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Description of key information

Based on a recent OECD guideline 422 study, a NOAEL of 50 mg/kg bw/day was identified in rats (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).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
repeated dose toxicity: oral, other
Remarks:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
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
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 H 25332 (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
Species:
rat
Strain:
Sprague-Dawley
Remarks:
RjHan: SD
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 mo
nitoring 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
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 oral exposure:
Dose formulation preparation:
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; see § Study plan adherence).
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

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
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
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 volumewas 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).
Positive control:
no
Observations and examinations performed and frequency:
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 remainupright, 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.
Sacrifice and pathology:
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.


Other examinations:
Estrous 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.
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).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
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.
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.
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:
no effects observed
Description (incidence and severity):
there were no effects on hematological parameters at any dose-level.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
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.
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.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test item-related morphological changes were confirmed in surviving F0 animals.
Other effects:
no effects observed
Description (incidence and severity):
Qualitative testis staging did not indicate any abnormalities in the integrity of the various cell types present.
Dose descriptor:
NOAEL
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, thyroid hormones results in males
Critical effects observed:
no

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 reportCiToxLAB France/Study No. 44021 VAS containing the data demonstrating the suitability of the method is currently being finalized.

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)
Executive summary:

Three groups of ten male and ten female Sprague-Dawley rats received the test item, Rhodiantal Original IBCH (batch No.ZRP16074GD), daily, by oral administration (gavage), before mating, during mating and, for the females, throughout gestation and until Day 14 p.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 14p.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.

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 15p.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 15p.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).

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).

 

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 notest item-relatedeffects on themean length of the estrous cycle or the mean number of cycles at any dose-level.

  

Pathology: No test item-related morphological changes were confirmed in surviving F0 animals.

  

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).         

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
reliability 1
System:
other:

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Oral route: One study is available for repeated dose toxicity by oral route and it is chosen as key study (CitoxLab 2018), reliability 1.

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 according to the OECD guideline 422.

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)

Justification for classification or non-classification

Oral route: Based on the NOAEL of 50 mg/kg bw/day identified from the key study, Rhodiantal original IBCH is not classified for repeated toxicity by oral route.