Phenol, 2-methoxy-, reaction products with 2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane, hydrogenated

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 16 may 2016 to 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

see below

Principles of method if other than guideline:

The deviations of the study plan are:
- Dose formulation preparation: no visual examination of dose formulation homogeneity was performed from Day 1 to Day 11 of treatment.
- Determination of Rhodiantal Original IBCH concentration in dose formulations: in Week 6 (21 October 2016), the result of the group 4 formulation prepared at 100 mg/mL was found to be at -18% compared to the nominal value. Without stability data, this dose formulation was not re analyzed. No error occurred in the determination of content or in the preparation process. Following this result, supplementary analysis was performed in Week 8 and the result was found to be compliant with acceptance criteria.
- Identification: males H24953 and H24955 (group 2) were identified on the tail due to an ear tattoo error.
- Environmental conditions:the temperature recorded in the animal room was outside the target range (lower than 20°C): from 18 to 19 September 2016: down to 17.9°C for 11 hours 32 minutes and 15 seconds; and on 20 September 2016: down to 18.6°C for 3 hours 17 minutes and 26 seconds. No impact on the animals was noted during the study.
- Water consumption: control male H24949 did not have free access to water on Day 1 for approximately 24 hours, as its bottle was incorrectly positioned.
- Administration: magnetic stirring of formulations throughout the dosing procedure was not documented due to an oversight.
- Clinical observations: no clinical examination was recorded for high dose group males on Day 41, detailed clinical examination was not documented on Day 57 (Week 09) of the study due to an oversight.
- Monitoring of estrous cycle: the estrous cycle of female H25337 (group 4) was determined on Day 14 p.p. instead of on Day 15 p.p. as described in the study plan, estrous cycle was missing for control female H25295 on Day 15 p.p.
- Laboratory investigations: samples for laboratory investigations were collected from the first five females euthanized on Day 15 p.p. in each group, instead of the first five females in each group as indicated in the study plan. the exact time of the fasting start was not documented on 3rd and 14th November 2016 (i.e. on Day 14 p.p. for the first F0 delivered females).
- Thyroid hormones (parent animals and pups): the plasma volume collected in the first tube 1 was lower than 125 µL for group 3 male H24969 and female H25316, a second tube of plasma for thyroid hormone evaluation was not prepared due to an insufficient volume of plasma as follows: on Day 4 p.p. for the pups from female H25295 (group 1) and the pups from female H25316 (group 3), and on Day 43 for male H24969 (group 3), the exact time of blood sampling was not documented for female H25313 (group 2) and blood sampling time recorded for F0 females on 04 November 2016 (i.e. Day 15 p.p.) was not consistent with the centrifugation time. Therefore, the time elapsed between blood collection and centrifugation could not be determined for these females, no results are available for some F0 females as coefficient of variation was not conform. Technical oversight was noted.
- Organ weight: for female H25323 (group 3, female sacrificed due to dead litter) and for female H25332 (group 4, female sacrificed due to the presence of palpable mass on one mammary gland), the weight of thyroids with parathyroids was recorded in excess of study plan requirements, as well as the body weight prior to euthanasia (female H25332).
- Preservation of tissues (parent animals and pups): for female H25337 (group 4), the tissues sampled were whitish in color and firm in consistency as they were most probably fixed in modified Davidson's fixative instead of 10% buffered formalin, one of two thyroids missing after weighing in group 3 male H24964.

These deviations were considered not to have compromised the validity or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL :
- Name: Rhodiantal Original IBCH.
- Purity: considered as 100%.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, protected from light.
- Stability under storage conditions: Stable during 2 years.
- Stability under test conditions: The test item dose formulations were prepared on each day of treatment, stored and delivered at room temperature and protected from light.
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: The corn oil is used for the preparation of test dose formulations. The dose formulations were kept under magnetic stirring for at least 38 minutes to ensure effective solubilization of the test item (homogeneity of the formulation was confirmed at visual examination).

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

RjHan: SD

Details on species / strain selection:

The rat was chosen because it is a rodent species accepted by Regulatory Authorities for this type of study.
The Sprague-Dawley strain was selected since background data from previous studies are available at Citoxlab France.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France.
- Age/Weight at study initiation: on the first day of treatment, the males were approximately 10 weeks old and had a mean body weight of 471 g (range: 433 g to 501 g) and the females were approximately 11 weeks old and had a mean body weight of 258 g (range: 232 g to 287 g). The males and the females were sexually mature and were not siblings. The females were virgin.
- Fasting period before study: no
- Housing: The animals were individually housed, except during mating and lactation, in polycarbonate cages (Tecniplast 2154, 940 cm²: 48 x 26.5 x 21 cm) with stainless steel lids and containing autoclaved sawdust (SICSA, Alfortville, France). Individual housing of animals was chosen in order to not jeopardize gestations and lactations, and to avoid aggressive behavior between males around mating. Toward the end of gestation and during lactation, autoclaved wood shavings (SICSA, Alfortville, France) were provided to females and their litter as nesting material. Each cage contained two objects (rat hut and Nylabone) for the environmental enrichment of the animals. The cages were placed in numerical order on the racks.
- Randomization: yes, Allocation to groups: during the acclimation period, the required number of animals (40 males and 40 females) was selected according to body weight and clinical condition. The animals were allocated to groups (by sex) using a stratification procedure based on body weight (these data are not presented), so that the average body weight of each group was similar. Only 40 out of 48 females with regular estrous were allocated to the groups (regularity of estrous cycles being confirmed 2 to 3 days before the beginning of the treatment period).
- Diet: All animals had free access to SSNIFF R/M-H pelleted maintenance diet, batch Nos. 9705482 and 7997891 (SSNIFF Spezialdiäten GmbH, Soest, Germany), which was distributed weekly.
- Water: The animals had free access to bottles containing tap water (filtered with a 0.22 µm filter).
- Acclimation period: yes , males were acclimated to the study conditions for 7 days before treatment, females were acclimated to the study conditions for 5 days before the beginning of estrous cycle monitoring during the pre-treatment period.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 30 - 70%
- Air changes (per hr): about 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12h/12h

IN-LIFE DATES: From: august 25th, 2016 to november 18th 2016

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks:

Used for preparation of test item dose formulations and administered to control group (control dose formulation)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil, other vehicle cited in the OCDE guideline.
- Amount of vehicle (if gavage): The test item was administered in the vehicle (corn oil) under a constant dosage volume of 5 mL/kg/day.
- Lot/batch no. (if required): MKBW9504V.

The test item was administered as a solution in the vehicle (according to visual aspect of dose formulations prepared for CiToxLAB France/Study No. 43826 TSR).
The required quantities were mixed progressively with the vehicle in order to obtain the desired concentration.
After addition of the vehicle, the dose formulations were kept under magnetic stirring for at least 38 minutes to ensure effective solubilization of the test item (homogeneity of the formulation was confirmed at visual examination).
The test item dose formulations were prepared on each day of treatment, stored and delivered at room temperature and protected from light. Control dose formulations were stored and delivered each day at room temperature.
No suspensions were observed on the formulations. In addition, regarding the results those formulations were considered as solutions.

Details on mating procedure:

Females were paired with males from the same dose level group. One female was placed with one male, in the latter's cage, during the night.
Confirmation of mating was made in the morning by checking for the presence of a vaginal plug or for sperm in a vaginal lavage.
The day of confirmed mating was designated Day 0 p.c.
Each female was placed with the same male until mating occurs or 14 days have elapsed. Any pair with no evidence of mating after 14 days was separated and the female was placed for a further 14 days with a different male from the same dose level group who had already mated.
The pre-coital time was calculated for each female.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The Gas Chromatography with FID detection (GC-FID) analytical method for the determination of Rhodiantal Original IBCH in dose formulation samples was provided by the Sponsor and this method was validated at CiToxLAB France prior to dose formulation analysis.
The concentration of the test item in samples of each control and test item dose formulation prepared for use in Weeks 1, 3 and 6 were determined. An additional measurement of the concentration was performed for the group 4 test item dose formulation in Week 8.

Duration of treatment / exposure:

The test item, Rhodiantal Original IBCH, was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before mating, during mating, and (for females) throughout gestation and until Day 14 post-partum, at dose levels of 50, 150 or 500 mg/kg/day.

Frequency of treatment:

daily.

Details on study schedule:

The dose formulations were administered daily according to the following schedule:
in the males:
- 2 weeks before mating,
- during the pairing period (up to 17 days),
- until euthanasia (6 weeks in total),

in the females:
- 2 weeks before mating,
- during the mating period (up to 17 days),
- during gestation,
- during lactation until Day 14 p.p. inclusive,
- until euthanasia for females with no delivery.

Day 1 corresponds to the first day of the treatment period.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

150 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 per sex and per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels were selected in agreement with the Sponsor, on the basis of the results of a previous study (CiToxLAB France/Study No. 43826 TSR performed in July 2016) performed in the same species. In this study, three groups of Sprague-Dawley rats received the test item daily, by oral administration (gavage) at dose levels of 100, 300 or 1000 mg/kg/day for 2 weeks. An additional group received the vehicle control, corn oil, under the same experimental conditions. The dosing volume was 5 mL/kg/day.
At 1000 mg/kg/day, ptyalism was observed in males and females together with piloerection in one male. Adverse body weight loss followed by a lower body weight gain and adverse lower food consumption were noted in males resulting in a lower final body weight. Females showed lower body weight gain over the first week of treatment accompanied by lower food intake over the whole study period. The thymus of one male was small. The relationship of this finding to test item administration was considered to be probably related to stress.
At 300 mg/kg/day, findings such as thin appearance, severe body weight loss and lower food consumption noted in males were considered not to be test item-related. Ptyalism was observed in males and females and lower body weight gain was recorded in females over the first week of treatment.
At 100 mg/kg/day, lower body weight gain was noted in males over the whole study period and in females during the first week of treatment, leading to a slightly lower final body weight in males. This correlated with lower food intake in males over the first week of treatment.

Consequently, the dose level of 1000 mg/kg/day was considered to exceed the Maximum Tolerated Dose (MTD) under the experimental conditions of the study.

Therefore, 500 mg/kg/day was selected as the high dose level. The low-dose and mid-dose were selected using a ratio representing approximately a 3-fold interval (i.e. 50 and 150 mg/kg/day).
- Dose selection rationale:
- Rationale for animal assignment (if not random):
- Fasting period before blood sampling for clinical biochemistry:
- Other:

Positive control:

no

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
General clinical observations: From arrival, each animal was observed at least once a day as part of routine examinations. From the start of the treatment period, each animal was observed at least once a day (see § Study plan adherence), at approximately the same time, for the recording of clinical signs.

Detailed clinical observations: Detailed clinical examinations were performed on all animals once before the beginning of the treatment period and then once a week until the end of the study.
Observations included (but were not limited to) changes in the skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behavior (e.g. self-mutilation, walking backwards) were also checked.


BODY WEIGHT: Yes
The body weight of each male was recorded on the first day of treatment (Day 1), then at least once a week until euthanasia including on the day before euthanasia.
The body weight of each female was recorded on the first day of treatment (Day 1), then once a week until mated and on Days 0, 7, 14 and 20 post-coitum (p.c.) and Days 1, 4, 8 and 13 p.p.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): yes
The quantity of food consumed by each male was measured once a week, from the first day of treatment until the start of the mating period.
The quantity of food consumed by each female was measured once a week, from the first day of treatment until the start of the mating period, during pregnancy for the intervals Days 0-7, 7-14 and 14-20 p.c. and during lactation for the interval Days 1-4, 4-8 and 8-13 p.p.
During the mating period, food consumption was not measured for males or females.
Food intake per animal and per day was calculated by noting the difference between the food given and that in the food-hopper the next time.


OTHER:
Morbidity and mortality:
Each animal was checked for mortality and morbidity once a day before the treatment period and at least twice a day during the treatment period, including weekends and public holidays.
Attention was paid to the following humane endpoints.
General endpoints: prolonged impaired locomotory difficulties that prevent the animal from reaching food or water, anorexia, body weight loss (e.g. 20%), severe dehydration (persistent skin fold), profuse blood loss, evidence to suggest irreversible organ failure (e.g. shown by clinical pathology results), prolonged absence of voluntary responses to external stimuli, persistent difficult/laboured breathing (e.g. dyspnea, tachypnea, severe abdominal breathing), prolonged inability to remain upright, convulsions, self-mutilation, prolonged diarrhea, abnormal body temperature, any other findings judged to be indicative of impending death, abnormal behavior (abnormal vocalisation, aggressiveness, posture, reaction to handling, movements, reluctance to move, absence of grooming), wounds or skin ulceration, bone fractures.
Specific endpoints: loss of proprioception, tremors, nystagmus, etc. (neurological symptoms), difficulty in premature delivery.
Any animal showing signs of poor clinical condition, especially if death appears imminent, was humanely euthanized. Any animal prematurely euthanized was subjected to a macroscopic post mortem examination (see § Animals prematurely euthanized or found dead).

Functional Observation Battery:
The first five males and the first five females to deliver from each group were evaluated with a Functional Observation Battery once at the end of the treatment period. For females, this was performed on Day 14 p.p. after euthanasia of the pups.
This included a detailed clinical examination, the assessment of reactivity to manipulation and to different stimuli and motor activity.
All animals were observed in the cage, in the hand and in the standard arena.
Detail clinical examination:
The following parameters were assessed and graded: in the cage: "touch escape" or ease of removal from the cage, in the hand: fur appearance, salivation, lacrimation, piloerection, exophthalmos, reactivity to handling, pupil size (presence of myosis or mydriasis), in the standard arena (2-minute recording): grooming, palpebral closure, defecation, urination, tremors, twitches, tonic and clonic convulsions, gait, arousal (hypo- and hyper-activity), posture, stereotypy, behavior, breathing, ataxia and hypotonia.
Reactivity to manipulation and different stimuli:
The following measurements, reflexes and responses were recorded: touch response, forelimb grip strength, pupillary reflex, visual stimulus response, auditory startle reflex, tail pinch response, righting reflex,
landing foot splay, at the end of observation: rectal temperature.
Motor activity: Finally, motor activity was measured once by automated infra-red sensor equipment over a 60-minute period.

Oestrous cyclicity (parental animals):

The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning until the females were mated:
during the 2 weeks before the treatment period (including the two supplementary females per group), from the beginning of the treatment period during the pre-mating and mating periods, until the females were mated, on Day 15 p.p. before euthanasia, to allow correlation with ovaries at histopathology (see § Study plan adherence).

Sperm parameters (parental animals):

Special emphasis was paid to the stages of spermatogenesis in the male gonads.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
On Day 4 p.p., the size of each litter was adjusted by randomly culling extra pups to obtain as nearly as possible four males and four females per litter. Whenever necessary, partial adjustment (for example five males and three females) was permitted. No cross-fostering was performed.
Standardization of litter size was considered to reduce the litter size-induced variability in the growth and development of the pups and thus increase the sensitivity of statistical analysis. This also ensured that any adverse effects on pup growth and development were not masked by a treatment-related reduction in litter size.


PARAMETERS EXAMINED
The total litter size and sex of each pup were recorded on Day 1 p.p. Any gross external malformations in pups were noted.
The litters were observed daily in order to note the number of live, dead and cannibalized pups.
The pups were observed daily for clinical signs, abnormal behavior and external abnormalities.
The body weight of each pup was recorded on Days 1, 4, 8 and 13 p.p.
The following physical development measurements were performed in pups of each litter: anogenital distance (AGD) on Day 4 p.p. (all pups before culling), number of nipples and of areolae in male pups: on Day 13 p.p.
The AGD was normalized to the cube root of body weight recorded on Day 4 p.p.


GROSS EXAMINATION OF DEAD PUPS:
yes, macroscopically

Postmortem examinations (parental animals):

SACRIFICE
On completion of the treatment period, after at least 14 hours fasting, all surviving parent animals were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and euthanized by exsanguination.
males: after the end of the pairing period (6 weeks of treatment in total), females: on Day 15 p.p.

The following F0 females were euthanized by the same way without overnight fasting (euthanasia by inhalation of carbon dioxide gas followed by cervical dislocation was used when gestation was suspected): females which did not deliver: on Day 24 or 25 p.c. (after a body weight recording to check for a possible un-noticed delivery), females with total litter loss.

During the gestation period, one female from the high-dose group was prematurely euthanized by inhalation of carbon dioxide gas followed by cervical dislocation and was submitted for a complete macroscopic post-mortem examination.

GROSS NECROPSY
A complete macroscopic post-mortem examination was performed on all F0 animals including any that was euthanized prematurely. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. Special attention was paid to the reproductive organs. The numbers of corpora lutea and implantation sites were recorded for females euthanized as scheduled on Day 15 post-partum and for any female euthanized on Day 24 or 25 p.c. due to no delivery.
For apparently non-pregnant females the presence of implantation scars on the uterus was checked using the ammonium sulphide staining technique.


HISTOPATHOLOGY / ORGAN WEIGHTS
The body weight of each F0 animal euthanized as scheduled (after the end of the mating period for males, on Day 24 or 25 p.c. for females which did not deliver or on Day 15 p.p. for females) was recorded before euthanasia. The organs of those animals specified in the Tissue Procedure Table were weighed wet as soon as possible after dissection (see § Study plan adherence). Thyroids with parathyroids of F0 animals and/or of pups were weighed after fixation.
The tissues of F0 animals specified in the Tissue Procedure Table were preserved in 10% buffered formalin (see § Study plan adherence) (except for the testes and epididymides which were fixed in modified Davidson's fixative).
All tissues required for microscopic examination were trimmed based on the RITA guidelines (Ruehl Fehlert et al., 2003; Kittel et al., 2004; Morawietz et al., 2004), embedded in paraffin wax, sectioned at a thickness of approximately four microns and stained with hematoxylin-eosin (except testes and epididymides which were stained with hematoxylin/PAS).
This tissue processing was performed at CiToxLAB France.
A microscopic examination was performed on:
all tissues listed in the Tissue Procedure Table from the first five euthanized as scheduled males and the first five females euthanized on Day 15 p.p. of the control and high-dose groups (groups 1 and 4),
all macroscopic lesions of all groups,
all tissues listed in the Tissue Procedure Table from all animals that were euthanized prematurely (at additional cost, will be performed after agreement of the Sponsor),
reproductive organs from any animal that did not conceive or from pregnant females that did not deliver, to investigate possible causes (at additional cost, will be performed after agreement of the Sponsor ).

Special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure .

Based upon the microscopic results of the high-dose group, other tissues of the low- and intermediate-dose groups were examined.

TISSUE PROCEDURE TABLE
Organs Organ
weights (b) Preservation
of tissues (c) Microscopic
examination (d)

Macroscopic lesions X (a) X (a)
Adrenals X X X
Brain (including medulla/pons cerebellar and cerebral cortex) X X X
Cecum X X
Colon X X
Cowper’s (i.e. bulbo-urethral) glands X (a) X (a)
Duodenum X X
Epididymides X (a) X (a) X (e)
Esophagus X X
Eyes with Harderian glands X X (eyes only)
Femoral bone with articulation X X
Glans penis X (a) X (a)
Gut-Associated Lymphoid Tissue (GALT) X X
Heart X X X
Ileum X X
Jejunum X X
Kidneys X X X
Levator ani plus bulbo cavernous muscle
complex X (a) X (a)
Liver X X X
Lungs with bronchi X X
Lymph nodes (mandibular and mesenteric) X X
Mammary gland area X (a) X
Ovaries (with oviducts) X (a) X (e)
Pituitary gland X X X
Prostate (dorso-lateral and ventral) X (a) X (e)
Rectum X X
Sciatic nerve X X
Seminal vesicles (including coagulating glands) X (a) X (e)
Skeletal muscle X X
Spinal cord (cervical, thoracic and lumbar) X X
Spleen X X X
Sternum with bone marrow X X
Stomach with forestomach X X
Testes X (a) X (a) X (e)
Thymus X X X
Thyroids with parathyroids X (f) X (f) X
Trachea X X
Urinary bladder X X
Uterus (horns and cervix) X (a) X (e)
Vagina X (a) X (e)
(a): all animals.
(b): the first five euthanized as scheduled males and the first five females euthanized on Day 15 p.p. of each group.
(c): the first five euthanized as scheduled males and the first five females euthanized on Day 15 p.p. of each group and all animals prematurely euthanized.
(d): the first five euthanized as scheduled males and the first five females euthanized on Day 15 p.p. of groups 1 and 4 and all animals prematurely euthanized.
(e): (d) + all animals that did not conceive or from pregnant females that did not deliver, to investigate possible causes.
(f): for all F0 animals and one pup/sex/litter sacrificed on Day 14 p.p.

Postmortem examinations (offspring):

SACRIFICE
Pups were euthanized by an intraperitoneal injection of sodium pentobarbital, followed by decapitation (on Day 4 p.p.) when blood samples were collected, (see § Laboratory investigations - Thyroid hormones) or deeply anesthetized by intraperitoneal injection of sodium pentobarbital and euthanized by exsanguination: pups not selected on Day 4 p.p.: on Day 4 p.p., surviving pups: on Day 14 p.p.
Found dead pups were submitted to a detailed external examination (including orifices and buccal cavity). This examination was done in addition to the daily observation for clinical signs, abnormal behavior and external abnormalities. A macroscopic post-mortem examination of the principal thoracic and abdominal organs was also performed. Particular attention was paid to the external genital organs, internal reproductive organs and to whether the pup was fed (e.g. presence of milk in the stomach). No tissues were preserved.

GROSS NECROPSY
Pups not selected on Day 4 p.p. were discarded without further examination.
Pups euthanized on Day 14 p.p. were submitted to a detailed external examination (including orifices and buccal cavity) after euthanasia (this examination was done in addition to the daily observation for clinical signs, abnormal behavior and external abnormalities) and to a macroscopic post-mortem examination of the principal thoracic and abdominal organs after euthanasia. Particular attention was paid to the external genital organs and internal reproductive organs.
No tissues were preserved except thyroids with parathyroids for one pup/sex/litter.


HISTOPATHOLOGY / ORGAN WEIGTHS
The body weight of one pup/sex/litter euthanized on Day 14 p.p. was recorded before euthanasia.
Thyroids with parathyroids of F0 animals and/or of pups were weighed after fixation.

Statistics:

Body weight, food consumption and reproductive data: Data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fisher’s exact probability test (proportions).
Hematology, blood biochemistry, hormones, anogenital distance and post-implantation loss data: Citox software was used to perform the statistical analyses of hematology, blood biochemistry, hormones, anogenital distance and post-implantation loss data.
Organ weight: PathData software was used to perform the statistical analysis of organ weight data (level of significance: 0.05 or 0.01).

Reproductive indices:

pre-implantation loss:
Number of corpora lutea - Number of implantation sites
_____________________________________________ x 100
Number of corpora lutea

post-implantation loss (calculated with Excel):
Number of implantation sites - Number of live pups
_____________________________________________ x 100
Number of implantation sites

mating index:
Number of mated animals
_____________________ x 100
Number of paired animals

fertility index:
Number of pregnant female partners
_______________________________ x 100
Number of mated pairs

gestation index:
Number of females with live born pups
________________________________ x 100
Number of pregnant females

Offspring viability indices:

live birth index:
Number of live born pups
_____________________ x 100
Number of delivered pups

viability index on Day 4 p.p.:
Number of surviving pups on Day 4 post-partum
_______________________________________ x 100
Number of live born pups

AGD/cube root of body weight ratio (calculated with Excel):
AGD
______________
³√Body weight

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Non-adverse test item treatment-related clinical signs, namely piloerection, round back and/or half-closed eyes, were transiently noted in 1/10 males given 150 mg/kg/day (in Week 4 and from Week 6), 1/10 females given 150 mg/kg/day (on Day 3 p.c.), 3/10 males given 500 mg/kg/day (in Weeks 2, 3, 5 and/or 6) and 6/10 females given 500 mg/kg/day (during the premating period). Ptyalism was observed from 50 mg/kg/day, few days to few weeks after the start of the study in a dose-related incidence and resulted from the oily consistence of the dose formulations.
These findings were considered to be related to the test item but of minor toxicological importance.

All the other clinical signs recorded during the study, i.e. cutaneous observations on various parts of the body (areas of hair loss, cutaneous lesions, scabs, nodosities), loss of teeth and/or abnormal growth of teeth, and chromodacryorrhea were considered to be unrelated to the test item treatment as they were present in both control and test item-treated animals, and/or were reported sporadically in only a few animals and/or are spontaneous findings in rats.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

there were no unscheduled deaths in the male groups or in the 50 mg/kg/day female group. Test item treatment-related sacrifices were recorded at the end of the gestation period for no delivery in 1/10 females given 150 mg/kg/day and 6/10 females given 500 mg/kg/day, and during the lactation period in 1/10 females from the 150 mg/kg/day group and in 1/10 females from the 500 mg/kg/day group because of deaths in the litters. The cause and mechanisms leading to the deaths in the litters remain unclear.

At 500 mg/kg/day, female H25332 was prematurely sacrificed on Day 19 p.c. due to a subcutaneous mass (2.0 x 2.0 cm in diameter, present at the left mammary gland number 2). Prior to sacrifice, the following clinical signs were also noted: piloerection on Day 7, ptyalism throughout the premating and gestation
periods, nodosity in the abdominal region from Day 0 p.c. to Day 2 p.c. and nodosity in the thoracic region from Day 3 p.c. to Day 15 p.c. At necropsy, this female was found to be pregnant with 12 live fetuses. Microscopically, the subcutaneous mass correlated with an adenocarcinoma. This isolated finding was
considered to be spontaneous (i.e. unrelated to the test item treatment).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

in males, when compared with controls, there were slightly to markedly lower body weight gains from 50 mg/kg/day (statistically significant from 150 mg/kg/day), and in females a slight body weight loss was seen from 150 mg/kg/day over the first week of the pre-mating period. A markedly lower body weight gain (statistically significant) was seen in females given 500 mg/kg/day during the gestation period and a slightly to markedly lower body weight gain in females from 150 mg/kg/day during the lactation period.
These variations resulted in adverse lower final body weights in males given 150 or 500 mg/kg/day (statistically significant: -16% and -19% vs. controls, respectively) and in females given 500 mg/kg/day at the end of the lactation period (statistically significant: -13% vs. controls with only 2/3 females having a lower final body weight). The final body weight was also affected in females given 150 mg/kg/day at the end of the lactation period (-3%) and in females given 500 mg/kg/day at the end of the gestation period ( 9%), but to a lesser extent.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

when compared with controls, there were adverse effects on mean food consumption in males given 150 or 500 mg/kg/day (statistically significant: down to -20% or -29% vs. controls, respectively), in females given 500 mg/kg/day during the lactation period (statistically significant: down to -52% vs. controls). The lower food consumption recorded in the 150 mg/kg/day female group during the lactation period and in the 500 mg/kg/day female group during the pre-mating period was considered to be of minor toxicological importance. There were no effects on food consumption at 50 mg/kg/day.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No changes that could be related to treatment were observed.

All the differences from controls, namely lower mean white blood cell count (including lower neutrophil, eosinophil, basophil, lymphocyte, monocyte and/or large unstained cell counts) in males and females at all dose levels, lower mean reticulocyte count in females given 150 or 500 mg/kg/day, lower mean cell hemoglobin in males given 50 mg/kg/day, and shortened activated partial thromboplastin time in males given 50 mg/kg/day and in one surviving female given 500 mg/kg/day, were of low magnitude and/or not dose-related. Therefore, these differences were considered to be unrelated to the test item treatment.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Dose-related lower mean urea level in females from 150 mg/kg/day and statistically significant, higher mean creatinine level and higher total protein level accompanied with higher mean calcium level in males given 500 mg/kg/day were noted. Marginally higher mean total bilirubin levels were also observed in both sexes at 500 mg/kg/day. These differences were not associated with other blood biochemistry or histological changes and were within the range of the Historical Control Data, they were therefore considered to be of minor toxicological importance.
All the other differences (namely higher mean bile acids levels in high-dose females, lower mean cholesterol level in intermediate-dose males, lower triglyceride levels in intermediate- and high-dose males and females and higher mean calcium, protein and albumin levels in low-dose females) were slight, not dose-related and/or resulted from higher mean control values. Therefore, they were considered to be of no toxicological significance.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no effects on Functional Observation Battery tests or motor activity data in any group.

There were no differences between test item-treated and control groups in motor activity (horizontal movements and rearing).
Higher mean numbers of horizontal movements and/or rearing noted in test item-treated males and females resulted from lower mean control values due to one isolated male (H24942: 195 horizontal movements and one rearing movement) and one isolated female (H25294: 173 horizontal movements and one incidence of rearing). These differences were therefore not considered to be test item treatment-related.

There were no test item treatment-related neurologic abnormalities. At 500 mg/kg/day, lower mean landing foot splay values (70 mm vs. 85 mm in controls) were recorded in males and lower mean rectal temperature (37.7°C vs. 38.6°C in controls) was noted in 1/2 surviving females. These findings were considered to be of no toxicological importance as they did not correlate with any other findings or were within physiological values.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Only two terminal sacrifice high-dose females (H25334 and H25337) were initially available for microscopic examination: five females did not get pregnant (H25328, H25330, H25331, H25335 and H25336), one failed to deliver (H25333) and two were prematurely euthanized for the presence of a mammary mass (H25332) or dead litter (H25338), as mentioned earlier. Microscopically, the mammary mass observed in animal H25332 correlated with an adenocarcinoma. No clear test item-related microscopic findings could be confirmed in these animals.
Ovarian atrophy was noted in the only two terminal high-dose females which were available for microscopic examination. Given the small number of animals, a test item-related effect cannot be excluded. In order to better investigate this question, the reproductive system (ovaries, oviducts, uterus and vagina) from all F0 females were examined. Minimal to slight ovarian atrophy (i.e. a decrease in the number and size of luteal bodies) was then observed in 3/10 females given the test item at 500 mg/kg/day. Given the incidence/severity and the absence of associated lesions in other reproductive organs, this finding was not considered to be directly related to the test item, but rather to the decrease in body weight and associated stress. In two of these three females, minimal or moderate atrophy of the thymus was also observed, which
further supports the hypothesis of a stress-related effect (see below).

Also upon examination of all reproductive organs in all F0 females, increased mucification of the vaginal epithelium (increased incidence of grade 3 cases) was observed in females given the test item at 150 mg/kg/day (5/10) and 500 mg/kg/day (3/10), when compared to controls (0/10) and females at 50 mg/kg/day (0/10). Of notice, there were no findings suggestive of endocrine disruption in other reproductive organs (ovary, oviducts and uterus), in females with grade 3 mucification, regardless of the test item dose (for instance, none of these animals presented the previously mentioned ovarian atrophy). Therefore, in the absence of a clear dose-relationship and concomitant lesions in other reproductive organs, this finding was not considered to be directly related to the test item, but likely due to alterations in the estrous cycle caused by decreased body weight and associated stress.
Qualitative testis staging did not indicate any abnormalities in the integrity of the various cell types present within the different stages of the spermatogenic cycle.

Lymphoid atrophy was observed in the thymus of two females at 150 mg/kg/day (slight or marked), three females at 500 mg/kg/day (two terminal sacrifice females and one prematurely dead; minimal to marked) and three males at 500 mg/kg/day (minimal). Based on the lack of similar findings in other lymphoid organs, as well as on its frequency and low magnitude (in the majority of the animals), this finding was considered as likely related to stress, rather than a direct effect of the test item.
Other microscopic findings noted in treated animals were considered incidental changes, as they also occurred in controls, were of low incidence, had no dose-relationship in incidence or severity, and/or are common background findings for the rat.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Thyroid hormones: In males, in all treated groups and when compared with controls, there was a decrease in mean T4 concentration correlating with a dose-related increase in mean TSH concentration (not statistically
significant). The differences in mean T4 values reached statistical significance from 150 mg/kg/day but were in the range of the Historical Control Data. These differences were considered to be test item treatment-related despite high standard deviations recorded in TSH values. In females, at 500 mg/kg/day and when compared with controls, there was a decrease in mean T4 concentration (not statistically significant). A relationship to the test item treatment was not excluded. At 50 mg/kg/day, lower mean TSH concentration was considered fortuitous in the absence of any dose relationship.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no test item-related effects on the mean length of the estrous cycle or the mean number of cycles at any dose-level.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Qualitative testis staging did not indicate any abnormalities in the integrity of the various cell types present within the different stages of the spermatogenic cycle.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Pairing, mating and fertility data:
All females mated. Males H24957 and H24971 (given 50 and 500 mg/kg/day, respectively) failed to mate.
At 500 mg/kg/day, effects were noted in the pre-coital time, the number of pregnant females and the number of females that delivered
(6.5 days vs. 2.0 days in controls, five pregnant females vs. ten pregnant control females and three females with live-born pups vs. ten in controls, respectively). These findings were considered to be test item treatment-related and adverse.
The other differences from controls (i.e. higher mean number of days taken to mate noted in females given 50 mg/kg/day, lower number of females with live-born pups in females given 150 mg/kg/day) were considered to be incidental as they were not dose-related, were due to isolated animals and/or were within
the range of the Historical Control Data.


Delivery data:
When compared with controls, there were no effects on the mean duration of gestation at 50 and 150 mg/kg/day, whereas gestation lasted 1 day longer in
females given 500 mg/kg/day.
Test item dose-related findings were noted in a dose-related manner at 150 and 500 mg/kg/day as follows:
- lower mean number corpora lutea (not statistically significant: -11 and -19%, respectively and outside the range of the Historical Control and reference Data)
- lower mean number of implantation sites [-14 and -28% (p<0.05), respectively and outside the range of the Historical Control and reference Data],
- higher mean pre-implantation loss (not statistically significant with high standard deviation values but in the range of the reference data),
- lower mean number of pups delivered [-17 and -47% (p<0.05), respectively and outside the range of the Historical Control and reference Data],
- higher mean number of post-implantation losses (not statistically significant with high standard deviation values and outside the range of the reference data at 500 mg/kg/day).
These differences were considered to be adverse as they were of high magnitude and/or statistically significant and/or outside the range of the Historical Control Data and/or reference Data, even though the limited number of dams given 500 mg/kg/day.
No test item treatment-related effects were observed on the delivery data at 50 mg/kg/day.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

parental systemic toxicity

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: body weight and body weight change, food consumption and thyroid hormones results in males .

2

Dose descriptor:

NOAEL

Remarks:

reproductive performance (mating, fertility and delivery)

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Prolonged pre-coital and gestation times and the lower fertility and gestation indexes at 500 mg/kg/day. Lower number of corpora lutea, implantation sites and pups delivered, and the higher pre- and post-implantation loss from 150 mg/kg/day.

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 500 mg/kg/day and when compared with controls, no clinical signs were noted in pups from the two surviving litters (11 pups in total). At 150 mg/kg/day and when compared with controls, an increase in clinical signs due to lack of maternal care (blackish discoloration of the abdomen, dehydration, generalized pallor and/or thinning of hair) was observed in pups along with an increased number of litters affected (3/9 litters with 11/88 affected pups vs. 2/10 with 3/118 affected pups in controls). These findings that could be due to lack of maternal care were considered to be adverse.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

at 150 and 500 mg/kg/day, when compared with controls, there were significant reductions in the viability index (statistically significant: 80.7% and 57.9% vs. 98.3% in controls, respectively), marked increases in the number of pups found dead and cannibalized as well as in the incidence of litters affected (4/9 and 1/3 vs. 1/10 in controls, respectively). These findings that could be resulted from an abnormal maternal behavior were considered to be adverse. There were no effects on the viability index at 50 mg/kg/day or on the live birth index at any dose level.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 150 and 500 mg/kg/day, when compared with controls, there was a moderate to marked dose-related lower mean body weight gain in male and female pups from Day 4 p.p. until the end of the lactation period (statistically significant in female pups at 500 mg/kg/day). This resulted in a moderately to markedly lower final mean body weight for males and females [150 mg/kg/day: -9% vs. controls in both sexes; 500 mg/kg/day: -24% and -22% vs. controls, for males and females (statistically significant in female pups at 500 mg/kg/day), respectively].
There were no effects on mean body weight or mean body weight change in pups at 50 mg/kg/day during the lactation period.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

The anogenital distance was considered to be unaffected by the test item treatment in both sexes at all dose levels taking into account the limited number of dams and pups at 500 mg/kg/day.
The statistically significant lower mean anogenital distance measured in male pups at 50 mg/kg/day group was not attributed to the test item treatment as the difference from controls was not dose-related and was of low magnitude.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No nipples or areolae were observed in any male pups.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no external abnormalities at any dose-level.
No test item-related macroscopic changes were observed in surviving or prematurely dead pups.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Pup sex ratio: there were no effects on the mean percentage of male fetuses (sex ratio) at 50 and 150 mg/kg/day. A relevant lower percentage of males was noted at 500 mg/kg/day (28.6 vs. 42.4% in controls). Despite the low number of pups and litters at this dose, a treatment related effect cannot be excluded.


Pup macroscopic post-mortem examination: there were no external abnormalities at any dose-level. There were no test item-related findings at post-mortem examination of the pups sacrificed as scheduled at any dose level (taking into account the limited number of dams and pups at 500 mg/kg/day). Nevertheless, absence of milk in the stomach observed in two pups from the same litter at 150 mg/kg/day, for which the remaining found dead pups were found autolyzed, was to be test item treatment-related and adverse as dead of the litter was the consequence of abnormal maternal behavior.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Clinical signs due to the absence of maternal care and lower viability index from 150 mg/kg/day. Lower pup body weight and lower percentage of males at 500 mg/kg/day.

Overall reproductive toxicity

Reproductive effects observed:

yes

Lowest effective dose / conc.:

50 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects in the absence of other toxic effects

Dose response relationship:

yes

Any other information on results incl. tables

Additional information on results:

CHEMICAL ANALYSIS OF THE DOSE FORMULATIONS

The test item concentrations in the administered dose formulations analyzed in Weeks 1, 3 and 6 remained within an acceptable range of variations (-8.2% to +3.0%) except for group 4 in Week 6 (see § Study plan adherence) when compared to the nominal values (± 10% of the nominal concentrations).

No test item was observed in the control dose formulation.

 In Week 8, a supplementary analysis of the dose formulation prepared for group 4 was performed following the previous results out of acceptance criteria in Week 06. The dose formulation was found at -8.3%.

The validation report CiToxLAB France/Study No. 44021 VAScontaining the data demonstrating the suitability of the method is currently being finalized.

Organ weights (Parents)

A statistically significant decrease in final body weight was observed in mid- (-16%) and high- (-20%) dose males when compared to controls. These changes were considered to be test item-related and adverse. In

addition, a statistically significant minimal increase in final body weight was noted in females at 50 mg/kg/day. Based on the low magnitude and lack of dose-correlation, a test item-related effect was dismissed.

There were no morphologic changes in organ weights considered to be related to the administered test item. Nevertheless, in treated males, statistically significant increases in relative weight were noted for the liver (+36%, at 500 mg/kg/day only), kidneys (up to +23%, from 150 mg/kg/day), brain (up to +23%, from 150 mg/kg/day), adrenal (+44%, at 500 mg/kg/day only), thyroid (+36%, at 500 mg/kg/day only), epididymides (up to +16%, from 150 mg/kg/day), penis (up to +19%, from 150 mg/kg/day), and testes (+19%, from 150 mg/kg/day). Based on the concurrent decreases in final body weight observed in these groups, the absence of similar changes in absolute weight, and the absence of significant macroscopic or microscopic correlates, these findings were considered as related to the reduction in final body weight, rather than a direct test item-related effect. Furthermore, also in treated males, decreases in absolute weight were noted for the heart (up to -18%, from 150 mg/kg/day), Levator ani plus bulbo cavernous muscle complex (-26%, at 500 mg/kg/day only) and Cowper’s glands (-19%, at 500 mg/kg/day only). These were statistically significant for the heart and Levator ani plus bulbo cavernous muscle complex (but not for the Cowper’s glands). All of these weight decreases were also likely related to the decrease in final body weight in the mentioned groups. An increase in absolute and relative pituitary weights (+33% and +23%, respectively, and statistically significant only for absolute weights) was observed in females at 50 mg/kg/day. Based on the lack of dose-relationship, a test item-related effect was dismissed. Other organ weight changes were not considered to be related to the test item as they were too small in amplitude, had no gross or microscopic correlates, and/or were not dose-related in magnitude.

Furthermore, they reflected the usual range of individual variations.

Organ weights (pups):

A decrease in final body weight was observed in mid- (up to -12%) and high- (up to -24%) dose pups of both sexes when compared to controls (statistically significant only at 150 mg/kg/day, in females). These

changes were considered to be test item-related and adverse. There were no changes in organ weights considered to be related to the administered test item. Nevertheless, in male pups, a statistically significant decrease in absolute weight was noted for the thyroid glands (-24%, at 150 mg/kg/day only). Based on the concurrent decrease in final body weight and given the lack of dose-relationship or consistency across gender, this finding was considered as related to the reduction in final body weight, rather than a direct test item-related effect.

TABLES:

Data on parents:

1) Mortality

Sex	Males				Females
Dose-level (mg/kg/day)	0	50	150	500	0	50	150	500
Pre-mating (females) or whole study (males)
Total surviving animals	10/10	10/10	10/10	10/10	10/10	10/10	10/10	10/10
Gestation
Humane reasons (mass)	-	-	-	-	0	0	0	1
No delivery	-	-	-	-	0	0	1	6
Total surviving animals	-	-	-	-	10/10	10/10	9/10	3/10
Lactation
Dead litter	-	-	-	-	0	0	1	1
Total surviving animals	-	-	-	-	10/10	10/10	8/10	2/10

-: not applicable

2) Clinical signs

Sex	Males				Females
Dose-level (mg/kg/day)	0	50	150	500	0	50	150	500
Pre-mating (females) or whole study (males)
Ptyalism	0	2	10	10	0	1	9	10
Piloerection	0	0	1	3	0	0	0	6
Round back	0	0	1	1	0	0	0	3
Half-closed eyes	0	0	1	0	0	0	0	0
Total affected animals	0/10	2/10	10/10	10/10	0/10	1/10	9/10	10/10
Gestation
Ptyalism					0	0	8	9
Piloerection					0	0	1	0
Total affected animals					0/10	0/10	8/10	9/9
Lactation
Ptyalism					0	0	5	2
Total affected animals					0/10	0/10	5/8	2/2

3) Body weight and body weight change

Sex	Males				Females
Dose-level (mg/kg/day)	0	50	150	500	0	50	150	500^a
Pre-mating (females) or whole study (males)
Day 1	473	473	472	466	249	265*	262	254
% from controls	/	0	0	-1	/	+6	+5	+2
Day 8	517	511	492*	464^#	255	269	261	250
% from controls	/	-1	-5	-10	/	+5	+2	-2
Day 15	554	539	499^#	476^#	264	276	271	268
% from controls	/	-3	-10	-14	/	+5	+3	+2
Day 22	573	551	508^#	489^#	/	/	/	/
% from controls	/	-4	-11	-15
Day 36	621	596	528^#	505^#	/	/	/	/
% from controls	/	-4	-15	-19
Day 42	637	611	534^#	517^#	/	/	/	/
% from controls	/	-4	-16	-19
Days 1 - 8	+45	+38	+20^#	-1^#	+6	+4	-1	-4*
Days 8 - 15	+37	+28	+8^#	+12^#	+9	+7	+10	+18**
Days 1 - 15	+82	+66	+27^#	+11^#	+15	+10	+9	+14
Days 1 - 42	+164	+139	+62^#	+52^#	/	/	/	/
Gestation
Day 0p.c.	/	/	/	/	264	284**	270	281
% from controls					/	+8	+2	+6
Day 20p.c.	/	/	/	/	423	440	413	386
% from controls					/	+4	-2	-9
Days 7-14p.c.	/	/	/	/	+44	+37	+36	+29*
Days 14-20p.c.	/	/	/	/	+88	+87	+84	+55*
Days 0-20p.c.	/	/	/	/	+159	+156	+144	+107**
Lactation
Day 1p.p.	/	/	/	/	324	342	319	308
% from controls					/	+6	-2	-5
Day 13p.p.	/	/	/	/	362	385	351	315*
% from controls					/	+6	-3	-13
Days 1-4p.p.	/	/	/	/	+11	+15	+3	-5
Days 1-13p.p.	/	/	/	/	+38	+43	+31	+7

/: not applicable;*: p<0.05; **: p<0.01 and^#: p<0.001;^a: n = 3 on Day 1p.p.and then n = 2.

4) Food consumption (g/animal/day)

Sex		Males				Females
Dose-level (mg/kg/day)		0	50	150	500	0	50	150	500^a
Pre-mating (females) or whole study (males)
Days 1 - 8		34	32	30	24^#	16	17	19	12
% from controls		/	-6	-12	-29	/	+6	+19	-25
Days 8 - 15		35	32	28**	26^#	17	19	21	18
% from controls		/	-9	-20	-26	/	+12	+24	+6
Gestation
Days 0 - 7		/	/	/	/	19	21	22	19
% from controls						/	+11	+16	0
Days 7 - 14		/	/	/	/	22	23	23	22
% from controls						/	+5	+5	0
Days 14 - 20		/	/	/	/	28	28	30	26
% from controls						/	0	+7	-7
Lactation
Days 1 - 4	/		/	/	/	36	36	30	21
% from controls						/	0	-17	-42
Days 4 - 8	/		/	/	/	49	53	42	29**
% from controls						/	+8	-14	-41
Days 8 - 13	/		/	/	/	64	67	56	31**
% from controls						/	+5	-13	-52

/: not applicable;**: p<0.01 and^#: p<0.001;^a: n = 3 on Day 1p.p.and then n = 2.

5) Motor activity

Sex	Male				Female
Dose-level (mg/kg/day)	0	50	150	500	0	50	150	500^a
Horizontal movements	517	518	576	599	533	532	507	608
Standard deviation	298.9	231.5	202.7	235.6	347.0	152.8	163.1	23.3
% from controls	-	0	+11	+16	-	0	-5	+14
Rearing	110	174	163	170	113	127	173	133
Standard deviation	61.2	76.7	23.7	93.5	94.0	43.8	84.8	9.2
% from controls	-	+58	+48	+55	-	+12	+53	+18

-: not applicable; ^a: n = two females.

6) Estrous cycle

Dose-level (mg/kg/day)	0	50	150	500
Number of cycles	3.0	3.0	3.1	3.1
Cycle length (days)	4.0	4.0	4.0	4.0
Number of females having a mean average cycle of 4-5 days	10	10	9	7
Number of days of diestrus	4.3	3.8	5.1	4.7
Number of days of proestrus	3.0	3.2	2.1	2.1

7) Paring, mating and fertility data

Dose-level (mg/kg/day)	0	50	150	500
Number of animals paired (M + F)	10 + 10	10 + 10	10 + 10	10 + 10
Number of males mated	10	9	10	9
Number of females mated	10	10	10	10
Mean number of days taken to mate (pre-coital time)	2.0	4.8	2.3	6.5*
Number of pregnant females	10	10	10	5*
Number of females with live born pups	10	10	9	3
Mating index (%)
. male	100	90	100	90
. female	100	100	100	100
Fertility index (%)
. male	100	100	100	55.6
. female	100	100	100	50.0
Gestation index (%)	100	100	90.0	60.0

M: male; F: female. *: p<0.05.

8) Delivery data

Dose-level (mg/kg/day)	0	50	150	500^a	HCD
Number of pregnant females	10	10	10	5*	71^b
Number of females which delivered	10	10	9	3**	70^b
Mean duration of gestation (days)	22.0	22.0	22.3	23.0	[21.0;21.4]^b
Mean number ofcorpora lutea	13.9	14.3	12.4	11.3	[13.8;16.0]^c
% from controls	/	+3	-11	-19	/
Standard deviation	1.3	2.4	2.4	2.1	/
Mean number of implantation sites	13.9	14.0	12.0	10.0*	[12.5;14.5]^c
% from controls	/	+1	-14	-28	/
Standard deviation	1.3	2.0	2.6	2.0	/
Mean pre-implantation loss (%)	0.0	1.7	3.8	11.4	[6.2;14.0]^c
Standard deviation	0.0	3.8	9.1	11.5	/
Mean number of pups delivered	11.8	11.7	9.8	6.3*	[13.7;14.7]^b
% from controls	/	-1	-17	-47	/
Standard deviation	2.9	2.5	3.3	4.7	/
Mean post-implantation loss (%)	15.4	16.9	20.4	41.4	[3.5;11.1]^c
Standard deviation	17.56	12.31	13.66	39.96	/

/: not applicable; a: n = three litters on Day 1p.p.and then n = two litters.

*: <0.05 and **: p<0.01.

b: HCD: Historical Control Data OCDE 416 (control data collected from three studies covering a period ranging from October 2009 to July 2012), [min.; max.].

c: HCD: Historical Control Data (control data collected from nine studies covering a period ranging from March 2014 to July 2016 with 388 females with live fetuses at termination/407 pregnant females at hysterectomy), [min.; max.]. Ref. Data: reference data collected from five GLP OECD 422 studies (i.e. Citoxlab France/Study Nos. 43937 RSR,44042 RSR, 44425 RSR, 44486 RSR and 44495 RSR)covering a period ranging from August 2016 to October 2017, [min.; max.].

Data on pups:

1) Mortality

Dose-level (mg/kg/day)	0	50	150	500^a
Number of pups (litters)	118 (10)	117 (10)	88 (9)	19 (3)
Live birth index (%)	100	100	100	100
Number of pups found dead or cannibalized (litters affected)	2 (1)	0 (0)	17 (4)	8 (1)
Viability index (Day 4p.p.%)	98.3	100.0	80.7^#	57.9^#

^a: n = three litters on Day 1p.p.and then n = two litters.

#: p<0.001 for the number of pups surviving on Day 4p.p.

2) Clinical signs

 

Dose-level (mg/kg/day)	0	50	150	500^a
Abnormal blackish color of abdomen, n (L)			1 (1)
Dehydration, n (L)			1 (1)
Generalized pallor, n (L)			1 (1)
Hematoma, n (L)	2 (2)
Swollen hidlimb, n (L)	1 (1)
Thinning of hair, n (L)			8 (1)
Scab, n (L)			1 (1)
Total pups affected, n (L)	2 (2)	0 (0)	11 (4)	0 (0)

( ): litter incidence, n: number of pups, L: litter.

^a: n = three litters on Day 1p.p.and then n = two litters.

3) Pup body weight

Sex	Male				Female
Dose-level (mg/kg/day)	0	50	150	500^a	0	50	150	500^a
Body weight
. Day 1p.p.	8.4	8.4	8.5	8.3	7.8	7.9	7.8	8.2
. Day 4p.p.post-culling	12.6	12.5	12.4	12.2	12.0	12.0	11.5	11.7
. Day 8p.p.	22.3	22.9	20.6	18.9	21.4	22.2	19.5	17.8
. Day 13p.p.	37.0	37.9	33.7	28.2	35.6	36.9	32.5	27.6*
Body weight change
. Days 4-8p.p.	+9.8	+10.5	+8.2	+6.7	+9.4	+10.2	+8.0	+6.1*
. Days 8-13p.p.	+14.6	+14.9	+13.1	+9.3	+14.2	+14.7	+13.0	+9.8**
. Days 4-13p.p.	+24.4	+25.4	+21.3	+16.0	+23.6	+24.9	+21.0	+15.9**

^a: n = three litters on Day 1p.p.and then n = two litters.

*: p<0.05 and **: p<0.01.

4) Sex ratio

Sex ratio on Day 0p.p.(% of males)

 

Dose-level (mg/kg/day)	0	50	150	500^a
. Day 0 p.p.	42.4	47.9	51.2	28.6

^a: n = three litters on Day 1p.p.and then n = two litters.

 

5) Macroscopic post-mortem examination of pups

Dose-level (mg/kg/day)	0	50	150	500^a
Liveborn pups	118	117	88	19
Culled Day 4 p.p.	38	39	17	2
Cannibalized pups	0	0	7	5
Dead pups
Number of pups examined (litter)	2 (1)	0	10 (2)	3 (1)
Autolysis	2 (1)	0 (0)	8 (2)	3 (1)
Stomach: absence of milk	0 (0)	0 (0)	2 (1)	0 (0)
Schedule sacrificed pups
Number of pups examined (litter)	78 (10)	78 (10)	54 (8)	9 (2)
Kidneys: dilated renal pelvis	0 (0)	0 (0)	1 (1)	0 (0)
Total pups affected (litters affected)	0 (0)	0 (0)	1 (1)	0 (0)

^a: n = three litters on Day 1p.p.and then n = two litters.

Tyroid hormones tables for parents and pups

	Thyroid hormones
Sex	F0 males
Dose-level (mg/kg/day)	0	50	150	500
T4 (ng/mL)	37.7	36.4	27.7**	29.1**
HCD	37.2 Minimum: 24.2 Maximum: 45.3
Standard deviation	4.27	6.14	4.44	5.89
n	10	10	10	10
% from controls	/	-3	-27	-23
TSH (pg/mL)	1284	1535	1643	2289
HCD	1604 Minimum: 827 Maximum: 2771
Standard deviation	693.8	1021.2	1240.0	1324.6
n	10	10	10	10
% from controls	/	+20	+28	+78

Statistically significant: **: p<0.01.

HCD: Historical Control Data.

n: number of animals with available values; /: not applicable

	Thyroid hormones
Sex	F0 females
Dose-level (mg/kg/day)	0	50	150	500
T4 (ng/mL)	28.9	26.0	26.0	13.9
Standard deviation	6.45	4.74	4.25	3.25
n	10	10	8	2
% from controls	/	-10	-10	-52
TSH (pg/mL)	1461	1174	1455	1394
Standard deviation	646.9	578.3	944.6	/
n	8	9	6	1
% from controls	/	-20	0	-5

n: number of animals with available values; /: not applicable

	Thyroid hormones
Sex	Pups, Day 14p.p.
Dose-level (mg/kg/day)	0	50	150	500
T4 (ng/mL)	37.5	35.9	36.2	32.9
Standard deviation	5.14	3.27	5.60	1.91
n	10	10	8	2
% from controls	/	-4	-3	-12
TSH (pg/mL)	2130	2079	1751	1186
Standard deviation	622.9	676.2	393.1	333.8
n	10	10	8	2
% from controls	/	-2	-18	-44

n: number of animals with available values; /: not applicable

Applicant's summary and conclusion

Conclusions:

Based on the experimental conditions of this study:
- the No Observed Adverse Effect Level (NOAEL) for parental systemic toxicity was considered to be 50 mg/kg/day (based on lower body weight and body weight change, lower food consumption in males and/or females from 150 mg/kg/day and thyroid hormones results in males from 150 mg/kg/day),
- the No Observed Adverse Effect Level (NOAEL) for reproductive performance (mating, fertility and delivery) was considered to be 50 mg/kg/day (based on the prolonged pre-coital and gestation times and the lower fertility and gestation indexes at 500 mg/kg/day, and on the lower number of corpora lutea, implantation sites and pups delivered, and the higher pre- and post-implantation loss from 150 mg/kg/day),
- the No Observed Adverse Effect Level (NOAEL) for toxic effects on progeny was considered to be 50 mg/kg/day (based on clinical signs due to the absence of maternal care, on the lower viability index from 150 mg/kg/day, and on the lower pup body weight and on the lower percentage of males at 500 mg/kg/day).

Executive summary:

Three groups of ten male and ten female Sprague-Dawley rats received the test item, Rhodiantal Original IBCH, daily, by oral administration (gavage), before mating, during mating and, for the females, throughout gestation and until Day 14p.p., at the dose-levels of 50, 150 or 500 mg/kg/day.The test item was administered in the vehicle (corn oil) under a constant dosage volume of 5 mL/kg/day. One other group of ten males and ten females received the vehicle alone and acted as a control group. 

The actual test item concentrations in the dose formulations prepared for use in Weeks 1, 3, 6 and 8 were determined using a validated GC-FID analytical method.

The animals were checked daily for clinical signs and mortality, and detailed clinical observations were conducted weekly. Body weight and food consumption were recorded weekly until mating and then at designated intervals throughout gestation and lactation (until Day 14 p.p.). Estrous cycle stage was determined each morning from 2 weeks before the treatment period until the females had mated and on Day 15 p.p. before euthanasia.

The animals were paired for mating after 2 weeks of treatment and the dams were allowed to litter and rear their progeny until Day 14 p.p.The total litter size and the number of pups of each sex were recorded after birth.The size of each litter was adjusted on Day 4 p.p. by culling extra pups to obtain as nearly as possible four males and four females per litter. Pups were observed daily for clinical signs, abnormal behavior and external abnormalities, and on Days 1, 4, 8 and 13p.p. they were weighed. The physical development of pups was assessed by measuring the anogenital distance on Day 4 p.p. (all pups before culling) and by counting the number of nipples and areolae in male pups on Day 13 p.p. A Functional Observation Battery (FOB, including motor activity) and laboratory investigations (hematology and blood biochemistry) were carried out on at least five males and five females from each group at the end of the study. Thyroid hormones (TSH and T4) were determined in F0 males at sacrifice, in F0 females sacrificed on Day 15 p.p.and in pups sacrificed on Day 14 p.p.

The males were sacrificed after completion of the mating period and dams were sacrificed on Day 15 p.p.

A full macroscopic post-mortem examination was performed, with particular attention accorded to the reproductive organs. Designated organs were weighed and selected tissue specimens were preserved.

A microscopic examination was performed on selected organs from the first five control and high-dose males sacrificed at the end of the treatment period, the first five control and high-dose females sacrificed on Day 15 p.p.,the female prematurely sacrificed, pregnant females that did not deliver, and all macroscopic lesions (all groups).

A macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed on pups sacrificed on Day 14 p.p.and on those found dead. Thyroids with parathyroids were preserved for one pup/sex/litter.

Actual concentrations of the test item in the dose formulations analyzed during the study remained within an acceptable range of variation (-8.3% to +3.0%) compared to the nominal values (nominal concentration± 10%), except for the high dose formulation in Week 6 for which the result was found to be -18% (i.e.82.0 mg/mL and 410 mg/kg/day for the dose-level).

Results:

Mortality: there were no unscheduled deaths in the male groups or in the 50 mg/kg/day female group. Test item treatment-related sacrifices were recorded at the end of the gestation period for no delivery in 1/10 females given 150 mg/kg/day and 6/10 females given 500 mg/kg/day, and during the lactation period in 1/10 females from the 150 mg/kg/day group and in 1/10 females from the 500 mg/kg/day group because of deaths in the litters.The cause and mechanisms leading to the deaths in the litters remain unclear.

Clinical signs: ptyalism was noted in a dose-related manner in males and females from 50 mg/kg/day. Piloerection, round back and/or half-closed eyes were noted in 1/10 males and 1/9 females given 150 mg/kg/day (during gestation) and in 3/10 males and 6/10 females given 500 mg/kg/day during the premating period. These findings were considered to be related to the test item but of minor toxicological importance. 

Functional Observation Battery and motor activity: there were no effects on Functional Observation Battery tests or motor activity data in any group. 

Body weight and body weight change: in males, when compared with controls, there were slightly to markedly lower body weight gains from 50 mg/kg/day (statistically significant from 150 mg/kg/day), and in females a slight body weight loss was seen from 150 mg/kg/day over the first week of the pre-mating period. A markedly lower body weight gain (statistically significant) was seen in females given 500 mg/kg/day during the gestation period and a slightly to markedly lower body weight gain in females from 150 mg/kg/day during the lactation period. These variations resulted in adverse lower final body weights in males given 150 or 500 mg/kg/day (statistically significant: -16% and -19%vs.controls, respectively) and in females given 500 mg/kg/day at the end of the lactation period (statistically significant: -13%vs.controls even if 2/3 females has lower final body weight). The final body weight was also affected in females given 150 mg/kg/day at the end of the lactation period (-3%) and in females given 500 mg/kg/day at the end of the gestation period (-9%), but to a lesser extent. 

Food consumption: when compared with controls, there were adverse effects on mean food consumption in males given 150 or 500 mg/kg/day (statistically significant: down to -20% or -29% vs. controls, respectively), in females given 500 mg/kg/day during the lactation period (statistically significant: down to -52% vs. controls). The lower food consumption recorded in the 150 mg/kg/day female group during the lactation period and in the 500 mg/kg/day female group during the pre-mating period was considered to be of minor toxicological importance. There were no effects on food consumption at 50 mg/kg/day. 

Hematology: there were no effects on hematological parameters at any dose-level. 

Blood biochemistry: when compared with controls, lower urea levels (statistically significant) were noted in females from 150 mg/kg/day. At 500 mg/kg/day, statistically significantly higher creatinine, total protein and calcium levels were observed in males and higher total bilirubin levels were observed in both sexes. All these changes were considered to be of minor importance. There were no effects on blood biochemistry parameters at 50 mg/kg/day. 

Thyroid hormones: in males,statistically significantly lower T4 levels together with higher TSH levels were observed in males from 150 mg/kg/day. At 500 mg/kg/day and when compared with controls, there was a decrease (not statistically significant) in mean T4 concentration in females and a decrease (not statistically significant) in mean T4 and TSH concentrations in pups for which a relationship to the test item treatment was not excluded.

Estrous cycle: there were no test item-related effects on the mean length of the estrous cycle or the mean number of cycles at any dose-level. 

Pairing, mating and fertility data: there were no effects on the mating index at any dose-level and no adverse effects at 50 and 150 mg/kg/day on pre-coital time (number of days taken to mate), fertility or gestation index. At 500 mg/kg/day, when compared with controls, there was a statistically significant, prolonged pre-coital time (6.5 days vs. 2.0 days in controls), a lower fertility index (55.6% (females) and 50% (males) vs. 100% in controls) and a lower gestation index (60%vs.100% in controls). These findings were considered to be adverse and test item treatment related. 

Delivery data: at 500 mg/kg/day, when compared with controls, a significant prolonged mean gestation day was noted (23.0 days vs. 22.0 days in controls). At 150 and 500 mg/kg/day, lower mean numbers of corpora lutea (12.4 and 11.3 vs. 13.9 in controls, respectively), lower mean numbers of implantation sites [12 and 10 (statistically significant) vs. 13.9 in controls, respectively], higher mean pre-implantation losses (3.8 and 11.4 (statistically significant) vs. 0.0 in controls, respectively), lower mean numbers of pups delivered [9.8 and 6.3 (statistically significant) vs. 11.8 in controls, respectively] and higher mean numbers of post-implantation loss (20.4 and 41.4 (statistically significant) vs. 15.4 in controls) were noted. All these dose‑related findings were considered to be adverse. There were no effects on delivery data at 50 mg/kg/day. 

Pup mortality and viability: at 150 and 500 mg/kg/day, when compared with controls, there were significant reductions in the viability index (statistically significant: 80.7% and 57.9% vs. 98.3% in controls, respectively), marked increases in the number of pups found dead and cannibalized as well as in the incidence of litters affected (4/9 and 1/3vs.1/10 in controls, respectively). These findings were considered to be adverse but can be a consequence of abnormal maternal behavior. There were no effects on the viability index at 50 mg/kg/day or on the live birth index at any dose-level.

Pup clinical signs and external abnormalities: there were no external abnormalities at any dose-level. At 500 mg/kg/day and when compared with controls, no clinical signs were noted in pups from the two surviving litters (11 pups in total). At 150 mg/kg/dayand when compared with controls, an increase in clinical signs due to lack of maternal care (blackish discoloration of the abdomen, dehydration, generalized pallor and/or thinning of hair) was observed in pups along with an increased number of litters affected (3/9 litters with 11/88 affected pups vs. 2/10 with 3/118 affected pups in controls). These findings were considered to be adverse and can be due to lack of maternal.

Pup body weight: at 150 and 500 mg/kg/day, when compared with controls, there were moderate to marked dose-related lower body weight gains [+21.1 and +15.8 g/animal/day (statistically significant) vs.24.0 g/animal/day in controls, respectively] in pups from Day 4 p.p.until the end of the lactation period. This resulted in moderately to markedly lower final body weights at 150 and 500 mg/kg/day [33.0 and 27.4 g (statistically significant) vs. 36.3 g in controls, respectively]. There were no effects on mean body weight or mean body weight change in pups at 50 mg/kg/day.

Pup sex ratio: there were no effects on the mean percentage of male fetuses (sex ratio) at 50 and 150 mg/kg/day. A relevant lower percentage of males was noted at 500 mg/kg/day (28.6 vs. 42.4% in controls). Despite the low number of pups and litters at this dose, a treatment related effect cannot be excluded. 

Pup development: there were no effects on the anogenital distance and no nipples or areolae in male pups at any dose-level.

Pup macroscopic post-mortem examination: there were no external abnormalities at any dose-level. There were no test item-related findings at post-mortem examination of the pups sacrificed as scheduled at any dose‑leve (taking into account the limited number of dams and pups at 500 mg/kg/day). Nevertheless,absence of milk in the stomach observed in two pups from the same litter at 150 mg/kg/day, for which the remaining found dead pups were found autolyzed, was to be test item treatment-related and adverse as dead of the litter was the consequence of abnormal maternal behavior.

Pathology: No test item-related morphological changes were confirmed in surviving F0 animals. No test item-related macroscopic changes were observed in surviving or prematurely dead pups.

  

Based on the experimental conditions of this study:

.          the No Observed Adverse Effect Level (NOAEL) for parental systemic toxicity was considered to be 50 mg/kg/day (based on lower body weight and body weight change, lower food consumption in males and/or females from 150 mg/kg/day and thyroid hormones results in males from 150 mg/kg/day),

.          the No Observed Adverse Effect Level (NOAEL) for reproductive performance (mating, fertility and delivery) was considered to be 50 mg/kg/day (based on the prolonged pre-coital and gestation times and the lower fertility and gestation indexes at 500 mg/kg/day, and on the lower number of corpora lutea, implantation sites and pups delivered, and the higher pre- and post-implantation loss from 150 mg/kg/day),

.          the No Observed Adverse Effect Level (NOAEL) for toxic effects on progeny was considered to be 50 mg/kg/day (based on clinical signs due to the absence of maternal care, on the lower viability index from 150 mg/kg/day,on the lower pup body weight and on the lower percentage of males at 500 mg/kg/day).