of 3 females were found not pregnant at necropsy: 2 in the mid-dose group and 1 in the high
dose group. The number of females with live pups on Day 4 post partum was: 10 each in the control and low dose groups (0 and 100 mg/kg bw/day), 8 in the mid-dose group (300 mg/kg bw/day) and 9 in the high dose group (1000 mg/kg bw/day).

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

Motor activity and sensory reaction to stimuli

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

Body weight and body weight gain
Means of body weight and body weight gain were comparable between control and treated groups both in males and females throughout the study.
The statistically significant decrease in body weight and/or body weight gain, detected in low dose group of males before pairing and during
mating, as well as the statistically significant increase in body weight gain of low dose group of females before pairing were considered of no
toxicological relevance since the changes were minimal and not dose-related.
In addition, decreases in body weight and body weight gain, noted during the post partum phase in control and treated females, were considered as a normal consequence of the parturition occurred in the females.

Food consumption

Food consumption was unaffected by treatment in both sexes during the study. The statistically decrease detected in the high dose group on Day 7
post coitum was minimal and not considered related to treatment.

Haematology

Reticulocytes were decreased in animals dosed with 1000 mg/kg/day (approximately 33%). Due to the absence of changes in the red cells
parameters, the above reticulopenia was considered toxicologically irrelevant. In addition, the statistically significant decrease of mean
corpuscular haemoglobin concentration, recorded in males dosed with 100 mg/kg/day and 1000 mg/kg/day (2%), was considered of no
toxicological importance due to its minimal severity. No changes were recorded in coagulation parameters.

Clinical chemistry

Statistically significant increase of glucose (50%) and potassium (14%) and decrease of cholesterol (27%), protein (9%), albumin (11%), calcium (3%)
and sodium (1%) were recorded in males dosed with 1000 mg/kg/day. No changes were recorded in treated females. Due to the slight severity of
the above changes, the recorded findings were not considered adverse.


Oestrus cycle, reproductive parameters, pairing combination and mating performance

Oestrous cycle and reproductive parameters (pre-coital intervals, copulatory and fertility indices) did not show relevant differences between treated and control groups.

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

Corpora lutea, implantations, pre-implantation loss and total litter size were similar between treated and control groups. The slight increase in
pre-birth loss percentage detected in mid- and high dose groups when compared to controls was not statistically significant and not dose-related. Gestation periods were similar in treated groups and controls.

Terminal body weight and organ weights

Terminal body weight was unaffected by treatment in both sexes. Some statistically significant differences were noted in the absolute and/or
relative organ weight of treated animals when compared to controls, such as:
– Higher absolute heart weight in low dose females (+11%).
– Lower absolute spleen weight in high dose females (-19%).
– Higher absolute and relative thymus weight in mid-dose females (+35% and +37% respectively).
– Higher relative kidneys weight in low dose males (+ 9%) and high dose females (13%).
All the above differences were of low magnitude and occurred without dosedependency. Therefore, they were considered unrelated to treatment.

Macroscopic observations
Detailed macroscopic observations have been reported for male and female animals from all groups. No remarkable changes were noted at post
mortem examination in treated animals when compared to controls.

Microscopic observations

Histopathological evaluation was performed on five randomly selected control and high dose males and females, as well as on all abnormalities
detected during post mortem observation. In addition, a detailed qualitative examination of the testes was performed in five randomly selected
control and high dose group males. No treatment-related findings were observed in high dose males and females. Seminiferous tubules were
evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular
layering in the germinal epithelium was noted. The lesions reported in control and treated animals were considered to be an expression of
spontaneous and/or incidental pathology, commonly seen in this species and age under our experimental conditions.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: general toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: fertility

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
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

No differences in total and live litter size, litter weight, mean pup weight and sex ratio were noted between groups at birth and on Day 4 post partum.

Clinical signs of pups

Apparently no food intake (milk), small appearance and pallor were the clinical signs noted in pups of the control and treated groups. A
malformation (acaudia) was detected in one foetus (female no. 96380039) of the low dose group. These clinical signs noted in pups were
considered unrelated to treatment.

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:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Oestrus cycle – Before pairing - Group summary data

 -----------------------------------------------------------------------------------------------------------------------------------

           Group 1                     Group 2                    Group 3                     Group 4

 Animal    Oestrus           Animal    Oestrus          Animal   Oestrus          Animal   Oestrus

 Number   Cycles            Number    Cycles           Number   Cycles         Number   Cycles

 -----------------------------------------------------------------------------------------------------------------------------------

96380001       2       96380021       1       96380041       3       96380061       3

96380003      4       96380023       2       96380043       3       96380063       3

96380005      2        96380025      2       96380045       2       96380065       3

96380007      4       96380027       0       96380047       2       96380067       2

96380009      2       96380029       3       96380049       2       96380069       2

96380011      3        96380031      3       96380051       2       96380071       2

96380013      2       96380033       3       96380053       2       96380073       2

96380015      3       96380035       2       96380055       4       96380075       2

96380017      2       96380037       4       96380057       3       96380077       4

96380019     1       96380039        2       96380059       1       96380079       2

    Means         2.5                                 2.2                            2.4                               2.5

 -----------------------------------------------------------------------------------------------------------------------------------

 Note: The number of oestrus cycles is based on the number of non sequential days the dams were in oestrus.

Reproductive parameters of males - Summary

 -----------------------------------------------------------------------------------------------------------------------------------

                              Group         1                 2                3                 4

 -----------------------------------------------------------------------------------------------------------------------------------

 

   Copulatory Index%             100.0            100.0            100.0         100.0

 

 -----------------------------------------------------------------------------------------------------------------------------------

 

   Fertility Index%                  100.0            100.0             80.0          90.0

 

 -----------------------------------------------------------------------------------------------------------------------------------

Reproductive parameters of females – Summary

 -----------------------------------------------------------------------------------------------------------------------------------

                              Group              1                 2                3                 4

 -----------------------------------------------------------------------------------------------------------------------------------

 

   Copulatory Index%                    100.0            100.0          100.0         100.0

 

 -----------------------------------------------------------------------------------------------------------------------------------

 

   Fertility Index%                        100.0            100.0           80.0          90.0

 

 -----------------------------------------------------------------------------------------------------------------------------------

Applicant's summary and conclusion

Conclusions:
On the basis of the results obtained in this study with Butyl diglycol methacrylate, the NOAEL (No Observed Adverse Effect Level) the NOAEL for
reproductive toxicity was found to be 1000 mg/kg/day for males and females.
Executive summary:

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test according to OECD Guideline 422 (22 March 1996) Butyl diglycol methacrylate was administered to 10 Hsd: Sprague Dawley SD rats/sex/dose orally by gavage at dose levels of 0 (control), 100, 300 and 1000 mg/kg bw/d. The treatment schedule included 2 weeks before pairing for males and during pairing with females until the day before necropsy, for a total of 29/30 days. Females were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until Day 3 post 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.

No animals died during the study. A total of 3 females were found not pregnant at necropsy: 2 in the mid-dose group and 1 in the high dose group. The number of females with live pups on Day 4 post partum was: 10 each in the control ad low dose groups, 8 in the mid-dose and 9 in the high dose groups.

No relevant clinical signs were seen throughout the whole study in treated animals of both sexes.

Clinical observations for neurotoxicity assessment (removal of animals from the home cage and open arena) did not reveal changes attributable to the test item.

Motor activity and sensory reaction to stimuli revealed in no relevant differences in all parameters investigated between

control and treated groups of both sexes.

No differences of toxicological significance in body weight and body weight gain were recorded in animals of both sexes compared to the control group, throughout the study.

Food consumption was unaffected by treatment in both sexes during the study.

Haematology: No changes of toxicological relevance were seen.

Clinical chemistry: No adverse findings were detected at any dose.

All females mated both in the control and treated groups. Oestrous cycle, precoital intervals, copulatory index and fertility index did not show intergroup differences.

Corpora lutea, implantations, pre-implantation and pre-birth loss percentages, total litter size and gestation length did not reveal any treatment-related effect. No differences in total and live litter size, litter weight, mean pup weight and

sex ratio were noted between groups at birth and on Day 4 post partum.

Clinical signs noted in pups throughout the study were considered unrelated to treatment.

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

Terminal body weight was unaffected by treatment in both sexes. Changes in organ weights were of slight severity and therefore considered of no toxicological significance.

Macroscopic observations: No remarkable changes were noted at post mortem examination in treated animals when compared to controls.

Microscopic observations: No treatment-related findings were observed in high dose males and females.

Conclusions:

This study is acceptable and satisfies the guideline requirement for a Screening reproductive study according to OECD 422 in rats.

On the basis of the results obtained in this study, the NOAEL (No Observed Adverse Effect Level) for general toxicity was considered to be 1000 mg/kg/day for males and females.

The NOAEL for reproductive toxicity was also found to be 1000 mg/kg/day for males and females.