Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19.09.2016 to 02.11.2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted on 28 July 2015.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Test material form:
solid
Specific details on test material used for the study:
98.408% 4-(Methacryloyloxy)benzophenone
0.581% Benzophenone acetate
0.054% 4-Hydroxy benzophenone

Expiry date 10 November 2016 (extended to 10 February 2017 as communication of the Sponsor of the 17 November 2016)
Storage conditions Room temperature protected from light

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS srl, San Pietro al Natisone (UD), Italy.
- Age at study initiation: 8 to 9 weeks
- Weight at study initiation: 199 to 213 g for females, 168 to 197 g for males
- Fasting period before study:
- Housing:
- Diet : commercially available laboratory rodent diet (4 RF 21,Mucedola S.r.l., Via G. Galilei, 4, 20019, SettimoMilanese (MI), Italy) was offered ad libitum throughout the study
- Water (e.g. ad libitum): yes except in the case of urinalysis investigations
- Acclimation period: 19 days

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

IN-LIFE DATES: from 28 September (day of allocation) to 15. November 2016

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Dose level:
Group 1: Control (vehicle)
Group 2: 70 mg/kg/day
Group 3: 200 mg/kg/day
Group 4: 600 mg/kg/day
Details on mating procedure:
- M/F ratio per cage: one male to one female
- Length of cohabitation: up to 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: single
- Any other deviations from standard protocol:no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis was performed to confirm that the proposed formulation procedure was acceptable and that the stability of the formulation was satisfactory. In the present study, samples of the formulations prepared during the study were also analysed to check the concentration and homogeneity (the first and the last week of treatment, where possible).
The software used for this activity was the Empower® Pro build No. 2154. Results of formulation analyses were within the acceptability limits.
Duration of treatment / exposure:
Males
Animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing,
through the mating period and thereafter through the day before necropsy (Day 49 of study).
Males were treated for a total of 48 days. 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 12 post partum (for
at least 50 days). 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 Days 1, 4 and 7
post partum. Thereafter individual dose volumes remained constant.
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
70 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
600 mg/kg bw/day (nominal)
No. of animals per sex per dose:
3 groups of 10 males and 10 females each. A similar constituted control group (Group 1) received the vehicle alone.
Control animals:
yes, concurrent vehicle
Details on study design:
The test item was administered orally by gavage to parental animals at 5 mL/kg body weight.
The dosages, selected in consultation with the Sponsor, were 100, 300 and 1000 mg/kg/day.

Examinations

Parental animals: Observations and examinations:
In vivo observations
Full records were maintained for all measurements and observations.
Mortality
Throughout the study, all animals were checked early in each working day and again 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 at least once aweek from the start of treatment until termination, 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). Changes in fur, skin, eyes, mucous membranes, occurrences of secretions and excretions were also recorded.

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) and for assessment of grip strength.
Measurements were performed using a computer generated random order. For the females, the tests were performed on Day 12 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 the females, the tests were performed on Day 12 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, 4, 7 and 13 post partum.

Food consumption
The weight of food consumed by each cage ofmales and females was recorded weekly during the pre-mating period starting from 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 Days 4, 7 and 13 post partum starting from Day 1 post partum.
Oestrous cyclicity (parental animals):
Vaginal smears
Females were evaluated for oestrous cyclicity 7 days before allocation to groups and animals that exhibited irregular cycle were not included in the study. Before the start of treatment a total of 5 females were replaced with spare animals from the batch initially ordered for the study.
Vaginal smears were taken daily in the morning starting from two weeks before pairing throughout the mating period until a positive identification of copulation was made.
The vaginal smear data were examined to determine the following:
1. anomalies of the oestrous cycle
2. the pre-coital interval (i.e., the number of nights paired prior to the detection of mating)
Sperm parameters (parental animals):
Parameters examined in male parental generations:
testis weight, epididymis weight, morphological evaluation of the seminiferous epithelium (staging of the spermatogenic cycle)
Litter observations:
As soon as possible after parturition was considered complete, all pups (live and dead) were counted, sexed and only live pups were identified. Live pups were individually weighed on Days 1, 4, 7 and 13 post partum. Pups killed or dying during the lactation period were weighed before the despatch to necropsy. Observations were performed once daily for all litters.
Postmortem examinations (parental animals):
Parental animals killed for humane reasons were killed under carbon dioxide asphyxiation.
Parental animals that had completed the scheduled test period and selected for blood collection were killed by exsanguination under isofluorane anaesthesia.

The males were killed after the mating, after 48 days of dosing.
Parental females
The females with live pups were killed on Day 13 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 adult animals 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:
– external and internal abnormalities;
– number of visible implantation sites (pregnant animals);
– number of corpora lutea (pregnant animals).
Uteri of females with no visible implantationswere immersed in a 10% solution of ammonium sulphide to reveal evidence of implantation.

Samples of all the tissues listed in Annex 1 of Study Protocol 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).

The tissues required for histopathological examination are listed Annex 1 of Study Protocol . 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). Themorphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was also performed.
The examination was restricted as detailed below:

Tissues specified in Annex 1 of Study Protocol from5 males and 5 females randomly selected (animals evaluated for clinical pathology) in the control and high dose group killed at term.
Tissues specified in Annex 1 of Study Protocol from all animals killed during the treatment period
All abnormalities in all groups
A detailed qualitative evaluation of testes was performed on 5 randomly selected control and high dose males. The evaluation took into account the tubular stages of the spermatogenic cycle, in order to identify treatment-related effects, such as: missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germcells and sloughing of spermatogenic cells into the lumen. Identification of the stages of the spermatogenic cycle was carried out as described by Leblond and Clermont, 1952 and referred to the comprehensive reviews on the subject Russell, 1990; Creasy, 1997; Creasy, 2002. The PAS-H stained sections were used to identify the spermatogenic stages.
Postmortem examinations (offspring):
All pups found dead in the cage were examined for external and internal abnormalities. Sex confirmation by gonadal inspection was also performed.
All live pups sacrificed at termination were examined for external abnormalities and sex confirmation by gonadal inspection.
Statistics:
Standard deviationswere 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 (non-continuous variables) was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non-parametric version of the Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test if ‘n’ was more than 5. 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:
Males
Copulatory Index (%)=no. of animals mated/no. of animals paired x 100
Fertility Index (%)=no. of males which induced pregnancy/no. of males 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
Offspring viability indices:
Pre-implantation loss was calculated as a percentage from the formula:

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

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)/Total litter size at birth x 100

Sex ratios were calculated at birth and on Day 4 post partum and were presented as the percentage of males per litter.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
Salivation was noted for a limited number of days in males and females receiving 600 mg/kg/day before pairing.
Thereafter no clinical signs were noted both in control and treated animals.
Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No treatment related effects were seen in animals of both sexes compared to the control group, throughout the study.
Haematological findings:
no effects observed
Urinalysis findings:
no effects observed
Description (incidence and severity):
No adverse findings were observed in urinalysis assessed in males only.
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No relevant differenceswere noted in all parameters investigated between control and treated groups at the end of the treatment period.
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Oestrous cycle, pre-coital intervals, copulatory index and fertility index did not show intergroup differences.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
Implantation sites, pre-implantation loss data, pre-natal loss data and gestation length of females:
No significant differences were observed for these parameters between the treated groups and the controls. All pregnant dams gave birth on Day 22 post coitum (mean value).

Litter data and sex ratio were unaffected by treatment, at birth, on Days 1, 4 and 13 post partum.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effect observed

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no compound-related effects. Pre-weaning clinical signs were comparable between treated and control groups.
Sexual maturation:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormone determination
Thyroid hormones in adult males.
No differences in hormone levels were described between the control and the treated parental males.
Thyroid hormones in male and female pups performed on Day 13 post partum.
Hormone levels in treated pups were similar to controls.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effect observed

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general toxicity and for reproductive and developmental toxicity was considered to be 600 mg/kg/day both 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 (28 July 2015) 4-{Methacryloyloxy)benzophenone was administered to 10 Hsd: Sprague Dawley SD rats/sex/dose orally by gavage at dose levels of 0 (control), 70, 200 and 600 mg/kg bw/d. The treatment schedule included 2 weeks before pairing, during pairing, post coitum and post partum periods up to day 3 post partum. Animals were administered for approximately 5 and 8 weeks for males and females, respectively. 

 

The purpose of this study was to generate information concerning toxic effects on Sprague Dawley rats of both sexes after repeated oral dosing with 4-(Methacryloyloxy)benzophenone,as well as any effects of the test item on male and female reproductive performance, such asgonadal function, conception, parturition and lactation of offspring. The dose levels used during the study were 70, 200 and 600 mg/kg/day.

Males

One control male was sacrificed on Day 14 of the study for poor health condition. On thebasis of the macroscopic and microscopic findings, the factor contributory to the death wasmainly attributed to a misdosing.

Males were treated for 2 weeks prior to pairing and during pairing with females until the daybefore necropsy, for a total of 48 days.

During the in-life phase, mortality check, clinical signs (including neurotoxicity assessment,motor activity and sensory reaction tostimuli), body weight, body weight gain, food consumptionand mating performance were evaluated.

Clinical pathology investigations (haematology, clinical chemistry, urinalysis), organ weights,macroscopic observations and histopathological examination were also performed. In

addition, thyroid hormone levels were determined in all adult males. The histopathologicalexamination was performed only on control and high dose groups (5 animals/sex/grouprandomly selected). It included identification of the stages of the spermatogenic cycle in the

same five control and high dose males.

No relevant signs of toxicological significance were observed in treated males, apart from atransient salivation, observed for a few days in the high dose animals.

Body weights and food consumption did not show any treatment related effects. Fertilityindex and copulatory index were unaffected by treatment. No effects considered treatmentrelated were observed in haematology or coagulation, clinical chemistry and urinalysis.

Hormone analysis did not showany relation to treatment. Some fluctuations in organweights,absolute and/or relative, were noted, but in the absence of histopathological correlated

findings, these changes were considered of no toxicological significance.

Females

Females were treated for 2weeks prior to pairing, during pairing and throughout the gestationand lactation periods until Day 12post partum.

During thein-lifephase, mortality check, clinical signs (including neurotoxicity assessment,motor activity and sensory reaction tostimuli), body weight, body weight gain, food

consumption and mating performance were evaluated. Clinical pathology investigations(haematology and clinical chemistry) and thyroid hormone determination in pups on Day 13post partumwere also evaluated. The histopathological examination was carried out in five

females of control and high dose groups, selected randomly. Clinical signs of pups, as well asnecropsy examination of decedent pups or pups sacrificed on Days 4 and 13post partum(including thyroid weight) were recorded. Litter data, sex ratios and gestation length were

recorded.

No relevant signs of toxicological significance were observed. As noted for males, salivation was recorded occasionally in treated females receiving 600 mg/kg/day. Body weights andfood consumption did not show changes of toxicological significance.

Concerning the reproductive parameters, no relevant differences were found in terms ofmating performance including the pre-coital interval (number of days paired to spermpositive day) and the copulatory evidence (the positive identification of mating i.e. the

presence of spermand/or copulation plugin situor in the cage), as well as fertility index.

The slight changes noted at clinical chemistry investigation (haematology, clinical chemistryand coagulation) were not considered treatment related. Some fluctuations in organ weights,absolute and/or relative, were noted but in the absence of histopathological correlated

findings, these changes were considered of no toxicological significance.

Conclusion

Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general toxicity and for reproductive and developmental toxicity was considered to be 600 mg/kg/day both for males and females.