Biphenyl-4,4'-diol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Three key studies are available for fertility investigation:

> A screening test according to OECD 422 (repeated dose/reproductive/developmental toxicity study) in Sprague-Dawley rats exposed by oral route is available and reports a NOAEL of 40 mg/kg bw/d for general toxicity on the basis of histologic changes noted in liver from males exposed at 200 mg/kg, and a NOAEL of 200 mg/kg bw/d for reproductive and develomental toxicity (the highest dose level tested).

In this study, treatment up to 200 mg/kg bw/d did not affect the reproductive performance of the parental animals.

> An Extended One Generation Reproductive Toxicity study was conducted in rats by oral route at the dose levels of 25, 75 and 225 mg/kg bw/day (on the basis of the results of a simplified OECD 422 study conducted at 200 and 250 mg/kg bw/day in order to select the dose levels of the OECD 443 study). The NOAEL for maternal toxicity was established at 75 mg/kg on the basis of adverse pathology findings in kidney, heart and glandular stomach. The NOAEL for reproduction was established at 225 mg/kg/day (the highest tested dose level) in absence of any effect on fertility or development.

In this study, treatment up to 225 mg/kg bw/d did not affect the reproductive performance of the parental animals.

> A 90-day repeated dose toxicity study in rats by oral route (gavage) according to OECD 408 guideline was conducted at the dose levels of 30, 125 and 500 mg/kg bw/day. The NOAEL was 125 mg/kg bw/day on the basis of histologic findings in kidneys of females and few males.

In this study, treatment up to 500 mg/kg did not affect the quality of the sperm in males or the oestrus cycles in females.

Link to relevant study records

Reference

Reference 1

Endpoint:

extended one-generation reproductive toxicity - with both developmental neuro- and immunotoxicity (Cohorts 1A, 1B without extension, 2A, 2B, and 3)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

other: The simplified OECD 422 was conducted as the preliminary experiment to the OECD 443 (for the selection of the dose levels.

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

25 July 2018.

Deviations:

no

Remarks:

None of the technical deviations that occurred during the experimental phase have impacted the results of the study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:
- Premating exposure duration for parental (P0) animals: 10 weeks.
- Basis for dose level selection: see details in the section “Study design” below.
- No extension of cohort 1B was implemented as the information available on potential reproduction toxicity and the results obtained with cohort 1A did not justify it.
- No F2 generation was generated as the results did not justify it.
- Cohorts 2A and 2B were included to evaluate developmental neurotoxicity.
- Cohort 3 was included to evaluate developmental immunotoxicity.
- The oral route of exposure was selected as this is a potential route of human exposure during manufacture, handling or use of the test substance as specified in the applicable guidelines.

Species:

rat

Strain:

Wistar

Remarks:

CRL: WI(Han)

Details on species / strain selection:

The rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Domaine des Oncins, 69210 Saint-Germain-Nuelles, France.
- Females nulliparous and non-pregnant: yes.
- Age at study initiation:
For the negative control and test material groups: (P) 6 weeks old; (F1) 3 weeks old (PND21).
For the F1 nulliparous and non-pregnant females as positive control of the TDAR (SRBC) Assay: 7 weeks old.
For the F1 nulliparous and non-pregnant females as positive control of the TDAR (KLH) Assay: 8 weeks old.
- Weight: the range of average weight of (P) animals at the initiation of dosing was 145.9-148.1 g for males and 135.7 – 138.7 g for females. The range of average weight of (F1) animals at the initiation of dosing was 59.5-67.8 g for males and 57.4 – 65.8 g for females.
- Fasting period before study: not specified.
- Housing: single or group housed. See also Table 3 in section "Any other information on materials and methods incl. tables".
- Diet: Reference No. A04C-10 (pellets), ad libitum, except during designated procedures. It is considered that there were no known contaminants in the feed that interfered with the objectives of the study.
- Water: filtered (0.2 μm) mains drinking water, ad libitum, except during designated procedures. It is considered that there were no known contaminants in the water that interfered with the outcome of the study.
- Acclimation period: 7 days before the start of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19°C to 25°C.
- Humidity (%):≥ 35%.
Both temperature and relative humidity were within the targets throughout the study.
- Air changes (per hr): 10 or more air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark (except during any designated procedures). Dimmed lighting appeared 15 minutes before the lights were switched on and disappeared 15 minutes after the lights were switched off.

OTHER: Animal Enrichment
For psychological/environmental enrichment, animals were provided with items such as a wooden gnaw block and shredded paper, except when interrupted by study procedures/activities.

IN-LIFE DATES: From: 02 February 2021 To: 19 August 2021.

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

The control was 0.5% (w/v) CMC (Batch No. 18E174115) 300-600 centipoises in purified water.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dose formulations were prepared according to Test Facility operating standard procedures on a weekly basis and divided into aliquots to be dispensed on each dosing occasion.
Storage conditions: temperature set to maintain +4°C
Any residual volumes from each dosing occasion were discarded.

VEHICLE:
The vehicle was Carboxymethylcellulose (CMC).
- Justification for use and choice of vehicle (if other than water): not specified.
- Concentration in vehicle: the concentration of test material in the vehicle were the following:
* Group 1 (negative control CMC): 0 mg/mL.
* Group 2 (test material 25 mg/kg/day): 5 mg/mL.
* Group 3 (test material 75 mg/kg/day): 15 mg/mL.
* Group 4 (test material 225 mg/kg/day): 45 mg/mL.
* Group 5 (positive control for immunotoxicity, cyclophosphamide 10 mg/kg/day): 0 mg test material/mL (2 mg cyclophosphamide/mL).
- Amount of vehicle (if gavage): the amount of dose formulation administered to the animals was 5 mL/kg/day.
- Lot/batch no. :
Negative control CMC: Batch N. 18E174115. - Purity: 0.5% (w/v) CMC 300-600 centipoises in purified water.
Positive control for TDAR assays: see below in the corresponding section.

Details on mating procedure:

- M/F ratio per cage: after 10 weeks of dosing animals were paired on the basis of 1 male and 1 female from the same group.
- Length of cohabitation: maximum of 14 days.
- Proof of pregnancy: vaginal plug or presence of sperm in vaginal smear, referred to as gestation day 0 (GD0).
- Mated females were separated from the males once mating had been confirmed and smearing
ceased or when the appearance of the female suggested pregnancy from an undetected
mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Sample collection and analysis: dose formulation samples were collected for analysis as indicated in Table 1 in the section “Any other information on materials and methods incl. tables”. All samples to be analyzed were transferred to the analytical laboratory at the Test Facility. Samples were stored and analyzed within the stability period as defined in the validation study.
- Analytical method: analyses were performed by UPLC-UV using a validated analytical procedure.
- Concentration analysis: formulations were analyzed for the assessment of achieved concentration of test material in formulations and verification of the absence of test material in the control formulation.
- Homogeneity analysis: the formulations of the test material at concentrations of 1 and 200 mg/mL in the vehicle are homogeneous (based on the analytical results of a previous study).
- Stability analysis: the Sponsor provided data that demonstrate that the test material formulation is stable when prepared and stored under the same conditions as those used in the present study, as follows: in a concentration range of 1-200 mg/mL for 11 days at room temperature and +4°C or
26 days at -20°C (based on the analytical results of a previous study).

Duration of treatment / exposure:

The treatment with the test material (or control item) was administered by gavage using a syringe with attached plastic gavage cannula and for the following animals/durations:
- (P) Males: 10 weeks before mating, throughout the mating period and up to the day before necropsy.
- (P) Females: 10 weeks before mating, throughout the mating period, during gestation (the first day of gestation is designated as GD0) and through weaning of their F1 offspring, at least 21 days after parturition, up to and including the day before scheduled necropsy (the day of birth is designated as LD0).
- Apparently unmated females for at least 23 days after the last day of the mating period.
- (F1) Males and Females: during lactation (up to PND21), pups were not dosed directly but could potentially be exposed to the test material in utero, via maternal milk or from exposure to maternal urine/feces. After weaning (PND21), F1 animals were dosed up to and including the day before scheduled necropsy. The F1 surplus and Cohort 2B animals were not dosed.

Frequency of treatment:

Daily.

Details on study schedule:

Additional details on study schedule:

> F0 GENERATION

- F0 males were dosed for 10 weeks prior to mating, during mating and up to the day before necropsy. Consequently F0-males were euthanised after at least 12 weeks of exposure.
- F0 females were dosed during 10 weeks prior to mating, during mating, gestation and lactation and up to the day before necropsy, i.e., on Lactation Day (LD) 21 to LD23 for females that delivered. Consequently F0 Females were euthanised after at least 16 weeks of exposure.

Throughout the study, animals were observed for general health/mortality and moribundity at least twice daily, at the beginning and end.

> F1 GENERATION

- The standardization of litters was performed: 8 pups from each litter of equal sex distribution (if possible) were selected. Selective elimination of pups, e.g. based upon body weight or anogenital distance (AGD), was not done. Whenever the number of male or female pups prevented having 4 of each sex per litter, partial adjustment (for example, 5 males and 3 females) was acceptable. No pups of either sex were eliminated when the litter size was below the culling target (8 pups/litter).
- On PND21, pups are selected randomly, and the selected F1 pups are randomly assigned to one of three cohorts of animals, as follows:
Cohort 1 (1A and 1B) = Reproductive/developmental toxicity testing
Cohort 2 (2A and 2B) = Developmental neurotoxicity testing
Cohort 3 = Developmental immunotoxicity testing

- F1 animals from Cohort 1A were sacrificed after clinical pathology evaluation between PND89 and PND99.
- F1 animals from Cohort 1B (KLH – TDAR assay) were sacrificed between PND92 and PND100.
- F1 animals from Cohort 2A were sacrificed after completion of the neurobehavioral tests between PND76 and PND90.
- F1 animals from Cohort 2B were not dosed directly and were necropsied on/before PND24 for neuro-histopathology and morphometric analysis.
- F1 animals from Cohort 3 (SRBC - TDAR assay) were sacrificed between PND54 and PND57 or at approximately 12 weeks of age for positive controls.
- Surplus offspring (10/sex/group) not included in any of the F1 cohorts were selected for thyroid hormone analysis and organ weights on PND21.

Dose / conc.:

0 mg/kg bw/day

Remarks:

Group 1 (negative control with vehicle alone).

Dose / conc.:

25 mg/kg bw/day

Remarks:

Group 2.

Dose / conc.:

75 mg/kg bw/day

Remarks:

Group 3.

Dose / conc.:

225 mg/kg bw/day

Remarks:

Group 4.

Dose / conc.:

0 mg/kg bw/day

Remarks:

Group 5 (positive control with administration of cyclophosphamide for the TDAR assays).

No. of animals per sex per dose:

- F0: 25 Males and 25 Females per dose.
- F1 cohorts 1A and 1B: 20 Males and 20 Females per dose (including the G5 group for the positive control).
- F1 cohorts 2A, 2B and 3: 10 Males and 10 Females per dose (including the G5 group for the positive control).
See also Tables 2 and 3 in the section “Any other information on materials and methods incl. tables”.
- F1 cohort 2B and F1 surplus animals were not dosed.
- Surplus offspring (10/sex/group) not included in any of the F1 cohorts were selected for thyroid hormone analysis and organ weights on PND21.

See also Table 2 in section "Any other information on materials and methods incl. tables".

Control animals:

yes

Details on study design:

- Dose selection rationale: dose levels were based on a simplified OECD 421 study in the Wistar rat (Charles River Study No. 20269996). In this study, 10 rat/sex were dosed at 0, 200 and 250 mg/kg/day by oral administration (gavage) from 14 days before mating, throughout mating, gestation and lactation and up to the day before necropsy for females or from 14 days before mating, throughout mating and up to the day before necropsy (28 days) for males.
Parental toxicity was limited to a dose-related slightly but statistically significantly lower mean body weight gain and food consumption during the first week of the premating period for both sexes in both treated groups without any impact on subsequent absolute mean body weight through to termination.
Test material-related preweaning developmental effects consisted of early postnatal pup mortality at 250 mg/kg/day including 2 females with total litter death and 1 female with 7 dead pups compared with no pup mortality in the control group. In addition, there was lower mean pup body weight at 250 mg/kg/day when compared with the control group (-7.4% and -9.2% for males and females, respectively) and with the historical control data range for both sexes on PND1. Mean pup body weight gain was then lower during the preweaning phase such that there was a dose-related lower mean pup body weight at 200 and 250 mg/kg/day on PND21 when compared with the control group (-6.3%/-9.8% and -12.3%/-13.7% for males/females, respectively).
Based on these findings, the dose of 250 mg/kg/day was considered to be above the maximum tolerated dose for pup viability and the dose of 200 mg/kg/day was associated with only minor parental and pup body weight changes.
- In addition, in the 90-day repeated dose toxicity study conducted in rats at 30, 125 and 500 mg/kg/day, microscopic kidney lesions were seen in most females and in a few males at 500 mg/kg bw/day. The NOAEL was established at 125 mg/kg bw/day.

For this extended 1-generation reproductive toxicity study, a high dose of 225 mg/kg/day was therefore selected to elicit a clear effect on pup body weight but limit the risk of pup mortality in order to ensure sufficient numbers for allocation to the different F1 cohorts. The low dose of 25 and intermediate dose of 75 mg/kg/day were selected to have a ratio of 3 between each dose level to demonstrate a dose-relationship for any potential test material-related findings.

- Animal assignment: at random.

- Fasting period before blood sampling for clinical biochemistry:
F0 and F1-cohort 1A animals were fasted overnight (about 16 hours) before blood sampling for clinical pathology evaluation and for thyroid hormone analysis.
F1 PND4 pups and surplus animals were not fasted before blood sampling for thyroid hormone analysis.

Positive control:

A positive control was used for the T-cell Dependent Antibody Response (TDAR) assays.
- Identification: cyclophosphamide monohydrate.
- Batch No.: MKCJ4697.
- Preparation: each day of dosing, dose formulation of 2.0 mg/mL cyclophosphamide was freshly prepared by dissolving the positive control item in sterile saline solution (0.9% NaCl batch No. 2105125.). No adjustment was made for specific gravity of the vehicle and no correction was made for the purity/composition of the positive control item. Dosing formulations of cyclophosphamide were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. Dosing formulations were kept at ambient temperature until dosing and were dosed within 6 hours after adding the vehicle to the positive control item.
The dosing formulations were gently inverted until dosing. Any residual volumes were discarded.

Parental animals: Observations and examinations:

F0 GENERATION

CAGE SIDE OBSERVATIONS: Yes.
- Time schedule: cage side observations were performed once daily on non-dosing days. During the dosing period, cage side observations were performed before and at least once after dosing. Animals were not removed from their cages during observation, unless necessary for identification or confirmation of possible findings.

ARENA OBSERVATIONS AND DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: animals were removed from their cages and observed for specific clinical signs in a standard arena and a detailed clinical observation was performed at the same time as arena observations i.e. once before the first administration of the test material and at least weekly thereafter (except during pairing or delivery).

BODY WEIGHT: Yes.
All females were weighed as follows:
- At least weekly during pre-mating and mating periods (only pre-mating data were reported).
- During the gestation period on GD0, 4, 7, 11, 14, 17 and 20.
- During the lactation period on LD1, 4, 7, 10, 14, 17 and 21.
All males were weighed at least weekly.
Non-pregnant females were also weighed weekly.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes.
Food consumption of each female was recorded for the following periods:
- Weekly during the pre-mating period starting from Day 1.
- During gestation: GD0 to 4, 4 to 7, 7 to 11, 11 to 14, 14 to 17 and 17 to 20.
- During lactation: LD1 to 4, 4 to 7, 7 to 10, 10 to 14, 14 to 17, 17 to 21.
Food consumption was recorded for each male weekly during the premating period starting from Day 1.
Food consumption during mating was not recorded for practical reasons.

WATER CONSUMPTION: No.

OTHER:

- Mortality/Moribundity Checks:
Throughout the study, animals were observed for general health/mortality and moribundity at least twice daily (except on days of receipt and study termination where frequency was at least once daily). Animals were not removed from their cages during observation, unless necessary for identification or confirmation of possible findings.

- Estrous cycle: see below, section "Oestrous cyclicity (parental animals)"

- Mating:
The day of mating was confirmed by the presence of sperm in the vaginal smear or a vaginal plug and were recorded and taken as GD0. Mated females were separated from the males once mating had been confirmed and smearing ceased or when the appearance of the female suggested pregnancy from an undetected mating.

- Pregnancy and Parturition:
Observations for pregnancy and parturition were made daily.
Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Palpation and/or body weight measurement could be used to aid in confirmation of pregnancy. Females were allowed to litter normally. LD0 for the dam and PND0 for the offspring was defined as the day when signs of parturition were observed (i.e., blood traces in the cage or pup observed) and used for recording of delivery. Cage debris of pregnant females were examined for evidence of premature delivery. Signs of difficult or prolonged parturition were recorded, if applicable. Deficiencies in maternal care, such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding, were recorded, if applicable.

- Hematology:
Blood samples were analyzed for the parameters listed in Table 5 in section “Any other information on materials and methods incl. tables”. In addition, a blood smear was prepared for each animal on each occasion and analyzed when necessary.

- Coagulation:
Blood samples were analyzed for Activated partial thromboplastin time and Prothrombin time.

- Clinical chemistry:
Blood samples were analyzed for the parameters listed in Table 6 in section “Any other information on materials and methods incl. tables”.

- Urinalysis:
Urine samples were processed and analyzed for the parameters specified in Table 7 in section “Any other information on materials and methods incl. tables”.

- Thyroid hormone analysis:
Blood samples were taken from 10 animals/sex/group on the day of scheduled necropsy. Samples were analyzed for Thyroxine (T4, µg/dL) and Thyroid-Stimulating Hormone (TSH, µIU/mL) in accordance with Standard Operating Procedure and using the Immulite 1000®.

Oestrous cyclicity (parental animals):

F0 generation:
Vaginal smears were taken daily to determine the stage of estrous for each female from 14 days prior to mating and until the identification of mating or separation from the male.
On the day of terminal necropsy, or for moribund animals, a vaginal lavage was also taken to determine the stage of estrous.

Sperm parameters (parental animals):

- Sperm analysis was performed for all surviving males of the F0 generation and all surviving males of the F1 Cohort 1A.
- Sperm analysis was performed using automated equipment (Hamilton Thorne Research IVOS). The left cauda epididymis was sampled and weighed and used for the assessment of caudal sperm reserves and sperm motility, including progressive motility.
- Sperm morphology was evaluated (at least 200 sperm per sample, where possible). The number of sperm with each type of abnormality was recorded.

Litter observations:

1) F1 GENERATION UNTIL WEANING

- Number and weight of pups on PND1, 4, 7, 14 and 21.
- General health/mortality and moribundity at least once daily (at the beginning of the working day) along with the mortality check of the dam. Pups were not removed from the cage during observation, unless necessary for confirmation of possible findings.
- Sex of pups on PND1 and 4.
- Anogenital Distance (AGD), measured for all live pups on PND1. The AGD was normalized to the cube root of body weight.
- Presence of areola/nipples examined on PND13 for all remaining males in each litter.
- Thyroid hormone analysis: Blood samples were taken from two F1 pups per litter (1 male and 1 female if possible) on PND4 for analysis of T4 content in accordance with Standard Operating Procedure and using the Immulite 1000®.


2) F1 GENERATION AFTER WEANING

The in-life procedures, observations, and measurements listed below were performed for all F1 animals from weaning (PND21) onwards, except for the animals from Cohort Surplus and Cohort 2B as these animals were necropsied on/before PND24.

CAGE SIDE OBSERVATIONS: Yes.
- Time schedule: cage side observations were performed at least once daily on non-dosing days. During the dosing period, cage side observations were performed before and at least once after dosing. Animals were not removed from their cages during observation, unless necessary for identification or confirmation of possible findings.

ARENA OBSERVATIONS AND DETAILED CLINICAL OBSERVATIONS: Yes.
- Time schedule: animals were removed from their cages and observed for specific clinical signs in a standard arena and a detailed clinical observation was performed at the same time as arena observations i.e. from Week 1 and at least weekly thereafter (except during pairing or delivery).

BODY WEIGHT: Yes.
- Time schedule for examinations: animals were weighed at least weekly from weaning onwards. This started on a specific date on which all pups were at least at PND21.

FOOD CONSUMPTION: Yes.
- Food consumption was quantitatively measured at least weekly from Day 1 and on the same day prior to necropsy as all cohorts were necropsied over several days.

WATER CONSUMPTION: No.

OTHER:

- Mortality/Moribundity Checks:
Throughout the study, animals were observed for general health/mortality and moribundity at least twice daily, at the beginning and end of the working day (except on days of receipt and study termination where frequency was at least once daily). Animals were not removed from their cages during observation, unless necessary for identification or confirmation of possible findings.

- Sexual development:
> Balanopreputial skinfold cleavage was monitored daily for all males from PND38. Examinations continued until balanopreputial skinfold cleavage was detected or up to the day of necropsy. Body weight was recorded on the day of acquisition of balanopreputial skinfold cleavage.
> Vaginal opening was monitored daily for all females from PND28 onwards. Vaginal opening was monitored by visual inspection of the vaginal area and body weight was recorded on the day of acquisition of vaginal patency.
> Daily vaginal lavage was performed for all Cohort 1A females starting on the day of onset of vaginal patency and until the first estrous was determined, in order to determine the time interval between these 2 events.
Daily vaginal lavage was also performed at least 2 weeks before necropsy. On the day of scheduled necropsy, a vaginal lavage was taken to determine the stage of estrous for each female.
If a vaginal thread was observed, it was recorded daily until no longer present.
> Sperm analysis was performed for all surviving males of the F1 Cohort 1A. Sperm analysis was performed using automated equipment (Hamilton Thorne Research IVOS). The left cauda epididymis was sampled and weighed and used for the assessment of caudal sperm reserves and sperm motility, including progressive motility. - Sperm morphology was evaluated (at least 200 sperm per sample, where possible). The number of sperm with each type of abnormality was recorded.

- Hematology:
Blood samples collected from F1 animals - cohort 1A were analyzed for the parameters listed in Table 5 in section “Any other information on materials and methods incl. tables”. In addition, a blood smear was prepared for each animal on each occasion and analyzed when necessary.

- Clinical chemistry:
Blood samples from F1 animals - cohort 1A were analyzed for the parameters listed in Table 6 in section “Any other information on materials and methods incl. tables”.

- Urinalysis:
Urine samples from F1 animals - cohort 1A were processed and analyzed for the parameters specified in Table 7 in section “Any other information on materials and methods incl. tables”.

- Thyroid hormone analysis:
Blood samples were taken from 10 animals /sex/group for F1 Cohort 1A animals on the day of scheduled necropsy and from 10 animals /sex/group for F1 Cohort surplus animals on PND21. Samples were analyzed for Thyroxine (T4, µg/dL) and Thyroid-Stimulating Hormone (TSH, µIU/mL) in accordance with Standard Operating Procedure using the Immulite 1000®. Blood samples were collected between 7.00 and 10.30 a.m. by aorta puncture under anaesthesia using isoflurane.

- DEVELOPMENTAL NEUROTOXICITY

1) The neurobehavioral tests are conducted in the cohort 2A: :
> Auditory Startle Reflex (Acoustic Startle) – Habituation:
An auditory reflex test was performed once between PND23-25 in a chamber equipped with a pressure sensor platform. The auditory reflex was monitored by an analysis system (StartleMonitor™ supplied by Kinder Scientific LLC, Poway, USA). The test sessions of auditory startle reflex consisted of a 5-minute acclimation period with a 65 ± 5-dB broadband background white noise.
> Functional Observation Battery (FOB)
Detailed clinical observations were performed once between PND63-75 (10 animals/sex/group) by Functional Observation Battery (FOB) in group-housed animals. FOB parameters were evaluated per Test Facility standard operating procedures (SOPs) and according to Table 4 in section “Any other information on materials and methods incl. tables”.
In a first step, the animal underwent neurobehavioral evaluation in the home cage with ranking of posture, palpebral closure, stereotypies, convulsions and tremors. In a second step, the ease of removal of the animal from the home cage and its reactivity to handling during removal and transfer to the open field was scored. In a third step, the animal was placed in a black open field and observed for 2 minutes in freely moving conditions. Arousal, vocalizations, respiration, gait, stereotypies, convulsions and tremors were ranked. The number of rearing occasions was counted.
At the end of the 2-minute observation period, defecation (aspect of feces) was ranked and the animals underwent a series of additional observations and tests (from the least to the most invasive).
1. The observer handled the animals and ranked fur appearance, lacrimation, salivation, exophthalmos, palpebral closure and erected fur (piloerection).
2. The animal was tested for various reflexes such as response to a touch of the body with a rod, startle response (using a clicker), tail pinch response and pupil response.
3. Body temperature was measured using a rectal probe.
4. The animal was ranked for body tone, forelimb grip strength and air righting reflex (from a height of 30 cm). Forelimb grip strength was measured with a metallic grid connected to a dynamometer. The measure was performed 3 times and the mean value was calculated and reported.
> Locomotor activity (Motor Monitor)
A locomotor activity test was performed once between PND63-75 in an open field in order to assess reactivity to a novel environment and motor activity. Activity was monitored by a photobeam analysis system (Motor Monitor supplied by Kinder Scientific LLC, Poway, USA) for 1 hour. The arena was divided by beams in width and length. The time and the number of beams interrupted by the animal were recorded. Motor activity was divided into 2 types of activity: ambulatory activity (large animal movements like walking) and fine movements (including grooming and head movements).

2) Neuro-histopathology and morphometric analysis in the cohorts 2A and 2B
See section "Postmortem examinations (Offsprings).

- DEVELOPMENTAL IMMUNOTOXICITY

a) Humoral Immunization with KLH (TDAR) - Cohort 1B and Positive Control Animals (cohort 1B, group 5)
Ten F1-animals of Cohort 1B per group and per sex (groups 1 to 4) and all positive control animals (group 5) associated with this cohort were immunized once at 11-12 weeks of age (10 weeks of age for positive control animals), via intravenous injection into the tail vein with 1.0 mL of 300 μg of KLH. This immunization was performed 2 to 4 hours after treatment of the positive control animals with cyclophosphamide.
For positive control animals, cyclophosphamide was administered at 7.5 mg/kg via intraperitoneal injection once daily on 5 consecutive days.
Blood samples were collected (from the jugular vein or other site if deemed necessary) before pre-immunization and on Day 5 post immunization and stored at -20°C until analysis.
Serum levels of KLH-specific IgM antibodies were performed at the Test Site using a validated method documented in the study report.
Positive animals were then euthanized without macroscopic examination.

b) Immunization with SRBC (TDAR) – Cohort 3 and Positive Control Animals
All F1-animals from Cohort 3 and all positive control animals associated with this cohort were immunized once, 5 days before scheduled necropsy (i.e., once between PND48-54) via intravenous injection into the tail vein (approximately 1 mL/min) with 0.5 mL of the 4 x 108 SRBC/mL in sterile PBS formulation.
This immunization was performed 2 to 4 hours after dosing of the positive control animals with cyclophosphamide.
The animals were restrained during the injection procedure (without sedation). On the day of SRBC injection, clinical sign observations were performed after this injection for animals of Cohort 3 and the positive control animals.
For positive control animals, cyclophosphamide was administered at 10 mg/kg via intraperitoneal injection, once daily on 5 consecutive days prior to necropsy (i.e., starting at approx. 7 weeks of age).
Blood samples were collected (from the jugular vein or other site if deemed necessary) from all animals of all groups (1 to 5), before pre-immunization and on Day 5 post immunization and stored at ≤-75°C until analysis.
Analysis of samples for the Rat Anti-SRBC IgM was performed at the Test Site using the commercially available kit Rat Anti-SRBC IgM ELISA kit/SRBCM-2 (from Life Diagnostics), based on a validated ELISA method of analysis (U/mL).This kit is marketed for research purpose and not for diagnostics.

c) Splenic Lymphocyte Subpopulation Analysis – F1 Cohort 1A
From 10 selected animals/sex/group of Cohort 1A, splenic lymphocyte subpopulation analysis was performed at termination. One male or 1 female per litter were selected representing a maximum number of litter. Half of the spleen sample was transferred to a sterile tube containing RPMI medium supplemented with 20% foetal bovine serum. Samples were analyzed at the Test Facility.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: all surviving animals were euthanized after successful mating, after a minimum of 12 weeks of dosing.
- Maternal animals: all surviving animals which delivered were euthanized on LD22-24. For the females which failed to deliver, the necropsy was scheduled approximately on GD26 for those with evidence of mating and approximately 26 days after the last day of the mating period for those without evidence of mating.

GROSS NECROPSY
Gross necropsy consisted of external and internal examinations including external surface, all orifices, cranial cavity, thoracic and abdominal cavities and organs and their contents, the carcass.
Special attention was paid to the organs of the reproductive system.
Any abnormalities observed were recorded and preserved in an appropriate fixative. The number of former implantation sites was recorded for all paired females. The uterus of all adult females was placed in ammonium sulphide solution in order to stain any previously undetected implantation sites. The number of corpora lutea was counted. Any foetuses were examined externally where possible and discarded.


HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 8 in section “Any other information on materials and methods incl. tables” below were weighed and prepared for microscopic examination.
Histopathological examinations were performed as follows:
- For all macroscopic lesions from all dose group animals.
- For all organs/tissues from all adult animals of Groups 1 (control) and 4 (high dose).
- Reproductive organs for males that failed to sire and females which failed to deliver.
Histology slides were prepared for the organs/tissues as indicated in Table 8.
Slide staining: haematoxylin and eosin.
Target organs were identified in Group 4 animals and included kidneys, heart, stomach, thymus, vagina, ovaries, uterus and cervix and were examined from all animals of Groups 2 and 3, each sex being considered separately.
The tissues identified for processing and microscopic examination are mentioned in Table 8.

Postmortem examinations (offspring):

Terminal procedures are summarized in Table 10 in section “Any other information on materials and methods incl. tables”.

GROSS EXAMINATION OF DEAD PUPS:

Pups (extra pups on PND4 or any moribund pups) were sacrificed by intraperitoneal injection of sodium pentobarbitone and were submitted to a macroscopic examination of the thoracic cavities.
Pups (including any found dead or sacrificed moribund) were necropsied. For any pups found dead or killed moribund, the stomach was examined for the presence of milk and defects or cause of death was evaluated, if possible.
Each pup was sexed and examined for external defects with special attention being paid to the external reproductive organs. There were no external abnormalities collected.

1) F1 COHORT SURPLUS ANIMALS

- The F1 Cohort surplus animals were sacrificed on PND21.
- Terminal body weight was recorded.
- All animals were subjected to a limited examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded and preserved.
- HISTOPATHOLOGY / ORGAN WEIGHTS: The organs identified for weighing and the representative samples of the tissues for collecting are mentioned in Table 9 in section “Any other information on materials and methods incl. tables”.

2) F1 GENERATION FROM WEANING ONWARDS

2) a) F1- Cohort 1A animals

- The terminal body weight was recorded.
- Necropsy of Cohort 1A was conducted on PND89 to PND99.
- All animals were submitted to necropsy procedures including an examination of external surface. all orifices, cranial cavity, thoracic and abdominal cavities and organs and their contents, the carcass.
- Special attention was paid to the organs of the reproductive system.
- Any abnormalities observed were recorded and preserved in an appropriate fixative.
- HISTOPATHOLOGY / ORGAN WEIGTHS: The organs identified for weighing and the representative samples of the tissues for collecting are mentioned in Table 11 in section “Any other information on materials and methods incl. tables”.
Histopathological examination was performed for all organs/tissues from all F1 Cohort 1A animals found dead or killed moribund during the study.
In the first instance, histopathological examination was performed for the control and high dose groups (Groups 1 and 4), unless otherwise indicated (see below). Examination was extended to kidneys for animals in the intermediate dose groups as it was identified as target organs.
From the 10 first animals/sex of all groups, bone marrow (sternum) was evaluated histopathologically, the lymph nodes were weighed and evaluated histopathologically.
From all females of Groups 1 and 4 (20 animals/group), ovarian follicle counts, quantitative evaluation of primordial and small growing follicles, as well as corpora lutea, was performed.
From 10 selected animals/sex/group, after determination of spleen weight, half of the spleen was used for splenic lymphocyte subpopulation analysis and the other half of the spleen was used for histopathology evaluation. From the remaining 10 animals/sex/group, the total spleen was used for histopathology. Where possible, 1 male or 1 female per litter and all litters represented by at least 1 pup were selected.
For the testicles of all males, detailed qualitative examination was performed taking into account the tubular stages of the spermatogenic cycle. In addition an additional slide was prepared and examined, stained with PAS (in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen).
For all females, haematoxylin and eosin stained step sections of ovaries and corpora lutea were prepared for quantitative evaluation of follicles.


2) b) F1- Cohort 1B animals

- The terminal body weight was recorded
- Necropsy of Cohort 1B was conducted on PND92 to PND100.
- All animals were submitted to necropsy procedures including an examination of external surface, all orifices, cranial cavity, thoracic and abdominal cavities and organs and their contents, the carcass. Special attention was paid to the organs of the reproductive system.
- Any abnormalities observed were recorded and preserved in an appropriate fixative.
- HISTOPATHOLOGY / ORGAN WEIGHTS: The organs identified for weighing and the representative samples of the tissues for collecting are mentioned in Table 11 in section “Any other information on materials and methods incl. tables”.
The reproductive organs of all Cohort 1B animals were processed to block stage. There was no further processing and histopathological examination required based on the results of Cohort 1A.

2) c) F1- Cohort 2A and 2B

- The terminal body weight was recorded
- Necropsy for Cohort 2A was conducted on PND76-90 and for Cohort 2B on PND21-22.
- Animals from Cohorts 2A and 2B were first anaesthetized using isoflurane and were subsequently sacrificed by whole body perfusion using 0.9% NaCl, followed by formalin. The animals were not deprived of food overnight before necropsy.
- All animals were subjected to a limited examination, with special attention paid to the reproductive organs.
- The organs identified for weighing and the representative samples of the tissues for collecting are mentioned in Table 12 in section “Any other information on materials and methods incl. tables”.
- HISTOPATHOLOGY / ORGAN WEIGHTS: after sacrifice of Cohort 2A and 2B animals, the cranium was removed, exposing the brain. The skull, including the brain, was placed in 10% buffered formalin for at least 7 days. The fixed brains were weighed. Subsequently, the brain was fixed in 10% buffered formalin together with selected peripheral nervous system tissues. Sections of the brains were also stained for myelin and cell bodies using Luxol Fast Blue and Cresyl Violet.
For morphometric analysis, 3 sections were taken from neocortical, hippocampal and cerebellar areas to ensure homologous sections were obtained.
- Morphometric (quantitative) analyses of central nervous system tissues were performed for Cohort 2A and 2B animals of Groups 1 and 4 in a first instance. Intermediate dose groups (Groups 2 and 3) were not further analysed. Analyses included measurements from neocortical, hippocampal and cerebellar areas selected.
- Specific histopathological examination of the brain and nervous system tissues was performed. Tissues are indicated in Table 12 in section “Any other information on materials and methods incl. tables” for animals in the control and high dose groups (Groups 1 and 4). Histopathological examination was not extended to animals in the intermediate dose groups.

2) d) F1- Cohort 3 and positive control animals

- Necropsy of Cohort 3 animals was conducted on PND53-59. - Positive control animals were euthanized on the same date(s) at approximately 8 weeks of age.
- These animals were not deprived of food overnight before necropsy.
- All animals were subjected to a limited examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded. In case of macroscopic abnormalities, gross lesions were preserved in the most appropriate fixative together with the identification marks.

Statistics:

Data from concurrent controls and historical data from control animals were used to assess effects.
The mean and standard deviation were calculated for all parameters where feasible, using the litter as the basic sample unit.
Body Weight Gains: Calculated between each scheduled interval and against the first body weight of each phase.
Food Consumption: Calculated between each scheduled interval and against the first body weight of each phase.
Additional body weight or food consumption intervals were also presented such as time points corresponding to the last body weight or food consumption recorded for each cohort to elucidate study results.
Data were processed, where appropriate, to give mean values and standard deviations using the litter as the basic sample unit.
See more details in paragraph 11 of section “Any other information on materials and methods incl. tables”.

Reproductive indices:

The following reproductive indices were calculated as follows:
- Pre-coital interval (in days) = Sum of days until successful insemination /Number of inseminated females
- Male and female copulation index (in %) = 100 x Number of inseminated females / Number of paired females
- Male and female fertility index (in %) = 100 x Number of pregnant females / Number of inseminated females
- Irregularity index = Mean standard deviation of length of the estrous cycle / square root (length of the estrous cycle)
- Days in estrus = 100 x Number of estrus days / Number of smears

Offspring viability indices:

The following reproductive indices were calculated as follows:
- Pre-birth loss (in %) = 100 x (Number of implantations – Number of offspring born) / Number of implantations
- Live birth index (in %) = 100 x Number of pups born alive / Number of pups born
- Viability index (in %) = 100 x Number of pups alive on PND4 / Number of pups alive at birth
- Lactation index (in %) = 100 x Number of pups alive on PND21 / Number of pups alive on PND4 (after culling)
- Sex ratio (proportion of male pups in %) = 100 x Number of males / Number of pups

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There was no test material-related clinical sign in any group for either sex.
Piloerection, red vaginal discharge and noisy breathing were noted for a few females amongst the treated and control groups and considered related to the littering since they were observed between LD1 and LD3.
One female in the control group (No. 36) had a mass at the abdomen noted from LD7.
Isolated clinical signs were noted amongst treated and control groups for males and/or females including bent tail, broken tail, hypersalivation, scabs, sores, chromodacryorrhea, erythema, and localised or extensive hairloss and were considered incidental and not test material-related.

Arena observations: Clinical signs noted during the arena observations were limited to an exaggerated startle response for 1 male (No. 67) and 1 female (No. 88) at 25 mg/kg/day, firm body for 2 males at 25 mg/kg/day (Nos. 71 and 75) and flacid body for 1 male at 225 mg/kg/day (No. 155). These isolated findings were considered incidental and not test material-related.

Mortality:

no mortality observed

Description (incidence):

There was no unscheduled death in any group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was slightly but statistically significantly lower mean body weight gain from Day 1 to Day 84 for males at 225 mg/kg/day when compared with the control group (-7%). This minor difference was mainly due to lower mean body weight gain towards the end of the dosing period (-41% between Days 58 and 84) but was considered not toxicologically significant in the absence of any effect on mean food consumption and any impact on mean terminal body weight.
There was non dose-related slightly lower mean body weight gain at 75 and 225 mg/kg/day for females during the lactation period (-30% and -27%, respectively) compared with the control group associated with slight reduction in mean food consumption at 225 mg/kg/day only (see Section 8.2.5) considered not toxicologically significant in the absence of dose-related trend or any differences on mean terminal body weight.
There was no test material-related effect on mean body weight gain in any female group during the pre-mating or gestation periods or for males at 25 and 75 mg/kg/day.
See also Tables 16, 17, 19 and 20 in section Any other information on results incl. tables.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There was sightly but statistically significantly lower mean food consumption at 225 mg/kg/day during the lactation period (-8%) when compared with the control group. This minor difference when compared with the control group was considered not toxicologically significant in the absence of any effect on mean terminal body weight. There was no test material-related effect on mean food consumption in any male group during the pre-mating period (Day 1 to Day 71) or for females during the pre-mating and gestation periods in any group and during the lactation period at 25 and 75 mg/kg/day.
See also Tables 18 and 21 in section Any other information on results incl. tables.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematology and coagulation:
There was no test material-related effect on the hematology and coagulation parameters that was considered adverse in any group for either sex.
There were higher mean neutrophil counts and lower mean lymphocyte counts for females given 225 mg/kg/day compared with control. This finding was considered non adverse in the absence of any associated clinical pathology or histopathology findings or any similar finding for the F1 generation.
Minor statistically significant differences arising between control and treated groups, such as lower monocyte count for males at 75 or 225 mg/kg/day, higher mean corpuscular hemoglobin concentration at 75 and 225 mg/kg/day, and higher mean neutrophil count and lower mean monocyte count at 225 mg/kg/day for females compared with the control group were considered not toxicologically relevant as they were of low magnitude, not dose-related and/or observed for a single gender.
See also Tables 22 and 23 in section "Any other information on results incl. tables".

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Administration of the test material to rats was associated with changes in clinical chemistry parameters at 225 mg/kg/day. These changes are presented in table 24 in section Any other information on results incl. tables.
At 225 mg/kg/day, there were slightly higher sodium concentrations for both sexes and slightly higher phosphorus, urea, and creatinine concentrations for females only compared with the control group.
At 225 mg/kg/day, tryglycerides and Alkaline Phosphatase (ALP) concentrations were slightly higher for both sexes and Alanine Transaminase (ALAT) and Aminotransferase (ASAT) concentrations were slightly higher for females only compared with the control group. Although the ALP and ASAT (for males) and ASAT and ALAT (for females) mean values were disproportionally influenced by 2 atypical animals with exceptionally high concentrations (Male No. 152 and Female No. 192) this correlated with slightly higher mean liver weight for both sexes when compared with the control group.
Minor statistically significant differences arising between the treated and control groups were noted including slightly lower albumin and protein concentrations at 225 mg/kg/day, higher cholesterol at 225 mg/kg/day and glucose concentrations at 75 and 225 mg/kg/day for females, lower cholesterol at 225 mg/kg/day and lower glucose concentration at 75 and 225 mg/kg/day for males compared with the control group. These changes were considered not toxicologically relevant as they were of low magnitude or noted in 1 gender only.
There were no test material-related effects amongst the serum clinical chemistry parameters at 25 and 75 mg/kg/day for either sex.
See also Tables 25 and 26 in section "Any other information on results incl. tables".

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

There was no test material-related effect on the mean T4 and TSH serum level that was considered toxicologically relevant in any group for either sex.
There was a statistically significantly lower mean T4 serum level for females at 225 mg/kg/day compared with the control group (x0.64) and a higher mean TSH serum level at 225 mg/kg/day for females compared with the control group (x4.88). The difference in TSH level did not reach any statistical significance in any group for adults.
These changes were not toxicologically relevant in the absence of any gender trend, any test item related effect on the thyroid weight, anogenital distance, nipple areola, reproduction and development, any histology examination of the thyroid glands, or similar findings for pups and considered related to the high variability of this parameter. As under the conditions of this study no adverse effect was observed that could be linked to the lower T4 and higher TSH values, this was not taken into account to determine the No Observed Adverse Effect Levels (NOAELs).
There was no differences in the mean TSH serum levels at 25 and 75 mg/kg/day for females
or in any group for males when compared with the control group.
See also Table 27 in section "Any other information on results incl. tables"

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test material-related effect on urine analysis parameters in any group for either sex.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test material-related microscopic findings were present in the kidney, heart, stomach, ovary, vagina, thymus and are summarized in Table 30 in section “Any other information on results incl. tables”.

In the kidney, bilateral tubular mineralization at the corticomedullary junction (minimal or mild) and multifocal cortical tubular dilatation (minimal or mild) were observed for some females at 75 and 225 mg/kg/day. These renal findings were considered test material-related. Of note, the tubular dilatation noted in Male No. 160 (225 mg/kg/day) was unilateral focal and was therefore not comparable to test material-related bilateral renal tubular dilatation present in females.

In the heart, aortic or myocardial mineralization (minimal or mild) was observed for some females at 75 and 225 mg/kg/day. This cardiac finding was considered test material-related.

In the stomach, mineralization of the glandular part (minimal or mild) was observed for some females at 75 and 225 mg/kg/day. It was associated with necrosis of the glandular part (minimal or mild) that grossly correlated with dark foci at necropsy for 2 females dosed at 225 mg/kg/day. These gastric findings were considered test material-related.

In the reproductive tract, diffuse epithelial mucification (minimal to moderate, characterized by the presence of prominent mucus vacuoles in the cytoplasm of superficial cells from the vaginal epithelium) in the vagina was observed at higher incidence and severity at 225 mg/kg/day compared with the control group. Ovarian atrophy (minimal) was present at 225 mg/kg/day and correlated with a lower ovary weight. These findings in the female reproductive tract suggested a delay in the post-gestation recovery of estrous cycling and were considered test material-related.

In the thymus, atrophy (minimal) and increased cortical apoptosis (mild) were noted in several females dosed at 225 mg/kg/day and were considered test material-related. Atrophy correlated with the lower thymus weight noted in some females dosed at 225 mg/kg/day.

In the thyroid gland of some females dosed at 225 mg/kg/day, a follicular cell adenoma was present in Female No. 195, and minimal bilateral diffuse follicular cell hypertrophy was observed in Female No. 184. These sporadic findings in the thyroid gland were considered incidental.

Other microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of Wistar Han rats, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of the test material.

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There was no test item material-related effect on mean estrous cycle length and regularity in any group.
See Table 31 in section "Any other information on results incl. tables".

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There was no test material-related effect on sperm count, motility or morphology in any group.
See also Tables 1 to 4 in the file "443-BPD-Attach-F0-Sperm analysis" in the attachments.

Reproductive performance:

no effects observed

Description (incidence and severity):

- Pre-coital time
The majority of mated females showed evidence of insemination within the first 4 days of pairing (approximate duration of a normal estrous cycle). The mean pre-coital interval was therefore comparable in all groups (i.e., 2.8, 2.7, 3.0 and 2.7 days in the control, 25, 75 and 225 mg/kg/day groups, respectively).

- Mating index
There was no test material-related effect on mating performance in any group. After 2 weeks of mating, all pairs of animals mated in all groups.

- Fertility index
There was no test material-related effect on fertility. All mated females became pregnant, with the exception of 2 (Nos. 26 and 35) and 1 (No. 200) females in each of the control and 225 mg/kg/day groups, respectively. The isolated case in the high dose group was not correlated with any microscopic finding of the reproductive organs and was therefore considered incidental.

- Number of implantations sites
There was no test material-related effect on the mean number of implantation sites. Mean values in the treated groups (13.3 in all groups) were comparable with the control mean value (13.8) and above the HCD mean (12.1).

- There was no test item-related effect on the gestation index and duration of gestation in any group.
There were 23, 25, 25 and 24 pregnant females in the control, 25, 75 and 225 mg/kg/day groups, respectively, all of which successfully completed delivery of liveborn pups with the exception of 1 female (No. 176) given 225 mg/kg/day with a total litter resorption. This isolated case was considered not test item-related in the absence of any differences on the mean percentage pre-birth loss between the 225 mg/kg/day and control groups.
The mean duration of gestation was approximately 22 (22.1-22.4) days in each group.

See also Tables 32 and 33 in section “Any other information on results incl. tables”.

For the F0 generation, there were no test item-related findings that were considered toxicologically significant on mean body weight gain, food consumption, clinical condition (including arena observation) or on the hematology, coagulation and urinalysis parameters for either sex in any group.

Higher creatinine and urea concentrations, minor ionic inbalance for females, and higher hepatic enzyme and trygliceride concentrations for both sexes were noted at 225 mg/kg/day compared with the control group and correlated with the pathology findings noted for the kidneys and liver (see below).

The differences in T4 and TSH serum levels were considered not toxicologically relevant in the absence of any gender trend, any test item-related effect on the thyroid weight, reproduction and development, any histology findings in the thyroid glands and any similar findings in the F1 generation (see below Results: F1 generation"). They are considered related to the high variability of this parameter.

At necropsy, there were test item-related findings for F0 females dosed at 225 mg/kg/day in the kidney (minimal to mild tubular dilatation and mineralization, correlating with higher kidney weight), heart (minimal-to-mild myocardial or aortic mineralization), stomach (minimal-to-mild mineralization and/or necrosis of the glandular part, grossly correlating with dark foci), thymus (minimal atrophy and mild increased cortical apoptosis, correlating with decreased organ weight). Mineralization in the kidney, heart and stomach was considered adverse, while other findings were considered secondary to test item-related systemic effects (ovary and thymus), or adaptative changes (liver and adrenal gland). The pathology findings noted on the kidneys were correlated with higher creatinine and urea concentrations and minor ionic inbalance. The higher mean liver weight at 225 mg/kg/day for both sexes correlated with slightly higher mean tryglyceride and hepatic enzyme concentrations when compared with the control group. Ovarian atrophy, lower ovaries weight compared with the control group, and vaginal mucification which suggested a delay in the post-gestation recovery of estrous cyclicity, were noted at necropsy for F0 females at 225 mg/kg/day and were considered probably secondary to test item-related systemic effects on the kidneys, stomach and heart. These findings were not correlated with any test item-related effect on fertility or estrous cycle data and no test item-related effect on the mean count of primordial/small growing follicles (Cohort 1A) and was therefore considered not adverse. In addition, there was no similar finding in the F1 generation.

There was no other test item-related effect on other reproductive parameters of the F0 generation including mating index, fertility index, estrous cyclicity, gestation index, gestation duration, pre-coital time, sperm quality, and pre-birth loss in any group.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 75 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other:

Remarks on result:

other: No remark.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

1) F1 pups (before weaning)
No clinical signs occurred amongst pups that were considered to be test material-related.
Incidental clinical signs consisted of occasional cyanotic/cold pups, weakness, thinness, incomplete hair growth, hair loss, sores, haematoma, lacrimation, partial or complete absence of an ear and scabs. In view of their nature and/or sporadic occurrence, including in the control, these findings were considered of no toxicological relevance.

2) F1-post weaning - Cohorts 1A, 1B, 2A and 3
There was no test material-related clinical sign in any group for either sex.
Isolated clinical signs were noted for males and/or females including bent tail, chromodacryorrhea, hypersalivation, lacrimation, localized hair loss, piloerection, scabs, swelling, incomplete hair growth, abscess, limping, malocclusion, red stained fur, scars and wheezing; these signs were considered incidental or related to the dosing procedure.
During arena observation, clinical signs noted were isolated and limited to minor differences in the tactile reflex, alertness and body tone observed sporadically at 75 and 225 mg/kg/day and considered incidental in the absence of any effect on the neurobehavioral tests.

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was no test item-related effect on the mortality of pups in any group at birth until the end of the study.
- At birth: see "Life birth index" and "Lactation index" in the section "Others" below.
- F1-before weaning: see "Viability index" and "Lactation index" in the section "Others" below.
- F1-post weaning - Cohorts 1A, 1B, 2A and 3:

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

1) F1 pups (before weaning)
There was slightly lower mean pup body weight gain at 225 mg/kg/day, leading to statistically significantly lower mean pup body weight compared with the concurrent control and Historical Control Data mean on PND21 (-8% and -4% for both sexes). However, the mean values (52.3 g and 50.6 g for males and females, respectively) remained within the historical control data range (49.7 to 57.6 for males and 48.8 to 56.0 for females), so this difference was considered not toxicologically significant.
There was no test material-related effect on mean pup body weight at 25 and 75 mg/kg/day.
See also Table 34 in section “Any other information on results incl. tables”.

2) F1-post weaning - Cohorts 1A, 1B, 2A and 3
For males, there was a test material-related slightly lower mean body weight gain after weaning throughout the dosing period for males (Day 1 to Day 70) at 225 mg/kg/day compared with the control group (-10%) leading to a lower mean terminal body weight (-11%) on Day 70 compared with the control group. This difference was considered not adverse in view of the low magnitude of the change and in the absence of any impact on the development such as sexual maturation.
For females, there was lower mean body weight gain during the first week post-weaning at 225 mg/kg/day considered not toxicologically relevant as it was followed by a recovery such that the mean terminal body weight was comparable with that of the control group. In addition there was no test material-related effect on the sexual maturation that was considered toxicologically significant.
There was no test material-related effect on mean body weight gain for females in any group or males at 25 and 75 mg/kg/day.
See also Tables 35 and 36 in section “Any other information on results incl. tables”.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

F1-post weaning - Cohorts 1A, 1B, 2A and 3:
Consistent with the slightly lower mean body weight gain observed, there was a slightly lower mean food consumption for males at 225 mg/kg/day compared with the control group (-8%) during the dosing period (Day 1 to Day 70). This difference was considered not adverse in view of the low magnitude of the change.
For females, slightly lower mean food consumption was noted at 225 mg/kg/day during the first 2 weeks post-weaning compared with the control group, considered not toxicologically significant in the absence of any difference on the mean terminal body weight.
There was no test material-related effect on mean food consumption for females in any group or males at 25 and 75 mg/kg/day.
See also Tables 37 and 38 in section “Any other information on results incl. tables”.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

F1-post weaning - Cohort 1A:
Administration of the test material to rats was associated with changes in hematology parameters at 225 mg/kg/day.
At 225 mg/kg/day, there were slightly higher mean corpuscular volume, mean corpuscular hemoglobin and reticulocyte count and lower white blood cells count including lower lymphocyte, monocyte and basophile counts for males, compared with the control group.
For females, there were slightly lower hematrocrit and hemoglobin concentration, slightly lower red blood cells count and slightly higher mean corpuscular hemoglobin. The difference correlated with spleen minimally increased extramedullary hematopoiesis at higher incidence at 225 mg/kg/day for males and females compared with the control groups, considered not adverse in view of the low magnitude of the change.
There were no changes in hematological parameters at 25 or 75 mg/kg/day for either sex.
See also Table 39 in section “Any other information on results incl. tables”.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

F1-post weaning - Cohort 1A:
There were no test material-related changes in serum clinical chemistry parameters in any group for either sex that were considered toxicologically relevant.
There was statistically significantly lower mean cholesterol serum concentration at 225 mg/kg/day for males (x0.48). In the absence of correlation with any microscopic finding on the liver or any increase in liver weight, this was considered not adverse.
Minor statistically significant differences arising between control and treated animals were noted including slightly higher sodium and alkaline phosphatase concentrations for both sexes at 225 mg/kg/day and a slightly lower mean globulin concentration in males at 225 mg/kg/day when compared with the control group. These changes were considered not toxicologically relevant as they were of low magnitude and/or observed in one sex only.
See also Tables 40 and 41 in section “Any other information on results incl. tables”.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

There was no test material-related effect on urine analysis parameters in any group for either sex. The variations noted in the pH were part of the background for this strain of rat and therefore considered not toxicologically relevant.

Sexual maturation:

effects observed, non-treatment-related

Description (incidence and severity):

F1-post weaning - Cohort 1A, 1B, 2A and 3:
There was no difference on sexual maturation that was considered toxicologically significant in any group for either sex.
> The time to attain sexual maturation (vaginal opening) was slightly longer for females at 225 mg/kg/day (37.1 days) compared with the control group (34.4 days) but this was likely related to the lower mean body weight at 225 mg/kg/day compared with the control group since mean body weight at sexual maturation was comparable (112 g) with that of the control group (114 g). In addition, the time to attain sexual maturation was within the Historical Control Data mean ± 2 Sd (28.0 to 40.4 days) so the difference was considered not toxicologically significant.
There were no differences in sexual maturation at 25 and 75 mg/kg/day for females, compared with the control group.
> There were no differences in sexual maturation in any group for males. The day of preputial cleavage was 51.6, 51.1, 51.6 and 52.0 for the control group and at 25, 75 and 225 mg/kg, respectively.

See also Table 42 in section “Any other information on results incl. tables”.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

There was no test material-related effect on the anogenital distance in any group.
See also Table 43 in section “Any other information on results incl. tables”.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Male pups did not present any areolae/nipples in any group.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

1) F1-post weaning - Cohort 1A, 1B and surplus:

When compared with controls, male and female groups dosed at 225 mg/kg/day had statistically significantly higher mean kidney weights in Cohort 1A and 1B when compared with the control groups; these effects were considered test material-related. In males, higher kidney weights correlated with gross enlargement and granular appearance.
There were no test material-related effect on the mean organ weights for the surplus animals in any group for either sex.
There were other isolated organ weight values that were statistically different from their respective control. There were, however, no patterns, trends or correlating data to suggest these values were toxicologically relevant. Thus, other organ weight differences observed were considered incidental and unrelated to administration of the test material.
See also Tables 44 and 45 in section “Any other information on results incl. tables”.

2) F1 - Cohorts 2A and 2B
- Brain dimension: There were no test material-related differences in the histomorphometric measurements of the brain. The differences were considered to be incidental and part of the biological individual variability.

- Brain Weight: There were no test material-related brain weight changes in males and females from Cohorts 2A and 2B.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

F1-animals

1) Cohort 1A:
In the kidney, granular appearance and/or enlargement noted for 2 males dosed at 225 mg/kg/day were histologically correlated with tubular basophilia, tubular dilatation and inflammatory cell infiltration. Granular appearance noted in one control male did not have any histopathologic correlate. Granular appearance and enlargement were considered test material-related in males dosed at 225 mg/kg/day.
See also Table 46 in section “Any other information on results incl. tables”.

2) Cohort 1B:
Enlarged kidneys (unilateral or bilateral) were noted for males and females dosed at 225 mg/kg/day with higher incidence compared with the control group.
See also Table 47 in section “Any other information on results incl. tables”.

3) Cohorts 1A, 1B, 2A, 2B, 3 and surplus animals:
Other gross findings in Cohorts 1A, 1B, 2A, 2B, 3 and for surplus animals were considered incidental, of the nature commonly observed in this strain and age of Wistar Han rats, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to administration of the test material.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

F1-animals

1) Cohort 1A
In the kidney, bilateral multifocal tubular basophilia (minimal to moderate) was observed at higher incidence and severity at 225 mg/kg/day for males and females compared with the control groups. Basophilic tubules were characterized by a thickened basement membrane and were associated with tubular dilatation (minimal to moderate) and chronic inflammatory cell infiltration (minimal to mild). These renal findings were considered test material-related.
In the spleen, minimally increased extramedullary hematopoiesis was observed at higher incidence at 225 mg/kg/day for males and females when compared with the control groups, and was considered test material-related.
Following quantitative microscopic evaluation of ovaries, there were no statistically significant differences in the mean numbers of primordial/small growing follicles and corpora lutea.
Other microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of Wistar Han rats, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of the test material.
See also Table 48 in section “Any other information on results incl. tables”.


2) Cohort 2A and 2B
- No test material-related microscopic findings were noted in the nervous system following detailed examination of the subregions of the brain (Cohorts 2A and 2B) and spinal cord, dorsal root ganglia and ventral root fibers, skeletal muscle, sciatic and tibial nerves, eye and optic nerves (Cohort 2A). The microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered not test material-related.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Other results on the F1 generation (DEVELOPMENTAL TOXICITY DATA):

1) GESTATION INDEX AND DURATION
See section above "Results: P0 (first parental generation) - Reproductive performance".

2) PRE-BIRTH LOSS
There was no test item-related effect on the pre-birth data in any group.
The mean percentage of pre-birth loss in treated groups (5.9%, 7.7% and 7.9% at 25, 75 and 225 mg/kg/day, respectively) was lower compared with the concurrent control group (8.3%) and the HCD mean (9.9%).
The mean numbers of pups delivered in the treated groups (12.5, 12.3 and 12.2 at 25, 75 and 225 mg/kg/day, respectively) were comparable with that of the concurrent control group (12.7) and above the historical control data mean (11.0).

3) LIFE BIRTH INDEX
There was no test item-related effect on the number of live offspring at birth in any group.
There were 4, 3, 2 and 5 stillborn pups in the control, 25, 75 and 225 mg/kg/day groups, respectively. The live birth index was consequently comparable in all groups (99.0%, 99.4% and 98.2% at 25, 75 and 225 mg/kg/day, respectively) compared with the control group (98.6%).

4) VIABILITY INDEX (F1 pups)
There was no test item-related effect in any group on the number of live offspring on PND4 (before culling) compared with the number of offspring on PND0 (viability index).
There were 4, 4, 5 and 10 dead, missing or cannibalized pups from 1, 3, 5 and 3 litters in the control, 25, 75 and 225 mg/kg/day groups, respectively, between PND0 and PND4. The viability index was consequently slightly lower at 225 mg/kg/day (96.4%) compared with the control group (98.6%) but remained within the HCD range (96.2% to 100%).

5) LACTATION INDEX (F1 pups)
There was no test item-related effect in any group on the number of live offspring at weaning compared with the number of offspring on PND4, post culling (lactation index).
There was only on dead pup at 75 mg/kg/day (from Female No. 126). This isolated finding in the intermediate dose group was incidental.

6) SEX RATIO (F1 pups)
There was no test item-related effect on the sex ratio in any group.

See also Tables 32 and 33 in section “Any other information on results incl. tables”.

7) THYROID HORMMONE ANALYSIS

1.a) F1 pups - before weaning - on PND 4:
There was no test material-related effect that was considered toxicologically significant on mean T4 serum levels for F1 PND4 pups in any group.
There were statistically significantly higher T4 serum levels at 25 and 225 mg/kg/day (x1.30 - x1.37) compared with the control group considered not toxicologically significant in the absence of any dose relationship or similar finding for PND21 pups and given that all values from the 225 mg/kg/day group remained within the control range with the exception of 2 pups.

1.b) F1 pups - before weaning - on PND 21
There was no test material-related effect that was considered toxicologically significant on mean T4 and TSH serum levels for F1-PND21 pups in any group.
There were slightly higher TSH serum levels in all group for both sexes (x1.39 to x1.79) compared with the control group (statistically significant for males only), considered not toxicologically significant in the absence of any changes in T4 serum levels, in the absence of any dose-relationship and in view of the low magnitude of the changes.
See also Table 60 in section “Any other information on results incl. tables”.

1.c) F1 pups - post weaning - Cohorts 1A
There was statistically significantly lower mean T4 serum level for males at 225 mg/kg/day when compared with the control group (x0.68).
There was no difference on mean T4 serum level for males at 25 and 75 mg/kg/day or for females in any group and no difference on the mean TSH serum level in any group for either sex that were considered toxicologically significant.
See also Table 61 in section “Any other information on results incl. tables”.

8) ESTROUS CYCLE DATA - Cohort 1A

There was no test material-related effect on mean estrous cycle length and regularity in any group. The mean duration from vaginal opening to first estrous was comparable in all groups.
See also Table 62 in section “Any other information on results incl. tables”.

9) SPERM ANALYIS - Cohort 1A

There was no test material-related effect on mean sperm counts, sperm morphology or motility, including the percentage of progressively motile sperm, in any group.
See also Tables 1 to 4 in the file "443-BPD-Attach-F1-Sperm analysis" in the attachments.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

F1-animals

1) Auditory Startle Reflex – Cohort 2A
There was no test material-related effect on the auditory startle reflex test in any group for either sex. The percentage of habituation to repeated acoustic stimuli was comparable between treated and control groups.
See also Tables 49 and 50 in section “Any other information on results incl. tables”.

2) Functional Observation Battery – Cohort 2A
There was no test material-related effect on the FOB parameters in any group for either sex.
See also Tables 51 and 52 in section “Any other information on results incl. tables”.

3) Motor Activity – Cohort 2A
There was no test material-related effect on motor activity (ambulatory or fine movements) in any group for either sex.
During the first 10 minutes of the test, there were no average differences in ambulatory or fine movements in treated groups compared with the control groups for either sex. Thereafter, there was a greater habituation at 225 mg/kg/day for both sexes for ambulatory and fine movements through to the end of the test compared with the control groups and the Historical Control Data range. However, all mean percentage values remained within the mean control value ± Sd. In addition, there were no test material-related findings in the FOB test, including the arena observations, no test material-related brain weight changes and no test material-related microscopic findings of the brain (including histomorphometric measurements). These differences were therefore considered related to the high inter-individual responses in the motor activity test and not test material-related.
See also Table 53 in section “Any other information on results incl. tables”.

Developmental immunotoxicity:

no effects observed

Description (incidence and severity):

1) Anti-KLH IgM Analysis (TDAR) – Cohort 1B
There was no test material-related effects for F1-Cohort B animals on the primary anti-KLH IgM response after immunization with KLH in any group for either sex.
See also Tables 54 and 55 in section “Any other information on results incl. tables”.

2) Anti-SRBC IgM Analysis (TDAR) - Cohort 3
There was no test material-related effects for F1-Cohort 3 animals on the primary anti-SRBC IgM response after immunization with SRBC in any group for either sex.
See also Tables 56 and 57 in section “Any other information on results incl. tables”.

3) Splenic Lymphocyte Subpopulation Analysis - Cohort 1A
There was no test material-related effect on the development of splenic subpopulation (assessed by flow cytometry) in any group for either sex.
See also Tables 58 and 59 in section “Any other information on results incl. tables”.

There were no test item-related effects that were considered toxicologically significant on the F1 litter parameters, pup weights, preweaning developmental landmarks (anogenital distance and areolae/nipple retention), neonatal and weanling serum thyroid hormone levels, organ weights or test item-related macroscopic findings for F1 pups that were found dead, culled on PND4, or examined at scheduled necropsy on PND21, i.e., there was no evidence of F1 postnatal toxicity.

For the F1 generation, there was no adverse test item-related effect on mean body weight gain, mean food consumption, clinical parameters, or sexual maturation.

There was no test item-related immunotoxicity (Cohorts 1A and 3), as evidenced by lymphocyte subpopulation analysis and KLH-IgM or SRBC-IgM responses in a TDAR assay.

Lastly, there was no test item-related neurotoxicity (Cohorts 2A and 2B), as indicated by auditory startle responsiveness at weaning or FOB and motor activity at adulthood, or brain morphometry, brain weight and neurohistopathology.

At necropsy, non adverse test item-related effects were observed in the kidney (tubular basophilia and dilatation, chronic inflammatory cell infiltration in both sexes, correlated with gross enlargement and/or granular appearance, and with higher kidney weight) and spleen (increased extramedullary hematopoiesis) of F1 males and females dosed at 225 mg/kg/day. The findings for the kidneys were considered non adverse in the absence of degenerative/necrotic renal changes and any clinical pathology correlate. There was no other test item-related pathology finding.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 225 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: No remark.

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

225 mg/kg bw/day

Treatment related:

no

1) Formulations analysis

All analyzed samples for formulations prepared at nominal concentrations of 5, 15 and 45 mg/mL of test material in vehicle (0.5% (w/v) CMC 300-600 centipoises in purified water), taken from each preparation, including the vehicle, on the first day of treatment and on Week 11 and Week 26 of treatment were within the acceptance criteria (± 15%). The deviations for the nominal concentrations ranged from -4.0% to 1.2%. Furthermore, the test material prepared as a suspension in the vehicle was homogenous as the RSD (Relative standard Deviation) on the 6 aliquots (top, middle, bottom) was ≤2.3%.

Finally, no test material was present in the vehicle sample.

Table 13: Formulations analysis – Day 1 results

Date of Formulation	Date of Analysis	Nominal Concentration (mg/mL)	Sampling	Experimental Concentration (mg/mL)	Mean Concentration (mg/mL)	Deviation from Nominal	Complies (a)
05-Feb-2021	08-Feb-2021	0	M	0.000	0.000	≤ 2 % LOQ	Yes
				0.000
		5	T	4.914	4.895	-2.1 %	Yes 0.7% RSD
				4.876
			M	4.975	4.960	-0.8 %
				4.946
			B	4.927	4.930	-1.4 %
				4.934
		15	M	15.06	15.02	0.1 %	Yes
				14.98
		45	T	44.53	43.65	-3.0 %	Yes 2.3% RSD
				42.76
			M	45.27	45.55	1.2 %
				45.83
			B	44.65	44.70	-0.7 %
				44.74

LOQ (Lower calibration standard): 5 μg/mL.

(a): Deviation from the nominal value complies when within the acceptable limits of ± 15% and RSD < 10%.

T: Top, M: Middle and B: Bottom of the formulation.

 

Table 14: Formulations analysis – Week 11 results

Date of Formulation	Date of Analysis	Nominal Concentration (mg/mL)	Experimental Concentration (mg/mL)	Mean Concentration (mg/mL)	Deviation from Nominal	Complies (a)
15-Apr-2021	19-Apr-2021	0	0.000	0.000	≤ 2 % LOQ	Yes
			0.000
		5	4.833	4.884	-2.3 %	Yes
			4.934
		15	14.67	14.75	-1.7 %	Yes
			14.83
		45	42.54	43.94	-2.3 %	Yes
			45.34

LOQ (Lower calibration standard): 5 μg/mL.

(a): Deviation from the nominal value complies when within the acceptable limits of ± 15%.

 

Table 15: Formulations analysis – Week 26 results

Date of Formulation	Date of Analysis	Nominal Concentration (mg/mL)	Experimental Concentration (mg/mL)	Mean Concentration (mg/mL)	Deviation from Nominal	Complies (a)
06-Aug-2021	11-Aug-2021	0	0.000	0.000	≤ 2 % LOQ	Yes
			0.000
		5	4.898	4.802	-4.0 %	Yes
			4.706
		15	14.74	14.76	-1.6 %	Yes
			14.78
		45	44.25	44.44	-1.3 %	Yes
			44.62

LOQ (Lower calibration standard): 5 μg/mL.

(a): Deviation from the nominal value complies when within the acceptable limits of ± 15%.

 

2) F0 - Body weights, body weight changes and food consumption

Table 16: Mean body weight (g) for males (F0) at Day 84

 

Day	0 mg/kg/day	25 mg/kg/day	75 mg/kg/day	225 mg/kg/day
84	390.30 I^1	390.81	379.48	373.96

¹ I - Automatic Transformation: Identity (No Transformation).

 

Table 17: Mean body weight change (g) for males (F0) for the period days 1-84

 

Period	0 mg/kg/day	25 mg/kg/day	75 mg/kg/day	225 mg/kg/day
Days 1-84	243.44 I^1	244.96	232.95	225.85 w^2

¹ I - Automatic Transformation: Identity (No Transformation).

² w - Test: Williams 2 Sided p < 0.05.

 

Table 18: Mean pre-mating food consumption of males (F0) for the period days 1-71

 

Period	0 mg/kg/day	25 mg/kg/day	75 mg/kg/day	225 mg/kg/day
Days 1-71	23.22 R^1	22.98	23.21	23.53

¹ R - Automatic Transformation: Rank.

 

Table 19: Mean body weight (g) for females (F0) at end of pre-mating (D71), end of gestation (GD20), end of lactation (LD21)

Day	0 mg/kg/day	25 mg/kg/day	75 mg/kg/day	225 mg/kg/day
D71	242.44 I^1	237.20	239.52	242.69
GD20	355.70 L^2	349.58	351.60	351.11
LD21	288.06 I^1	282.86	286.05	279.56

¹ I - Automatic Transformation: Identity (No Transformation).

² L - Automatic Transformation: Log.

Table 20: Mean body weight change (g) for females (F0) for the periods Days 1-71 (pre-mating), GD0-GD20 (gestation) and LD1-LD21 (lactation)

Day	0 mg/kg/day	25 mg/kg/day	75 mg/kg/day	225 mg/kg/day
Days 1-71	103.74 I^1	101.50	101.43	103.98
GD0-GD20	112.08 R^2	107.14	107.80	108.72
LD1-LD21	21.90 R, k3	24.37	15.28	15.93

¹ I - Automatic Transformation: Identity (No Transformation).

² R - Automatic Transformation: Rank.

³ R,k - Automatic Transformation: Rank, (All Groups) Test: Kruskal-Wallis p < 0.05.

 

Table 21: Mean food consumption (g) for females (F0) for the periods Days 1-71 (pre-mating), GD0-GD20 (gestation) and LD1-LD21 (lactation)

Day	0 mg/kg/day	25 mg/kg/day	75 mg/kg/day	225 mg/kg/day
Days 1-71	17.55 R^1	17.18	17.12	17.41
GD0-GD20	22.47 I^2	21.81	22.02	23.49
LD1-LD21	53.77 R^1	53.40	53.14	49.44 SS^3

¹ R - Automatic Transformation: Rank.

² I - Automatic Transformation: Identity (No Transformation).

³ SS - Test: Shirley 2 Sided p < 0.01.

 

3) F0 - Hematology

Table 22: Group mean hematology results – F0 males - Monocytes counts

Day 85 relative to start date:

Group	M.Abs. Giga/L	M (%)
1	0.233	3.80
2	0.191	3.56
3	0.159*	2.60*
4	0.167*	3.27*

M: Monocytes.

* 5% significance level.

 

Table 23: Group mean hematology results – F0 females – extract of results

Day 22 relative to litter date:

Group	MCHC g/L	N.Abs. Giga/L	N(%)	L.Abs. Giga/L	L(%)	M.Abs. Giga/L	M (%)
1	320.8	2.565	42.37	2.883	50.26	0.303	4.99
2	322.7	2.411	41.59	2.739	52.61	0.226	3.97
3	329.8*	2.069	36.29	3.027	57.16	0.218	4.26
4	327.4*	4.805**	70.48**	1.340**	23.61**	0.187	2.96*

MCHC: mean corpuscular hemoglobin concentration.

N: Neutrophil.

L: Lymphocyte.

M: Monocytes.

* 5% significance level.

** 1% significance level.

 

4) F0 - Clinical chemistry

 

Table 24: Toxicologically Relevant Changes in Serum Clinical Chemistry Parameters in the 225 mg/kg/day Group – Fold changes relative to control group

	Males	Females
Na (mmol/L)	X1.01	X1.01
Triglycerides (mmol/mL)	X1.46	X1.70
ALP (IU/L)	X2.10	X1.48
ASAT (IU/L)	-	X1.76
ALAT (IU/L)	-	X2.15
Chol (mmol/L)	-	X 1.76
Cl (mmol/L)	-	X0.95
P (mmol/L)	-	X 1.39
Urea (mmol/L)	-	X2.19
Creat (mcmol/L)	-	X1.54

-: Absence of change.

Numerical values indicate fold changes of treated group value relative to control group mean value.

 

Table 25: F0 males – Group mean serum clinical parameters – extract of results

Day 85 relative to start date

 

Group	Na mmol/L	Gluc mmol/L	Chol mmol/L	Trigs mmol/L	T.Bili µmol/L	ALP BC IU/L	ASAT IU/L
1	142.8	8.597	1.506	1.095	3.01	67.6	98.2
2	144.4	7.896	1.416	1.183	2.81	71.6	84.6
3	143.4	7.081**	1.173**	1.253	2.62**	72.5	90.1
4	144.9*	7.118**	0.639**	1.598*	2.58**	142.2**	93.3*

Na: sodium – Gluc: glucose - Chol: cholesterol - Trigs: Triglycerides - T.Bili: Total Bilirubin.

ALP BC: Alkaline phosphatase - ASAT: Aspartate aminotransferase.

* 5% significance level.

** 1% significance level.

 

Table 26: F0 females – Group mean serum clinical parameters – extract of results

Day 22 relative to litter date

Group	Na mmol/L	Cl  mmol/L	P mmol/L	Gluc mmol/L	Urea mmol/L	Creat µmol/L	Chol mmol/L
1	140.1	99.2	2.979	5.773	9.080	47.8	2.056
2	140.5	100.3	2.839	6.078	9.300	48.0	2.055
3	139.6	99.6	2.786	6.617*	10.440	50.1	2.299
4	142.2**	94.4*	4.131**	7.839**	19.880**	73.4**	3.623**
Group	Trigs mmol/L	Prot g/L	Alb g/L	A.G ratio	ALP BC IU/L	ASAT IU/L	ALAT IU/L
1	2.901	60.83	34.16	1.30	82.9	101.7	27.2
2	1.925	61.67	34.12	1.24	113.6	92.7	25.3
3	2.795	62.87	34.54	1.22	90.5	95.3	25.6
4	4.940	54.53*	29.28**	1.17*	122.5*	178.8	58.6

Na: sodium – Cl: Chloride – P: Phosphorus - Gluc: glucose – Urea : urea nitrogen – Creat: creatinine – Chol: cholesterol - Trigs: Triglycerides - Prot: Total protein – Alb : Albumin – A.G ratio : Albumin/Globulin ratio - ALP BC: Alkaline phosphatase - ASAT: Aspartate aminotransferase – ALAT: Alanine aminotransferase.

* 5% significance level.

** 1% significance level.

 

5) Thyroid Hormone analysis

Table 27: F0 - Summary of T4 and TSH values

Animals	Hormone	Group 1 0 mg/kg/day	Group 2 25 mg/kg/day	Group 3 75 mg/kg/day	Group 4 225 mg/kg/day
F0-Males Mean	T4 µg/dL	5.63	5.66	5.80	5.07
	TSH µIU/mL	0.092	0.108	0.159	0.143
F0-Females Mean	T4 µg/dL	4.66	4.66	3.67	2.97 *
	TSH µIU/mL	0.464	0.289	0.501	2.265

*p ≤ 0.05.

 

6) F0 - Gross pathological findings

Table 28: Summary of gross pathological findings - F0 animals

	Males				Females
Group No.	1	2	3	4	1	2	3	4
Dose (mg/kg bw/day)	0	25	75	225	0	25	75	225
Number of Animals per Group	25	25	25	25	25	25	25	25
Liver (Number Examined) Enlargement	(25) 0	(25) 0	(25) 0	(25) 0	(25) 1	(25) 0	(25) 2	(25) 5
Stomach (Number Examined) Dark (depressed) focus, single/several/many, glandular mucosa	(25) 0	(25) 0	(25) 1	(25) 2	(25) 3	(25) 4	(25) 6	(25) 6

 

7) F0 – Organ weights

Table 29: Summary of Organ Weight Data – Scheduled Euthanasia (F0-animals)

	Male			Females
Group No.	2	3	4	2	3	4
Dose (mg/kg/day)	25	75	225	25	75	225
Number of Animals per Group	25	25	25	25	25	23
Liver (Number Weighed)^a	(25)	(25)	(25)	(25)	(24)	(23)
Absolute value	-7	-3	+6	0	+2	+14
% of body weight	-7	0	+12	+3	+3	+18
Gland, adrenal (Number Weighed)	(25)	(25)	(25)	(25)	(24)	(23)
Absolute value	+1	-3	+11	+7	+2	+22
% of body weight	+1	0	+18	+9	+3	+27
Kidney (Number Weighed)	(25)	(25)	(25)	(25)	(24)	(23)
Absolute value	-2	0	0	-1	-1	+9
% of body weight	-2	+3	+7	+1	0	+14
Thymus (Number Weighed)	(25)	(25)	(25)	(25)	(24)	(23)
Absolute value	+4	-8	-8	+2	-5	-22
% of body weight	+4	-5	-2	+5	-4	-20
Ovary (Number Weighed)	NA	NA	NA	(25)	(24)	(23)
Absolute value				-5	-8	-13
% of body weight				-2	-7	-9

^a All values expressed as percent difference of control group means.

NA: Not Applicable.

Based upon statistical analysis of group means, values highlighted in bold are significantly different from control group – P ≤ 0.05.

 

8) F0 – Histopathological findings: non-neoplastic

Table 30: Summary of microscopic findings – scheduled euthanasia (F0)^a Numbers in parentheses represent the number of animals with the finding.

	Males				Females
Group No.	1	2	3	4	1	2	3	4
Dose (mg/kg/day)	0	25	75	225	0	25	75	225
Number of Animals per Group	25	25	25	25	25	25	25	25
Kidney (Number Examined)	25	0	0	25	23	25	25	23
Mineralization, tubular, corticomedullary	(0)^a	(0)	(0)	(0)	(0)	(0)	(1)	(8)
Minimal	0	0	0	0	0	0	1	4
Mild	0	0	0	0	0	0	0	4
Dilatation, tubular	(0)	(0)	(0)	(1)	(0)	(0)	(1)	(10)
Minimal	0	0	0	1	0	0	1	6
Mild	0	0	0	0	0	0	0	4
Heart (Number Examined)	25	0	0	25	23	25	25	23
Mineralization, aortic	(0)	(0)	(0)	(0)	(0)	(0)	(1)	(4)
Minimal	0	0	0	0	0	0	1	2
Mild	0	0	0	0	0	0	0	2
Mineralization, myocardial	(0)	(0)	(0)	(0)	(0)	(0)	(0)	(2)
Minimal	0	0	0	0	0	0	0	1
Mild	0	0	0	0	0	0	0	1
Stomach (Number Examined)	25	0	1	25	23	25	25	23
Mineralization, glandular part	(0)	(0)	(0)	(0)	(0)	(0)	(2)	(4)
Minimal	0	0	0	0	0	0	2	3
Mild	0	0	0	0	0	0	0	1
Necrosis, glandular part	(0)	(0)	(0)	(0)	(0)	(0)	(0)	(2)
Minimal	0	0	0	0	0	0	0	1
Mild	0	0	0	0	0	0	0	1
Ovary (Number Examined)	NA	NA	NA	NA	23	25	25	23
Atrophy					(0)	(0)	(0)	(5)
Minimal					0	0	0	5
Vagina (Number Examined)					23	25	25	23
Mucification, epithelial					(2)	(4)	(2)	(12)
Minimal					1	3	1	3
Mild					1	1	1	7
Moderate					0	0	0	2
Thymus (Number Examined)	25	0	0	25	23	25	25	23
Atrophy	(0)			(0)	(0)	(0)	(0)	(3)
Minimal	0			0	0	0	0	3
Apoptosis, increased, cortical	(0)			(0)	(0)	(0)	(0)	(4)
Mild	0	0	0	0	0	0	0	4

NA: Not Applicable.

 

Table 31: F0 - mean estrous cycle data – Pre-mating period

Parameter	Cycle length (days)	Irregularity index	Percentage of estrus days	Percentage of females acyclic or with acyclic period
Group 1: 0 mg/kg/day MEAN	4.1	0.1	24.0	20
SD	0.2	0.2	4.0
N	20	20	20
Group 2: 25 mg/kg/day MEAN	4.0	0.2	26.7	16
SD	0.2	0.2	4.7
N	21	21	21
Group 3: 75 mg/kg/day MEAN	4.0	0.1	27.2*	8
SD	0.2	0.2	4.0
N	23	23	23
Group 4: 225 mg/kg/day MEAN	4.0	0.1	27.9**	16
SD	0.2	0.2	4.0
N	21	21	21

* p ≤ 0.05.

** p ≤ 0.01.

 

9) F0 – Reproduction performance

Table 32: F0 Summary of Mating Performance and Fertility

GROUP	1	2	3	4
Dose Level	0 mg/kg/day	25 mg/kg/day	75 mg/kg/day	225 mg/kg/day
Number of females :
Paired	25	25	25	25
Inseminated	25	25	25	25
Not pregnant	2	0	0	1
Total Litter Resorption	0	0	0	1
Mistimed Pregnancy	0	1	0	0
Pregnant	23	25	25	24
Females with live pups	23	25	25	23
Pre Coital interval :
MEAN	2.8	2.7	3.0	2.7
S.D.	1.3	1.1	1.2	0.9
N	25	24	25	25
COPULATION INDEX (%)	100	100	100	100
FERTILITY INDEX (%)	92	100*	100*	96

*p ≤ 0.05.

 

Table 33: F0 Summary of Delivery and litter data

Sex: Female		0 mg/kg/day	25 mg/kg/day	75 mg/kg/day	225 mg/kg/day
Females Completing Delivery [CHSQFS]	N+ve	23	25	25	23
with Liveborn Pups [CHSQFS]	N+ve	23	25	25	23
with Stillborn Pups [CHSQFS]	N+ve	1	1	1	3
with all Stillborn Pups [CHSQFS]	N+ve	0	0	0	0
with all Dead PND 21 [CHSQFS]	N+ve	0	0	0	0
Gestation Length (Days) [GEN AN]	Mean	22.2     R¹	22.4	22.3	22.1
Number of Implantation Sites [GEN AN]	Mean	13.8     R¹	13.3	13.3	13.3
Pre-Birth Loss (%) [GEN AN]	Mean	8.33   R¹	5.93	7.73	7.94
Pups Delivered/Litter [GEN AN]	Mean	12.7     R¹	12.5	12.3	12.2
Live Pups PND 0 [GEN AN]	Mean	12.5     R¹	12.4	12.2	12.0
Live Pups PND 1 [GEN AN]	Mean	12.3     R¹	12.3	12.2	11.5
Live Pups Precull [GEN AN]	Mean	12.3     R¹	12.2	12.0	11.5
Live Pups Postcull [GEN AN]	Mean	7.9     R¹	7.9	7.9	7.6
Live Pups PND 7 [GEN AN]	Mean	7.9     R¹	7.9	7.9	7.6
Live Pups PND 14 [GEN AN]	Mean	7.9     R¹	7.9	7.9	7.6
Live Pups PND 21 [GEN AN]	Mean	7.9     R¹	7.9	7.9	7.6
Dead, Miss., Cannib. PND 0 [CHSQFS]	Sum	4	3	2	5
Dead, Miss., Cannib. PND 1-4 [CHSQFS]	Sum	4	4	5	10
Dead, Miss., Cannib. PND 5-21 [CHSQFS]	Sum	0	0	1	0
Dead, Miss., Cannib. PND 0-21 [CHSQFS]	Sum	8	7	8	15
Live Birth Index (%)	-	98.6	99.0	99.4	98.2
Viability Index (PND 0-4) (%)	-	98.6	98.7	98.4	96.4
Weaning Index (PND 4-21) (%)	-	100.0	100.0	99.5	100.0
Sex Ratio PND 1 - % Males [CHSQFS]	Mean	51.7	47.7	48.2	48.2
Sex Ratio PND 21 - %  Males [CHSQFS]	Mean	48.0	49.1	48.5	51.3

[CHSQFS] - Chi-Squared & Fisher's Exact.

[GEN AN] - Generalised Anova/Ancova Test.

¹ R - Automatic Transformation: Rank.

N+ve : number of positive.

 

10) F1- Body weight and weight changes

Table 34: Mean F1 Pup Body Weights at 225 mg/kg/day vs. Historical Control Data (HCD) Mean or Control Group

	HCD	Control	Group 4
		0 mg/kg/day	225 mg/kg/day
	Pup Wt (g)	Pup Wt (g)	Pup Wt (g)	Group 4 vs. HCD/Control Mean (%)
Males D1	7.0	6.8	6.7	-4/-1
Males D21	54.2	56.9	52.3	-4/-8
Females D1	6.7	6.5	6.3	-6/-3
Females D21	52.6	55.1	50.6	-4/-8

Wt: Weight.

D: Day.

HCD : Historical Control data.

 

Table 35: Mean body weight (g) – F1 post weaning Males cohorts 1A, 1B, 2A and 3

Day(s) Relative to Start Date	0 mg/kg/day	25 mg/kg/day	75 mg/kg/day	225 mg/kg/day
1	67.80 L^1	67.79	67.20	59.46 www^5
8	111.29 I^2	109.99	107.44 w^4	88.90 www^5
15	161.31 R^3	160.76	157.84	136.62 SSS^6
22	205.09 I^2	203.66	201.97	182.98 www^5
28	236.57 I^2	234.23	238.36	218.42 w^4
29	247.22 R^3	248.30	244.96	224.63 SSS^6
36	284.80 R^3	285.96	282.87	264.43 SSS^6
43	311.24 R^3	313.26	308.72	289.62 SSS^6
50	333.36 R^3	336.58	334.13	308.57 SSS^6
56	350.30 I^2	345.79	346.92	333.20
57	349.54 I^2	357.64	348.42	317.31 www^5
64	366.10 I^2	370.84	362.08	328.98 www^5
70	377.34 I^2	380.17	372.87	337.39 www^5

¹ L - Automatic Transformation: Log.

² I - Automatic Transformation: Identity (No Transformation).

³ R - Automatic Transformation: Rank.

⁴ w - Test: Williams 2 Sided p < 0.05.

⁵ www - Test: Williams 2 Sided p < 0.001.

⁶ SSS - Test: Shirley 2 Sided p < 0.001.

 

Table 36: Mean body weight (g) – F1 Females post weaning cohorts 1A, 1B, 2A and 3

Day(s) Relative to Start Date	0 mg/kg/day	25 mg/kg/day	75 mg/kg/day	225 mg/kg/day
1	65.84 L^1	64.18	63.92 w^6	57.36 www^7
5	-	-	-	57.70
8	101.57 R^2	99.79	99.59	81.69 SSS^8
15	134.18 I^3	131.67	134.06	118.07 www^7
22	157.39 I,a^4	151.72 d^5	156.33	145.71 www^7
28	172.75 I^3	167.84	166.42	158.64 w^6
29	177.82 I^3	171.58	176.52	164.39 www^7
36	196.60 I^3	189.89 w^6	192.83 w^6	181.23 www^7
43	206.93 I^3	200.16	204.28	192.79 www^7
50	214.30 I^3	211.18	214.44	204.83 ww^9
56	229.40 I^3	218.40	223.84	210.55
57	225.20 I^3	222.43	225.50	214.32 ww^9
64	232.07 I^3	227.86	230.91	221.38 ww^9
70	234.05 I^3	230.18	235.26	225.48

¹ L - Automatic Transformation: Log.

² R - Automatic Transformation: Rank.

³ I - Automatic Transformation: Identity (No Transformation).

⁴ I,a - Automatic Transformation: Identity (No Transformation), (All Groups) Test: Analysis of Variance p < 0.

⁵ d - Test: Dunnett 2 Sided p < 0.05.

⁶ w - Test: Williams 2 Sided p < 0.05.

⁷ www - Test: Williams 2 Sided p < 0.001.

⁸ SSS - Test: Shirley 2 Sided p < 0.001.

⁹ ww - Test: Williams 2 Sided p < 0.01.

 

 

11) F1- Food consumption

Table 37: Mean food consumption (g/animal/day) – F1 post weaning Males cohorts 1A, 1B, 2A and 3

Day(s) Relative to Start Date	0 mg/kg/day	25 mg/kg/day	75 mg/kg/day	225 mg/kg/day
1 -> 8	15.32 R,kk ^1	16.09 ddd^3	15.51	11.80 SSS^4
8 -> 15	20.57 R^2	20.62	20.56	17.24 SSS^4
15 -> 22	22.75 R^2	22.82	22.94	22.02 SSS^4
22 -> 28	23.08 R^2	23.13	23.20	22.34 S^5
22 -> 29	24.45 R,k^6	24.44	24.28	23.79
1 -> 29	20.78 R^2	21.09	20.81	18.79 SSS^4
29 -> 36	26.02 R,k^6	26.58 d^7	26.35 d^7	25.50 S^5
36 -> 43	25.24 R,kk^1	26.08 dd^8	25.54	24.57 SS^9
43 -> 50	25.57 R^2	25.71	25.71	24.25 SSS^4
50 -> 57	25.34 R^2	25.67	25.57	23.75 SSS^4
1 -> 57	23.09 I^2	23.50	23.40	21.45 SSS^4
57 -> 64	24.60 R,kk^1	25.43 d^7	25.46 d^7	23.42 SSS^4
1 -> 64	23.26 R^2	23.72	23.63	21.67 SSS^4
64 -> 70	24.75 R^2	24.12	24.82	23.01 SSS^4
1 -> 70	23.79 R^2	23.72	23.92	21.82 SSS^4

¹ R,kk - Automatic Transformation: Rank, (All Groups) Test: Kruskal-Wallis p < 0.01.

² R - Automatic Transformation: Rank.

³ ddd - Test: Dunnett Non-Parametric 2 Sided p < 0.001.

⁴ SSS - Test: Shirley 2 Sided p < 0.001.

⁵S - Test: Shirley 2 Sided p < 0.05.

⁶ R,k - Automatic Transformation: Rank, (All Groups) Test: Kruskal-Wallis p < 0.05.

⁷ d - Test: Dunnett Non-Parametric 2 Sided p < 0.05.

⁸ dd - Test: Dunnett Non-Parametric 2 Sided p < 0.01.

⁹ SS - Test: Shirley 2 Sided p < 0.01.

 

Table 38: Mean food consumption (g/animal/day) – F1 post weaning Females cohorts 1A, 1B, 2A and 3

Day(s) Relative to Start Date	0 mg/kg/day	25 mg/kg/day	75 mg/kg/day	225 mg/kg/day
1 -> 8	13.22 R,k ^1	12.88 d^4	13.20	9.85 SSS^7
8 -> 15	15.58 R,k^1	15.64	15.89 dd^5	14.18 SSS^7
15 -> 22	16.89 R,kk^2	16.52dd^5	17.38 S^6	16.33 S^6
22 -> 28	16.10 R^3	15.95	15.29	15.67
22 -> 29	17.11 R^3	16.97	17.19	16.94
1 -> 29	15.68 R,kk^2	15.42	15.94	14.55 SSS^7
29 -> 36	18.22 R^3	18.05	18.48	17.76 SS^8
36 -> 43	17.93 R^3	17.99	18.08	17.63 S^6
43 -> 50	18.09 R,kk^2	19.89 dd^5	18.42	18.06
50 -> 57	18.21 R^3	18.30	18.93	18.28
1 -> 57	16.84 R^3	16.92	17.11	16.21 SSS^7
57 -> 64	18.35 R^3	18.77	19.11 S^6	18.82 SS^8
1 -> 64	17.00 R^3	17.10	17.34	16.50 SSS^7
64 -> 70	18.18 R,kkk^9	17.41 ddd^10	18.65	17.96
1 -> 70	17.27 R,kk^2	17.09	17.64 d^4	16.52 SSS^7

¹ R,k - Automatic Transformation: Rank, (All Groups) Test: Kruskal-Wallis p < 0.05.

² R,kk - Automatic Transformation: Rank, (All Groups) Test: Kruskal-Wallis p < 0.01.

³ R - Automatic Transformation: Rank.

⁴ d - Test: Dunnett Non-Parametric 2 Sided p < 0.05.

⁵ dd - Test: Dunnett Non-Parametric 2 Sided p < 0.01.

⁶S - Test: Shirley 2 Sided p < 0.05.

⁷ SSS - Test: Shirley 2 Sided p < 0.001.

⁸ SS - Test: Shirley 2 Sided p < 0.01.

⁹ R,kkk - Automatic Transformation: Rank, (All Groups) Test: Kruskal-Wallis p < 0.001.

¹⁰ ddd - Test: Dunnett Non-Parametric 2 Sided p < 0.001.

 

12) F1- Haematological findings

Table 39: F1 post weaning - cohort 1A - Toxicological Relevant Changes in Haematology between 225 mg/kg/day and Control Groups

	Males	Females
MCV (fL)	X1.03	-
MCH (pg)	X1.04	X1.04
Reti. (Giga/L)	X1.15	-
Reti. (%)	X1.17	-
WBC (Giga/L)	X0.76	-
Lymphocytes (Giga/L)	X0.73	-
Monocytes (Giga/L)	X0.57	-
Basophils (Giga/L)	X0.38	-
RBC (T/L)	-	X0.93
Hb (g/L)	-	X0.97
HCT (L/L)	-	X0.95

-: Absence of change.

Numerical values indicate fold changes of treated group value relative to control group mean value.

MCV: Mean corpuscular volume.

MCH: Mean corpuscular haemoglobin.

Reti. : Reticulocytes.

WBC: White blood cells.

RBC: Red blood cells.

Hb: Hemoglobin.

HCT: Hematocrit.

 

12) F1 - Clinical biochemistry findings

Table 40: F1 males post weaning - cohort 1A - Group Mean Serum Clinical Chemistry Parameters – extract of results

Group	Dose level mg/kg/day	Na mmol/L	Chol. mmol/L	Glob. g/L	ALP BC IU/L
1	0	141.9	1.456	28.94	110.2
2	25	142.5	1.611	28.23	97.1
3	75	141.6	1.336	29.39	101.9
4	225	142.8*	0.712**	27.08**	135.7*

Na: sodium.

Chol: cholesterol.

Glob: globulin.

ALP BC: Alkaline phosphatase.

*p ≤ 0.05.

**p ≤ 0.01.

Table 41: F1 females post weaning - cohort 1A - Group Mean Serum Clinical Chemistry Parameters – extract of results

Group	Dose level mg/kg/day	Na mmol/L	ALP BC IU/L
1	0	142.4	58.8
2	25	142.2	60.3
3	75	143.0	57.4
4	225	143.6**	79.5**

Na: sodium.

ALP BC: Alkaline phosphatase.

**p ≤ 0.01.

 

13) F1 – Sexual maturation

Table 42: F1 females - cohorts 1A, 1B, 2A and 3 – Mean sexual maturation data – extract of results

	0 mg/kg/day	25 mg/kg/day	75 mg/kg/day	225 mg/kg/day
Day of vaginal opening (Days)	34.4 R^1	34.0	34.5	37.1 SSS^2
Body weight (g)	113.71 R^1	113.44	116.65	111.82

¹ R - Automatic Transformation: Rank.

² SSS - Test: Shirley 2 Sided p < 0.001.

 

 

14) F1- Anogenital distance

Table 43: F1 animals - Mean Pup Anogenital Distance (normalized to the cube root of body weight)

Dose level	Value	Males	Females
0 mg/kg/day	Mean SD N	1.8 0.2 23	1.0 0.1 23
25 mg/kg/day	Mean SD N	1.9 0.2 25	1.0 0.2 25
75 mg/kg/day	Mean SD N	1.9 0.1 25	0.9 0.1 25
225 mg/kg/day	Mean SD N	1.9 0.2 23	0.9 0.1 22

 

 

15) F1- organ weight findings

Table 44: F1 cohort 1A Summary of Organ Weight Data - Scheduled Euthanasia

	Males			Females
Group No.	2	3	4	2	3	4
Dose (mg/kg/day)	25	75	225	25	75	225
Number of Animals per Group	20	20	20	19	20	20
Kidney (Number Weighed)^a	(20)	(20)	(20)	(19)	(20)	(20)
Absolute value	+6	+8	+14	+1	+1	+12
% of body weight	+4	+9	+27	+2	+2	+19

^a All values expressed as percent difference of control group means.

Based upon statistical analysis of group means, values highlighted in bold are significantly different from control group - p ≤ 0.05.

 

Table 45: F1 cohort 1B Summary of Organ Weight Data - Scheduled Euthanasia

	Males			Females
Group No.	2	3	4	2	3	4
Dose (mg/kg/day)	25	75	225	25	75	225
Number of Animals per Group	20	20	20	20	20	20
Kidney (Number Weighed)^a	(10)	(10)	(10)	(10)	(10)	(10)
Absolute value	0	+3	+11	-3	+2	+18
% of body weight	-1	+4	+24	-1	+2	+23

^a All values expressed as percent difference of control group means.

Based upon statistical analysis of group means, values highlighted in bold are significantly different from control group - p ≤ 0.05.

 

16) F1 gross pathology findings

Table 46: F1 cohort 1A - Summary of Gross Pathology Findings – Scheduled Euthanasia

Sex	Males				Females
Group No.	1	2	3	4	1	2	3	4
Dose (mg/kg/day)	0	25	75	225	0	25	75	225
Number of Animals per Group	20	20	20	20	20	19	20	20
Kidney (Number Examined)	20	20	20	20	20	19	20	20
Granular appearance	1	0	0	2	0	0	0	0
Enlarged	0	0	0	1	0	0	0	0

 

 

Table 47: F1 cohort 1B - Summary of Gross Pathology Findings – Scheduled Euthanasia

Sex	Males				Females
Group No.	1	2	3	4	1	2	3	4
Dose (mg/kg/day)	0	25	75	225	0	25	75	225
Number of Animals per Group	20	20	20	20	20	20	20	20
Kidney (Number Examined)	0	0	0	6	2	0	3	13
Enlarged (right and/or left)	0	0	0	5	1	0	3	13

 

17) F1 histopathology findings

Table 48: F1 cohort 1A - Summary of Microscopic Findings - Scheduled Euthanasia

Sex	Males				Females
Group No.	1	2	3	4	1	2	3	4
Dose (mg/kg/day)	0	25	75	225	0	25	75	225
Number of Animals per Group	20	20	20	20	20	19	20	20
Kidney (Number Examined)	20	20	20	20	20	19	20	20
Basophilia, tubular	(3)^a	(6)	(7)	(20)	(1)	(0)	(1)	(17)
- Minimal	3	6	7	2	1	0	1	8
- Mild	0	0	0	14	0	0	0	9
- Moderate	0	0	0	4	0	0	0	0
Dilatation, tubular	(0)	(0)	(0)	(16)	(0)	(0)	(0)	(8)
- Minimal	0	0	0	11	0	0	0	7
- Mild	0	0	0	4	0	0	0	1
- Moderate	0	0	0	1	0	0	0	0
Inflammatory cell infiltration, chronic	(1)	(0)	(0)	(12)	(0)	(0)	(0)	(3)
- Minimal	1	0	0	10	0	0	0	3
- Mild	0	0	0	2	0	0	0	0
Spleen (Number Examined)	20	20	20	19	20	19	20	20
Extramedullary hematopoiesis, increased	(4)	(3)	(3)	(10)	(2)	(0)	(1)	(6)
- Minimal	4	3	3	10	2	0	1	6

^a Numbers in parentheses represent the number of animals with the finding.

 

18) F1 – Behaviour (functional findings)

Auditory startle reflex

Table 49: F1 - cohort 2A males - Group Mean Auditory Startle Reflex – Habituation

 

	Block No. 1	Block No. 2		Block No. 3		Block No. 4		Block No. 5
Group	Mean	Mean	% diff. vs B1*	Mean	% diff. vs B1*	Mean	% diff. vs B1*	Mean	% diff. vs B1*
Group 1	0.1957	0.1764	-9.9%	0.1830	-6.7%	0.1536	-21.7%	0.1542	-21.0%
Group 2	0.2147	0.1744	-18.1%	0.1741	-17.3%	0.1777	-15.4%	0.1543	-26.6%
Group 3	0.1740	0.1541	-9.3%	0.1426	-16.2%	0.1378	-19.5%	0.1352	-21.0%
Group 4	0.1750	0.1540	-10.3%	0.1415	-18.6%	0.1396	-17.8%	0.1225	-28.1%

* % difference versus Block 1.

Group 1: 0 mg/kg/day – Group 2: 25 mg/kg/day - Group 3: 75 mg/kg/day - Group 4: 225 mg/kg/day.

 

 

Table 50: F1 - cohort 2A females - Group Mean Auditory Startle Reflex – Habituation

	Block No. 1	Block No. 2		Block No. 3		Block No. 4		Block No. 5
Group	Mean	Mean	% diff. vs B1*	Mean	% diff. vs B1*	Mean	% diff. vs B1*	Mean	% diff. vs B1*
Group 1	0.2177	0.1786	-16.4%	0.1729	-18.9%	0.1749	-19.3%	0.1635	-23.5%
Group 2	0.2184	0.1991	-6.9%	0.1895	-11.7%	0.1848	-13.1%	0.1801	-15.4%
Group 3	0.1847	0.1820	-1.0%	0.1650	-9.3%	0.1620	-10.6%	0.1452	-19.7%
Group 4	0.1714	0.1395	-18.3%	0.1439	-15.0%	0.1395	-16.4%	0.1329	-21.4%

* % difference versus Block 1.

Group 1: 0 mg/kg/day – Group 2: 25 mg/kg/day - Group 3: 75 mg/kg/day - Group 4: 225 mg/kg/day.

 

Functional Observation Battery (FOB)

Table 51: F1 - cohort 2A males – summary of neurobehavioral evaluation – PND 63-75

Group	1	2	3	4
	Mean	Mean	Mean	Mean
Home cage observations
Posture/Body carriage	/	/	/	/
Convulsions	/	/	/	/
Stereotypy	/	/	/	/
Tremors	/	/	/	/
Palpebral closure/Ptosis	/	/	/	/
Handling
Ease of removal	/	/	/	/
Handling reactivity	/	/	/	/
Op. field observations
Rearing	8.4	6.5	6.8	6.0
Arousal/alertness	/	/	/	/
Gait/Mobility	/	/	/	/
Vocalizations	/	/	/	/
Tremors	/	/	/	/
Respiration	/	/	/	/
Defecation	-0.9	-0.8	-0.6	-1.0
Stereotypy	/	/	/	/
Convulsions	/	/	/	/
Appearance	/	/	/	/
Salivation	/	/	/	/
Lacrimation	/	/	/	/
Palpebral closure/Ptosis	/	/	/	/
Exophtalmos	/	/	/	/
Erected fur (piloerection)	/	/	/	/
Touch response/tactile reflex	/	/	/	/
Startle response	/	/	/	/
Tail pinch response	-0.2	-0.1	0.1	/
Pupil response	/	/	/	/
Body temperature (°C)	37.0	37.0	36.9	37.2
Body tone	/	/	/	/
Grip strength	427.1	556.0	569.2	540.8
Air righting reflex	/	/	/	/

Group 1: 0 mg/kg/day – Group 2: 25 mg/kg/day - Group 3: 75 mg/kg/day - Group 4: 225 mg/kg/day.

/: Normal observation.

Op. field: open field.

 

 

Table 52: F1 - cohort 2A females – summary of neurobehavioral evaluation – PND 63-75

Group	1	2	3	4
	Mean	Mean	Mean	Mean
Home cage observations
Posture/Body carriage	/	/	/	/
Convulsions	/	/	/	/
Stereotypy	/	/	/	/
Tremors	/	/	/	/
Palpebral closure/Ptosis	/	/	/	/
Handling
Ease of removal	/	/	/	/
Handling reactivity	0.1	/	0.2	0.1
Op. field observations
Rearing	3.8	6.3	8.1	6.0
Arousal/alertness	0.2	0.1	/	/
Gait/Mobility	/	/	/	/
Vocalizations	/	/	/	/
Tremors	0.1	/	/	/
Respiration	/	/	/	/
Defecation	-1.0	-1.0	-1.0	-1.0
Stereotypy	/	/	/	/
Convulsions	/	/	/	/
Appearance	/	/	/	/
Salivation	/	/	/	0.1
Lacrimation	/	/	/	/
Palpebral closure/Ptosis	/	/	/	/
Exophtalmos	/	/	/	/
Erected fur (piloerection)	/	/	/	/
Touch response/tactile reflex	/	/	/	0.1
Startle response	/	/	/	0.1
Tail pinch response	-0.1	/	-0.1	-0.1
Pupil response	/	/	/	/
Body temperature (°C)	37.7	37.8	37.9	38.0
Body tone	/	/	/	/
Grip strength	463.6	512.4	521.7	587.7
Air righting reflex	/	/	/	/

Group 1: 0 mg/kg/day – Group 2: 25 mg/kg/day - Group 3: 75 mg/kg/day - Group 4: 225 mg/kg/day.

/: Normal observation.

Op. field: open field.

 

 

 

Table 53: F1 - cohort 2A – Motor activity - % of Habituation at 225 mg/kg/day versus HCD range

	HCD Range					225 mg/kg/day
Intervals	2	3	4	5	6	2	3	4	5	6
Male Fine Movements	-27 to -42	-41 to -59	-47 to -59	-57 to -73	-62 to -65	-49	-87	-95	-90	-81
Female Fine Movements	-30 to -41	-39 to -55	-35 to -66	-48 to -72	-57 to -77	-60	-81	-85	-87	-94
Male Ambulation	-36 to -64	-45 to -71	-57 to -70	-60 to -83	-65  to -74	-74	-93	-99	-100	-89
Female Ambulation	-39 to -52	-41 to -66	-45 to -78	-56 to -81	-49 to -80	-78	-93	-92	-92	-98

HCD: Historical Control Data.

Bold: % below the HCB range.

 

19) F1 – Anti-KLH IgM Analysis (TDAR) – Cohort 1B

Table 54: F1 - cohort 1B females – summary of Anti-KLH IgM (adjusted to pre-dose)

Time-point	Group 1		Group 2	Group 3	Group 4	Cyclophos-phamide 10 mg/kg/day	Global p-value	Test
1	Mean	0.0	0.0	0.0	0.0	0.0	1.0000	(KW)
	StD	0.00	0.00	0.00	0.00	0.00
	N	10	10	10	10	10
5	Mean	22.5	24.2	23.8	21.2	0.4 ***	0.0001 ***	(KW)   (DR)
	StD	10.62	11.27	11.98	13.10	0.75
	N	10	10	10	10	10

Group 1: 0 mg/kg/day – Group 2: 25 mg/kg/day - Group 3: 75 mg/kg/day - Group 4: 225 mg/kg/day.

(L): Logarithmic transformation; (S): Student; (WMS): Wilcoxon-Mann-Whitney; (A): ANOVA; (KW): Kruskall-Wallis; (D): Dunnett; (DR): Dunnett on Ranks.

*, ** or ***: p-value<0.05, 0.01 or 0.001, respectively.

 

Table 55: F1 - cohort 1B males – summary of Anti-KLH IgM (adjusted to pre-dose)

Time-point	Group 1		Group 2	Group 3	Group 4	Cyclophos-phamide 10 mg/kg/day	Global p-value	Test
1	Mean	0.0	0.0	0.0	0.0	0.0	1.0000	(KW)
	StD	0.00	0.00	0.00	0.00	0.00
	N	10	10	10	10	10
5	Mean	26.3	32.5	19.2	14.6	-0.1***	<0.0001 ***	(KW)   (DR)
	StD	18.16	19.86	14.56	12.62	0.31
	N	10	10	10	10	10

Group 1: 0 mg/kg/day – Group 2: 25 mg/kg/day - Group 3: 75 mg/kg/day - Group 4: 225 mg/kg/day.

(L): Logarithmic transformation; (S): Student; (WMS): Wilcoxon-Mann-Whitney; (A): ANOVA; (KW): Kruskall-Wallis; (D): Dunnett; (DR): Dunnett on Ranks.

*, ** or ***: p-value<0.05, 0.01 or 0.001, respectively.

 

20) F1 – Anti-SRBC IgM Analysis (TDAR) – Cohort 3

Table 56: F1 - cohort 3 males – summary of Anti-SRBC IgM values

	Value	Group 1	Group 2	Group 3	Group 4	Group 5
Pre-Immunization  a-SRBC IgM U/mL	Mean	339	280	316	339	436
	St Dev	134.6	104.3	193.5	138.2	269.0
	N	10	10	10	10	9
Post-Immunization a-SRBC IgM U/mL	Mean	11080	6823	13145	12263	327 ***
	St Dev	5524.4	2203.8	14968.8	8709.9	178.3
	N	10	10	10	10	10

Group 1: 0 mg/kg/day – Group 2: 25 mg/kg/day - Group 3: 75 mg/kg/day - Group 4: 225 mg/kg/day.

Group 5: Positive control animals treated with cyclophosphamide.

***: p-value<0.001

 

Table 57: F1 - cohort 3 females – summary of Anti-SRBC IgM values

	Value	Group 1	Group 2	Group 3	Group 4	Group 5
Pre-Immunization  a-SRBC IgM U/mL	Mean	271	283	287	466	285
	St Dev	83.1	117.8	94.3	313.2	132.7
	N	10	10	10	10	10
Post-Immunization a-SRBC IgM U/mL	Mean	13158	11710	9973	14166	436 ***
	St Dev	8712.2	8519.1	4587.9	8296.1	96.6
	N	10	10	10	10	10

Group 1: 0 mg/kg/day – Group 2: 25 mg/kg/day - Group 3: 75 mg/kg/day - Group 4: 225 mg/kg/day.

Group 5: Positive control animals treated with cyclophosphamide.

***: p-value<0.001

 

21) F1 – Splenic Lymphocyte Subpopulation Analysis - Cohort 1A

Table 58: F1 – Cohort 1A Splenic Lymphocyte Subpopulation Summary - mean values

Dose level	Group 1		Group 2		Group 3		Group 4
Gender	M	F	M	F	M	F	M	F
All leucocytes	82.30	87.17	86.16	84.60	85.79	86.77	86.00	85.11
Viability	88.23	88.34	88.54	88.07	87.05	88.47	87.85	90.23
All T-lymphocytes	21.80	25.94	24.01	23.20	23.54	25.76	21.95	26.01
T-helper lymphocytes	12.62	15.21	13.99	13.14	13.36	14.79	12.58	14.34
T-cytotoxic	8.68	10.06	9.37	9.38	9.63	10.32	8.79	11.00
B-lymphocytes	36.29	30.24	35.15	32.15	33.09	30.04	32.69	32.20
Natural killer cells	3.10	3.65	3.53	3.75	3.19	3.59	3.53	3.06
NKT-lymphocytes	2.10	2.39	2.44	2.42	2.21	2.43	2.42	2.46

Group 1: 0 mg/kg/day – Group 2: 25 mg/kg/day - Group 3: 75 mg/kg/day - Group 4: 225 mg/kg/day.

M: males - F/ Females.

 

Table 59: F1 – Cohort 1A Immunophenotyping Fold Changes to Control Animals (Group 1)

Group	Group 1		Group 2		Group 3		Group 4
Gender	M	F	M	F	M	F	M	F
All leucocytes	1.00	1.00	1.05	0.97	1.04	1.00	1.04	0.98
Viability	1.00	1.00	1.00	1.00	0.99	1.00	1.00	1.02
All T-lymphocytes	1.00	1.00	1.10	0.89	1.08	0.99	1.01	1.00
T-helper lymphocytes	1.00	1.00	1.11	0.86	1.06	0.97	1.00	0.94
T-cytotoxic	1.00	1.00	1.08	0.93	1.11	1.03	1.01	1.09
B-lymphocytes	1.00	1.00	0.97	1.06	0.91	0.99	0.90	1.06
Natural killer cells	1.00	1.00	1.14	1.03	1.03	0.98	1.14	0.84
NKT-lymphocytes	1.00	1.00	1.16	1.01	1.05	1.02	1.15	1.03

Group 1: 0 mg/kg/day – Group 2: 25 mg/kg/day - Group 3: 75 mg/kg/day - Group 4: 225 mg/kg/day.

M: males - F/ Females.

 

22) F1 – Thyroid hormone analysis

Table 60: F1 – pups before weaning - Summary of T4 and TSH values

Animals	Hormone	Group 1 0 mg/kg/day	Group 2 25 mg/kg/day	Group 3 75 mg/kg/day	Group 4 225 mg/kg/day
F1-PND4 Pups Mean	T4 µg/dL	1.11	1.52**	1.09	1.44*
F1-PND21 Male pups Mean	T4 µg/dL	4.52	4.47	4.68	4.52
	TSH µIU/mL	0.043	0.072*	0.063**	0.077**
F1-PND21 Female pups Mean	T4 µg/dL	4.62	4.18	4.67	4.93
	TSH µIU/mL	0.051	0.081	0.054	0.071

*p ≤ 0.05.

**p ≤ 0.01.

 

Table 61: F1 cohort 1A – post weaning - Summary of T4 and TSH values

Animals	Hormone	Group 1 0 mg/kg/day	Group 2 25 mg/kg/day	Group 3 75 mg/kg/day	Group 4 225 mg/kg/day
F1-cohort 1A males Mean	T4 µg/dL	7.00	6.50	6.95	4.79***
	TSH µIU/mL	0.071	0.098	0.100	0.112
F1-cohort 1A females Mean	T4 µg/dL	4.01	3.57	4.19	4.70
	TSH µIU/mL	0.075	0.054	0.053	0.098

***p ≤ 0.001.

 

21) F1 – Cohort 1A – Estrous cycle data

Table 62: F1 cohort 1A – Mean time between the day of vaginal opening and the first estrus

Dose level	Value	Time between the day of vaginal opening and the first estrous (days)
Group 1: 0 mg/kg/day	MEAN	3.2
	SD	1.8
	N	20
Group 2: 25 mg/kg/day	MEAN	2.9
	SD	2.1
	N	19
Group 3: 75 mg/kg/day	MEAN	2.7
	SD	1.7
	N	20
Group 4: 225 mg/kg/day	MEAN	2.6
	SD	1.8
	N	20

 

 

Conclusions:

In conclusion, based on the results of this extended one generation reproductive toxicity study (including Cohorts 1, 2 and 3), the following No Observed Adverse Effect Levels (NOAELs) were established for the substance:

- General Toxicity (F0): At least 75 mg/kg/day (based on adverse pathology findings including renal tubular dilatation and mineralization correlated with higher kidney weight, myocardial or aortic mineralization in the heart, mineralization and/or necrosis of the glandular stomach at 225 mg/kg/day for females).

- Reproductive Toxicity (F0): At least 225 mg/kg/day.

- General Toxicity (F1): At least 225 mg/kg/day.

- Developmental Toxicity (F1): At least 225 mg/kg/day.

Executive summary:

This GLP Study following the OECD guideline 443 was carried out to provide an evaluation of the pre and postnatal effects of the test material, on development as well as a thorough evaluation of systemic toxicity in adult male and pregnant and lactating female Wistar Han rats and their offspring.

Detailed examination of key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, and physical and functional development until adulthood, was expected to identify specific target organs in the offspring.

In addition, the study provided and/or confirmed information about the effects of the test material on the integrity and performance of the adult male and female reproductive systems.

Specifically, but not exclusively, the following parameters were considered: gonadal function, estrous cyclicity, epididymal sperm maturation, mating behaviour, fertility, pregnancy, parturition, and lactation. Developmental neurotoxicity and developmental immunotoxicity assessments were also performed to characterize potential effects on those systems.

 

STUDY DESIGN

The test material was administered by daily oral gavage at dose levels of 25, 75 and 225 mg/kg/day to groups of 25 males and 25 females Wistar rats (F0 generation). F0 males were dosed for 10 weeks prior to mating, during mating and up to the day before necropsy. F0 females were dosed during 10 weeks prior to mating, during mating, gestation and lactation and up to the day before necropsy, i.e., on Lactation Day (LD) 21 to LD23 for females that delivered. A fourth group of 25 rats/sex received the same dose volume (5 mL/kg/day) of the control material (0.5% CMC in purified water). The inseminated females were allowed to litter, and development of the offspring was observed up to weaning on Postnatal Day (PND) 21.

The following parameters and endpoints were evaluated in all F0 animals: mortality, clinical signs (including arena observations), body weights, body weight changes, food consumption, clinical pathology, thyroid hormone levels, estrous cycles, mating performance and fertility, delivery and litter data, sperm quality, macroscopic findings, organ weights and histology findings. F0-males were euthanised after at least 12 weeks of exposure and F0 females after at least 16 weeks of dosing.

The following parameters and endpoints were evaluated in all F1 offspring until weaning (PND21): mortality, clinical signs, body weights, number of pups, pup sex ratio and anogenital distance and areola/nipple retention for male pups.

From weaning, the selected F1-pups were allocated to 5 cohorts (1A, 1B, 2A, 2B and 3) of 20 males and 20 females for the first 2 cohorts, and 10 males and 10 females for the last 3 cohorts. All F1-animals, except Cohort 2B and F1 surplus animals, were dosed by daily oral gavage (once a day) from PND21 up to the day before necropsy at the same dose levels of 25, 75 and 225 mg/kg/day. A fourth group received the same dose volume (5 mL/kg/day) of the control material (0.5% CMC in purified water). Selected F1 animals (with the exception of Cohort 2B) were observed for mortality, clinical signs (including arena observations), body weights, body weight changes, food consumption, clinical pathology (Cohort 1A), thyroid hormone levels (PND4 pups, surplus pups and Cohort 1A), sexual maturation, estrous cycles (Cohort 1A), neurobehavioral tests (Cohort 2A), macroscopic findings, organ weights and histology findings. Additionally, spleen samples were taken for splenic lymphocyte subpopulation analysis from 10 rats/sex/group from F1 Cohort 1A animals. Humoral response was also evaluated following SRBC administration for Cohort 3 animals and KLH administration for Cohort 1B animals (TDAR assays). F1 animals from Cohort 2B were not dosed directly and were necropsied on/before PND24 for neuro-histopathology and morphometric analysis.

F1 animals from Cohort 3 were sacrificed between PND54 and PND57 or at approximately 12 weeks of age for positive controls. F1 animals from Cohort 2A were sacrificed after completion of the neurobehavioral tests between PND76 and PND90. F1 animals from Cohort 1A were sacrificed after clinical pathology evaluation between PND89 and PND99. F1 animals from Cohort 1B were sacrificed between PND92 and PND100.

Surplus offspring (10/sex/group) not included in any of the F1 cohorts were selected for thyroid hormone analysis and organ weights on PND21.

 

RESULTS

All formulations at 5, 15 and 45 mg/mL used on Day 1, and in Week 11 and Week 26 of treatment were in agreement with acceptance criteria. Homogeneity was confirmed from the formulations used on Day 1. No test material was detected in the vehicle samples.

 

F0 generation:

There was no test material-related death in any group.

There was no test material-related clinical sign in any group.

There was no test material-related effect in any group on mean body weight gain or food consumption for either sex that was considered toxicologically significant.

There was no test material-related effect in any group on estrous cyclicity, mating performance (including copulation and pre-coital interval), fertility (including sperm quality), and gestation length.

Higher creatinine and urea concentrations, minor ionic unbalance for females and higher hepatic enzymes and triglyceride concentrations were noted for both sexes at 225 mg/kg/day compared with the control group and correlated with the pathology findings noted for the kidneys and liver.

There were no test material-related effects on T4 and TSH levels that were toxicologically significant in any group for either sex. Changes in hormone serum levels for F0 females and F1 males at 225 mg/kg/day were noted but considered not toxicologically relevant in the absence of any gender trend, any test item‑related effect on the thyroid weight, anogenital distance, nipple areola, reproduction and development, any histology findings in the thyroid glands, or similar findings for pups. They are considered related to the high variability of this parameter. As under the conditions of this study no adverse effect was observed that could be linked to the lower T4 and higher TSH values,  this was not taken into account to determine the No Observed Adverse Effect Levels (NOAELs).

At necropsy, there were test material-related findings for females dosed at 225 mg/kg/day in the kidney (minimal to mild tubular dilatation and mineralization, correlating with higher kidney weight), heart (minimal to mild myocardial or aortic mineralization), stomach (minimal to mild mineralization and/or necrosis of the glandular part, grossly correlating with dark foci), thymus (minimal atrophy and mild increased cortical apoptosis, correlating with lower organ weight when compared with the control group). Mineralization in the kidney, heart and stomach was considered adverse, while other findings were considered secondary to test material-related systemic effects (ovary, adrenal gland and thymus), or adaptative changes (liver). The pathology findings noted on the kidneys were correlated with higher creatinine and urea concentrations and minor ionic unbalance. The higher mean liver weight at 225 mg/kg/day for both sexes correlated with slightly higher mean tryglyceride and hepatic enzyme concentrations when compared with the control group. Ovarian atrophy, lower ovaries weight when compared with the control group and vaginal mucification which suggested a delay in the post-gestation recovery of estrous cycle were noted at necropsy at 225 mg/kg/day and were considered probably secondary to test material-related systemic effects noted in kidneys, stomach and heart. These findings were not correlated with any test material-related effect on fertility or estrous cycle data (F0 and F1 generation) and no test material-related effect on the mean count of primordial/small growing follicles (Cohort 1A) and was therefore considered not adverse. In addition, there was no similar finding in the F1 generation.

 

F1 generation:

There was no test material-related effect on postnatal survival of the F1 offspring in any group. Mean live litter size was comparable in all groups between birth and weaning.

There were no test material-related effects that were considered toxicologically significant on clinical condition, body weight and anogenital distance of the pups, or any evidence of areolae/nipple retention for the male offspring at any dose level.

There were no test material-related effects on T4 and TSH levels for the F1-pups on PND4 and PND21.

There was no test material-related effect on the mean age at attainment of balanopreputial cleavage and vaginal opening or on the duration from vaginal opening to first estrus in any group.

There was no test material-related clinical sign in any group for either sex.

There was a test material-related slightly lower mean body weight gain and reduced mean food consumption throughout the dosing period for males at 225 mg/kg/day compared with the control group leading to slightly, lower mean terminal body weight compared with the control group considered not adverse due to the low magnitude of the change and in the absence of any developmental effects.

There were no test material-related effects on the hematology, coagulation, serum clinical chemistry, urinary parameters or on the thyroid hormone serum levels that were considered toxicologically significant at any dose for either sex (Cohort 1A).

There were no test material-related effects on estrous cyclicity or on sperm quality in any group (Cohort 1A).

There was no test material-related immunotoxicity (Cohorts 1A and 3), as evidenced by the lymphocyte subpopulation analysis or KLH-IgM and SRBC-IgM responses in the TDAR assay.

There were no test material-related findings amongst the auditory startle test (at weaning), and FOB and motor activity, brain morphometry, brain weight and neurohistopathology indicative of any neurotoxicity (Cohorts 2A and 2B).

At necropsy, non-adverse test material-related effects were observed for the kidney and spleen (slightly higher extramedullary hematopoiesis compared with the control group) of males and females dosed at 225 mg/kg/day. The findings for the kidneys were considered non adverse in the absence of any degenerative/necrotic renal changes and any clinical pathology correlate.

 

CONCLUSION

In conclusion, based on the results of this extended one generation reproductive toxicity study (including Cohorts 1, 2 and 3), the following No Observed Adverse Effect Levels (NOAELs) were established for the substance:

General Toxicity (F0): At least 75 mg/kg/day (based on adverse pathology findings including renal tubular dilatation and mineralization correlated with higher kidney weight, myocardial or aortic mineralization in the heart, mineralization and/or necrosis of the glandular stomach, kidneys and heart at 225 mg/kg/day for females).

Reproductive Toxicity (F0): At least 225 mg/kg/day.

General Toxicity (F1): At least 225 mg/kg/day.

Developmental Toxicity (F1): At least 225 mg/kg/day.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted in accordance with OECD Test guideline No 422 and with the principles of GLP

Reason / purpose for cross-reference:

reference to same study

Remarks:

This study (OECD 422) investigates both the fertility toxicity and the developmental toxicity.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Principles of method if other than guideline:

Study was in accordance with the range-finding assay described in OECD method 422.
Males were treated for 42 days, with a satellite high dose group retained for a further 15 days to demonstrate recovery after treatment.
The females were treated for 14 days prior to mating and then through gestation to lactation day 4. A high dose satellite recovery group of females was also retained for fifteen days following completion of treatment.

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Purity: 99.96%

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Crj:CD(SD)IGS rats used but no details provided regarding supplier
- Age at study initiation: (P) x wks; (F1) x wks No data
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g No data
- Fasting period before study: No data
- Housing: No data

- Diet (e.g. ad libitum): no data
- Water (e.g. ad libitum): no data
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data
- Humidity (%): No data
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): No data

IN-LIFE DATES: From: not stated To: not stated

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% sodium carboxymethylcellulose solution

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water):0.5% sodium carboxymethylcellulose solution used as suspending agent

Details on mating procedure:

- M/F ratio per cage: 1:1.

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No information provided

Duration of treatment / exposure:

Males were dosed for 42 days prior to mating; the females were treated for 14 days prior to mating and then until day 4 of lactation. The female satellite animals were treated or 42 days. The recovery period for males and satellite females was 14 days. The males were terminated on day 43 of treatment (day 15 of recovery) and the females were terminated on day 5 of lactation, satellite females on day 15 of recovery and the offspring were terminated 4 days after birth.

Frequency of treatment:

Once daily for up to 42 days

Details on study schedule:

As a screening study this investigation was limited to the parental generation and F1 offspring only. The repeated adminstration phase for males (42 days) and females (14 days) prior to mating provides some subacute toxicity data and the treatment of females through gestation gives some reproductive and developmental toxicity information.

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

8 mg/kg bw/day (actual dose received)

Dose / conc.:

40 mg/kg bw/day (actual dose received)

Dose / conc.:

200 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

twelve; an additional five rats per sex were allocated to the satellite groups for retention through the recovery phase.

Control animals:

yes, concurrent vehicle

Details on study design:

No further details provided

Positive control:

Not applicable

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at weekly intervals - assessment in homecage, then in observers hands and then outside the homecage
Assessments included posture, sleeping, locomotion,vocalisations, tremors and convulsions, response to capture and handling, salivation, grading of heart beat, body temperature, exophthalmus and pupil size, exhibition of any discoloration of fur,skin or lacrimation. Outside the cage the assessment of posture, grooming, vocalisations, occurrence of straub tail, gait, tremor, convulsion pilo-erection and palpebral opening; exploration and respiratory rate and any exhibiion of stereotypy or bizarre behaviour.

BODY WEIGHT: recorded on days 1, 7, 14, 21, 28, 35 and 42 of treatment and days 1, 7, and 14 of recovery for males.
Females were weighed on days 1, 7, and 14 of treatment; days 0, 7, 14 and 20 of pregnancy and days 0 and 4 of lactation

Food consumption was recorded for the males and for the satellite females (treated at 200 mg/kg) on days 1-2, 7-8, 14-15, 29-30, 35-36 and 41-42 and then days 6-7 and 13-14 of the recovery phase.


Urinalysis was performed on Day 31 and 32 for males and females respectively
Necropsy was completed on termination of treatment or after completion of the recovery phase or day 5 of lactation

The reproductive performance of the parents was recorded for the twelve pairings per treatment group; the number of pairs copulating provided the copulation index and the number of pregnant females was used to calculate the fertility index. the mean number of days from pairing to copulation was also calculated

Oestrous cyclicity (parental animals):

All females examined. The number of rats showing a 4 day cycle in the pre-treatment phase was recorded, together with number with 5-day cycle. Mean cycle duration was calculated

Sperm parameters (parental animals):

no data

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring on day 0 of lactation:
number of pups, stillbirths, live births, birth index and live birth index

Bodyweights recorded on day 0 and 4 of lactation

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

Necropsy of live pups surviving to lactation day 4- examination of external and visceral changes

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals following day 42 of treatment or on day 15 of the recovery phase.
- Maternal animals: All surviving animals on day 5 of lactation or on day 15 for the satellite animals in the recovery phase.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in the attached Table were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.
- These animals were subjected to macroscopic postmortem examinations for external and visceral changes

Statistics:

No data

Reproductive indices:

The reproductive performance of the parents was recorded for the twelve pairings per treatment group; the number of pairs copulating provided the copulation index and the number of pregnant females was used to calculate the fertility index. the mean number of days from pairing to copulation was also calculated

Offspring viability indices:

Number of newborn, delivery index, number of live newborn, birth index and live bith index calculated on day 0 of lactation. On day 4 of lactation the number of live pups, viability index and sex ratio were calculated.

Clinical signs:

no effects observed

Description (incidence and severity):

daily observations showed no adverse reactions; detailed clinical assessment revealed no treatment related changes

Body weight and weight changes:

no effects observed

Description (incidence and severity):

bodyweights for males and females showed no significant differences from controls at any timepoint. Food consumption showed no significant treatment related differences

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

bodyweights for males and females showed no significant differences from controls at any timepoint. Food consumption showed no significant treatment related differences

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

liver histopathology in males at 200 mg/kg bw.d

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No significant effects recorded

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Oral administration of 4,4'-biphenyldiol at dose levels of 8, 40 or 200 mg/kg bw/day did not cause death or a moribund condition in any animals. None of the scores obtained during detailed clinical observations differed in a biologically significant manner between the control and the compound-treated groups.
No apparent changes were observed in general clinical conditions, except that urine in the 200 mg/kg-treated group became cloudy with elapse of time after excretion. Urinalysis was performed on treatment days 31 and 32 in male and female rats, respectively; calcium oxalate-like urinary crystal sediments were found in males given 200 mg/kg and in females given 40 or 200 mg/kg. Turbidity was enhanced at dose levels of 40 or 200 mg/kg, and urinary specific gravity was decreased in these females. These changes in urine parameters were not found when urinalysis was performed 11 days after cessation of the treatment (on day 11 of recovery) in either sex for the satellite animals.
Body weight, weight gain and food consumption were not affected by treatment at any dose level in either sex. No animals showed any abnormality in functional parameters after the final treatment.
At necropsy on termination of the treatment, no apparent effects of the compound were found on haematological or blood-biochemical examination at any dose level of the compound in either sex.
No effects of the compound were apparent in the female organs, including their weights or macroscopic or microscopic findings.
In contrast, 200 mg/kg of the compound exerted effects on male livers, with significant increase in relative weight, darkening or enlargement of the macroscopic appearance, development of centrilobular hepatocyte hypertrophy and reduction in occurrence of periportal fatty change. However, no animals showed any abnormality in functional observations on day 14 of the recovery period. At necropsy on day 15 of recovery, changes in the liver were not found in males given 200 mg/kg. In females given 200 mg/kg for 42 days and killed for necropsy on day 15 of recovery (satellite group), there were similarly no abnormal findings.

The treatment did not affect parameters of reproductive performance, such as the estrous cycle, ovulation, mating, implantation, delivery or lactational condition at any dose level. No adverse effects of the compound were observed on viability, morphology or growth of offspring.

Key result

Dose descriptor:

NOAEL

Remarks:

General toxicity

Effect level:

40 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Liver effects at 200 mg/kg bw/day (urinary turbidity at 40 and 200 mg/kg bw/d is not considered to be of toxicological significance).

Key result

Dose descriptor:

NOEL

Remarks:

Reproductive toxicity

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No evidence of reproductive toxicity was seen in this study at the highest dose level tested of 200 mg/kg bw/d

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

not specified

There were no adverse effects of administration on offspring viability, morphology or growth.

Key result

Dose descriptor:

NOEL

Remarks:

Developmental toxicity

Generation:

F1

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No evidence of developmental toxicity was seen in this study at the highest dose level tested of 200 mg/kg bw/d

Key result

Reproductive effects observed:

no

The no observed adverse effect level (NOAEL) for repeat dose toxicity of 4,4'- biphenyldiol is considered to be 40 mg/kg bw/d in both sexes. The NOAEL for reproductive toxicity is considered to be 200 mg/kg bw/d, in the absence of any relevant effects.

Summary of oestrous cycle data for females treated with 4,4’-biphenyldiol

	4,4’-biphenyldiol
	0 (0.5% CMC Na solution, 5 mL/kg bw	8 mg/kg bw/day	40 mg/kg bw/day	200 mg/kg bw/day
Number of females examined	12	12	12	12
Number of females with 4-day cycle in pre-treatment period	12	12	12	12
Number of females in treatment period with 4-day cycle or 5 day cycle	12 0	11 1	12 0	12 0
Mean length of oestrous cycle (days)	4.0	4.0	4.0	4.0
Number of vaginal oestrous during mating phase	1.0	1.0	1.0	1.0

 

Reproductive performance of rats treated with 4,4’-biphenyldiol

	4,4’-biphenyldiol
	0 (0.5% CMC Na solution, 5 mL/kg bw	8 mg/kg bw/day	40 mg/kg bw/day	200 mg/kg bw/day
Number of pairs examined	12	12	12	12
Number of pairs copulated	11	11	12	12
Copulation index	91.7	91.7	100	100
Number of pregnant females	11	11	10	12
Fertility index	100	100	83.3	100
Mean number of pairing days to copulation	2.5 ± 1.1	2.6 ± 1.2	2.7 ± 0.8	2.7 ± 1.2

 

Development of pups up to day 4 of lactation

	4,4’-biphenyldiol
	0 (0.5% CMC Na solution, 5 mL/kg bw	8 mg/kg bw/day	40 mg/kg bw/day	200 mg/kg bw/day
Number of pregnant females	12	12	10	12
Number of pregnant females with live newborns	11	11	10	12
Gestation index	100	100	100	100
Gestation length in days	22.4 ± 0.5	22.4 ± 0.5	22.0 ± 0.0	22.4 ± 0.5
Number of corpora lutea	15.9 ± 1.6	16.5 ± 1.1	16.2 ± 1.2	15.2 ± 1.5
Number of implantations	15.5 ± 1.7	14.3 ± 3.6	16.0 ± 1.6	14.7 ± 1.72
Implantation index	97.1 ± 4.3	86.9 ± 21.6	98.7 ± 4.2	96.7 ± 5.3
Day 0 of lactation
Number of newborns	14.5 ± 2.3	13.8 ± 4.0	15.1 ± 1.7	14.0 ± 1.8
Delivery index	93.2 ± 1.1	94.6 ± 12.2	94.4 ± 4.8	95.5 ± 5.2
Number of live newborns	14.3 ± 2.3	13.8 ± 4.0	14.6 ± 1.6	12.8 ± 2.9
Birth index	92.0 ± 7.6	94.6 ± 12.2	91.5 ± 8.1	88.8 ± 20.1
Live birth index	98.7 ± 3.0	100.0 ± 0.0	97.0 ± 7.5	93.3 ± 20.8
Sex ratio on Day 0	54.8 ± 14.5	49.4 ± 17.4	52.3 ± 15.1	51.6 ± 14.7
Day 4 of lactation
Number of live pups	13.9 ± 2.1	13.6 ± 4.0	14.5 ± 1.5	12.4 ± 4.2
Viability index	97.7 ± 4.0	98.8 ± 2.6	99.4 ± 2.0	91.7 ± 28.9
Sex ratio on Day 4	54.4 ± 14.9	49.3 ± 17.9	52.1 ± 15.9	50.8 ± 15.2

 

Pup bodyweights up to Day 4 of lactation

	4,4’-biphenyldiol
	0 (0.5% CMC Na solution, 5 mL/kg bw	8 mg/kg bw/day	40 mg/kg bw/day	200 mg/kg bw/day
Day 0 of lactation
Number of live newborns
Male	7.7 ± 2.1	6.9 ± 3.0	7.6 ± 2.1	6.5 ± 2.2
Female	6.5 ± 3.0	6.9 ± 3.3	7.0 ± 2.6	6.3 ± 2.7
Bodyweight of live newborn (g)
Male	6.8 ± 0.8	6.9 ± 0.8	6.5 ± 0.5	6.6 ± 0.8
Female	6.6 ± 0.6	6.6 ± 0.7	6.0 ± 0.4	6.2 ± 0.8
Day 4 of lactation
Number of live pups
Male	7.5 ± 2.3	6.8± 3.1	7.5± 2.2	6.8± 2.0
Female	6.4 ± 2.2	6.8± 3.2	7.0 ± 2.6	6.7± 2.5
Bodyweight of pups (g)
Male	10.6 ± 1.7	10.7 ± 1.1	10.2 ± 1.1	10.8 ± 1.0
Female	10.3 ± 1.4	10.4 ± 1.4	9.8 ± 1.0	10.0 ± 0.9

 

Pup morphology findings

	4,4’-biphenyldiol
	0 (0.5% CMC Na solution, 5 mL/kg bw	8 mg/kg bw/day	40 mg/kg bw/day	200 mg/kg bw/day
Dead pups
Number of dead pups examined	3	1	6	12
External examinations	3	1	6	12
Visceral examinations	0	0	5	1
Number of pups with external changes	0	0	0	0
Number of pups with visceral changes	0	0	0	0
Live pups
At birth (day 0)
Number of newborns examined	157	152	146	154
Number of newborns with external changes	0	0	0	0
At necropsy (day 4 of lactation)
Number of pups examined	153	150	145	149
Number of pups with external changes	0	0	0	0
Number of pups with visceral changes	0	0	0	0

 

Conclusions:

The treatment up to 200 mg/kg bw/d did not affect the reproductive performance of parental animals or the early development of their offspring. The no observed adverse effect dose level (NOAEL) for repeat dose toxicity of 4,4'-biphenyldiol is considered to be 40 mg/kg/day for both sexes of animals. The NOAEL for reproductive toxicity is considered to be 200 mg/kg bw/d, in the absence of any findings.

Executive summary:

A combined repeat dose and reproductive/developmental toxicity screening test was conducted in rats according to the OECD Test Guideline 422 with 4,4'-biphenyldiol. No effects on fertillity or reproductive parameters (oestrus cyclicity, mating, implantation or lactation) were observed at any dose level. In the absence of any effects on reproductive parameters, a NOAEL for reproductive toxicity of 200 mg/kg bw/d can be determined. A NOAEL for general toxicity of 40 mg/kg bw/d can be determined based on liver effects at the highest dose level. Effects of treatment on urinalysis parameters seen at 40 and 200 mg/kg bw/d are not considered to be of toxicological significance.

 

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

225 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

This study is very reliable: it is a recent OECD 443 study (2022) in compliance with the current guideline and GLP.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

Four studies are available for the investigation of the developmental effect.

 

> A screening test according to OECD 422 (repeated dose/reproductive/developmental toxicity study) in Sprague-Dawley rats exposed by oral route is available and reports a NOAEL of 40 mg/kg bw/d for general toxicity and a NOAEL of 200 mg/kg bw/d for develomental toxicity (the highest dose level tested).

In this study, treatment up to 200 mg/kg bw/d did not affect the the developmental parameters assessed.

 

> A developmental toxicity study according to OECD 414 (prenatal developmental toxicity study) in Wistar pregnant rats exposed by the oral route is available.

Firstly a Dose Range Finding study was conducted at 250, 500 and 750 mg/kg/day. Severe toxicity was observed in one and all females given 500 and 750 mg/kg/day, respectively. Therefore in the main experiment, the substance was administered by daily gavage at dose levels of 25, 75 and 250 mg/kg/day to groups of 22 mated female Wistar rats from G 6 to G 20 of gestation inclusive.

A transient reduced body weight gain at 75 mg/kg/day and persistent reduced body weight gain and food consumption at 250 mg/kg/day were observed.

Slightly lower mean placental and foetal weights were seen at the highest group (250 mg/kg bw/d) in the presence of maternal toxicity. These findings were likely related to the maternal toxicity rather than a direct effect of the test item.

Therefore a NOAEL of 75 mg/kg bw/d for maternal toxicity and a NOAEL of 250 mg/kg bw/d for embryo-foetal toxicity (the highest dose level tested) were reported.

In this study, treatment up to 250 mg/kg bw/d was considered not to affect the prenatal embryo-foetal development.

 

> A prenatal toxicity study in rabbits conducted according to the OECD 414 guideline, at the dose levels of 20, 40 and 60 mg/kg administered by oral route.

The NOAEL for maternal toxicity was established at 40 mg/kg on the basis of the reduction of the food consumption at 60 mg/kg over the period G6-G29, correlated with a slight decrease in the terminal BW gain. 

The NOAEL for prenatal toxicity was 60 mg/kg in absence of any relevant effects on fetuses.

In this study, treatment up to 60 mg/kg bw/d was considered not to affect the prenatal embryo-foetal development.

 

> An Extended One Generation Reproductive Toxicity study was conducted in rats by oral route at the dose levels of 25, 75 and 225 mg/kg bw/day (on the basis of the results of a simplified OECD 422 study conducted at 200 and 250 mg/kg bw/day in order to select the dose levels of the OECD 443 study). 

In the OECD 443 study, the NOAEL for maternal toxicity was established at 75 mg/kg on the basis of adverse pathology findings in kidney, heart and glandular stomach at 225 mg/kg/day in females.

A NOAEL of 225 mg/kg bw/day was reported for developmental toxicity in absence of any effect on the F1 generation.

In this study, treatment up to 225 mg/kg bw/d did not affect the development of the F1 generation. 

 

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Deviations:

no

Remarks:

None of the technical deviations that occurred during the experimental phase have impacted the results of the study.

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Wistar

Remarks:

CRL: WI(Han)

Details on test animals or test system and environmental conditions:

See the same study in the section 7.8.1 "'Toxicity to reproduction".

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

See the same study in the section 7.8.1 "'Toxicity to reproduction".

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

See the same study in the section 7.8.1 "'Toxicity to reproduction".

Details on mating procedure:

See the same study in the section 7.8.1 "'Toxicity to reproduction".

Duration of treatment / exposure:

See the same study in the section 7.8.1 "'Toxicity to reproduction".

Frequency of treatment:

See the same study in the section 7.8.1 "'Toxicity to reproduction".

Duration of test:

See the same study in the section 7.8.1 "'Toxicity to reproduction".

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1 (negative control with vehicle alone).

Dose / conc.:

25 mg/kg bw/day (nominal)

Remarks:

Group 2

Dose / conc.:

75 mg/kg bw/day (nominal)

Remarks:

Group 3

Dose / conc.:

225 mg/kg bw/day (nominal)

Remarks:

Group 4

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 5 (positive control with administration of cyclophosphamide for the TDAR assays).

No. of animals per sex per dose:

See the same study in the section 7.8.1 "'Toxicity to reproduction".

Control animals:

yes, concurrent vehicle

Details on study design:

See the same study in the section 7.8.1 "'Toxicity to reproduction".

Maternal examinations:

See the same study in the section 7.8.1 "'Toxicity to reproduction".

Ovaries and uterine content:

See the same study in the section 7.8.1 "'Toxicity to reproduction".

Blood sampling:

See the same study in the section 7.8.1 "'Toxicity to reproduction".

Fetal examinations:

1) EXAMINATION OF F1 UNTIL WEANING

- Litter size, clinical signs and mortality: The litter sizes were recorded on PND1.
The litters were observed daily in order to note the number of live, dead and cannibalized pups.
- Litter size adjustment: On PND4, the size of each litter was adjusted by randomly culling extra pups to obtain as nearly as possible five males and five females per litter. Whenever necessary, partial adjustment (for example six males and four females) was permitted.
- Clinical signs and mortality: The pups were observed daily for clinical signs, abnormal behavior and external abnormalities (including oral cavity and orifices).
- Body weight: The weight of each live pup was recorded on PND 1, 4, 7, 14 and 21.
- Sex: The sex of each pup was externally determined on PND1 and 4.
- Pups development: The following physical development measurements were performed in pups of each litter:
> anogenital distance (AGD) on PND4 (The AGD was normalized to the cube root of body weight recorded on PND4),
> number of nipples and of areolae in male pups: on PND13.

2) EXAMINATION OF F1 FROM WEANING ONWARDS

- Clinical signs: at least weekly for clinical signs.
- Body weight: at least weekly.
- Food consumption: at least weekly.
- Vaginal opening: daily for all females from PND28 onwards
- Balanopreputial separation: daily for all males from PND38 onwards
- Stage of estrus determination: starting on the day of onset of vaginal patency and until the first estrus is determined. (for females from cohort 1A only)
- Neurotoxicity (Neurobehavioral Tests): for 10 Animals/Sex/Group from cohort 2A only (see the same study in the section 7.8.1 "Toxicity to reproduction", part "F1 Generation".
- Immunotoxicity: for all F1 animals from the Cohort 3 and Positive Control Animals (see the same study in the section 7.8.1 "Toxicity to reproduction", part "F1 Generation").
- Hematology, Serum clinical chemistry and Urine analysis for 10/sex/group (randomly selected) from F1 cohort 1A only, on the day of necropsy
- Thyroid hormone analysis:
1/ analysis of T4 and TSH for 10/sex/group from F1-animals cohort 1A and from Surplus pups , on the day of necropsy between 7.00 and 10.30 am
2/ analysis of T4 only for 2 pups/litter on PND4 between 7.00 and 10.30 am

EXAMINATION OF F1 AT THE NECROPSY
- Cohort Surplus (necropsy at PND 21): All animals were weighted and subjected to a limited examination, with special attention being paid to the reproductive organs. The organs identified for weighting and tissues for collecting are described in the same study in the section 7.8.1 "Toxicity to reproduction", part "F1 Generation".

- Cohort 1A (necropsy at PND89-99): All animals were weighted. The organs identified for weighting and tissues for collecting, as well as microscopic examination are described in the same study in the section 7.8.1 "Toxicity to reproduction", part "F1 Generation".
A sperm analysis was performed for all the surviving males of the cohort 1A.
From 10 selected animals/sex/group of Cohort 1A, splenic lymphocyte subpopulation analysis was performed (see the same study in the section 7.8.1 "Toxicity to reproduction", part "F1 Generation").

- Cohort 1B (necropsy at PND92-100): All animals were weighted and subjected to a limited examination, with special attention being paid to the reproductive organs. The organs identified for weighting and tissues for collecting are described in the same study in the section 7.8.1 "Toxicity to reproduction", part "F1 Generation".

- Cohorts 2A (necropsy at PND76-90) and 2B (necropsy at PND21-22): All animals were weighted and subjected to a limited examination, with special attention being paid to the reproductive organs. A morphometric analysis was done on the brain. Organs related to nervous system identified for weighing and the representative samples of the tissues for collecting as well as microscopic examination are described in the same study in the section 7.8.1 "Toxicity to reproduction", part "F1 Generation".

- Cohort 3 (necropsy at PND53-59): All animals were subjected to a limited examination, with special attention being paid to the reproductive organs. See the same study in the section 7.8.1 "Toxicity to reproduction", part "F1 Generation".

Statistics:

See the same study in the section 7.8.1 "'Toxicity to reproduction".

Indices:

See the same study in the section 7.8.1 "'Toxicity to reproduction".

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

See the same study in the section 7.8.1 "'Toxicity to reproduction".

Mortality:

no mortality observed

Description (incidence):

See the same study in the section 7.8.1 "'Toxicity to reproduction".

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See the same study in the section 7.8.1 "'Toxicity to reproduction".

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

See the same study in the section 7.8.1 "'Toxicity to reproduction".

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

See the same study in the section 7.8.1 "'Toxicity to reproduction".

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

See the same study in the section 7.8.1 "'Toxicity to reproduction".

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

See the same study in the section 'Toxicity to reproduction'.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

See the same study in the section 7.8.1 "'Toxicity to reproduction".

Behaviour (functional findings):

not examined

Description (incidence and severity):

See the same study in the section 7.8.1 "'Toxicity to reproduction".

Immunological findings:

not examined

Description (incidence and severity):

See the same study in the section 7.8.1 "'Toxicity to reproduction".

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

See the same study in the section 7.8.1 "'Toxicity to reproduction".

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

See the same study in the section 7.8.1 "'Toxicity to reproduction".

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See the same study in the section 7.8.1 "'Toxicity to reproduction".

Histopathological findings: neoplastic:

not examined

Details on results:

See the same study in the section 7.8.1 "'Toxicity to reproduction".

Number of abortions:

no effects observed

Description (incidence and severity):

See results in the table 33 in the same study in the section 7.8.1 "'Toxicity to reproduction".

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There was no test item-related effect on the pre-birth data in any group.
The mean percentage of pre-birth loss in treated groups (5.9%, 7.7% and 7.9% at 25, 75 and 225 mg/kg/day, respectively) was lower compared with the concurrent control group (8.3%) and the HCD mean (9.9%).
See results in the tables 32 and 33 in the same study in the section 7.8.1 "'Toxicity to reproduction".

Total litter losses by resorption:

effects observed, non-treatment-related

Description (incidence and severity):

One female (No. 176) given 225 mg/kg/day with a total litter resorption. This isolated case was considered not test item-related in the absence of any differences on the mean percentage pre-birth loss between the 225 mg/kg/day and control groups.
See results in the tables 32 and 33 in the same study in the section 7.8.1 "'Toxicity to reproduction".

Early or late resorptions:

no effects observed

Description (incidence and severity):

There was no test item-related effect on the early or late resorption in any group.
See results in the table 33 in the same study in the section 7.8.1 "'Toxicity to reproduction".

Dead fetuses:

no effects observed

Description (incidence and severity):

The mean numbers of pups delivered in the treated groups (12.5, 12.3 and 12.2 at 25, 75 and 225 mg/kg/day, respectively) were comparable with that of the concurrent control group (12.7) and above the historical control data mean (11.0).
See results in the tables 32 and 33 in the same study in the section 7.8.1 "'Toxicity to reproduction".

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

There was no test item-related effect on the duration of gestation in any group.
The mean duration of gestation was approximately 22 (22.1-22.4) days in each group.
See results in the table 33 in the same study in the section 7.8.1 "'Toxicity to reproduction".

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

There were 23, 25, 25 and 24 pregnant females in the control, 25, 75 and 225 mg/kg/day groups, respectively, all of which successfully completed delivery of liveborn pups with the exception of 1 female (No. 176) given 225 mg/kg/day with a total litter resorption. This isolated case was considered not test item-related in the absence of any differences on the mean percentage pre-birth loss between the 225 mg/kg/day and control groups.
See results in the tables 32 and 33 in the same study in the section 7.8.1 "'Toxicity to reproduction".

Details on maternal toxic effects:

See results and tables in the same study in the section 7.8.1 "'Toxicity to reproduction".

Key result

Dose descriptor:

NOAEL

Effect level:

>= 75 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other:

Remarks on result:

other: For more details, see the same study in the section 7..8.1 "Toxicity to reproduction"

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

> Pups body weight at birth (D1):

There was no statistically significant differences in the pups body weight at D1 in the treated groups compared to the control group.
The mean body weight of the male pups at day 1were 6.83, 6.86, 6.97 and 6.65 g in the control, 25, 75 and 225 mg/kg groups respectively.
The mean body weight of the female pups at day 1 were 6.53, 6.53, 6.53 and 6.28 g in the control, 25, 75 and 225 mg/kg groups respectively.

> Pups Body Weight During Lactation (F1 Pups):

There was slightly lower mean pup body weight gain at 225 mg/kg/day, leading to statistically significantly lower mean pup body weight compared with the concurrent control and HCD mean on PND21 (-8% and -4% for both sexes). However, the mean values (52.3 g and 50.6 g for males and females, respectively) remained within the historical control data range (49.7 to 57.6 for males and 48.8 to 56.0 for females), so this difference was considered not toxicologically significant.
There was no test item-related effect on mean pup body weight at 25 and 75 mg/kg/day.

> Body Weight and Body Weight Change - Cohorts 1A, 1B, 2A and 3 - Post-Weaning:

For males, there was a test item-related slightly lower mean body weight gain after weaning throughout the dosing period for males (Day 1 to Day 70) at 225 mg/kg/day compared with the control group (-10%) leading to a lower mean terminal body weight (-11%) on Day 70 compared with the control group. This difference was considered not adverse in view of the low magnitude of the change and in the absence of any impact on the development such as
sexual maturation.
For females, there was lower mean body weight gain during the first week post-weaning at 225 mg/kg/day considered not toxicologically relevant as it was followed by a recovery such
that the mean terminal body weight was comparable with that of the control group. In addition there was no test item-related effect on the sexual maturation that was considered toxicologically significant.
There was no test item-related effect on mean body weight gain for females in any group or males at 25 and 75 mg/kg/day.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There was no test item-related effect on the number of live offspring at birth in any group.
The number of live pups were 12.5, 12.4, 12.2 and 12.0 in the control, 25, 75 and 225 mg/kg groups respectively.
See results and table 33 in the same study in the section 7.8.1 "'Toxicity to reproduction".

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There was no test item-related effect on the sex ratio in any group.
See results and table 33 in the same study in the section 7.8.1 "'Toxicity to reproduction".

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There was no test item-related effect on the number of litter size and weights.
The litter size were 12.7, 12.5 12,3 and 12.2 in the control, 25, 75 and 225 mg/kg groups respectively.
See results and table 33 in the same study in the section 7.8.1 "'Toxicity to reproduction".

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

See results and table 43 in the same study in the section 7.8.1 "'Toxicity to reproduction".

Changes in postnatal survival:

effects observed, non-treatment-related

Description (incidence and severity):

There was no test item-related effect in any group on the number of live offspring on PND4 (before culling) compared with the number of offspring on PND0 (viability index).
There were 4, 4, 5 and 10 dead, missing or cannibalized pups from 1, 3, 5 and 3 litters in the control, 25, 75 and 225 mg/kg/day groups, respectively, between PND0 and PND4. The viability index was consequently slightly lower at 225 mg/kg/day (96.4%) compared with the control group (98.6%) but remained within the HCD range (96.2% to 100%).
See results and tables in the same study in the section 7.8.1 "'Toxicity to reproduction".

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

> There were no test item-related effects that were considered toxicologically significant on clinical condition or body weight
> There were no test-item related effects on the anogenital distance of the pups, or any evidence of areolae/nipple retention for the male offspring at any dose level.
> There were no test item-related effects on the hematology, serum clinical chemistry, urinary parameters (cohort 1A)
> There were no test item-related effects on T4 and TSH levels for the F1-pups on PND4 and PND21
> There was no test item-related effect on the mean age at attainment of balanopreputial cleavage and vaginal opening or on the duration from vaginal opening to first estrus in any group.
> There were no test item-related effects on estrous cyclicity or on sperm quality in any group (Cohort 1A).
> There was no test item-related immunotoxicity (Cohorts 1A and 3), as evidenced by the lymphocyte subpopulation analysis or KLH-IgM and SRBC-IgM responses in the TDAR assay.
> There were no test item-related findings amongst the auditory startle test (at weaning), and FOB and motor activity, brain morphometry, brain weight and neurohistopathology indicative of any neurotoxicity (Cohorts 2A and 2B).
> At necropsy, non adverse test item-related effects were observed for the kidney and spleen (slightly higher extramedullary hematopoiesis compared with the control group) of males and females dosed at 225 mg/kg/day. The findings for the kidneys were considered non adverse in the absence of any degenerative/necrotic renal changes and any clinical pathology correlate.


> For more details and results on the parameters in F1 generation, see the same study in the section 7.8.1, "Toxicity to reproduction", "Results - F1 generation".

Key result

Dose descriptor:

NOAEL

Effect level:

>= 225 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: For more details, see the same study in the section 7..8.1 "Toxicity to reproduction"

Key result

Developmental effects observed:

no

Conclusions:

In conclusion, based on the results of this extended one generation reproductive toxicity study (including Cohorts 1, 2 and 3), the following No Observed Adverse Effect Levels (NOAELs) were established for the substance:

- General Toxicity (F0): At least 75 mg/kg/day (based on adverse pathology findings including renal tubular dilatation and mineralization correlated with higher kidney weight, myocardial or aortic mineralization in the heart, mineralization and/or necrosis of the glandular stomach at 225 mg/kg/day for females).

- Reproductive Toxicity (F0): At least 225 mg/kg/day.

- General Toxicity (F1): At least 225 mg/kg/day.

- Developmental Toxicity (F1): At least 225 mg/kg/day.

Executive summary:

This GLP Study following the OECD guideline 443 was carried out to provide an evaluation of the pre and postnatal effects of the test material, on development as well as a thorough evaluation of systemic toxicity in adult male and pregnant and lactating female Wistar Han rats and their offspring.

Detailed examination of key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, and physical and functional development until adulthood, was expected to identify specific target organs in the offspring.

In addition, the study provided and/or confirmed information about the effects of the test material on the integrity and performance of the adult male and female reproductive systems.

Specifically, but not exclusively, the following parameters were considered: gonadal function, estrous cyclicity, epididymal sperm maturation, mating behaviour, fertility, pregnancy, parturition, and lactation. Developmental neurotoxicity and developmental immunotoxicity assessments were also performed to characterize potential effects on those systems.

 

STUDY DESIGN

The test material was administered by daily oral gavage at dose levels of 25, 75 and 225 mg/kg/day to groups of 25 males and 25 females Wistar rats (F0 generation). F0 males were dosed for 10 weeks prior to mating, during mating and up to the day before necropsy. F0 females were dosed during 10 weeks prior to mating, during mating, gestation and lactation and up to the day before necropsy, i.e., on Lactation Day (LD) 21 to LD23 for females that delivered. A fourth group of 25 rats/sex received the same dose volume (5 mL/kg/day) of the control material (0.5% CMC in purified water). The inseminated females were allowed to litter, and development of the offspring was observed up to weaning on Postnatal Day (PND) 21.

The following parameters and endpoints were evaluated in all F0 animals: mortality, clinical signs (including arena observations), body weights, body weight changes, food consumption, clinical pathology, thyroid hormone levels, estrous cycles, mating performance and fertility, delivery and litter data, sperm quality, macroscopic findings, organ weights and histology findings. F0-males were euthanised after at least 12 weeks of exposure and F0 females after at least 16 weeks of dosing.

The following parameters and endpoints were evaluated in all F1 offspring until weaning (PND21): mortality, clinical signs, body weights, number of pups, pup sex ratio and anogenital distance and areola/nipple retention for male pups.

From weaning, the selected F1-pups were allocated to 5 cohorts (1A, 1B, 2A, 2B and 3) of 20 males and 20 females for the first 2 cohorts, and 10 males and 10 females for the last 3 cohorts. All F1-animals, except Cohort 2B and F1 surplus animals, were dosed by daily oral gavage (once a day) from PND21 up to the day before necropsy at the same dose levels of 25, 75 and 225 mg/kg/day. A fourth group received the same dose volume (5 mL/kg/day) of the control material (0.5% CMC in purified water). Selected F1 animals (with the exception of Cohort 2B) were observed for mortality, clinical signs (including arena observations), body weights, body weight changes, food consumption, clinical pathology (Cohort 1A), thyroid hormone levels (PND4 pups, surplus pups and Cohort 1A), sexual maturation, estrous cycles (Cohort 1A), neurobehavioral tests (Cohort 2A), macroscopic findings, organ weights and histology findings. Additionally, spleen samples were taken for splenic lymphocyte subpopulation analysis from 10 rats/sex/group from F1 Cohort 1A animals. Humoral response was also evaluated following SRBC administration for Cohort 3 animals and KLH administration for Cohort 1B animals (TDAR assays). F1 animals from Cohort 2B were not dosed directly and were necropsied on/before PND24 for neuro-histopathology and morphometric analysis.

F1 animals from Cohort 3 were sacrificed between PND54 and PND57 or at approximately 12 weeks of age for positive controls. F1 animals from Cohort 2A were sacrificed after completion of the neurobehavioral tests between PND76 and PND90. F1 animals from Cohort 1A were sacrificed after clinical pathology evaluation between PND89 and PND99. F1 animals from Cohort 1B were sacrificed between PND92 and PND100.

Surplus offspring (10/sex/group) not included in any of the F1 cohorts were selected for thyroid hormone analysis and organ weights on PND21.

 

RESULTS

All formulations at 5, 15 and 45 mg/mL used on Day 1, and in Week 11 and Week 26 of treatment were in agreement with acceptance criteria. Homogeneity was confirmed from the formulations used on Day 1. No test material was detected in the vehicle samples.

 

F0 generation:

There was no test material-related death in any group.

There was no test material-related clinical sign in any group.

There was no test material-related effect in any group on mean body weight gain or food consumption for either sex that was considered toxicologically significant.

There was no test material-related effect in any group on estrous cyclicity, mating performance (including copulation and pre-coital interval), fertility (including sperm quality), and gestation length.

Higher creatinine and urea concentrations, minor ionic unbalance for females and higher hepatic enzymes and triglyceride concentrations were noted for both sexes at 225 mg/kg/day compared with the control group and correlated with the pathology findings noted for the kidneys and liver.

There were no test material-related effects on T4 and TSH levels that were toxicologically significant in any group for either sex. Changes in hormone serum levels for F0 females and F1 males at 225 mg/kg/day were noted but considered not toxicologically relevant in the absence of any gender trend, any test item‑related effect on the thyroid weight, anogenital distance, nipple areola, reproduction and development, any histology findings in the thyroid glands, or similar findings for pups. They are considered related to the high variability of this parameter. As under the conditions of this study no adverse effect was observed that could be linked to the lower T4 and higher TSH values,  this was not taken into account to determine the No Observed Adverse Effect Levels (NOAELs).

At necropsy, there were test material-related findings for females dosed at 225 mg/kg/day in the kidney (minimal to mild tubular dilatation and mineralization, correlating with higher kidney weight), heart (minimal to mild myocardial or aortic mineralization), stomach (minimal to mild mineralization and/or necrosis of the glandular part, grossly correlating with dark foci), thymus (minimal atrophy and mild increased cortical apoptosis, correlating with lower organ weight when compared with the control group). Mineralization in the kidney, heart and stomach was considered adverse, while other findings were considered secondary to test material-related systemic effects (ovary, adrenal gland and thymus), or adaptative changes (liver). The pathology findings noted on the kidneys were correlated with higher creatinine and urea concentrations and minor ionic unbalance. The higher mean liver weight at 225 mg/kg/day for both sexes correlated with slightly higher mean tryglyceride and hepatic enzyme concentrations when compared with the control group. Ovarian atrophy, lower ovaries weight when compared with the control group and vaginal mucification which suggested a delay in the post-gestation recovery of estrous cycle were noted at necropsy at 225 mg/kg/day and were considered probably secondary to test material-related systemic effects noted in kidneys, stomach and heart. These findings were not correlated with any test material-related effect on fertility or estrous cycle data (F0 and F1 generation) and no test material-related effect on the mean count of primordial/small growing follicles (Cohort 1A) and was therefore considered not adverse. In addition, there was no similar finding in the F1 generation.

 

F1 generation:

There was no test material-related effect on postnatal survival of the F1 offspring in any group. Mean live litter size was comparable in all groups between birth and weaning.

There were no test material-related effects that were considered toxicologically significant on clinical condition, body weight and anogenital distance of the pups, or any evidence of areolae/nipple retention for the male offspring at any dose level.

There were no test material-related effects on T4 and TSH levels for the F1-pups on PND4 and PND21.

There was no test material-related effect on the mean age at attainment of balanopreputial cleavage and vaginal opening or on the duration from vaginal opening to first estrus in any group.

There was no test material-related clinical sign in any group for either sex.

There was a test material-related slightly lower mean body weight gain and reduced mean food consumption throughout the dosing period for males at 225 mg/kg/day compared with the control group leading to slightly, lower mean terminal body weight compared with the control group considered not adverse due to the low magnitude of the change and in the absence of any developmental effects.

There were no test material-related effects on the hematology, coagulation, serum clinical chemistry, urinary parameters or on the thyroid hormone serum levels that were considered toxicologically significant at any dose for either sex (Cohort 1A).

There were no test material-related effects on estrous cyclicity or on sperm quality in any group (Cohort 1A).

There was no test material-related immunotoxicity (Cohorts 1A and 3), as evidenced by the lymphocyte subpopulation analysis or KLH-IgM and SRBC-IgM responses in the TDAR assay.

There were no test material-related findings amongst the auditory startle test (at weaning), and FOB and motor activity, brain morphometry, brain weight and neurohistopathology indicative of any neurotoxicity (Cohorts 2A and 2B).

At necropsy, non-adverse test material-related effects were observed for the kidney and spleen (slightly higher extramedullary hematopoiesis compared with the control group) of males and females dosed at 225 mg/kg/day. The findings for the kidneys were considered non adverse in the absence of any degenerative/necrotic renal changes and any clinical pathology correlate.

 

CONCLUSION

In conclusion, based on the results of this extended one generation reproductive toxicity study (including Cohorts 1, 2 and 3), the following No Observed Adverse Effect Levels (NOAELs) were established for the substance:

General Toxicity (F0): At least 75 mg/kg/day (based on adverse pathology findings including renal tubular dilatation and mineralization correlated with higher kidney weight, myocardial or aortic mineralization in the heart, mineralization and/or necrosis of the glandular stomach, kidneys and heart at 225 mg/kg/day for females).

Reproductive Toxicity (F0): At least 225 mg/kg/day.

General Toxicity (F1): At least 225 mg/kg/day.

Developmental Toxicity (F1): At least 225 mg/kg/day.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

FROM 04 NOVEMBER 2020 TO 27 JULY 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)

Version / remarks:

25 June 2018.

Deviations:

yes

Remarks:

None of the deviations, a temporary lack of access to water for 1 animal and an unsexed fetus (control group) due to an error, were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Remarks:

(Crl:KBL[NZW]).

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratoires France - ESD, 01400 Châtillon sur Chalaronne, France.
- Age at study initiation: 18 to 20 weeks old.
- Weight at study initiation: between 2820 g and 4355 g.
- Fasting period before study: not specified.
- Housing: single housing, in composite plastic and metal cages.
- Diet: pelleted complete diet (Diet Reference No. 3409, KLIBA), sterilized by irradiation - approximately 200 g/animal/day.
- Water: municipal tap water, softened and filtered (0.2 μm) - freely available to each animal (except during designated procedures).
Remark: on Gestation Day (GD) 26, Female No. 11 (dose group 40 mg/kg/day) and Females Nos. 6 to 10 (dose group 20 mg/kg/day) were found without access to drinking water due to the rack purge being left open the previous day. These animals had potentially no access to drinking water for a maximum period of 21 hours and 40 minutes. This isolated event had no impact on the outcome of the study.
- Acclimation period: 6 days before the start of dosing.

ENVIRONMENTAL CONDITIONS
Environmental conditions were within the targets throughout the study:
- Temperature (°C): 17°C to 23°C.
- Humidity (%): ≥ 35%.
- Air changes (per hr): 10 or more air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark (except during any designated procedures).

IN-LIFE DATES: From: 04 November 2020 To: 09 December 2020.

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Dose formulations were prepared weekly and divided into aliquots to be dispensed on each dosing occasion.
- Dose formulations were prepared at appropriate concentrations to meet dose level requirements: 0 mg/mL (control group), 4 mg/mL (low dose group), 8 mg/mL (mid dose group) and 12 mg/mL (high dose group).
- They were stored at +4°C, in accordance with Sponsor data indicating stability in a concentration range of 1-200 mg/mL for 11 days at room temperature and +4°C or 26 days at -20°C.
- Formulations were maintained under continuous stirring for at least 15 minutes before and throughout the dosing procedure.

VEHICLE (CMC- Carboxymethyl cellulose):
- Justification for use and choice of vehicle if other than water: not specified.
- Concentration in vehicle: the concentrations of the test material in the vehicle were 0 mg/mL, 4 mg/mL, 8 mg/mL and 12 mg/mL.
- Amount of vehicle (if gavage): the dose volume administered to the animals was 5 mL/kg.
- Lot/batch no.: 18E174115.
- Purity: the vehicle was prepared as 0.5% [w/v] CMC 300-600 centipoises in purified water.
- Storage conditions: +4°C.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected on GD6 for analysis.
- For groups 1 and 3, samples were taken from the middle stratum for concentration analyses.
- For groups 2 and 4, samples were taken from the bottom, middle and top stratum for concentration and homogeneity analyses. The averaged result from homogeneity analysis served as concentration verification.
All samples to be analyzed were transferred to the analytical laboratory at the Test Facility and stored at room temperature for analysis within known formulation stability period (see the section “Details on exposure » above). Analyses were performed by UPLC-UV using a validated analytical procedure.

Results: all analyzed samples for formulations prepared at nominal concentrations of 4, 8 and 12 mg/mL of Biphenyl-4,4'-diol in vehicle taken from each preparation, including the vehicle, on GD6, were in agreement with the acceptance criteria (± 15% of the nominal concentration).
The deviations from the nominal concentrations ranged from 0.9% to 4.9%.
Furthermore, the test material prepared as a suspension in the vehicle was homogenous as the RSD (Relative Standard Deviation) on the 6 aliquots was ≤ 1.2%.
Finally, no test material was present in the vehicle sample.
See also Tables 1 and 2 in the section “Any other information on materials and methods incl. tables”.

Details on mating procedure:

Females were mated by the supplier and delivered on GD0 (day of mating).

Duration of treatment / exposure:

FRom day G6 to day G28 (gestation period)

Frequency of treatment:

Once daily.

Duration of test:

The pregnant females (mated by the supplier) were delivered to the test facility on GD0, administered the test material from GD6 to GD28 and euthanized on GD29.

Dose / conc.:

0 mg/kg bw/day

Remarks:

Group 1 (control group).

Dose / conc.:

20 mg/kg bw/day

Remarks:

Group 2.

Dose / conc.:

40 mg/kg bw/day

Remarks:

Group 3.

Dose / conc.:

60 mg/kg bw/day

Remarks:

Group 4.

No. of animals per sex per dose:

22 females per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: the doses were selected based on the results of a dose-range finding prenatal developmental toxicity study in the New Zealand White (NZW) rabbit (same strain Crl:KBL(NZW)) conducted at the same test facility (reference: Study No. 20261929 "A Non-GLP Dose Range Finding Prenatal Developmental Toxicity Study of Biphenyl-4,4’-Diol by the Oral (Gavage) Route in the Rabbit", Mounier R, February 2021). In this study, 6 pregnant female NZW rabbits per group were dosed once daily (by gavage) at 0 (CMC vehicle), 50, 75 and 90 mg/kg/day from GD6 to GD29. The doses of 75 and 90 mg/kg/day were associated with 1 and 2 aborted females, respectively, likely due to excessive maternal toxicity observed in both groups (overall mean body weight loss and a marked reduction in mean food consumption during the dosing period). These doses were therefore considered to be above the maximum tolerated dose.
At 50 mg/kg/day, there was slightly lower mean body weight gain and slightly lower mean food consumption when compared with the control group but both mean values were comparable or above the historical control data means. There was no sign of a direct test material-related effect on prenatal development at any dose level. Consequently a high dose between 50 and 75 mg/kg/day was considered suitable for this main prenatal developmental toxicity study in the NZW rabbit and 60 mg/kg/day was selected.
- Animal assignment: at random.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes.
- Time schedule: cage side observations were performed at least once daily on non-dosing days. During the dosing period, cage side observations were performed before and at least once after dosing.

DETAILED CLINICAL OBSERVATIONS: Yes.
- Time schedule: the animals were removed from their cages and a detailed clinical observation was performed at least on each weighing day.

BODY WEIGHT: Yes.
- Time schedule for examinations: all animals were weighed on GD0, GD3, GD6, GD9, GD12, GD15, GD18, GD21, GD24, GD27 and GD29.

FOOD CONSUMPTION: Yes.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes.
Food consumption of each animal was recorded daily from the day of arrival to GD29. The mean (g/animal/day) was calculated for the periods GD0 to GD3, GD3 to GD6, GD6 to GD9, GD9 to GD12, GD12 to GD15, GD15 to GD18, GD18 to GD21, GD21 to GD24, GD24 to GD27, GD27 to GD29 and GD6 to GD29.

WATER CONSUMPTION: No.

POST-MORTEM EXAMINATIONS: Yes.
- Sacrifice on gestation day 29.
- Organs examined: the ovaries and uterus were removed and examined, including examination of the placentae.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes.
Examinations included:
- Gravid uterus weight: Yes. The uterus of apparently non-pregnant females was placed in ammonium
sulphide solution in order to stain any previously undetected implantation sites.
- Number of corpora lutea: Yes.
- Number of implantations, early resorptions and late resorptions: Yes. Implantations were classified as: Live fetuses, dead fetuses, early resorptions, late resorptions and empty implantation sites (in case of abortion).

Blood sampling:

No blood sampling was performed.

Fetal examinations:

- External examinations: Yes [all per litter].
- Soft tissue examinations: Yes [all per litter].
- Skeletal examinations: Yes [all per litter].
- Head (visceral and skeletal) examinations: Yes [approximately half per litter].
- Anogenital distance of all live rodent pups: No.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two-sided tests and were reported at the 1% and 5% levels, unless otherwise noted.
The pairwise comparisons of interest are listed below:
- Group 2 vs. Group 1.
- Group 3 vs. Group 1.
- Group 4 vs. Group 1.
Analyses were performed according to the matrix showed in Table 3 included in the section “Any other information on materials and methods incl. tables” when possible but excluded any group with less than 3 observations.
Parametric / Non-Parametric:
Levene’s test was used to assess the homogeneity of group variances. Groups were then compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if at least one of these tests was significant.
If the overall F-test or Kruskal-Wallis test was found significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively.
Non parametric:
The groups were compared using an overall Kruskal-Wallis test. If the overall Kruskal-Wallis test was found significant, then the above pairwise comparisons were conducted using Dunn’s test.
Incidence:
A Fisher’s exact test was used to conduct pairwise group comparisons of interest. Datasets were compared using a Dunn’s test.

Indices:

The following parental indices and litter calculations were included, where applicable:
- Pre-Implantation Loss (in %): 100 x (Number of corpora lutea - Number of implants)/Number of corpora lutea.
- Post-Implantation Loss (in %): 100 x (Number of implants - Number of live foetuses)/Number of implants.
- Sex Ratio (% Males): 100 x (Number of male fetuses)/Total number of fetuses.
- Litter % of Fetuses With Abnormalities: 100 x (Number of fetuses in litter with a given finding)/Number of fetuses in litter examined.

Historical control data:

Historical control data were provided to allow comparison with concurrent controls.
These data were values compiled from undosed and control New Zealand White (Crl: KBL[NZW]) rabbits in previous studies performed in the period 2013 to 2018.
These data included: maternal body weight, maternal food consumption, caesarean data, malformations and fetal examinations.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Consistent with lower mean food consumption at 60 mg/kg/day (see the section on food consumption below), there was a higher incidence of days with reduced fecal output in this group during the dosing period compared with the control group.
There were 14, 15 and 17 females at 20, 40 and 60 mg/kg/day, respectively, with pink traces observed around urine-soaked food pellets compared with none in the concurrent control. This finding, observed only when urine was in contact with food pellets, was considered not toxicologically relevant.
One female in the 20 mg/kg/day group had decreased activity and was noted lying down during dosing on GD23. This isolated finding in the low dose group was considered incidental. Thin fur covering/fur loss was occasionally noted among treated and control groups and was considered incidental or related to the pregnancy status of the females. Other incidental clinical signs were noted such as vocalization, skin lesions, scabs, broken nail, swollen and pale, reduced size or soft feces.

Mortality:

no mortality observed

Description (incidence):

There was no mortality until the scheduled euthanasia on GD29 except for one female from the control group that aborted on GD25 and was subsequently euthanized the same day (see section "number of abortions). This isolated finding in the control group was incidental.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was a test material-related slightly lower mean body weight gain at 60 mg/kg/day compared with the control group (-9%) and the historical control data (HCD) mean (-25%) over the dosing period (GD6 to GD29). However, this difference of body weight gain was not statistically significant through to termination so this change was considered not adverse.
Mean body weight gain at 20 and 40 mg/kg/day was higher than that of the control (but again the differences were not statistically significant).
See also Table 4 in section "Any other information on results incl. tables" and Figure 1 in the section "Overall remarks, attachments".

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There was a test material-related lower mean food consumption during the dosing period (GD6 to GD29) at 60 mg/kg/day when compared with the control group (-10%) and the historical control data (HCD) mean (-13%). The difference was considered adverse in view of the magnitude of the change (≥ 10% when compared with both the HCD mean and the control group).
Mean food consumption at 20 and 40 mg/kg/day was generally higher than that of the concurrent control and consistent with the mean HCD value.
See also Tables 5 and 6 in section "Any other information on results incl. tables".

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There was no test material-related effect on mean gravid uterus weight in any group. Slightly lower mean gravid uterus weight in all treated groups, principally at 60 mg/kg/day (-15%), compared with the concurrent control group, was due to incidental differences in the mean live litter size.
See also Table 7 in section "Any other information on results incl. tables".

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There was no test material-related macroscopic finding in any group. Cysts on the oviducts were noted for 4, 5 and 3 females at 20, 40 and 60 mg/kg/day, respectively. These non dose-related findings are part of the background of changes in this strain of rabbit and were considered incidental. One female in each of the 20 mg/kg/day and control groups had dark foci on the lungs as well as adherent and abnormal consistency of the lungs for the female given 20 mg/kg/day. These isolated findings were considered incidental.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Pink traces were observed for a number of females around urine-soaked food pellets in all treated groups during the dosing period compared with none in the control. This finding observed in a non-dose-related manner and only when urine was in contact with food pellets was considered not toxicologically relevant.

Number of abortions:

effects observed, non-treatment-related

Description (incidence and severity):

One female from the control group aborted on GD25 and was subsequently euthanized. Body weight gain and food consumption for that animal from GD21 to GD24 were comparable with that of the other control group females and clinical signs noted were limited to thin fur cover and reduced fecal output observed on GD24 or GD25. This isolated finding in the control group was incidental.

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

Due to incidental differences in the mean number of corpora lutea and the percentage pre-implantation loss, the mean number of implantations was slightly lower in each of the treated groups, principally at 60 mg/kg/day, compared with the concurrent control. However, values in the treated groups were consistent with the Historical Control Data (HCD).
Moreover, there was no test material-related effect on the percentage of post-implantation loss in any group.
See also Table 8 in section "Any other information on results incl. tables".

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

There were 19/21, 20/22, 22/22 and 21/22 pregnant females at the terminal caesarean in the control, 20, 40 and 60 mg/kg/day groups, respectively. All of the females had viable fetuses. See also Table 8 in section "Any other information on results incl. tables".

Key result

Dose descriptor:

NOAEL

Effect level:

>= 40 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

food consumption and compound intake

Key result

Dose descriptor:

NOAEL

Effect level:

>= 40 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There was no test material-related effect on mean fetal weight in any group. See also Table 8 in section "Any other information on results incl. tables".

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There was no test material-related effect on sex ratio in any group. See also Table 8 in section "Any other information on results incl. tables".

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

Consistent with the incidentally lower mean number of uterine implantations, mean live litter size was slightly lower in the treated groups (10.1, 9.5 and 9.2 live fetuses at 20, 40 and 60 mg/kg/day, respectively) when compared with the concurrent control group (10.4 live fetuses) but was equivalent to the HCD mean (9.3 live fetuses).
See also Table 8 in section "Any other information on results incl. tables".

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There was no test material-related external malformation in any group. One fetus in the 40 mg/kg/day group had hyperflexion of the forepaws and 1 fetus in the 20 mg/kg/day had an omphalocele. Both malformations are present in the Historical Control Data (7 occurrences each 2016-2018) and in the absence of similar findings in the high dose group, these isolated cases were considered incidental.
See also Tables 9 and 10 in section "Any other information on results incl. tables".

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There was no test material-related skeletal malformation and the incidences of other skeletal variations did not suggest any association with the test material.
There were 4 (4) and 1 (1) fetuses (litters) in the 20 and 40 mg/kg/day, respectively, with malformed thoracic vertebrae with or without branched, fused or absent rib(s). Multiple abnormalities of the thoracic vertebrae, generally with associated rib changes, are commonly observed in the historical control data (15 cases 2016-2018) and in the absence of similar findings in the 60 mg/kg/day group, these findings were considered incidental.
Fusion of the nasal bones was also noted for 1 fetus in the 40 mg/kg/day. This isolated finding from a single litter in the mid dose group was considered incidental.
See also Tables 9 and 10 in section "Any other information on results incl. tables".

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There was no test material-related visceral malformations and the incidences of other visceral variations did not suggest any association with the test material. There were 1 (1) and 2 (2) fetuses (litters) in the 0 and 60 mg/kg/day, respectively, with a retroesophageal subclavian artery. This finding, noted also in the concurrent control group, is also present in the Historical Control Data (HCD) at a comparable incidence (2 cases in the same study and 7 in total 2016-2018) and therefore considered incidental.
One fetus at 20 mg/kg/day had a small ovary. This isolated finding from a single litter in the low dose group was considered incidental.
One fetus at 60 mg/kg/day had malpositioned kidney. This malformation is present in the HCD (2 occurrences in 2016-2018) so this isolated finding was considered incidental.
See also Tables 9 and 10 in section "Any other information on results incl. tables".

Key result

Dose descriptor:

NOAEL

Effect level:

>= 60 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: no remark

Key result

Developmental effects observed:

no

Lowest effective dose / conc.:

60 mg/kg bw/day

Treatment related:

no

Body weight and weight changes:

Table 4: Mean body weight gains (GD6 to GD29)

	Group (mg/kg/day)
	0	20	40	60
Mean Value (g)	321.1	360.4	423.9	290.6
Versus Control group	-	+12%	+32%	-9%
Versus mean HCD value (389 g)	-17%	-7%	+8%	-25%

- : not applicable.

HCD: Historical Control Data (2018).

 

 

Food consumption:

Table 5: Mean food consumption over the treatment period: GD6 to GD29

	Group (mg/kg/day)
	0	20	40	60
Mean Value (g/animal/day)	130.6	136.1	141.6	118.0
Versus Control group	-	+4%	+8%	-10%
Versus mean HCD value (135.8 g/day)	-4%	0%	+4%	-13%

- : not applicable.

HCD: Historical Control Data (2018).

 

 

Table 6: Mean food consumption over the various sub-periods

Day(s) relative to Mating	Group 1 0 mg/kg /day	Group 2 0 mg/kg /day	Group 3 0 mg/kg /day	Group 4 0 mg/kg /day
0-3 (G)	115.51 (37.22)	102.07 (34.30)	97.64 (45.33)	108.25 (40.85)
3-6 (G)	158.84 (28.23)	154.67 (19.01)	146.67 (52.01)	147.24 (26.38)
6-9 (G)	171.23 (18.12)	165.85 (21.25)	159.42 (41.19)	142.46** (30.42)
9-12 (G)	162.47 (25.58)	160.93 (20.43)	155.83 (33.50)	138.90* (34.58)
12-15 (G)	128.95 (39.91)	126.83 (47.30)	133.68 (41.30)	100.71 (46.68)
15-18 (G1)	128.72 (41.30)	138.22 (56.81)	147.76 (35.79)	112.13 (40.87)
18-21 (G1)	122.05 (54.00)	142.72 (41.79)	153.05 (37.29)	127.46 (31.91)
21-24 (G)	108.81 (29.67)	130.50 (31.83)	132.52* (34.43)	112.54 (29.19)
24-27 (G)	103.37 (30.31)	105.07 (31.94)	118.68 (37.67)	96.10 (26.72)
27-29 (G)	115.42 (30.67)	110.20 (36.21)	127.30 (39.11)	110.95 (24.86)
6-29 (G)	130.59 (25.35)	136.12 (25.23)	141.63 (22.79)	117.95 (24.16)

(G) - Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01

(G1) - Kruskal-Wallis & Dunn

(number) : standard deviation.

 

Gravid uterus weight:

Table 7: Mean gravid uterus weight

	Group 1 0 mg/kg /day	Group 2 0 mg/kg /day	Group 3 0 mg/kg /day	Group 4 0 mg/kg /day
Gravid Uterus Weight (g) (G)	591.98 (80.55)	545.81 (80.54)	556.93 (82.15)	505.53** (94.31)

(G) - Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01

(number) : standard deviation.

 

Litter data:

Table 8: Summary of ovarian and uterine observations and litter observations

 

Day(s) relative to Mating	Group 1 0 mg/kg /day	Group 2 0 mg/kg /day	Group 3 0 mg/kg /day	Group 4 0 mg/kg /day
Female with live fetuses [f]	19 (100%)	20 (100%)	22 (100%)	21 (100%)
Number of Corpora Lutea [k]	11.8 (2.0)	11.1 (2.1)	10.9 (1.5)	10.7 (1.7)
Number of Implantations [k]	11.3 (1.7)	10.5 (2.4)	10.3 (1.5)	9.7 (2.2)
Pre-implantation Loss (%) [k]	4.16 (4.99)	5.49 (7.46)	5.34 (6.48)	9.39 (13.78)
Total Number of Resorptions [k]	0.9 (1.1)	0.5 (0.7)	0.8 (1.4)	0.5 (0.9)
Number of Early Resorptions [k]	0.2 (0.4)	0.3 (0.6)	0.6 (1.0)	0.4 (0.7)
Number of Late Resorptions [k]	0.7 (1.1)	0.2 (0.5)	0.2 (0.5)	0.1 (0.4)
Total Number of Fetuses [k]	10.4 (1.8)	10.1 (2.3)	9.5 (1.9)	9.2 (2.2)
Number of Live Fetuses [k]	10.4 (1.8)	10.1 (2.3)	9.5 (1.9)	9.2 (2.2)
Number of Live Male Fetuses [k]	5.1 (2.5)	5.0 (1.7)	5.2 (1.7)	4.4 (2.3)
Number of Dead Fetuses [k]	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)
Post-implantation Loss (%) [k]	7.84 (9.97)	4.09 (6.11)	8.28 (14.70)	5.42 (9.07)
Live Male Fetus/Litter (%) [k]	48.49 (19.58)	49.58 (15.52)	56.09 (16.78)	47.46 (19.29)
Number of Live Female Fetuses [k]	5.2 (2.1)	5.1 (2.0)	4.2 (1.9)	4.8 (2.2)
Mean Fetal Weight all (g) [G]	37.58 (4.52)	38.11 (5.05)	39.89 (4.73)	38.52 (5.22)
Mean Fetal Weight males (g) [G]	38.00 (4.61)	38.55 (4.73)	40.67 (5.96)	38.78 (5.69)
Mean Fetal Weight females (g) [G]	37.39 (5.44)	37.86 (5.85)	39.34 (5.46)	38.14 (5.28)

(number) : standard deviation

[f] - Fisher's Exact

[G] - Anova & Dunnett

[k] - Kruskal-Wallis & Dunn

 

Fetuses data:

Table 9: Foetal examinations design

The numbers of fetuses (litters) submitted to the different examinations were as follows:

Group No. and Dose Level (mg/kg/day)	Group 1 0 mg/kg /day	Group 2 0 mg/kg /day	Group 3 0 mg/kg /day	Group 4 0 mg/kg /day
External examination	197 (19)	201 (20)	208 (22)	193 (21)
Visceral examination: Body Head	197 (19) 96 (19)	201 (20) 94 (20)	208 (22) 99 (22)	193 (21) 91 (21)
Skeletal examination: Body Head	197 (19) 101 (19)	201 (20) 107 (20)	208 (22) 109 (22)	193 (21) 102 (21)

 

 

Table 10: Summary of malformations – Individual descriptions

Dose Level (mg/kg/day)	Female No.	Fetus No.	Malformation(s)#
0	22	7	Domed head Phocomelia Misshapen brain Misshapen olfactory lobes Bent humeri, radii, ulnae, femurs and ilia
	67	7	Retroesophageal subclavian artery
	68	8	Malformed lumbar vertebrae
20	8	2	Small left ovary
	9	5	Malformed thoracic vertebrae
	28	5	Branched ribs Absence of 1 or more thoracic vertebrae
		6	Branched and absent ribs Fused vertebrae Malformed thoracic vertebrae
	29	10	Omphalocele
	51	1	Fused ribs Malformed thoracic vertebrae
40	11	1	Malformed thoracic vertebrae
	33	1	Fused nasal bones
	34	4	Branched rib
	79	5	Fused ribs
	81	3	Hyperflexed forepaws
60	17	6	Retroesophageal subclavian artery
	84	5	Retroesophageal subclavian artery
	86	8	Malpositioned kidney

#: Including external, visceral and skeletal examinations.

 

 

Conclusions:

In conclusion, daily oral (gavage) administration of the test material at doses of 20, 40 and 60 mg/kg/day in 0.5% CMC at a dose volume of 5 mL/kg, in the pregnant New Zealand White rabbit, from GD6 to GD28, was associated with slightly lower mean body weight gain and food consumption with reduced fecal output at 60 mg/kg/day, considered adverse in view of the magnitude of the change (≥ 10% when compared with both the Historical Control mean Data and the control group).
There was no sign of maternal toxicity at 20 or 40 mg/kg/day and no test-material related prenatal toxicity in any group.
No Observed Adverse Effect Levels (NOAELs) were therefore set at 40 mg/kg/day for maternal toxicity and 60 mg/kg/day for prenatal toxicity.

Executive summary:

A GLP study following the OECD guideline 414 was performed on New Zealand White pregnant female rabbits. The test material was prepared in 0.5% Carboxymethyl cellulose (CMC) and administered orally (gavage) at a dose volume of 5 mL/kg/day at dose levels of 0, 20, 40 and 60 mg/kg bw /day from implantation to the day before caesarean section (from Gestation Day (GD) 6 to GD28).

The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, food consumption, maternal gross necropsy findings, litter parameters and fetal morphology.

All analyzed samples for formulations prepared at nominal concentrations of 4, 8 and 12 mg/mL were within the acceptance criteria for accuracy and homogeneity. No test material was present in the vehicle sample.

There was no test material-related unscheduled death in any group.

Consistent with the lower mean food consumption noted at 60 mg/kg/day, there was a higher incidence of reduced fecal output in this group during the dosing period when compared with the control.

Pink traces were observed for a number of females around urine-soaked food pellets in all treated groups during the dosing period compared with none in the control. This finding observed in a non-dose-related manner and only when urine was in contact with food pellets was considered not toxicologically relevant.

Slightly lower mean body weight gain and food consumption noted at 60 mg/kg/day were considered adverse in view of the magnitude of the change (≥ 10% when compared with both the Historical Control mean Data and the control group).

There was no test material-related macroscopic finding in any group.

There was no test material-related effect on the mean gravid uterus weight in any group.

There were 19, 20, 22 and 21 pregnant females at the terminal caesarean examinations in the control, 20, 40 and 60 mg/kg/day groups, respectively, all of which had viable fetuses.

The mean numbers of corpora lutea and uterine implantations were incidentally slightly lower in the treated groups compared with the control.

There was no test material-related effect on the post-implantation data in any group.

There was no test material-related effect on the mean fetal weight or the fetal sex ratio in any group.

There was no test material-related external, visceral or skeletal malformation or variation in any group.

In conclusion, daily oral (gavage) administration of the test material at doses of 20, 40 and 60 mg/kg/day in 0.5% CMC at a dose volume of 5 mL/kg in the pregnant New Zealand White rabbit from GD6 to GD28 was associated with slightly lower mean body weight gain and food consumption with reduced fecal output at 60 mg/kg/day considered adverse in view of the magnitude of the change (≥ 10% when compared with both, the HCD mean and the control group).

There was no sign of maternal toxicity at 20 or 40 mg/kg/day and no test-material related prenatal toxicity in any group.

No Observed Adverse Effect Levels (NOAELs) were therefore set at 40 mg/kg/day for maternal toxicity and 60 mg/kg/day for prenatal toxicity.

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted in accordance with OECD Test guideline No 422 and with the principles of GLP

Reason / purpose for cross-reference:

reference to same study

Remarks:

This study (OECD 422) investigates both the fertility toxicity and the developmental toxicity.

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Purity: 99.96%

Species:

rat

Strain:

Crj: CD(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Crj:CD(SD)IGS rats used but no details provided regarding supplier
- Age at study initiation: (P) x wks; (F1) x wks No data
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g No data
- Fasting period before study: No data
- Housing: No data

- Diet (e.g. ad libitum):
- Water (e.g. ad libitum):
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data
- Humidity (%): No data
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): No data

IN-LIFE DATES: From: not stated To: not stated

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% sodium carboxymethylcellulose solution

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water):0.5% sodium carboxymethylcellulose solution used as suspending agent

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No information provided

Details on mating procedure:

- M/F ratio per cage: 1:1.

Duration of treatment / exposure:

Males were dosed for 42 days prior to mating; the females were treated for 14 days prior to mating and then until day 4 of lactation. The female satellite animals were treated or 42 days. The recovery period for males and satellite females was 14 days. The males were terminated on day 43 of treatment (day 15 of recovery) and the females were terminated on day 5 of lactation, satellite females on day 15 of recovery and the offspring were terminated 4 days after birth.

Frequency of treatment:

Once daily for up to 42 days

Duration of test:

6 weeks

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

8 mg/kg bw/day (actual dose received)

Dose / conc.:

40 mg/kg bw/day (actual dose received)

Dose / conc.:

200 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

twelve; an additional five rats per sex were allocated to the satellite groups for retention through the recovery phase.

Control animals:

yes, concurrent vehicle

Details on study design:

No further details provided.
As a screening study this investigation was limited to the parental generation and F1 offspring only. The repeated adminstration phase for males (42 days) and females (14 days) prior to mating provides some subacute toxicity data and the treatment of females through gestation gives some reproductive and developmental toxicity information.

The males and females were dosed for up to 42 days by oral gavage and this subacute exposure was used to address the short-term toxicology endpoints. Clinical signs, bodyweight gain, food consumption haematology, clinical chemistry and urinalysis parameters were recorded together with a limited functional observation battery, organ weights and macroscopic and microscopic examinations at termination.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at weekly intervals - assessment in homecage, then in observers hands and then outside the homecage
Assessments included posture, sleeping, locomotion,vocalisations, tremors and convulsions, response to capture and handling, salivation, grading of heart beat, body temperature, exophthalmus and pupil size, exhibition of any discoloration of fur,skin or lacrimation. Outside the cage the assessment of posture, grooming, vocalisations, occurrence of straub tail, gait, tremor, convulsion pilo-erection and palpebral opening; exploration and respiratory rate and any exhibiion of stereotypy or bizarre behaviour.

A functional assessment was completed on the final day of treatment to assess various reflex responses.

BODY WEIGHT: recorded on days 1, 7, 14, 21, 28, 35 and 42 of treatment and days 1, 7, and 14 of recovery for males.
Females were weighed on days 1, 7, and 14 of treatment; days 0, 7, 14 and 20 of pregnancy and days 0 and 4 of lactation
Bodyweights and bodyweight gains are reported.

Food consumption was recorded for the males and for the satellite females (treated at 200 mg/kg) on days 1-2, 7-8, 14-15, 29-30, 35-36 and 41-42 and then days 6-7 and 13-14 of the recovery phase.

Urinalysis was performed on Day 31 and 32 for males and females respectively. Parameters assessed included colour, turbidity, pH, protein, glucose, ketone and bilirubin levels, occult blood, urobilinogen and presence in urinary sediment of crystals by incidence and type/shape and urinary specific gravity.

Clinical pathology in the form of standard haematology and biochemical parameters were assessed on Day 43 of treatment and on Day 15 of the recovery phase for male rats and on Day 5 of lactation for the females.

Organ weights were recorded on Day 43 of treatment and on Day 15 of the recovery phase for male rats and on Day 5 of lactation for the females.


Necropsy was completed on termination of treatment or after completion of the recovery phase or on day 5 of lactation. Macroscpic findings were recorded at each scheduled necropsy. Histopathological findings were documented for tissues sampled following each of the scheduled necropsy events. The list of tissues examined was in accordance with the test guideline.

The reproductive performance of the parents was recorded for the twelve pairings per treatment group; the number of pairs copulating provided the copulation index and the number of pregnant females was used to calculate the fertility index. the mean number of days from pairing to copulation was also calculated

All females examined for oestrous cyclicity . The number of rats showing a 4 day cycle in the pre-treatment phase was recorded, together with number with 5-day cycle. Mean cycle duration was calculated

Ovaries and uterine content:

No data

Fetal examinations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring on day 0 of lactation:
number of pups, stillbirths, live births, birth index and live birth index

Bodyweights recorded on day 0 and 4 of lactation

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

Necropsy of live pups surviving to lactation day 4- examination of external and visceral changes

Statistics:

No data

Indices:

Number of newborn, delivery index, number of live newborn, birth index and live bith index calculated on day 0 of lactation. On day 4 of lactation the number of live pups, viability index and sex ratio were calculated.

Historical control data:

No data

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Oral administration of 4,4'-biphenyldiol at dose levels of 8, 40 or 200 mg/kg bw/day did not cause death or a moribund condition in any animals. None of the scores obtained during detailed clinical observations differed in a biologically significant manner between the control and the compound-treated groups.
No apparent changes were observed in general clinical conditions, except that urine in the 200 mg/kg-treated group became cloudy with elapse of time after excretion. Urinalysis was performed on treatment days 31 and 32 in male and female rats, respectively, calcium oxalate-like urinary crystal sediments were found in males given 200 mg/kg and in females given 40 or 200 mg/kg. Turbidity was enhanced at dose levels of 40 or 200 mg/kg, and urinary specific gravity was decreased in these females. These changes in urine parameters were not found when urinalysis was performed 11 days after cessation of the treatment (on day 11 of recovery) in either sex for the satellite animals.
Body weight, weight gain and food consumption were not affected by treatment at any dose level in either sex. No animals showed any abnormality in functional parameters after the final treatment.
At necropsy on termination of the treatment, no apparent effects of the compound were found on haematological or blood-biochemical examination at any dose level of the compound in either sex.

The reproductive and developmental toxicity indices evaluated in this screening study indicated no adverse effects on the dams. No effects were apparent in the female oestrous cycle either during the pre-treatment, mating or treament phases. The reproductive indices included the numbers of pairings examined, copulating and the number of resulting pregnant females and the length of pairing time required before copulation. There were no changes between control and treated groups.

Key result

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

External malformations:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no adverse effects of administration on offspring viability, morphology or growth.
Pup development indices were evaluated to assess the duration of gestation, numbers of corpora lutea and implantations, the numbers of pups born, numbers of live born and sex ratio for the liveborn pups and survival and viability of pups to day 4 of lactation was also evaluated . None of the comparisons between treated and control groups revelaed any significant changes in these indices or any statistically significant effects. Similarly no effects were apparent when newborn or neonate (lactation day 4) pup weights were assessed.

Morphological evaluations for external and visceral changes showed no effects on the live pups at birth or those that survived to lactation Day 4. The dead pups examined (3 controls and 1, 6 or 12 pups in the 8, 40 or 200 mg/kg bw/day groups) also had no external and visceral changes .

Key result

Dose descriptor:

NOAEL

Effect level:

>= 200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effect observed

Key result

Developmental effects observed:

no

Combined repeat dose and reproductive/developmental toxicity screening test for 4,4’-biphenyldiol

	4,4’-biphenyldiol mg/kg bw/day
	0	8	40	200
No. of females examined	12	12	12	12
No. of females showing 4-day cycle in pre-treatment phase	12	12	12	12
No. of females showing 4-day cycle in treatment phase	12	11	12	12
No. of females showing 4 and 5-day cycle in treatment phase	0	1	0	0
Mean length of oestrous cycle in days	4.0±0.0	4.0±0.1	4.0±0.0	4.0±0.0
Number of vaginal oestrous during mating period	1.0±0.0	1.0±0.0	1.0±0.0	1.0±0.0

 

	4,4’-biphenyldiol mg/kg bw/day
	0	8	40	200
No. of pairs examined	12	12	12	12
No. of pairs copulated	11	11	12	12
Copulation index	91.7	91.7	100.0	100.0
No. of pregnant females	11	11	10	12
Fertility index	100.0	100.0	83.3	100.0
Number of pairing days  to copulation	2.5±1.1	2.6±1.2	2.7±0.8	2.7±1.2

 

  Development of pups up to Day 4 of lactation

	4,4’-biphenyldiol mg/kg bw/day
	0	8	40	200
No. of pregnant females	11	11	10	12
No. of pregnant females with live neonates	11	11	10	12
Gestation index	100	100	100	100
Gestation length in days	22.4±0.5	22.4±0.5	22.0±0.0	22.4±0.5
Number of corpora lutea	15.9±1.6	16.5±1.1	16.2±1.2	15.2±1.5
Number of implantations	15.5±1.7	14.3±3.6	16.0±1.6	14.7±1.7
Implantation Index	97.1±4.3	86.9±21.6	98.7±4.2	96.7±5.3
Day 0 of lactation
Number of newborn	14.5±2.3	13.8±4.0	15.1±1.7	14.0±1.8
Delivery index	93.2±6.9	94.6±12.2	94.4±4.8	95.5±5.2
Number of live newborn	14.3±2.3	13.8±4.0	14.6±1.6	12.8±2.9
Birth index	92.0±7.6	94.6±12.2	91.5±8.1	88.8±20.1
Live birth index	98.7±3.0	100.0±0.0	97.0±7.5	93.3±20.8
Sex ratio on day 0	54.8±14.5	49.4±17.4	52.5±15.1	51.6±14.7
Day 4 of lactation
Number of live pups	13.9±2.1	13.6±4.0	14.5±1.5	12.4±4.2
Viability index	97.7±4.0	98.8±2.6	99.4±2.0	91.7±28.9
Sex ratio on day 4	54.4±14.9	49.3±17.9	52.1±15.9	50.8±15.2

The vehicle control was 5 ml/kg of 0.5% CMC Na solution.
Gestation index      (number of pregnant females with live newborns/number of pregnant females) x 100,%
Implantation index (number of implantations/number of corpora lutea) x 100,%
Delivery index        (number of newborns/number of implantations) x 100,%
Birth index             (number of live newborns/number of implantations) x 100,%
Live birth index      (number of live newborns/number of newborns) x 100,%
sex ratio (day 0)     (number of male live newborns / number of live newborns) x 100,%
viability index        (number of live pups on day 4 of lactation/ number of live newborns) x 100,%
sex ratio (day 4)     (number of male live pups on day 4 of lactation / number of live pups on day 4 of lactation) x 100,%

Bodyweight of pups up to Day 4 of lactation

	4,4’-biphenyldiol mg/kg bw/day
	0	8	40	200
Day 0 of lactation
Number of live newborn
male	7.7±2.1	6.9±3.0	7.6±2.1	6.5±2.2
female	6.5±2.5	6.9±3.3	7.0±2.6	6.3±2.7
Birthweight of live newborn
Males	6.8±0.8	6.9±0.8	6.5±0.5	6.6±.0.8
females	6.6±0.6	6.6±0.7	6.0±0.4	6.2±0.8
Day 4 of lactation
Number of live pups
Males	7.5±2.3	6.8±3.1	7.5±2.2	6.8±2.0
females	6.4±2.2	6.8±3.2	7.0±2.6	6.7±2.5
Birthweight of pups
Males	10.6±1.7	10.7±1.1	10.2±1.1	10.8±1.0
females	10.3±1.4	10.4±1.4	9.8±1.0	10.0±0.9

Morphological findings of pups

	4,4’-biphenyldiol mg/kg bw/day
	0	8	40	200
Dead pups
Number of dead pups examined	3	1	6	12
External observations	3	1	6	12
Visceral observations	0	0	5	1
No. of pups with external changes	0	0	0	0
No. of pups with visceral changes	0	0	0	0
Live pups at birth (Day 0)
Number of live neonates examined	157	152	146	154
Number with external changes	0	0	0	0
At necropsy on Day 4
Number of pups examined	153	150	145	149
No. of pups with external changes	0	0	0	0
No. of pups with visceral changes	0	0	0	0

Conclusions:

Treatment with up to 200 mg/kg bw/d did not affect early offspring development. The no observed adverse effect level (NOAEL) for repeat dose toxicity of 4,4'-biphenyldiol is considered to be 40 mg/kg bw/d. In the absence of any effects on the parameters measured. the NOAEL for developmental toxicity is considered to be 200 mg/kg/ bw/d.

Executive summary:

A combined repeat dose and reproductive/developmental toxicity screening test was conducted in rats according to the OECD Test Guideline 422 with 4,4'-biphenyldiol. No effects on developmental parameters were observed at any dose level. In the absence of any effects on developmental parameters, a NOAEL for developmental toxicity of 200 mg/kg bw/d can be determined. A NOAEL for general toxicity of 40 mg/kg bw/d can be determined based on liver effects at the highest dose level. Effects of treatment on urinalysis parameters seen at 40 and 200 mg/kg bw/d are not considered to be of toxicological significance.

Reference 4

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 November 2016 - 6 April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to the OECD Test Guideline No. 414.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

January 2001

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

August 1998

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

2008

GLP compliance:

yes

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI (Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Number of animals in the study: 88 virgin mated females. The females were mated at the supplier with a documented day of mating. They were received at the Test Facility on day 1 of gestation (G0).
- Source: Charles River Laboratories France, Domaine des Oncins, 69210 Saint-Germain-Nuelles, France.
- Age at mating: 10 to 13 weeks.
- Weight at mating: 197 to 255 g.
- Acclimatisation period before study: 6 days between animal arrival and the start of treatment
- Housing: One air-conditioned room in a barrier protected unit. Females were individually housed in plastic cages meeting European directive 2010/63/EU requirements.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 6 days between animal arrival and the start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): >35 %
- Air changes (per hr): At least 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light (artificial)/12 hours dark

IN-LIFE DATES: From: 28 February to 03 March 2017 (G0 Animal arrivals) To: 24 March 2017 (last caesarean)

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5 % [w/v] Carboxymethylcellulose 300-600 centipoises in water for injection

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Rate of preparation: Weekly
- Storage of formulations (including vehicle control): Refrigerated (between +2 and +8 °C).
- Homogeneity of the test item in the vehicle: The formulations at concentrations of 1 and 200 mg/mL in the vehicle are homogeneous (based on the analytical results in a specific study).
- Stability of the test item in the vehicle: 11 days at room temperature (between +17 and +27 °C) and refrigerated (5±4 °C), and 26 days frozen (between -15 and -25 °C) (based on the analytical results of a specific study).

VEHICLE
- Concentration in vehicle: 5, 15 and 50 mg/mL

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of formulations:
4x1 g samples were taken from each formulation, including the vehicle, on a single occasion during the treatment of the main study phase only.
The samples were stored frozen (between -25 and -15 °C).
The first set of samples (2x1 g) was dispatched on dry ice to the Test Site for analysis.

Details on mating procedure:

The females were mated at the supplier with a documented day of mating. They were received at the Test Facility on day 1 of gestation (G0).

Duration of treatment / exposure:

From G 6 to G 20 inclusive

Frequency of treatment:

Once daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group number: 1 (Control)

Dose / conc.:

25 mg/kg bw/day (nominal)

Remarks:

Group number: 2 (Low dose)

Dose / conc.:

75 mg/kg bw/day (nominal)

Remarks:

Group number: 3 (Intermediate dose)

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

Group number: 4 (High dose)

No. of animals per sex per dose:

22

Control animals:

yes, concurrent vehicle

Details on study design:

- Group 1 animals (Control) received the vehicle (0.5 % [w/v] Carboxymethylcellulose).
- Volume of administration: 5 mL/kg/day. Individual dose volumes were adjusted using the latest body weight.
- Rationale for the dose selection: the doses for the main study phase were selected based on the DRF phase where oral (gavage) administration of the test item in the female rat from G 6 to G 20 was above the maximum tolerated dose (i.e. severe body weight loss, negligible food consumption, poor clinical condition and renal changes) at 500 and 750 mg/kg/day. Dose of 250 mg/kg/day was associated with reduced body weight change and food consumption throughout the dosing period.
Consequently, in the main phase, a high dose of 250 mg/kg/day was selected in order to obtain acceptable maternal toxicity. The low and intermediate doses selected were 25 and 75 mg/kg/day in order to identify a possible No Observed Adverse Effect Level.

Maternal examinations:

OBSERVATIONS:
- Morbidity/mortality: All animals were observed at least twice daily.
- Clinical signs: The animals were observed daily during the study. To detect any abnormalities in appearance, behaviour or other signs of reaction to treatment, the animals were observed before and twice after dosing. A full clinical examination was performed on each weighing day.
- Body weight: All animals were weighed individually on G 0, G 6, G 9, G 12, G 15, G 18 and G 21.
- Food consumption: Individual food consumption was measured for the periods G 0 to G 6, G 6 to G 9, G 9 to G 12, G 12 to G 15, G 15 to G 18 and G 18 to G 21.

TERMINAL EXAMINATIONS:
- Necropsy: All females were killed on G 21. They were dissected and examined for macroscopic pathological changes. Abnormal organs were sampled and preserved in 10 % neutral formalin but not examined further.
The ovaries and uterus of each female were removed and examined as below.

Ovaries and uterine content:

The ovaries and uterus of each female were removed and examined including examination of the placentae.
The following data were recorded:
- pregnancy status
- number of corpora lutea
- number of implantations
- number and distribution of live foetuses
- number and distribution of embryonic/foetal deaths, classified as follows:
> early: only placenta visible at termination
> late: both placenta and embryonic tissue visible at termination
> dead foetus.
- gravid uterus weight
- individual foetal weights
- individual placental weights
- foetal sex.

Fetal examinations:

- Each foetus was examined for external defects.
- All live foetuses were killed by an oral administration of sodium pentobarbitone.
- Approximately one half of each litter was submitted to fresh visceral examination of the body (abdominal and thoracic cavities) and then processed for skeletal examination. The skeletal examinations were performed following maceration of the soft tissues with aqueous potassium hydroxide, staining of the skeleton with Alizarin red then passage into glycerol.
- The remaining half of the foetuses in each litter was fixed in Harrison’s fluid for subsequent examination of the head only by serial sectioning. The remaining carcass was retained fixed but not examined further in the first instance.
- Soft tissue and skeletal examinations were performed using a binocular microscope.

Statistics:

Statistical analyses were performed by the Provantis data acquisition system, where appropriate, as follows:
- The best transformation for the data (none, log or rank) was determined depending upon
> the normality of the data distribution tested by the Shapiro-Wilk's test
> the homogeneity of the variances across groups tested by the Bartlett's test.
- Non- or log-transformed data were analysed by parametric methods.
- Rank transformed data were analysed using non-parametric methods.
- Data were then analysed to test for a dose-related trend to detect the lowest dose at which there is a significant effect, based on the Williams test for parametric data or the Shirley's test for non-parametric data.
- Homogeneity of means was assessed by analysis of variances (ANOVA) for parametric data or Kruskal-Wallis test for non-parametric data.
- If no trend was found and means were not homogeneous, the data were analysed by parametric or non-parametric Dunnett's test to look for significant differences from the control group.
- The number of resorptions, number of dead foetuses and all litter-based percentages were analysed using non-parametric methods, i.e. Kruskal-Wallis test followed by non-parametric Dunnett’s test if the Kruskal-Wallis is significant.
- Selected incidence data were analysed using a chi2 test for all groups followed by Fisher’s two-tailed test with Bonferroni correction for each treated group versus the control if the chi2 is significant.
Microsoft Excel 2010® was employed to present certain results.

Indices:

For each group, the following parameters were calculated:
- Pre-implantation loss (in %) = [(Number of corpora lutea - Number of implantations) / (Number of corpora lutea)] x 100
- Post-implantation loss (in %) = [(Number of implantations - Number of viable foetuses) (Number of implantations)] x 100

Historical control data:

Historical control data are provided in the study report.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Partly closed eyes were noted for 2 females given 250 mg/kg/day on G 7.
Other clinical signs noted were either associated with pregnancy/parturition (piloerection at the end of the gestation period) or incidental (erythema, localized hairloss, torn claw, scabs and/or sores).

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was a dose-related lower mean body weight gain for females given 75 and 250 mg/kg/day during the first three days of treatment (G 6 to G 9) compared with the concurrent control.
Thereafter, recovery was noted for females given 75 mg/kg/day and the effect persisted up to the end of treatment (up to G 21) for females given 250 mg/kg/day (-24 % body weight gain between G6 and G21 when compared with control).
As a consequence, absolute terminal mean body weight (including mean carcass weight of the dams after correction for gravid uterus weight) was lower in the 250 mg/kg/day group compared with the concurrent control (-7 %).
This effect on mean body weight gain in the high dose group impacting terminal body weight was considered adverse.

The lower mean body weight gain noted between G 18 and G 21 for females given 25 and 75 mg/kg/day compared with the concurrent control, was considered incidental since values remained comparable with the historical control data.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There was a slightly lower mean food consumption for females given 250 mg/kg/day during the whole treatment period (G 6 to G 21) compared with the concurrent control.
As a consequence to this change, a lower mean body weight gain during the overall dosing period and a lower mean terminal body weight in the high dose group was noted.
This effect was therefore considered adverse.

The slightly lower mean food consumption noted between G 18 and G 21 for females given 75 mg/kg/day compared with the concurrent control, was considered incidental since values remained comparable with the historical control data.

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No test item-related effect on mean gravid uterus, kidneys and liver weights in any group

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test item-related macroscopic abnormalities in any group

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The pre-implantation data (mean numbers of corpora lutea, implantation sites and the percentage pre-implantation loss) were comparable in all groups.
There was no test item-related effect on embryo-foetal survival; the percentage post-implantation loss was comparable in all groups.
The mean live litter size was comparable in all groups.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

75 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were slightly lower mean placental and foetal weights in the 250 mg/kg/day group compared with the concurrent control and lower dose groups. However, the mean foetal weight value in the high dose group was comparable with the historical control data (range from 4.9 to 5.1g) and the change was of low magnitude.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There was no test item-related effect on mean foetal sex ratio in any group.

Changes in litter size and weights:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were 1, 2, 2 and 0 foetus(es) from as many litter(s) with external malformations in the control, 25, 75 and 250 mg/kg/day groups, respectively.
The foetus from the control group (dam no. 106) had a proboscis and one foetus from the 75 mg/kg/day group (dam no. 156) had a microstomia and micrognathia.
One foetus from the 25 mg/kg/day group (dam no. 124) had malrotated hindlimbs, a thread-like tail and an anal atresia. The other foetus from the 25 mg/kg/day group (dam no. 131) had an anasarca.
The last foetus from the 75 mg/kg/day group (dam no. 162) had polydactyly.
These isolated findings, not observed in the 250 mg/kg/day group, were considered incidental.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were 0, 1, 2 and 0 foetus(es) from as many litter(s) with skeletal malformations in the control, 25, 75 and 250 mg/kg/day groups, respectively.
The foetus with the thread-like tail and anal atresia observed externally in the 25 mg/kg/day group (dam no. 124) had a malformed vertebral column.
The foetus with the micrognathia observed externally in the 75 mg/kg/day group (dam no. 156) had a malformed skull.
Polydactyly observed externally in the foetus from the 75 mg/kg/day group (dam no. 162) were confirmed at the skeletal examination (presence of one additional claw).
These isolated findings, not observed in the 250 mg/kg/day group, were considered incidental.
Less severe skeletal anomalies and variations did not suggest any association with the test item and are part of the background of changes noted for this strain of rat.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were 1, 1, 0 and 0 foetus from as many litter with visceral malformations in the control, 25, 75 and 250 mg/kg/day groups, respectively.
The foetus with the anasarca observed externally in the 25 mg/kg/day group (dam no. 131) had malformed great blood vessels. This isolated finding, not observed in the higher dose groups, was considered incidental.
The control foetus with a proboscis observed externally (dam no. 106) had a malformed head.
Less severe visceral anomalies and variations did not suggest any association with the test item and are part of the background of changes noted for this strain of rat.

Key result

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: No evidence of a direct effect of the test item (slightly lower mean placental and foetal weights in the 250 mg/kg/day group, likely related to the maternal toxicity).

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

250 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Conclusions:

Under the experimental conditions of the study (according to the guideline OECD 414), the oral administration of 4,4'-biphenyldiol to the pregnant Wistar rat from G 6 to G 20 at doses of 25, 75 and 250 mg/kg/day was associated with a transient reduced body weight gain at 75 mg/kg/day and persistent reduced body weight gain and food consumption at 250 mg/kg/day. These changes had an impact on the mean terminal body weight in the high dose group and were therefore considered as adverse. The No Observed Adverse Effect Level (NOAEL) for maternal toxicity was therefore considered to be 75 mg/kg/day.

Evidence of embryo-foetal effect was restricted to slightly lower mean placental and foetal weights in the 250 mg/kg/day group. The mean foetal weight in the high dose group was comparable with the historical control data and the change was of low magnitude. As this was not correlated with morphological development changes, these findings were likely related to the maternal toxicity rather than a direct effect of the test item. The NOAEL for embryo-foetal toxicity was therefore considered to be 250 mg/kg/day.

Executive summary:

A GLP compliant-study based on the OECD guidelilne No. 414 was conducted in rat in order to provide information on the effect of the test item, 4,4'-biphenyldiol, on prenatal development.

The test item, 4,4'-biphenyldiol, was administered by daily gavage at dose levels of 25, 75 and 250 mg/kg/day to groups of 22 mated female Wistar rats from G 6 to G 20 of gestation inclusive. A control group received a similar volume (5 mL/kg) of the vehicle 5 % [w/v] Carboxymethylcellulose n water for injection).

Parameters monitored included morbidity/mortality, clinical signs, body weight and food consumption. The females were submitted to a caesarean examination on G 21 and litter parameters were recorded.

At necropsy, the females were examined macroscopically and the gravid uterus, the placenta, the kidneys and the liver were weighed. All live foetuses were

weighed, sexed and examined for external abnormalities. Half of the foetuses were examined internally prior to processing for skeletal examination. The remaining foetuses were preserved for fixed-visceral examination of the head by the modified Wilson-Barrow technique.

There was no unscheduled death in any group.

Partly closed eyes were noted for 2 females given 250 mg/kg/day on G 7 only.

There was a statistically significant lower mean body weight gain and food consumption in the 250 mg/kg/day group during the whole treatment period compared with the control. These changes impacted the mean terminal body weight in the high dose group and were therefore considered as adverse. A slightly lower mean body weight gain was also noted at 75 mg/kg/day during the first three days of treatment (G 6 to G 9) compared with the control.

There were no test item-related effect on macroscopic observations, mean gravid uterus, kidneys and liver weights in any group.

There were 22, 20, 22 and 22 pregnant females in the control, 25, 75 and 250 mg/kg/day groups, respectively, at the terminal caesarean examinations, all of which had viable foetuses.

There were no test item-related effect on embryo-foetal survival and sex ratio in any group.

There were statistically significant slightly lower mean placental and foetal weights in the 250 mg/kg/day group compared with the control. However, the mean foetal weight value in the high dose group was comparable with the historical control and the change was of low magnitude.

There were 1, 2, 2 and 0 malformed foetus(es) from as many litter(s) in the control, 25, 75 and 250 mg/kg/day groups, respectively. None of which were attributable to the test item.

To conclude under the experimental conditions of this study:

The No Observed Adverse Effect Level (NOAEL) for maternal toxicity was therefore considered to be 75 mg/kg/day.

The NOAEL for embryo-foetal toxicity was therefore considered to be 250 mg/kg/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

225 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The quality of all the database is good.
All the studies are compliant to GLP and conducted according to OECD guidelines.

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

Justification for selection of oral route:

Oral route is a potential route of human exposure during manufacture, handling or use of the test substance as specified in the applicable guidelines.

Justification for classification or non-classification

No classification for reproductive or developmental toxicity is proposed in the absence of relevant findings in the current available data.