Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

Toxicity to Reproduction (Screening): In accordance with Column 2 of ANNEX VIII of the REACH regulation, screening for reproductive/developmental toxicity study does not need to be conducted as a pre-natal developmental toxicity study (OECD 414/GLP) in rats is available.

Extended one-generation reproductive toxicity: In accordance with Column 2 of ANNEX IX of the REACH regulation, the extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies (OECD 408/GLP) that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity.

Link to relevant study records

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Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity
Endpoint:
screening for reproductive / developmental toxicity
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the study does not need to be conducted because a pre-natal developmental toxicity study is available
Reason / purpose for cross-reference:
data waiving: supporting information
Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Toxicity to Reproduction (Screening):In accordance with Column 2 of ANNEX VIII of the REACH regulation, screening for reproductive/developmental toxicity study does not need to be conducted as a pre-natal developmental toxicity study (OECD 414/GLP) in rats is available.

Extended one-generation reproductive toxicity:In accordance with Column 2 of ANNEX IX of the REACH regulation, the extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies (OECD 408/GLP) that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity.

Effects on developmental toxicity

Description of key information

Pre-natal developmental toxicity study (rat, oral):

NOAEL for maternal/foetotoxicity toxicity: 300 mg/kg bw/day (OECD 414/GLP)

NOAEL for embryotoxicity/teratogenicity: 1000 mg/kg bw/day (OECD 414/GLP)

Link to relevant study records

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Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 June 2020 - 02 February 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material:Batch number: Willing New Materials Technology Co., Ltd.; 23191022201
- Expiration date of the lot/batch: 21 October 2020
- Purity:97%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25℃) protected from humidity (in a tightly closed container)
Species:
rat
Strain:
Wistar
Remarks:
Hannover Wistar, (Crl:WI(Han))
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Address: Sandhofer Weg 7, D-97633, Sulzfeld, Germany) from SPF colony.
- Age at study initiation: Young adult female rats, nulliparous and non-pregnant, 11 weeks old at mating.
- Weight at study initiation: 188-257 g (the variation did not exceed ± 20% of the mean weight at the start of the treatment).
- Housing: Animals were housed in Type II and/or Type III polycarbonate cages. Deep wood sawdust bedding (SAFE 3/4-S Hygienic Animal Bedding and SAFE natural crinklets nest building material produced by J. Rettenmaier & Söhne GmbH+Co.KG (Address: Holzmühle 1, D-73494 Rosenberg, Germany) was also added to the cages) allowed digging and other normal rodent activities. Nesting material and certified cardboard hiding tunnels (GLP Mini Fun Tunnels, LBS (Serving Biotechnology) Ltd, Address: Unit 20, Gatwick Business Park, Kennel Lane, Hookwood, Surrey, RH6 0AH UK) were also provided to the animals. During the acclimation period animals were group housed (2 females/cage). After mating, the sperm-positive females were housed individually.
- Diet (e.g. ad libitum): The animals were provided with ssniff® SM R/M “Complete Feed for Rats and Mice – Breeding and Maintenance” produced by ssniff Spezialdiäten GmbH (Address: Ferdinand Gabriel Weg 16, D-59494 Soest, Germany), ad libitum. The food was routinely analysed, it was is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
- Water (e.g. ad libitum):Animals received tap water from the municipal supply, as for human consumption from a 400 or 500-mL bottle, ad libitum.The quality control analysis of the water was performed once every three months and microbiological assessment is performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8200 Veszprém, József Attila u. 36., Hungary). The water was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
- Acclimation period: Environmental acclimation period for the study was at least 30 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1-24.6°C (target range: 19-25°C)
- Humidity (%): 27-72% (target range: 30-70%)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light):12 hours daily, from 6.00 a.m. to 6.00 p.m.

Route of administration:
oral: gavage
Vehicle:
other: 1% (w/v) aqueous methylcellulose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle (1% MC), as a visibly stable solution at the appropriate concentrations according to the dose level and volume selected in the Pharmacy of Charles River Laboratories Hungary Kft. Formulations were prepared fresh prior to administration to animals or at the appropriate frequency to allow their use according to stability assessment results. For the formulation step, the calculated amount of test item was added into a clean glass beaker and was filled up to the required volume with the vehicle (the first mixing step was prepared using a mortar and pestle, then the mixture was analytically transferred into the beaker). The suspension was stirred by magnetic stirrer until homogeneity was reached. A constant volume of 5 mL/kg body weight was administered to all dose groups, including the controls. The individual volume of the treatment was based on the most recent* individual body weight of the animals.

*Note: In case of a Low dose animal (#2510), a body weight value not fitting into the profile of later values was recorded on GD 8. The administration was performed according to the actual body weight in all cases. A possible technical error could not be excluded, but as no malformation / variation was recorded in this litter, this fact had no impact on the results of the study.


VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on the available information provided by the Sponsor as well as results of the trial formulation, 1% (w/v) aqueous methylcellulose solution (abbreviation 1% MC) was selected as vehicle for this study.
- Concentration in vehicle: 1% (w/v)
- Lot/batch no. (if required): 7105702
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
According to the stability assessment results of the analytical method development and analytical method validation studies performed at the Test Facility (Study codes: 19/306-316AN), the test item was stable in the selected vehicle for at least seven days when stored at room temperature.

Sample collection for formulation analysis was performed immediately after formulation preparation in the Pharmacy of the Test Facility by a responsible member of the Analytical Department. Samples were collected twice (at the beginning and near the end of the treatment period). On each sampling occasion, top, middle and bottom duplicate samples were taken from test item formulations for concentration and homogeneity measurement, one set to analyse (which could be collected in replicates as practical) and one set as a back-up, if required for any confirmatory analyses. Similarly, duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement.

Analysis of the formulations for concentration of test item was performed using a validated analytical method in the Analytical Department of the Test Facility (Study code: 19/306-316AN). The density of the formulations was determined at the first analytical sampling by using three parallels in the Analytical Department of the Test Facility. Analysis of the formulations for concentration and/or homogeneity of test item was performed in the Analytical Department of the Test Facility on two occasions* (at the beginning and near the end of the treatment period).

*Note: Due to technical issue, one of the duplicate samples of the Low dose formulation (20 mg/mL concentration sample) collected at the first sampling occasion (24 June 2020) was accidentally lost, therefore, the measurement was repeated on 25 June 2020 from the back-up sample in case of this concentration (the samples were stored at room temperature until the re-measurement).

Acceptance criterion of the concentration analysis was 100 ± 15% of the nominal concentration.
Acceptance criterion of the homogeneity was that the coefficient of variation (CV) of replicates (top, middle and bottom of test formulations) must be less than 15%.


Details on mating procedure:
The oestrous cycle of female animals was examined a day before start of pairing (except of the first mating day), and then daily, up to the day of mating. On the first week of the mating, approximately half of the animals was examined daily for their oestrous cycles. After acclimatisation the females, according to their oestrous cycle, were paired with males for approximately 2 hours (1 male: 1 female) in the morning, until at least 24 sperm-positive females/group were obtained (on the last mating day, all the extra mated females were included in the study as agreed by the Study Monitor resulting 25 sperm-positive females/group). After the daily mating period, a vaginal smear was prepared and stained with 1% aqueous methylene blue solution. The smear was examined with a light microscope; the presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (GD 0). Sperm-positive females were separated and caged individually.

The sperm-positive, assumed pregnant females were allocated to each experimental group (on each mating day) in such a way that the group averages of the body weight were as similar as possible. The software PROVANTIS v9 was used in order to verify homogeneity/variation among/within groups.

On GD 13 and/or 14 the sperm-positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat, which is considered to confirm implantation).




Duration of treatment / exposure:
14 days (GD 6-19)
Frequency of treatment:
Daily
Duration of test:
20 days (Day 20 - Caesarean section and necropsy)
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Controll (vehicle)
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Low dose
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Mid dose
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High Dose
No. of animals per sex per dose:
25 mated female animals/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were set by the Study Monitor based on the available information of the chemical nature, characteristics of the test item, and available data and information from previous experimental work, including the results of a Dose Range Finding (DRF) study (Study code: 19/306-105PE). Based on those results, 1000 mg/kg bw/day was selected as the High dose for this study. The Mid and Low dose levels selected were 300 and 100 mg/kg bw/day. Refer to Supporting, RL1, rat/Willing, 2020/Developmental toxicity/teratogenicity (DRF).002
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General (routine) clinical observations were made twice daily (before treatment and end of each working day). Only one clinical observation was made in the afternoon on those days when detailed clinical observation was made in the morning. Furthermore, only one detailed clinical observation was made on necropsy days.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made on all animals at the onset of treatment (GD 6), then weekly and at necropsy.
Pertinent behavioural changes and all signs of toxicity including mortality were recorded including onset, degree and duration of signs as applicable. Signs evaluated were include, but not be limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), bizarre behaviour (e.g. self-mutilation, walking backwards) were recorded. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

BODY WEIGHT: Yes
- Time schedule for examinations:Body weight of each animal was recorded with precision of 1 g on GD 0, 3, 6, 8, 10, 12, 14, 16, 18 and 20. Body weight gain was calculated for each interval, including GD0-6, GD6-20 and GD0-20.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Food consumption was measured with precision of 1 g on GD 0, 3, 6, 8, 10, 12, 14, 16, 18 and 20. Food consumption was calculated for each interval, including GD0-6, GD6-10, GD6-20.

POST-MORTEM EXAMINATIONS: Yes
All animals were subjected to a necropsy and a macroscopic examination. Before expected delivery, on GD 20, Caesarean section was performed on each treated dam.

The weight of the thyroid gland with parathyroid glands for all dams were measured with a precision of at least 0.001 g. As a paired organ, it was weighed together. Absolute organ weights were measured, and relative organ weights to the body weights were calculated and reported. The organs were retained as detailed in the next section. The ovaries and uterus were removed, and the pregnancy status was ascertained. The uterus including the cervix was weighed and examined for early and late embryonic or foetal deaths and for the number of live foetuses.

The dams’ viscera were examined macroscopically for any structural abnormalities or pathological changes; the thyroid gland with parathyroid glands from all animals and all the gross findings were retained in 10% buffered formalin solution for possible future evaluation.

The retained thyroids were embedded in paraffin wax, sections were cut at 4-6 µm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope. Parathyroid glands were examined histologically only if present in routine sections. No additional histopathology evaluation was performed.

OTHER: For thyroid hormone analysis (study phase number 48092 ABR), blood samples were taken from all dams at termination by venepuncture (from v. sublingualis) into 1.2-mL tubes containing K3-EDTA as anticoagulant. The levels of the thyroid hormones (triiodothyronine, T3 and thyroxine, T4) and thyroid stimulating hormone (TSH) were determined respectively in plasma by LC-MS/MS or Luminex MAP® technology at Charles River Laboratories Evreux, France. For more details on the analytical procedure, refer to below.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included: The number of corpora lutea in each ovary and of implantation sites in each uterine horn*, the number of live foetuses, early and late embryonic death and foetal death were counted; the number and percentage of pre- and post-implantation losses were calculated. The degree of resorption was described in order to estimate the relative time of death of the conceptus. The placentas were examined macroscopically.
*Note: In case of a Mid dose female (#3520), no corpora lutea and no implantation site was recorded.
Fetal examinations:
The anogenital distance of each living foetus were measured, and the sex of each foetus were assigned based on the distance. The sex of the foetuses was confirmed by examining the internal sex organs*. Thereafter, the foetuses were individually identified, the weight was measured; approximately half of each litter was subjected to detailed visceral examination, and the other half was processed for skeletal examination.
*Note: Due to technical error, this step was not performed on two necropsy days (refer to deviations below).

After ensuring humane death, each fully developed foetus was weighed individually (with a precision of ±0.01 g) and subjected to external examination, plus an additional examination of the great arteries and thymus (for foetuses selected for visceral examination).

Approximately half of each litter (with as equal number of males as females as possible) was subjected to detailed visceral examination, and the other half was processed for skeletal examination.

For the foetuses subjected to visceral examination, the abdominal and thoracic region were opened, and the thymus and great arteries were freshly examined by means of a dissecting microscope. The rest of the body was fixed in Sanomya mixture: then after fixation, the body was micro-dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method.

For the foetuses subjected to skeletal examination, the abdominal region was opened, and the viscera and skin of foetuses were removed, and the cadaver was fixed in Alcian-blue - acetic acid – ethanol/isopropanol mixture. After fixation, the skeletons were stained by KOH-Alizarin red-S method and examined by means of a dissecting microscope.

All abnormalities (external, soft tissue and skeletal malformations, and variations) found during the foetal examinations were recorded.

Statistics:
See below
Indices:
Post-implantation loss: Number of implantations-Number of live foetuses x100/Number of implantations

Sex distribution: Number of male (female) foetuses x100/Number of foetuses

External abnormalities/litter: Number of foetuses with abnormality x100/Number of foetuses

Visceral abnormalities/litter: Number of foetuses with abnormality x100/Number of foetuses

Skeletal abnormalities/litter: Number of foetuses with abnormality x100/Number of foetuses
Historical control data:
Historical control data was provided for the relevant parameters (2015-2020). Refer to results and Appendix 9.
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs were observed during the study. All female animals were symptom-free during the study. Summary tables are presented in Appendices 3.1.1 and 3.1.2.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortality was seen during the study. Summary tables are presented in Appendices 3.1.1 and 3.1.2.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A test item-related adverse effect on body weight or body weight gain was observed in the High dose group (1000 mg/kg bw/day). No test item-related effect on body weight parameters was observed in animals in the Mid dose (300 mg/kg bw/day) or Low dose (100 mg/kg bw/day) groups when compared to controls.

Reduced body weight and body weight gain was observed in the High dose group when compared to control data (absolute and corrected by uterine weight) as detailed in Table 2. The absolute body weight gain of the High dose dams in the treatment period (GD6-20) was 22% lower than control (p<0.01), while the net (corrected) body weight gain during the treatment period was by 65% lower than the relevant control value (p<0.01). These changes were considered as a test item-related adverse effect. Summary tables are presented in Appendices 3.2.1-3.2.3.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A test item-related adverse effect on food consumption was observed in the High dose group (1000 mg/kg bw/day); no effect was noted in the Mid or Low dose groups (300 and 100 mg/kg bw/day, respectively), when compared to controls.

Reduced daily food consumption (by 12%) was observed in the High dose dams (GD6-20; p<0.01) when compared to the Control (Table 3). This fact was considered as a test item-related adverse effect, and most probably contributed to the body weight gain mentioned above. Summary tables are presented in Appendix 3.3.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
During the macroscopic examination of dams, the thyroid gland with parathyroid glands were retained from all animals and the organ weights recorded. There were no statistical differences in the organ weights between the control and the test item treated groups. The weight of gravid uterus was similar within groups, no test item effect was noted. Summary tables are presented in Appendix 3.7
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test item related observations were recorded for any animals in the study. For pregnant females, no test item-related macroscopic findings were observed in any dose groups. A single, clear cyst (approx. 1 mm diameter) was observed in a Mid dose dam (#3513), this observation was considered incidental or common background. In case of the non-pregnant Mid dose female (#3520), small thyroid/parathyroid glands were observed.Summary tables are presented in Appendix 3.7.

Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No test item related changes were recorded during the histopathology evaluation. All changes, like ectopic thymus, ultimobranchial cyst, multinucleated giant cells, cytoplasmatic vacuolation in the parathyroid seen in the thyroid/parathyroid glands of Control or test item treated animals were without any meaningful differences in severity or incidence and regarded as incidental or common background. The microscopic examination of the thyroid/parathyroid glands of the non-pregnant Mid dose female (#3520) did not reveal any microscopic changes. Histopathology data are presented in Appendix 3.8.1 and 3.8.2 (summary tables).
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Blood samples were taken from all dams at termination and 100 blood samples were successfully analysed for thyroid hormones. There were no statistically significant changes in the concentration of the T3, T4 and TSH level between the Control and any test item treated groups. Thyroid hormone data are presented in Appendix 3.9 (summary table).
Number of abortions:
not examined
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
The pre-implantation loss in the Low dose group (100 mg/kg bw/day) was statistically significantly lower (p<0.01), which led to the fact that the total intrauterine mortality was significantly lower and hence, the numbers of implantations and viable foetuses were significantly higher than the Control group. However, all those trends had no biological relevance, all these values were within the historical control range, therefore not considered as test item related effect. Relevant data are shown in Appendices 3.4. (summary tables). Historical control data are shown in Appendix 9.
Total litter losses by resorption:
not examined
Early or late resorptions:
no effects observed
Description (incidence and severity):
No test item-related effect was noted during the intrauterine evaluation in any dose groups. Relevant data are shown in Appendices 3.4. (summary tables)
Dead fetuses:
no effects observed
Description (incidence and severity):
The number of dead foetuses were comparable with control. Relevant data are shown in Appendices 3.4. (summary tables).
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
One hundred females (25 for each group) were mated successfully in the study. The number of confirmed pregnant, evaluated dams was 25, 25, 24 and 25 in the Control, Low, Mid and High dose groups, respectively. Relevant data are shown in Appendices 3.4 (summary table).
Other effects:
no effects observed
Description (incidence and severity):
No abnormalities were observed in the placentas of any dams in the Control, Low, Mid, or High dose groups. Relevant data are shown in Appendix 3.5.2 (summary data).
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Abnormalities:
effects observed, non-treatment-related
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
A slight test item-related effect was seen in foetal body weight in the High dose group (1000 mg/kg bw/day); no effect was noted in Mid and Low dose groups (300 and 100 mg/kg bw/day, respectively).

Reduced body weights (by 4.2%, significant at p<0.05; litter mean values) were recorded in the High dose group (1000 mg/kg bw/day). Although the weights in treated groups were not below the expected historical control range (mean ± SD 3.38 ± 0.27), the dams did have significantly lower body weight (5.4% below control corrected weight) and lower food intake (12% below control); therefore the foetal weight differences were considered to be possibly treatment related, but in the presence of significant maternal toxicity, not a direct effect of the test item. Summary tables are presented in Appendix 3.5.1;. Historical control data are shown in Appendix 9.

Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
The mean number of viable foetuses was significantly (p<0.05) increased at 100 mg/kg bw/day; all other dose groups were comparable to the control. Relevant data are shown in Appendices 3.4. (summary tables).
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The number of male and female foetuses within a group was varied within normal limits. The sex ratio of male and female foetuses was similar in all dose group, no test item effect was identified on this parameter. Summary tables are presented in Appendix 3.4.
Changes in litter size and weights:
effects observed, non-treatment-related
Description (incidence and severity):
There was higher number of foetuses with retarded body weights in the Low and High dose groups, while lower number of runts were recorded in the Mid dose group when compared to control; the same trends were seen when the number of affected litters are evaluated (Table 6). However, none of those differences were statistically significant, there was no dose response and the observed values were just outside the maximum of the historical control range (5.6%) when the foetal incidence was evaluated, and within the historical control range (maximum of 41.7%) when the litter-based incidence was evaluated, thus no test item effect was concluded.
Summary tables are presented in Appendix 3.5.1;. Historical control data are shown in Appendix 9.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
There were no external variations or malformations in the study. Foetopathology report is shown in Appendix 7. Historical control data are summarized in Appendix 9. Relevant data are shown in Appendix 3.5.2 (summary tables)
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Based on the skeletal findings the number of intact / variant / malformed foetuses were comparable with the control in the Low, Mid and High dose groups (100, 300 and 1000 mg/kg bw/day, respectively). All data was considered to be in the normal range and there was no dose related trend.

For all the skeletal variations, the foetal or litter-based incidence in the test item treated groups was comparable with the current study control or historical control values or were considered incidental ascribed to individual variability and not related to treatment. Therefore, they were considered as biologically not relevant findings and not related to the test item treatment.

Only one malformation (pelvic gridle) was recorded in the test item treated groups, for two Mid dose foetuses in one litter (#3504/6 and #3504/10) and one High dose foetus (#4504/10). This is a frequently seen malformation in the historical control data. The frequency was comparable with the control group results of recent studies*. Thus, these malformations were considered as not being a test item related effect.
*Note: The same findings were recorded in recent studies of 18/288-105P (3/110 foetuses in 2/22 Control litters), 18/296-105P (2/114 foetuses in 2/24 Control litters) and 19/211-105P (4/123 foetuses in 4/24 Control litters).

Bent scapula was observed only in one foetus of the Control group; this was considered as an irrelevant finding, from the study point of view.
In case of several variations (incomplete ossification of skull (3 or more bones), wavy ribs, unossified sternebra (3 or more), dumbbell or asymmetric vertebrae ossification (2 or more), unossified metatarsal (limbs)) the foetal or litter-based incidence in the test item treated groups was comparable with the current study control or historical control values. Therefore, they were considered as biologically not relevant findings and not related to the test item treatment.
Some of the observed variations (dumbbell shaped vertebrae, misaligned vertebrae (lumbar transverse process), bipartite ossification of a vertebrae, bipartite ossification and misshapen sternebra) were considered incidental findings based on the isolated occurrence (recorded only for one foetus of the Low dose group in case of the vertebrae findings or one High dose foetus in case of the sternum finding), these were not related to the treatment.

In case of unossified hyoid body in the skull, increased incidence was observed in the Mid dose group when compared to the Control group or the historical control data, while the incidence was the same in the Low dose group and no occurrence was noted in the High dose group. No statistical significance was detected, and there was no dose response. Based on these data, the observed values were considered not related to the test item treatment.

In case of interrupted ribs (or costal cartilage), increased incidence was observed in the Low and Mid dose groups when compared to the Control group or the historical control data, but the incidence in the High dose groups was the same as in the Control. No statistical significance was detected, and there was no dose response. Based on these data, the observed values were considered not related to the test item treatment.

Foetopathology report is shown in Appendix 7. Historical control data are summarized in Appendix 9. Relevant data are shown in Appendix 3.5.2 (summary tables).

Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Based on the visceral findings the number of intact / variant / malformed foetuses were comparable with the control in the Low, Mid and High dose groups (100, 300 and 1000 mg/kg bw/day, respectively).

For all the visceral findings, the foetal or litter-based incidence in the test item treated groups was comparable with the current study control or historical control values or were considered incidental ascribed to individual variability and not related to treatment. Therefore, they were considered as biologically not relevant findings and not related to the test item treatment.

A malformation, small testis, was observed in a Low dose foetus (#2524/6). Although this finding was not included in the historical control database, based on the lack of dose response and its incidental occurrence, it was not considered as a test item related effect.

Short brachiocephalic trunk was noted only in two Low dose foetuses (2509/2 and #2517/2). The study control group did not show this variation, but it was included in the historical control database although at a slightly lower frequency, however based on the isolated occurrence and lack of dose response, it was not considered as a test item related effect.

In case of thymic cord variation, the number of affected litters and foetuses was slightly increased in the High dose group, but this variation was included in the study control and the difference compared to that level was statistically not significant. Furthermore, the observed litter-based frequencies (12.5% in the Mid dose group and 24.0% in the High dose group) were within historical control range of individual control groups (range: 4.2-30.4%). Based on these data, the observed values were considered not related to the test item treatment.

Foetopathology report is shown in Appendix 7. Historical control data are summarized in Appendix 9. Relevant data are shown in Appendix 3.5.2 (summary tables)
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Sex of the foetuses was determined by visual observation of the anogenital distance before the visceral examination. These results were later confirmed by internal examination of the reproductive organs with a 100% external to internal correspondence. When evaluating the litter mean data, no statistically significant differences were seen between control and test item treated groups. The distribution of the individual results of male / female foetuses in the control and test item treated groups were also similar. Thus, no effect on anogenital distance was concluded (the slight, but statistically significant (p<0.05) decrease in the group mean values of the Mid and High male foetuses compared to control were considered as having less relevance).

Summary tables are presented in Appendix 3.5.1; Historical control data are shown in Appendix 9.
Dose descriptor:
NOAEL
Remarks:
embryotoxicity
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Based on the lack of any test item-related intrauterine effect in any treatment group.
Dose descriptor:
NOAEL
Remarks:
foetotoxicity
Effect level:
300 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Based on the effect on foetal weight at 1000 mg/kg bw/day in the presence of maternal toxicity.
Dose descriptor:
NOAEL
Remarks:
teratogenicity
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Based on the lack of treatment-related malformations in any dose group.
Abnormalities:
no effects observed
Developmental effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
no
Relevant for humans:
no

Dose formulation analysis

The analysis showed that the mean concentration of all formulations was found to be in the range of 95-102% of their nominal concentrations and were found to be homogenous. No test item was detected in the vehicle control formulations. Based on these analytical results, test item formulations were considered suitable for the study purposes.

Formulation occasion

Nominal concentration (mg/mL)

Measured concentration
(mg/mL)

Percentage of the nominal concentration
(%)

RSD
(%)

24 June 2020

Control

not detected

-

-

20*

18.92 ± 0.378

95

1.9

60

57.51 ± 0.484

96

0.8

200

194.66 ± 1.87

97

0.9

15 July 2020

Control

not detected

-

-

20

19.70 ± 0.300

98

1.4

60

60.34 ± 0.499

101

0.8

200

203.15 ± 3.40

102

1.6

 *Note: Due to technical issue one of the taken duplicate samples of the 20 mg/mL concentration sample was lost,

therefore, the measurement was repeated on 25 June 2020 from the back up sample in case of this concentration

(the samples were stored at room temperature until the re-measurement).

 

Conclusions:
In conclusion, WILLING TiBTD, when administered daily by oral gavage to pregnant Hannover Wistar rats from Gestation Day (GD) 6 to 19 at 100, 300 and 1000 mg/kg bw/day induced significant body weight and food consumption effects at the 1000 mg/kg bw/day dose level. No embryotoxic effect was recorded, but foetotoxicity (slightly reduced foetal body weight in the presence of significant maternal toxicity) was observed at 1000 mg/kg bw/day. There were no test item-related effects on external, visceral or skeletal development of foetuses in the study.

The following NOAELs were derived: NOAEL for maternal/ foetotoxicity toxicity:300 mg/kg bw/day; NOAEL for embryotoxicity/ teratogenicity: 1000 mg/kg bw/day
Executive summary:

In a prenatal developmental toxicity in rats (OECD 414/GLP), WILLING TiBTD (97%) was administered to pregnant Crl:WI (Han) rats (25/dose) by oral gavage in 1% (w/v) aqueous methylcellulose at dose levels of 0, 100 (low dose), 300 (mid dose) and 1000 (high dose) mg/kg bw/day daily for 14 days (GD 6-19).  

All test item formulations were within the range of 95-102% of nominal concentration and were found to be homogenous. No test item was detected in the vehicle control samples. Test item formulations were considered suitable stable for the study purposes.

No mortality or clinical observations were recorded in any of the dose groups. Test item-related adverse effects on body weight parameters and food consumption were observed in the High dose group (1000 mg/kg bw/day), when compared to controls. No test item-related effects were noted in the Mid dose (300 mg/kg bw/day) or Low dose (100 mg/kg bw/day) groups.

No test item-related macroscopic or microscopic findings were present at necropsy in the evaluated adult animals. There were no statistically significant changes in the thyroid hormone concentrations of T3, T4 and TSH between the groups. There were no statistical differences in the thyroid organ weights between the Control and the Dose groups. There were no statistically significant differences in the intrauterine parameters in the test item treated animals when compared to the controls. No abnormalities were observed in the placentas of any dams in the Control, Low, Mid, or High dose groups.

Reduced foetal body weights were recorded in the High dose group (1000 mg/kg bw/day); the differences were considered to be possibly treatment related, but in the presence of significant maternal toxicity, not a direct effect of the test item (i.e. secondary non-specific consequence of maternal toxicity effects). No effect was noted in Mid and Low dose groups (300 and 100 mg/kg bw/day, respectively). There were no test item-related effects on external, visceral or skeletal development of the foetuses in the study. Foetal malformations observed in the study were all considered to be incidental. They showed no dose dependency and thus were not regarded as test item-related effects.

No endocrine disrupter effect was identified in the study (based on the lack of effects on thyroid hormones and thyroid histopathology for dams, and anogenital distance for foetuses).

In conclusion, WILLING TiBTD, when administered daily by oral gavage to pregnant Hannover Wistar rats from Gestation Day (GD) 6 to 19 at 100, 300 and 1000 mg/kg bw/day induced significant body weight and food consumption effects at 1000 mg/kg bw/day dose level. No embryotoxic effect was recorded, but foetotoxicity (slightly reduced foetal body weight in the presence of significant maternal toxicity) was observed at 1000 mg/kg bw/day. There were no test item-related effects on external, visceral or skeletal development of the foetuses in the study.

The following NOAELs were derived:

NOAEL for maternal toxicity:300 mg/kg bw/day (based on the effects on body weight and food consumption at 1000 mg/kg bw/day).

NOAEL for embryotoxicity: 1000 mg/kg bw/day (based on the lack of any test-item related intrauterine effect in any treatment group.)

NOAEL for foetotoxicity: 300 mg/kg bw/day (based on the effect on foetal weight at 1000 mg/kg bw/day in the presence of maternal toxicity.)

NOAEL for teratogenicity: 1000 mg/kg bw/day (based on the lack of treatment-related malformations in any dose group.)

Endpoint:
developmental toxicity
Remarks:
Dose Range Finding Study for Prenatal Developmental Toxicity (Gavage) in Pregnant Hannover Wistar Rats
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
13 March 2020- 12 June 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: DRF study in rats for OECD 414
Qualifier:
according to guideline
Guideline:
other: DRF study in rats for OECD414
Version / remarks:
The details in the study report results relevant to study number 19/306-105P (A GLP Prenatal Developmental Toxicity of WILLING TiBTD by Oral (Gavage) in Pregnant Hannover Wistar Rats).
GLP compliance:
no
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material:Batch number: Willing New Materials Technology Co., Ltd.; 23191022201
- Expiration date of the lot/batch: 21 October 2020
- Purity: :97%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25℃) protected from humidity (in a tightly closed container)

Species:
rat
Strain:
Wistar
Remarks:
Hannover Wistar, (Crl:WI(Han))
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Sandhofer Weg 7, D-97633 Sulzfeld, Germany) from SPF colony
- Age at study initiation: Young adult female rats, nulliparous and non-pregnant, at least 12 weeks old
- Weight at study initiation: 193-260 g (the variation did not exceed ± 20% of the mean weight)
- Housing: Animals were housed individually. Type II polycarbonate cages (38x23x18 cm) were used during mating and gestation period and Type III polycarbonate cages (38x38x18) were used during the acclimatisation period. Deep wood sawdust (SAFE 3/4-S Hygienic Animal Bedding produced by J.Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany) was use as bedding to allow digging and other normal rodent activities. Nest building material (SAFE crinklets natural nesting material produced by J.Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany)) was also added into the cages. Certified cardboard tunnel tubes were provided to the animals for environmental enrichment.
- Diet (e.g. ad libitum) and Water (e.g. ad libitum): The animals were received ssniff® SM "Autoclavable Complete Diet for Rats/Mice – Breeding and Maintenance" (Batch number: 207 63596 /232 58297, Expiry date: 30 September 2020 / 30 June 2020) Ssniff Spezialdiäten GmbH, D-59494 Soest, Germany) and tap water (in water bottles) as for human consumption, ad libitum. The diet and drinking water were routinely analysed and are considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
- Acclimation period:at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0-23.8°C (target: 22 ± 3°C)
- Humidity (%): 20-70% (target: 30-70%)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light):12 hours daily, from 6.00 a.m. to 6.00 p.m.

Route of administration:
oral: gavage
Vehicle:
other: 1% (w/v) aqueous methylcellulose solution
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle (1% MC), as a visibly stable solution at the appropriate concentrations according to the dose level and volume selected in the Pharmacy of Charles River Laboratories Hungary Kft. Formulations were prepared fresh prior to administration to animals or at the appropriate frequency to allow their use according to stability assessment results. A constant volume of 5 mL/kg body weight was administered to all dose groups, including the control. The individual volume of the treatment was based on the most recent individual body weight of the animals.


VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on the available information provided by the Sponsor as well as results of the trial formulation, 1% (w/v) methylcellulose solution (abbreviation 1% MC) was selected as vehicle for this study.
- Concentration in vehicle: 1% (w/v)
- Lot/batch no. (if required): 7105702
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulations were prepared fresh prior to administration to animals or at the appropriate frequency to allow their use according to stability assessment results. Stability of the test item in the vehicle was assessed in the conditions employed on the study (concentration range and storage conditions of the dose formulations pending use, according to Charles River Laboratories Hungary Kft. study codes and 19/306-316AN). During the method validation 7 days of stability of the Test Item in the selected vehicle was obtained. The Test Item proved to be stable on room temperature in the concentration range of 10-210 mg/mL.

To verify the concentration and homogeneity of the Test Item in formulations, representative samples (including back-up samples for possible confirmatory analysis, if necessary) were taken and analysed from each concentration on one occasion during the study. Similarly, one representative sample (from the middle) was taken from Control sample to justify the absence of the Test Item (no homogeneity was required for the Control). Analysis of test item formulations for concentration and/or homogeneity was performed in the Analytical Laboratory of Charles River Laboratories Hungary Kft.

Acceptance criteria of the concentration analysis was 100 ± 15% of the nominal concentration.

Acceptance criteria of the homogeneity: RSD% (relative standard deviation) of replicates (top, middle and bottom of Test Item formulations) must be less than 15% of the nominal concentration.


Details on mating procedure:
The oestrus cycle of female animals was examined shortly before start of pairing. After acclimation, the females were paired according to their oestrus cycle with males in the morning for approximately 2 hours (1 male - 1 female) until at least 6 sperm positive females/group were attained. After the daily mating period, a vaginal smear was prepared and stained with 1% aqueous methylene blue solution. The smear was examined with a light microscope; the presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (gestation day 0, GD 0). Sperm positive females were separated and caged individually.

The sperm-positive, assumed pregnant females were allocated to each experimental group (on each mating day) controlled by SPSS/PC software, according to the actual body weight verifying the homogeneity/variability between/within the groups. Females paired with the same male were allocated to different groups on the same mating day.

On GD13 and/or 14 the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat, which is considered to confirm implantation).
Duration of treatment / exposure:
14 days (GD 6-19)
Frequency of treatment:
Daily
Duration of test:
20 days (Day 20 - Caesarean section and necropsy)
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control (Vehicle)
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Low dose
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Mid dose
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High Dose
No. of animals per sex per dose:
6 mated females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were set by the Study Monitor in agreement with the Study Director based on the available data provided by the Sponsor and on the results of a 14-day repeated dose range finding study, with the aim of inducing toxic effects but no death or suffering at the highest dose (1000 mg/kg bw/day). As the dose levels after 14-daily administration did not result in clear adverse effects and were considered well tolerated, the top dose for the current study is set at 1000 mg/kg bw/day, with subsequent doses calculated by a factor of approximately 3 (100, 300 mg/kg bw/day).
Maternal examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day). Clinical observations were made at least twice daily at the beginning and end of the working day as practical, or before treatment and when the peak of the clinical observations, if any, is observed after treatment, or once, before necropsy, as applicable. Pertinent behavioural changes and all signs of toxicity including mortality were recorded including onset, degree and duration of signs as applicable. Signs evaluated were include, but not be limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), bizarre behaviour (e.g. self-mutilation, walking backwards) will also be recorded. Special attention will be directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

BODY WEIGHT: Yes
- Time schedule for examinations:The body weight of each animal was recorded with precision of ±1 g on GD 0, 3, 6, 8,10, 12, 14, 16, 18 and 20.

FOOD CONSUMPTION : Yes
Food was measured with precision of ± 1 g on GD 0, 3, 6, 8, 10, 12, 14, 16, 18 and 20. Food consumption was also calculated for each interval, including GD 0-6, GD 6-20 and GD 0-20.

POST-MORTEM EXAMINATIONS: Yes
Before expected delivery, on GD 20, Caesarean section was performed on each treated dam. The dams’ viscera were examined macroscopically for any structural abnormalities or pathological changes; all the gross findings were retained in 10% buffered formalin solution (or modified Davidson fixative, in case of the eyes, in any abnormalities noted). Furthermore, the stomachs of the dams were weighed and retained in 10% buffered formalin solution. No histopathology evaluation was performed in the study.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included: The ovaries and uterus were removed and the pregnancy status ascertained. The uterus including the cervix was weighed and examined for early and late embryonic or foetal deaths and for the number of live foetuses. If no implantation sites were evident but corpora lutea were present, the uterus was stretched and held in front of a light source to clearly identify the implantation sites. Uteri that appear non-gravid were further examined to confirm the non-pregnant status (i.e. by ammonium sulphide staining or a suitable alternative method). The number of corpora lutea in each ovary and implantation sites in each uterine horn, the number of live foetuses, early and late embryonic death and foetal death were counted, the number and percentage of pre- and post-implantation losses were calculated. The degree of resorption was described in order to estimate the relative time of death of the conceptus. The placentas were examined macroscopically.
Fetal examinations:
Each live foetus was weighed individually (accuracy ±0.01 g) and subjected to external examination.

All external abnormalities found during the foetal examinations were recorded; any additional observations might be recorded as noted during caesarean section and necropsy.
Statistics:
Group mean and standard deviation was calculated for numerical data. Statistical analysis was performed using SAS 9.2 (built in Provantis System).

The following decision tree were automatically applied within the validated Provantis system for statistical evaluation of numeric data:

The normality and heterogeneity of variance between groups were checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test will be carried out. If the obtained result is positive, Dunnett (Multiple Range) test were used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis is the best option when the normality and heterogeneity assumptions implicit in the tests were adequate.

If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach was not valid and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate.

The limit for growth retarded foetuses was calculated from the average body weight of the vehicle control foetuses. A foetus was considered as growth retarded if the deviation from the mean control values were greater than minus two-fold standard deviation of all control foetuses.

Non-pregnant females, females with no implantation, or ≤ 5 implantation sites, and females showing signs of abnormal pathology and/or misdosing were excluded from statistical analysis; in-life individual data was reported as applicable.

Indices:
Pre-implantation loss: Number of corpora lutea-Number of implantations x100/Number of corpora lutea

Post-implantation loss: Number of implantations-Number of live foetuses x100/Number of implantations

Sex distribution: Number of male (female) foetuses x100/Number of foetuses

External abnormalities/litter: Number of foetuses with abnormality x100/Number of foetuses
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs in the Control and in any of the dose groups. Clinical observations are shown in Appendices 3.1.A-3.1.B (summary tables).
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
None of the animals died during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No body weights were statistically lower than controls. Statistically significant lower body weight gains were seen at the High dose level sporadically during the treatment period (GD6-20), with about 36% lower overall weight gain. A lower gain, not statistically significant was seen in the Mid dose group. These small changes were considered to be a test item related effect. There were no significant body weight differences in the Low dose group. Detailed body weight data of the evaluated animals are presented in Appendices 3.2.1-3.2.3 (summary tables).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Lower food consumption, not reaching statistical significance, was observed transiently in the Low and Mid groups, the High dose remained lower throughout the treatment period and was considered to be a test item related effect. Food consumption data of the evaluated animals are presented in Appendix 3.3 (summary tables).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
There was no thickening of the non-glandular mucosal region observed for any of the animals. Necropsy data are presented in Appendices 3.6.1-3.6.2 (summary tables)
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
not examined
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The preimplantation loss and early and the late embryonic loss values of the test item treated groups were comparable with control. There was no statistically significant difference in the post-implantation loss between the test item treated and control groups. Relevant data are shown in Appendices 3.4. (summary tables).
Total litter losses by resorption:
not examined
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
The number of total intrauterine mortality compared to number of corpora lutea was significantly higher (p<0.05) in the Mid dose group compared to the Control group. No significant changes were observed in the other intrauterine parameters. It was considered that there were no effects of treatment. A summary of intrauterine evaluations is shown in Table 4. Relevant data are shown in Appendices 3.4. (summary tables).
Dead fetuses:
no effects observed
Description (incidence and severity):
The number of dead foetuses were comparable with control. Relevant data are shown in Appendices 3.4. (summary tables).
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
Twenty-four females (6 for each group) were mated in the study. The number of confirmed pregnant evaluated dams was 6 in the Control and in the Low dose groups and 5 in the Mid and in the High dose groups. Relevant data are shown in Appendices 3.4 (summary table).
Other effects:
no effects observed
Description (incidence and severity):
No abnormalities were observed in the placenta in any of the study groups. Relevant data are shown in Appendix 3.5 (summary table).
Dose descriptor:
other: 0,100,300,1000 mg/kg bw/day
Effect level:
0 mg/kg bw/day (nominal)
Basis for effect level:
other: dose levels for main test identified
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The mean foetal bodyweight (male and female) was significantly decreased in case of Mid (p<0.05) and High dose animals (p<0.01) compared to the Control group. Taking into account the data of the last five similar pre-natal studies (3.445 ± 0.414 (mean ±2 SD), the mean foetal body weight in the Mid dose group was in the normal range, therefore the statistical difference in the Mid dose group is not considered to be a test item related effect. The slightly reduced foetal body weight in the High dose group was considered to be a secondary effect due to the observed maternal toxicity (lower food consumption and/or lower body weight parameters) in the High dose group dams. Relevant data are shown in Appendices 3.4, 3.5 (summary tables).
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Relevant data are shown in Appendices 3.4, 3.5 (summary tables).
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Relevant data are shown in Appendices 3.4, 3.5 (summary tables).
Changes in litter size and weights:
effects observed, non-treatment-related
Description (incidence and severity):
Mean foetal weight per litter was significantly lower (p<0.05) in the High dose group; the low and mid-dose groups were comparable to the control mean value. Again, this was considered to be a secondary effect due to the observed maternal toxicity (lower food consumption and/or lower body weight parameters) in the High dose group dams. The number of litters with runts (body weight > 2SD below the controls) was similar in all groups.Relevant data are shown in Appendices 3.4, 3.5

Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No external abnormalities were observed in any of the study groups. Relevant data are shown in Appendix 3.5 (summary table)
Skeletal malformations:
not examined
Visceral malformations:
not examined
Dose descriptor:
other:
Remarks:
0,100,300,1000 mg/kg bw/day
Effect level:
0 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: base level for main test identified
Abnormalities:
no effects observed
Developmental effects observed:
no

Samples for test item concentration determination of the dosing formulations were collected once during the treatment period, the test item concentration was analysed at the Test Site using a HPLC-UV method. The mean concentration of all formulations were found to be in the range of 104-106% of their nominal concentrations (10 and 210 mg/mL). The stability of the test item in 1% methyl cellulose at 10 and 210 mg/mL at room temperature was 7 days. No test item was detected in the vehicle control formulations. Based on these results, test item formulations were considered suitable for the study purposes.

Conclusions:
In conclusion, WILLING TiBTD, when administered daily by oral gavage to pregnant Hannover Wistar rats from gestation days GD6 to GD19 up to 1000 mg/kg bw/day, was associated with slightly lower body weight gain in the High dose group, but no other sign of maternal toxicity (no mortality and no local effects in the stomach). No embryotoxicity or teratogenicity effect was identified in this preliminary study.

In selecting dose levels for the following OECD TG 414 study, 1000 mg/kg bw/day was considered suitable as the High dose. The Mid and Low dose levels selected by the Study Monitor for the main OECD TG 414 study were 300 and 100 mg/kg bw/day.

Executive summary:

In a dose range finding study (no guideline) for a prenatal developmental toxicity in rats (OECD 414/GLP), WILLING TiBTD (97%) was administered to pregnant Crl:WI (Han) rats (6/dose) by oral gavage in 1% (w/v) aqueous methylcellulose at dose levels of 0, 100 (low dose), 300 (mid dose) and 1000 (high dose) mg/kg bw/day daily for 14 days (GD 6-19).  

All formulations proved to be homogeneous. The mean concentration of all formulations was found to be in the range of 104-106% of their nominal concentrations.

Twenty-four females (6 for each group) were mated in the study. The number of confirmed pregnant evaluated dams was 6 in the Control and in the Low dose groups and 5 in the Mid and in the High dose groups. None of the animals died during the study. There were no clinical signs in the Control and any of the dose groups. Body weights were not statistically lower than control. Statistically significant lower body weight gains were seen at the High dose level sporadically during the treatment period, with about 36% lower overall weight gain during the treatment period. A lower gain, not statistically significant was seen in the of Mid dose group. These small changes were considered to be a test item related effect. There were no significant body weight differences in the Low group.  Lower food consumption, not reaching statistical significance, was observed transiently in the Low and Mid groups, the High dose remained lower and was considered to be a test item related effect.

There was no thickening of the non-glandular mucosal region observed for any of the animals.

The number of total intrauterine mortality compared to number of corpora lutea was significantly higher (p<0.05) in the Mid dose group compared to the Control group. No significant changes were observed in the other intrauterine parameters. It was considered that there were no effects of treatment. There was no statistically significant difference in foetal death in any test item treated groups compared to the control. The mean number of corpora lutea was comparable with the control in all test item treated groups. No significant differences were noted in in the number of implantations of the test item treated groups when compared to the control. The preimplantation loss and early and the late embryonic loss values of the test item treated groups, and the number of dead foetuses were comparable with control. There was no statistically significant difference in the postimplantation loss between the test item treated and control groups.

The mean foetal bodyweight was significantly decreased in case of Mid (p<0.05) and High dose animals (p<0.05 and p<0.01) compared to the Control group. However, when analysed as mean foetal weight per litter (the more relevant measure) there was a significantly lower weight (p<0.05) in the High dose group; the low and mid-dose groups were comparable to the control mean value. The slightly reduced foetal body weight in the High dose group was considered to be a secondary effect due to the observed maternal toxicity (lower food consumption and/or lower body weight parameters) in the High dose group dams.

No toxicologically relevant adverse effects of the test item were observed on the rest of the foetal parameters, number of retarded foetuses (runts), and in the sex distribution between the test item treated and control groups. The number of litters with runts (body weight > 2SD below the controls) was similar in all groups. Placentas were normal for all observed animals.

There was no test item related effect on the external development of foetuses in the study (no visceral or skeletal evaluations were made in this preliminary study).

In conclusion, WILLING TiBTD, when administered daily by oral gavage to pregnant Hannover Wistar rats from gestation days GD6 to GD19 up to 1000 mg/kg bw/day, was associated with slightly lower body weight gain in the High dose group, but no other sign of maternal toxicity (no mortality and no local effects in the stomach). There were no effects on numbers of foetuses. The body weight of the pups at 1000 mg/kg bw/day was slightly lower than controls, which was considered to be secondary to the lower dam weight gain. No embryotoxicity or teratogenicity effect was identified in this preliminary study.

In selecting dose levels for the following OECD 414 study, 1000 mg/kg bw/day was considered suitable as the High dose. The Mid and Low dose levels selected for the main OECD TG 414 study were 300 and 100 mg/kg bw/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The key study was an OECD 414/GLP compliant study and was assigned a Klimisch score of 1. The overall quality of the database is high.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

 Developmental toxicity

There is one 14 day dose range finding oral study in rats and one oral pre-natal developmental toxicity study in rats available. 

 

Dose range finding study

In a dose range finding study (no guideline) for a prenatal developmental toxicity in rats (OECD 414/GLP), WILLING TiBTD (97%) was administered to pregnant Crl:WI (Han) rats (6/dose) by oral gavage in 1% (w/v) aqueous methylcellulose at dose levels of 0, 100 (low dose), 300 (mid dose) and 1000 (high dose) mg/kg bw/day daily for 14 days (GD 6-19).  

All formulations proved to be homogeneous. The mean concentration of all formulations was found to be in the range of 104-106% of their nominal concentrations.

Twenty-four females (6 for each group) were mated in the study. The number of confirmed pregnant evaluated dams was 6 in the Control and in the Low dose groups and 5 in the Mid and in the High dose groups. None of the animals died during the study. There were no clinical signs in the Control and any of the dose groups. Body weights were not statistically lower than control. Statistically significant lower body weight gains were seen at the High dose level sporadically during the treatment period, with about 36% lower overall weight gain during the treatment period. A lower gain, not statistically significant was seen in the of Mid dose group. These small changes were considered to be a test item related effect. There were no significant body weight differences in the Low group.  Lower food consumption, not reaching statistical significance, was observed transiently in the Low and Mid groups, the High dose remained lower and was considered to be a test item related effect.

There was no thickening of the non-glandular mucosal region observed for any of the animals.

The number of total intrauterine mortality compared to number of corpora lutea was significantly higher (p<0.05) in the Mid dose group compared to the Control group. No significant changes were observed in the other intrauterine parameters. It was considered that there were no effects of treatment. There was no statistically significant difference in foetal death in any test item treated groups compared to the control. The mean number of corpora lutea was comparable with the control in all test item treated groups. No significant differences were noted in in the number of implantations of the test item treated groups when compared to the control. The preimplantation loss and early and the late embryonic loss values of the test item treated groups, and the number of dead foetuses were comparable with control. There was no statistically significant difference in the postimplantation loss between the test item treated and control groups.

The mean foetal bodyweight was significantly decreased in case of Mid (p<0.05) and High dose animals (p<0.05 and p<0.01) compared to the Control group. However, when analysed as mean foetal weight per litter (the more relevant measure) there was a significantly lower weight (p<0.05) in the High dose group; the low and mid-dose groups were comparable to the control mean value. The slightly reduced foetal body weight in the High dose group was considered to be a secondary effect due to the observed maternal toxicity (lower food consumption and/or lower body weight parameters) in the High dose group dams.

No toxicologically relevant adverse effects of the test item were observed on the rest of the foetal parameters, number of retarded foetuses (runts), and in the sex distribution between the test item treated and control groups. The number of litters with runts (body weight > 2SD below the controls) was similar in all groups. Placentas were normal for all observed animals.

There was no test item related effect on the external development of foetuses in the study (no visceral or skeletal evaluations were made in this preliminary study).

In conclusion, WILLING TiBTD, when administered daily by oral gavage to pregnant Hannover Wistar rats from gestation days GD6 to GD19 up to 1000 mg/kg bw/day, was associated with slightly lower body weight gain in the High dose group, but no other sign of maternal toxicity (no mortality and no local effects in the stomach). There were no effects on numbers of foetuses. The body weight of the pups at 1000 mg/kg bw/day was slightly lower than controls, which was considered to be secondary to the lower dam weight gain. No embryotoxicity or teratogenicity effect was identified in this preliminary study. In selecting dose levels for the following OECD 414 study, 1000 mg/kg bw/day was considered suitable as the High dose. The Mid and Low dose levels selected for the main OECD TG 414 study were 300 and 100 mg/kg bw/day.

OECD 414 main study

In a prenatal developmental toxicity in rats (OECD 414/GLP), WILLING TiBTD (97%) was administered to pregnant Crl:WI (Han) rats (25/dose) by oral gavage in 1% (w/v) aqueous methylcellulose at dose levels of 0, 100 (low dose), 300 (mid dose) and 1000 (high dose) mg/kg bw/day daily for 14 days (GD 6-19).  

All test item formulations were within the range of 95-102% of nominal concentration and were found to be homogenous. No test item was detected in the vehicle control samples. Test item formulations were considered suitable stable for the study purposes.

No mortality or clinical observations were recorded in any of the dose groups. Test item-related adverse effects on body weight parameters and food consumption were observed in the High dose group (1000 mg/kg bw/day), when compared to controls. No test item-related effects were noted in the Mid dose (300 mg/kg bw/day) or Low dose (100 mg/kg bw/day) groups.

No test item-related macroscopic or microscopic findings were present at necropsy in the evaluated adult animals. There were no statistically significant changes in the thyroid hormone concentrations of T3, T4 and TSH between the groups. There were no statistical differences in the thyroid organ weights between the Control and the Dose groups. There were no statistically significant differences in the intrauterine parameters in the test item treated animals when compared to the controls. No abnormalities were observed in the placentas of any dams in the Control, Low, Mid, or High dose groups.

Reduced foetal body weights were recorded in the High dose group (1000 mg/kg bw/day); the differences were considered to be possibly treatment related, but in the presence of significant maternal toxicity, not a direct effect of the test item (i.e. secondary non-specific consequence of maternal toxicity effects). No effect was noted in Mid and Low dose groups (300 and 100 mg/kg bw/day, respectively). There were no test item-related effects on external, visceral or skeletal development of the foetuses in the study. Foetal malformations observed in the study were all considered to be incidental. They showed no dose dependency and thus were not regarded as test item-related effects.

No endocrine disrupter effect was identified in the study (based on the lack of effects on thyroid hormones and thyroid histopathology for dams, and anogenital distance for foetuses).

In conclusion, WILLING TiBTD, when administered daily by oral gavage to pregnant Hannover Wistar rats from Gestation Day (GD) 6 to 19 at 100, 300 and 1000 mg/kg bw/day induced significant body weight and food consumption effects at 1000 mg/kg bw/day dose level. No embryotoxic effect was recorded, but foetotoxicity (slightly reduced foetal body weight in the presence of significant maternal toxicity) was observed at 1000 mg/kg bw/day. There were no test item-related effects on external, visceral or skeletal development of the foetuses in the study.

The following NOAELs were derived:

NOAEL for maternal toxicity:300 mg/kg bw/day (based on the effects on body weight and food consumption at 1000 mg/kg bw/day).

NOAEL for embryotoxicity: 1000 mg/kg bw/day (based on the lack of any test-item related intrauterine effect in any treatment group.)

NOAEL for foetotoxicity: 300 mg/kg bw/day (based on the effect on foetal weight at 1000 mg/kg bw/day in the presence of maternal toxicity.)

NOAEL for teratogenicity: 1000 mg/kg bw/day (based on the lack of treatment-related malformations in any dose group.)

Justification for classification or non-classification

Based on the available information in the dossier, the substance WILLING TiBTD (CAS No. 3064-73-1) does not need to be classified for reproductive toxicity when the criteria outlined in Annex I of 1272/2008/EC are applied.

Additional information