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Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

There is one reliable OECD 421 study performed according to GLP (Spézia, 2020) showing alerts on fertility parameters.

 The test item was administered daily by oral gavage to male and female Sprague-Dawley rats for 15 days before mating, during mating, and (for females) throughout gestation and until Day 13p.p.at dose levels of 0, 80, 250 or 800 mg/kg/day.

 

Under the experimental conditions of the study:

. the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 250 mg/kg/day, based on thyroid follicular hypertrophy associated with low T4 concentration and decreased mean number ofcorpora lutea/implantation sites in females at 800 mg/kg/day,

. the No Observed Adverse Effect Level (NOAEL) for reproductive performance (mating, fertility and delivery) was considered to be 250 mg/kg/day based on decreased fertility index at 800 mg/kg/day,

. the No Observed Adverse Effect Level (NOAEL) for F1 development was considered to be 250 mg/kg/day based on marked decreases in body weight on Days 8-13p.p. (males and females).

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 March 2019 - 27 May 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
Name: dibenzylbenzene, ar-methyl derivative
Synonyms: JARYTHERM DBT; Jarytherm® DBT; DIBENZYLBENZENE, AR-METHYL DERIVATIVE; Dibenzylbenzene, ar-methyl derivative; Dibenzyltoluene
CAS No.: 53585-53-8
Batch No.: B529183511
Description: liquid; colorless to yellow; slightly aromatic
Storage conditions: at room temperature, in a dry and well-ventilated place
Purity: 98.3% (according to CoA); considered as 100% (UVCB)
Correction factor: None
Expiry date: 18 December 2020.
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: breeder: Janvier, Le Genest-Saint-Isle, France
- Age: on the first day of treatment, the animals were approximately 10 weeks old
- Mean body weight: on the first day of treatment, the males had a mean body weight of 394 g (range: 367 g to 431 g) and the females had a mean body weight of 279 g (range: 259 g to 321 g)
- Fasting period before study: no
- Housing: F0 animals were individually housed, except during mating (males + females) and lactation (females + pups), in polycarbonate cages (Tecniplast 2154, 940 cm2)
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: males were acclimated to the study conditions for a period of 7 days before treatment and females were acclimated to the study conditions for a period of 5 days before the beginning of estrous cycle monitoring during the pre-treatment period.
- Parental females selected according to their estrous cyclicity checked before initiation of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): about 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 13 March 2019 to 02 April 2019.
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING FORMULATIONS:
- Emulsion in the vehicle
- Concentration in vehicle: 16, 50 and 160 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day
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 occurred.
The pre-coital time was calculated for each female.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Type of method: Gas Chromatography with Flame Ionization Detection (GC-FID)
Test item concentrations: remained within an acceptable range of variations (-9.1% to +10.9%) when compared to the nominal values
Homogeneity: the dose formulations containing the test item and prepared at 0.5 mg/mL and 200 mg/mL in 0.5% (w/v) carboxymethylcellulose 400-800cPs + 0.5% (w/v) Tween 80 in drinking water treated by reverse osmosis were found to be homogeneous after preparation.
Stability: standard solutions of the test item prepared at 20.0 µg/mL in diluent were found to be stable for 6 days when they were stored at room temperature.
Duration of treatment / exposure:
In the males:
- 2 weeks before mating,
- during the mating period,
- until sacrifice (at least ---- weeks in total),

In the females:
- 2 weeks before mating,
- during the mating period,
- during pregnancy,
- during lactation until Day 13 post-partum inclusive,
- until sacrifice for females which had not delivered.
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
80 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
800 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 animals
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for dose selection:
The dose levels were selected in agreement with the Sponsor, on the basis of the results of the following studies:
- OECD Test Guideline 408: In this GLP study, three groups of ten/sex Sprague-Dawley rats received JARYLEC DBT at dose levels of 5, 50 or 500 mg/kg/day for at least 120 days. Another group of ten/sex rat received the vehicle only (10% gum arabic solution) and acted as a control group. There was no treatment-related mortality, clinical signs or, findings at ophthalmologic, hematology, urinalysis or macroscopic examinations. At 500 and 50 mg/kg/day, the test item was associated with changes suggesting liver enzyme induction. At 50 mg/kg/day there were no noticeable findings,
- a preliminary dose-range finding study in pregnant rats. This study was conducted with dose levels of 100, 500 and 1000 mg/kg/day. The test item was administered to eight female rats per dose once daily by gavage from throughout gestation and lactation up to Day 4 p.p. Significantly decreased weight gains during the first week of treatment (Days 0 to 7 p.c.) in pregnant rats at all dose levels. Food consumption was significantly decreased at 1000 mg/kg/day from Days 1 to 7 and 1 to 20 p.c. The pre-birth loss index in the high dose group was increased. In the F1 generation at 1000 mg/kg/day, the mean litter size was significantly reduced at birth and at Day 4 p.c. and in correspondence, the number of males was significantly decreased. The pup loss index at birth and the number of litters with dead pups was found to be increased at 1000 mg/kg/day,
- OECD Test Guideline 414: The test item was administered to pregnant Wistar rats via oral gavage from Day 6 to 15 p.c. At 25 and 150 mg/kg/day, no maternal toxicity was observed. At 1000 mg/kg/day, clear maternal toxicity, such as reduced body weight gain, was observed. At 25 mg/kg/day, no effects on offspring were observed. At 150 mg/kg/day, only a very slight delay of ossification occurred. Treatment of the females at 1000 mg/kg/day led to fetal growth retardation and increased resorptions. Under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) for maternal toxicity was considered to be 150 mg/kg/day. The No Observed Adverse Effect Level (NOAEL) for developmental toxicity was considered to be 150 mg/kg/day.

Therefore, 800 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. 80 and 250 mg/kg/day).

- Rationale for animal assignment: stratified procedure.
Positive control:
no (not required)
Parental animals: Observations and examinations:
MORTALITY/MORBIDITY:
- Time schedule: 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.

CLINICAL SIGNS:
- Time schedule: from arrival, each animal was observed once a day as part of routine examinations. From the start of the treatment period, each animal was observed once a day, at approximately the same time, for the recording of clinical signs.

BODY WEIGHT:
- Time schedule: the body weight of each male was recorded once before the beginning of the treatment period, on the first day of treatment (Day 1), then once a week until euthanasia. The body weight of each female was recorded once before the beginning of the treatment period, 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 on the day of euthanasia for females which did not deliver) and on Days 1, 4, 8 and 13 p.p.

FOOD CONSUMPTION:
- Time schedule: 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, during gestation 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.

THYROID HORMONES:
- Time schedule: at termination in males --- ajouter les femelles si leurs analyses ont été faites---

REPRODUCTION (apart from indices):
- Pre-coital time and duration of gestation were recorded for each female.

Oestrous cyclicity (parental animals):
The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning (between 8 and 10 am) (see § Study plan adherence):
- during the 2 weeks of the pre-treatment period (including for the two supplementary females per group, whose data are not presented in the study report),
- from the beginning of the treatment period during the pre-mating and mating periods, until the females were mated,
- on the day and before euthanasia.
Litter observations:
STANDARDISATION OF LITTERS: No

PARAMETERS EXAMINED:
The following parameters were examined in F1 offspring:
- number and sex of pups,
- number of live, dead and cannibalized pups,
- presence of gross anomalies, weight gain, clinical signs.

GROSS EXAMINATION OF DEAD PUPS:
- external and internal abnormalities.

THYROID HORMONES:
- at termination on Day 13 post-partum from at least 2 pups/litter
--- ajouter les pups à PND 4 si leurs analyses ont été faites ----
Postmortem examinations (parental animals):
SACRIFICE
- males: after the end of the mating period (at least 4 weeks of treatment in total),
- females: on Day 14 p.p.,
- females which did not deliver: on Days 25 or 26 p.c. (after a body weight recording to check for a possible un-noticed delivery; euthanasia by inhalation of carbon dioxide gas followed by cervical dislocation was used when gestation was suspected).

GROSS NECROPSY
F0 animals
A macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed on all F0 animals including euthanized prematurely. 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 14 p.p. and when possible for females euthanized 25 days after the end of the mating period with no evidence of mating and for females euthanized on Days 25-26 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.

PRESERVATION OF TISSUES
The tissues of F0 animals specified in the Tissue Procedure Table were preserved in 10% buffered formalin (except for the testes and epididymides which were fixed in Modified Davidson's fixative).
Thyroids with parathyroids of the selected pup/sex/litter euthanized on Day 13 p.p. were preserved in 10% buffered formalin.

PREPARATION OF HISTOLOGICAL SLIDES
All tissues required for microscopic examination were trimmed based on the RITA guidelines, when applicable, 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.
Other specific staining may be performed as necessary and will be specified in a study plan amendment.
If microscopic examination of thyroids with parathyroids of the selected pup/sex/litter is required (at additional cost and to be documented in a study plan amendment), these organs will be weighed (fixed) before processing for preparation of histological slides.

HISTOPATHOLOGY
A microscopic examination was performed on:
- all tissues listed in the Tissue Procedure Table from all animals of the control and high-dose groups (groups 1 and 4) euthanized as scheduled (at the end of the mating period for males or on Day 14 p.p. for females),
- all macroscopic lesions of all groups.

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

Based upon the results of the microscopic examination of the high-dose group and after the agreement of the Sponsor, other tissues of the low- and intermediate-dose groups may be examined (at additional cost and to be documented in a study plan amendment).

ORGAN WEIGHTS:
The body weight of each F0 animal euthanized as scheduled (after the end of the mating period for males or on Day 14 p.p. for females) was recorded before euthanasia. For these animals, the organs specified in the Tissue Procedure Table were weighed wet as soon as possible after dissection, or after fixation for thyroids with parathyroids (when applicable).
The body weight of one selected pup/sex/litter euthanized on Day 13 p.p. was recorded before euthanasia.
Thyroids with parathyroids of the selected pups were weighed after fixation if a microscopic examination was requested (at additional cost and to be documented by a study plan amendment).
The ratio of organ weight to body weight (recorded immediately before euthanasia) was calculated.
Postmortem examinations (offspring):
SACRIFICE:
Pups were euthanized by an intraperitoneal injection of sodium pentobarbital (or by decapitation under isoflurane anesthesia on Day 4 p.p. when blood sampled, see § Thyroid hormones), followed by exsanguination when the thyroids were sampled (see § Preservation of tissues):
- pups whose mother dies: as soon as possible,
- pups not selected on Day 4 p.p.: on Day 4 p.p.,
- surviving pups: on Day 13 p.p.

GROSS NECROPSY:
Pups not selected on Day 4 p.p. were discarded without further examination.
Pups euthanized on Day 13 p.p. were submitted to a detailed external examination (including orifices and buccal cavity) after euthanasia. Particular attention was paid to the external genital organs. Then, they were discarded without any further examination, or after sampling of thyroids with parathyroids for the selected pups.

PRESERVATION OF TISSUES
The tissues of F0 animals specified in the Tissue Procedure Table were preserved in 10% buffered formalin (except for the testes and epididymides which were fixed in Modified Davidson's fixative).
Thyroids with parathyroids of the selected pup/sex/litter euthanized on Day 13 p.p. were preserved in 10% buffered formalin.

HISTOPATHOLOGY: No.

ORGAN WEIGTHS: No.
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 Fischer exact probability test (proportions).
Hormones, anogenital distance, nipples/areolae, live birth index(es), sex ratio and post-implantation loss
CITOX software was used to perform the statistical analysis of these 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 = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea
Post-implantation loss = 100 * (Number of implantation sites - Number of live pups) / Number of implantation sites
Mating index = 100 * (Number of mated animals / Number of paired animals)
Fertility index = 100 * (Number of pregnant female partners / Number of mated pairs)
Gestation index = 100 * (Number of females with live born pups / Number of pregnant females)
Offspring viability indices:
Live birth index = 100 * (Number of live born pups on Day 1 post-partum/ Number of delivered pups)
Viability index on Day 4 post-partum = 100 * (Number of surviving pups on Day 4 post-partum / Number of delivered pups)
Lactation index on Day 13 post-partum = 100 * (Number of surviving pups on Day 13 post-partum / Number of surviving pups on Day 4 post-partum (after culling))
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related clinical signs were observed during pre-mating, gestation and lactation period in males and females at any dose level except for ptyalism which is a common observation after gavage procedure.
All other clinical signs recorded during the study, cutaneous observations (areas of hair loss, cutaneous lesions, scabs), chromodacryorrhea, reflux at dosing, eye pallor/discoloration, general aspects (round back, piloerection, pallor of extremities) and reddish nasal or vaginal discharge, were considered to be unrelated to the test item treatment as they were present both in control and test item-related animals, and/or were reported sporadically in only a few animals. In addition, these findings are common observations in these experimental conditions for this strain of animals.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were no unscheduled deaths in males during the study.

Pre-mating period:
Female P29898 given 800 mg/kg/day was prematurely euthanized on Day 10. Signs of poor clinical conditions (emaciated appearance, dehydration, round back, piloerection and/or cold to the touch from Day 8) were observed prior to death. Necropsy findings (heart with a white discoloration, harderian glands with many punctiform discolorations, lungs with a diffuse red discoloration and a forestomach wall with a diffuse white deposit) did not correlate with microscopically findings. Therefore, a technical issue during the gavage procedure cannot be excluded.

Gestation period:
There were no unscheduled deaths in females during the gestation period.

Lactation period:
One female given 80 mg/kg/day was prematurely euthanized on Day 2 p.p. due to difficulties to deliver. Round back and piloerection were observed before euthanasia. At necropsy, this female had five dead fetuses with placentae (right horn) and five placentae + seven scars (left horn). In the absence of any dose level relationship, a test item treatment relationship was considered unlikely.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
See tables 1 to 1ter.
Premating (males and females) and post-mating (males) periods:
There were no effects during the premating (males and female) and post-mating (males) periods on mean body weight and mean body weight change.

Gestation period:
At 80 and 250 mg/kg/day, there were no test item-related effects during the gestation period on mean body weight and mean body weight change.
At 800 mg/kg/day and when compared with controls, there were lower mean body weight (-8%, p<0.05) on Day 20 p.c. and lower mean body weight change (+17 g vs. +36 g in controls, p<0.001) on Days 0 to 7 p.c. Taking into account the amplitude of these changes and in the absence of any associated clinical findings and effects on mean food consumption during the gestation period, a test item relationship was considered to be unlikely.

Lactation period:
There were no effects during the lactation period on mean body weight and mean body weight change.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
See tables 2 and 2bis
Premating period:
There were no test item-related effects during the pre-mating period on mean food consumption.
At 800 mg/kg/day and when compared with controls, there were lower mean food consumption (-14%, p<0.05) on Days 1 to 8. Taking into account the amplitude of these changes and in the absence of any associated clinical findings and effects on body weight during the premating period, a test item relationship was considered to be unlikely.

Gestation period:
There were no effects during the gestation period on mean food consumption.

Lactation period:
At 800 mg/kg/day and when compared with controls, there was a lower mean food consumption (down to 29%, p<0.001) during the lactation period. This finding was considered to be not adverse in the absence of associated clinical signs and effects on mean body weight or mean weight change during the lactation period. However, a test item relationship cannot be excluded.
At 250 and 80 mg/kg/day, there were no effects on mean food consumption during the lactation period.
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
See table 5.
There were no test item-related microscopic changes except minimal dose-related thyroid gland hypertrophy in males treated at = 80 mg/kg/day.
The hypertrophy of follicular cells was associated with a decreased size of follicles and decreased colloid in the lumen of follicles, correlating with the decrease thyroid gland weight at 800 mg/kg/day.
This correlated also with the T4 decreased concentration at 800 mg/kg/day.
It is noteworthy that this correlated also with individual TSH increase since there was an individual matching between the hypertrophy and intra-group high TSH concentration, although the overall TSH concentrations remained within the range of historical control data.
The other occasional findings were considered to be part of the spontaneous background.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
THYROID HORMONES:
See tables 3 and 3bis.
At 250 and 80 mg/kg/day, when compared with controls or Historical Control Data, there were no adverse effects on mean TSH and T4 concentrations.

In F0 males, from 250 mg/kg/day and when compared with controls, there were increases in mean TSH concentrations (up to +38% at 800 mg/kg/day, not statistically significant) associated with lower mean T4 concentrations at 800 mg/kg/day (-22%, p<0.001). When compared with Historical Control Data, all values remained within the ranges. Therefore, while a test-item relationship cannot be excluded, these findings were considered not to be adverse.
In Day 13 p.p. pups and when compared with controls, there were no effects on mean TSH concentrations. However, there were lower T4 concentrations from 250 mg/kg/day (down to -44% at 800 mg/kg/day). At 800 mg/kg/day, when compared with Historical Control Data, mean T4 concentration was below the lower limit. Therefore, this finding was considered to be adverse.

Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related effects on estrous cycle during the pre-mating period.
At 250 mg/kg/day and when compared with controls, there was an incidental higher number of days of proestrus (3.9 vs. 2.8, p<0.05). In the absence of a dose level relationship, a test item effect was considered to be unlikely.
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
See table 6.
Pairing, mating and fertility data
There were no effects on mating index and all groups had comparable pre-coital time intervals (number of days taken to mate).
At 800 mg/kg/day and when compared with controls, there was a lower fertility index (77.8% vs. 100%) as a consequence of two non pregnant females (P29887 and P29888). In this strain and species, fertility index is usually ranging from 91.7 to 100% (Two generation reproduction toxicity studies and Reproduction screening toxicity screening tests, April 2010 to December 2017, n = 7 studies), therefore a test item treatment relationship cannot be excluded.
At 250 and 80 mg/kg/day, there were no effects on fertility index.

Reproductive and litter data
See table 7.
In all test item-treated groups and when compared with controls, there were no effects on mean duration of gestation.
At 800 mg/kg/day and when compared with controls, there was a lower mean number of pups delivered (9.1 vs. 14.9, p<0.001) has a consequence of lower mean numbers of corpora lutea (13.3 vs. 17.4, p<0.001) and implantation sites (11.9 vs. 17.2, p<0.001). When compared with Historical Control Data, the values were below the lower limits of the ranges. Therefore, these findings were considered to be test item treatment-related and adverse.
At 250 and 80 mg/kg/day, there were no effects on reproductive and litter data.
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
other: based on thyroid follicular hypertrophy associated with low T4 concentration and decreased mean number of corpora lutea/implantation sites in females at 800 mg/kg/day, and decrease fertility index
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
800 mg/kg bw/day (nominal)
System:
female reproductive system
Organ:
thyroid gland
other: decreased mean number of corpora lutea/implantation sites in females
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
800 mg/kg bw/day (nominal)
System:
other: males
Organ:
thyroid gland
Treatment related:
yes
Dose response relationship:
yes
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
At 800 mg/k/day and when compared with controls, one litter had 8 pups with emaciated appearance/dehydration on Days 6-10 p.p. This finding was no longer observed from Day 11 p.p. up to termination of Day 13 p.p. While a test item treatment relationship cannot be excluded, this finding was considered to be not adverse based on reversibility.
At 250 and 80 mg/kg/day, there were no test item treatment-related findings.
All other findings (hematoma and scab) recorded in pups during the lactation period are common observations in this species and strain of rats maintained in the experimental condition of this study.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
See tables 8 and 9.
There were no effects on the incidences of pups found dead or cannibalized.
There were no test item-related effects on pup viability.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 800 mg/kg/day, in both sexes and when compared with controls, there were lower mean body weight after culling (down to -27% on Day 13 p.p., p<0.001) as a consequence of lower mean body weight gain (+9.6 g vs. +14.6 g). These effects were also observed from 250 mg/kg/day in females with a decrease in mean body weight (-11% on Day 13 p.p.) as a consequence of lower mean body weight gain (+12.7 g vs. +14.5 g, p<0.05). Taking into account the amplitudes of the effects these findings were considered to be adverse at 800 mg/kg/day. There were no effects at 80 mg/kg/day.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
See table 5.
There were no test item-related microscopic changes in the thyroid glands.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Thyroid hormones
In Day 13 p.p. pups and when compared with controls, there were no effects on mean TSH concentrations. However, there were lower T4 concentrations from 250 mg/kg/day (down to -44% at 800 mg/kg/day). At 800 mg/kg/day, when compared with Historical Control Data, mean T4 concentration was below the lower limit. Therefore, this finding was considered to be adverse.

Sex ratio
See table 11.
There were no effects on sex ratio (mean percentage of males) at any dose level.

Anogenital distance
See tables 12 and 12bis.
In both sexes, there were no test item treatment effects on mean anogenital distance (and normalized AGD).
At 800 mg/kg/day and when compared with controls, there were apparent increases in mean AGD and normalized AGD both in male and female pups (with statistical significances in males). When compared with Historical Control Data, the values remained within the ranges. Therefore, a test-item relationship was considered to be unlikely.

Areolae and nipples
See table 13.
There were no test item-related areolae or nipples in male pups on Day 12 p.p.
In the control and 800 mg/kg/day groups, pup No. 3 from one dam and pup No 1 from another dam had an areola. Therefore, this finding was considered to represent spontaneous background in this species and strain. Any relationship to the test item treatment in the high-dose group was considered to be unlikely.
Key result
Dose descriptor:
NOAEL
Effect level:
80 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Reproductive effects observed:
yes
Lowest effective dose / conc.:
800 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects in the absence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
not specified

Table 1: Body weight and body weight changes (pre-mating and post-mating periods)

Sex

Female

Dose level (mg/kg/day)

0

80

250

800

Mean body weight (g)

. Day 0p.c.

298

297

(0)

301

(+1)

304

(+2)

. Day 7p.c.

334

332

(-1)

333

(0)

321

(-4)

. Day 14p.c.

375

374

(0)

371

(-1)

357

(-5)

. Day 20p.c.

468

471

(+1)

459

(-2)

430*

(-8)

Mean body weight change (g)

. Days 0 to 7p.c.

+36

+35

+32

+17#

. Days 7 to 14p.c.

+41

+42

+38

+36

. Days 14 to 20p.c.

+93

+97

+88

+74

. Days 14 to 20p.c.

+170

+174

+158

+126#

(): in brackets, percentage (%) differencevs.controls.

Statistical significance: *: p<0.05, #: p<0.001.

Table 1bis: Body weight and body weight changes (gestation period)

Sex

Female

Dose level (mg/kg/day)

0

80

250

800

Mean body weight (g)

. Day 0p.c.

298

297

(0)

301

(+1)

304

(+2)

. Day 7p.c.

334

332

(-1)

333

(0)

321

(-4)

. Day 14p.c.

375

374

(0)

371

(-1)

357

(-5)

. Day 20p.c.

468

471

(+1)

459

(-2)

430*

(-8)

Mean body weight change (g)

. Days 0 to 7p.c.

+36

+35

+32

+17#

. Days 7 to 14p.c.

+41

+42

+38

+36

. Days 14 to 20p.c.

+93

+97

+88

+74

. Days 14 to 20p.c.

+170

+174

+158

+126#

(): in brackets, percentage (%) differencevs.controls.

Statistical significance: *: p<0.05, #: p<0.001.

Table 1ter: Body weight and body weight changes (lactation period)

Sex

Female

Dose level (mg/kg/day)

0

80

250

800

Mean body weight (g)

. Day 1p.p.

356

361

356

334

. Day 4p.p.

371

374

372

344

. Day 8p.p.

388

390

390

362

. Day 13p.p.

404

407

406

381

Mean body weight change(g)

. Days 1 to 4p.p.

+15

+13

+16

+10

. Days 4 to 8p.p.

+17

+16

+18

+18

. Days 8 to 13p.p.

+16

+17

+15

+20

. Days 1 to 13p.p.

+49

+45

+50

+47

No statistically significant differencesvs.controls.


 Table 2: Food consumption (pre-mating period)

Sex

Male

Female

Dose level (mg/kg/day)

0

80

250

800

0

80

250

800

. Days 1 to 8

34

34

(0)

35

(+3)

35

(+3)

22

24

(+9)

22

(0)

19*

(-14)

. Days 8 to 15

36

35

(-3)

36

(0)

38

(+6)

22

24

(+9)

24

(+9)

21

(-5)

. Days 1 to 15

35

35

(0)

35

(0)

37

(+6)

22

24

(+9)

23

(+5)

19

(-14)

(): in brackets, percentage (%) differencevs.controls.

Statistical significance: *: p<0.05.

Table 2bis: Food consumption (gestation period)

Sex

Female

Dose level (mg/kg/day)

0

80

250

800

. Days 0 to 7p.c.

25

26

29

23

. Days 7 to 14p.c.

29

28

29

26

. Days 14 to 20p.c.

34

36

33

33

. Days 0 to 20p.c.

30

30

31

28

No statistically significant differencesvs.controls.

Table 2ter: Food consumption (lactation period)

Sex

Female

Dose level (mg/kg/day)

0

80

250

800

. Days 1 to 4p.p.

46

48

(+4)

44

(-4)

33#

(-28)

. Days 4 to 8p.p.

62

61

(-2)

57

(-8)

44#

(-29)

. Days 8 to 13p.p.

77

75

(-3)

70

(-9)

55#

(-29)

. Days 1 to 13p.p.

62

61

(-2)

57

(-8)

44#

(-29)

(): in brackets, percentage (%) differencevs.controls.

Statistical significance: #: p<0.001.

Table 3: Thyroid hormones

Mean TSH concentrations (ng/mL)

Dose level (mg/kg/day)

0

80

250

800

HCD

[min.; max.]

. F0 males at termination

2.63

2.73

(+4)

4.11

(+57)

3.63

(+38)

[0.63; 4.42]

. Day 13p.p.pups

1.46

1.36

(-7)

1.54

(+6)

1.47

(+1)

HCD: Historical Control Data (n = 40 males).

(): in brackets, percentage (%) differencevs.controls.

No statistically significant differencesvs.controls.

Table 3bis: Thyroid hormones

Mean T4 concentrations (ng/mL)

 

Dose level (mg/kg/day)

0

80

250

800

HCD

[min.; max.]

. F0 males at termination

35.09

34.71

(-1)

33.98

(-3)

27.25**

(-22)

[25.6; 54.2]

. Day 13p.p.pups

34.64

31.64

(-9)

28.53*

(-18)

19.53**

(-44)

HCD: Historical Control Data (n = 40 males).

(): in brackets, percentage (%) differencevs.controls.

Statistical significancevs.controls: **: p<0.01.

Table 4: Organ weights

Sex

Male

Female

Dose-level (mg/kg/day)

80

250

800

80

250

800

Examined animals/total number of animals

10/10

10/10

10/10

9/10

10/10

9/10

- Final body weight

-1

-1

-4

+2

+1

-8

- Thyroid glands

. absolute

-8

-8

-15*

+5

+9

0

. relative-to-body

-7

-7

-12*

+2

+8

+9

Statistically significant from controls: *: p<0.05

Table 5: Microscopic examination

Sex

Male

Female

Group

1

2

3

4

1

2

3

4

Dose-level (mg/kg/day)

0

80

250

800

0

80

250

800

Number of animals

10

10

10

10

10

9

10

9

Thyroid glands : hypertrophy of follicular cells

 

 

. grade 1

-

3

5

9

na

na

na

na

-: not present; na:not applicable.

Table 6: Pairing, mating and fertility data

Dose level (mg/kg/day)

0

80

250

800

Number of animals paired (M+F)

10+10

10+10

10+10

9+9(a)

Number of males mated

10

10

10

9

Number of females mated

10

10

10

9

Mean number of days taken to mate(b)

2.5

2.8

3.9

3.2

Mating index (%)

100

100

100

100

Number of pregnant females

10

10

10

7

Fertility index (%)

100

100

100

77.8

M: male; F: female.

(a): female P29898 was prematurely euthanized before mating (male P26640 was therefore not paired).

(b): no statistical significancevs.controls.

Table 7: Reproductive and litter data

Dose level

(mg/kg/day)

0

80

250

800

HCD

[min.; max.]

Number of pregnant females

10

10

10

7

30

Number of females which delivered

9

9

8

7

30

Mean duration of gestation (days)

22.0

22.0

21.9

22.0

[22.0; 22.2]

Mean number ofcorpora lutea

17.4

17.4

16.4

13.3#

[14.0; 16.6]

Mean number of implantation sites

17.2

16.9

15.4

11.9#

[13.4; 14.4]

Mean pre-implantation loss (%)

1.3

3.2

6.9

11.3

[4.0; 14.7]

Mean number of pups delivered

14.9

13.8

13.4

9.1#

[10.7; 12.3]

Mean post-implantation loss (%)

13.5

17.9

13.7

20.8

[16.7; 22.5]

HCD: Historical Control Data (OECD 421/422, Jan-2016 to Dec-2017, n = 3 studies).

Statistical significancevs.controls:#: p<0.001.

Table 8: Pups mortality

Dose level (mg/kg/day)

0

80

250

800

Number of litters

9

9

8

7

Number of pups found dead

5

2

0

2

Number of pups cannibalized

1

1

0

3

Table 9: Pups clinical signs

(Number of pups affected per group)

Dose level (mg/kg/day)

0

80

250

800

Emaciated appearance

 

1 (1): Day 4p.p.

 

8 (1):Days 6-10p.p.

Hematoma (head)

 

 

2 (1): Days 1-2p.p.

 

Dehydration

 

 

 

8 (1):Days 6-10p.p.

Scab (abdomen and/or tail)

1 (1): Day 1p.p.

3 (1): Days 12-13p.p.

1 (1): Day 13p.p.

 

Number of litters

9

9

8

7

Number of affected litters [%]

1 [11]

2 [22]

2 [25]

1 [14]

(): in brackets, number of litters.

Table 10: Pups viability

Dose level (mg/kg/day)

0

80

250

800

Live birth index (%)

96.5

97.5

100.0

91.8

Day 4p.p. viability index (%)

95.7

97.5

100.0

91.8

Day 13p.p. lactation index (%)

100.0

100.0

100.0

100.0

No statistical significancevs.controls.

Table 11: Pup body weight

Dose level (mg/kg/day)

0

80

250

800

Mean body weight (males and females)

. Day 1p.p.

7.9

8.0

(+1)

7.6

(-4)

7.4

(-6)

. Day 4p.p. (preculling)

11.2

11.2

(0)

10.7

(-4)

10.5

(-6)

. Day 8p.p.

21.1

20.3

(-4)

19.3

(-9)

16.6**

(-21)

. Day 13p.p.

35.7

34.3

(-4)

32.6

(-9)

26.2#

(-27)

Mean body weight (males)

. Day 1p.p.

8.1

8.1

(0)

7.8

(-4)

7.6

(-6)

. Day 4p.p. (preculling)

11.4

11.4

(0)

11.0

(-4)

10.7

(-6)

. Day 8p.p.

21.6

20.5

(-5)

19.9

(-8)

16.9#

(-22)

. Day 13p.p.

36.3

34.5

(-5)

33.7

(-7)

26.6#

(-27)

Mean body weight (females)

. Day 1p.p.

7.6

7.7

(+1)

7.3

(-4)

7.1

(-7)

. Day 4p.p. (preculling)

10.8

11.0

(-2)

10.3

(-5)

10.1

(-6)

. Day 8p.p.

20.6

20.1

(-2)

18.6

(-10)

16.1**

(-22)

. Day 13p.p.

35.0

34.0

(-3)

31.3

(-11)

25.6#

(-27)

(): in brackets, percentage (%) differencevs.controls.

Statistical significancevs.controls: **: p<0.01;#: p<0.001.

Table 11bis: Pup body weight change

Dose level (mg/kg/day)

0

80

250

800

Body weight changes (males and females)

. Days 1 - 4p.p.

+3.3

+3.3

+3.1

+3.0

. Days 4 - 8p.p. (preculling)

+9.9

+9.2

+8.7

+6.1#

. Days 8 - 13p.p.

+14.6

+13.9

+13.3

+9.6#

Body weight changes (males)

. Days 1 - 4p.p.

+3.3

3.3

+3.2

+3.1

. Days 4 - 8p.p. (preculling)

+10.0

+9.2

+8.9

+6.2#

. Days 8 - 13p.p.

+14.8

+14.0

+13.8

+9.7#

Body weight changes (females)

. Days 1 - 4p.p.

+3.2

+3.3

+3.0

+3.0

. Days 4 - 8p.p. (preculling)

+9.8

+9.2

+8.3

+6.0#

. Days 8 - 13p.p.

+14.5

+13.9

+12.7*

+9.5#

Statistical significancevs.controls: *: p<0.05;#: p<0.001.

Table 12: Sex ratio

Dose level (mg/kg/day)

0

80

250

800

Day 1p.p. viability index (%)

57.0

59.5

56.7

48.0

Day 13p.p. lactation index (%)

51

54

53

50

No statistical significancevs.controls.

Table 13: Anogenital distance

AGD in male pups

Dose level
(mg/kg/day)

0

80

250

800

HCD

[min.; max.]

AGD

5.28

5.46

(+3)

5.40

(+2)

6.01**

(+14)

[4.14; 6.55]

AGD / (BW)1/3

2.64

2.72

(+3)

2.73

(+3)

3.06**

(+16)

[2.16; 3.26]

HCD: Historical Control Data (Anogenital Distance in F1 generation on Day 1p.p., n = 54 male pups).

AGD: anogenital distance on Day 1p.p.

(BW)1/3: cube root of body weight recorded on Day 1p.p.

(): in brackets, percentage (%) differencevs.controls.

Statistical significancevs.controls: **: p<0.0

Table 13bis: Anogenital distance

AGD in female pups

Dose level

(mg/kg/day)

0

80

250

800

HCD

[min.; max.]

AGD

3.15

3.44

(+9)

3.46

(+10)

3.59

(+14)

[1.85; 4.71]

AGD / (BW)1/3

1.61

1.75

(+9)

1.74

(+8)

1.87

(+16)

[0.92; 2.37]

HCD: Historical Control Data (Anogenital Distance in F1 generation on Day 1p.p., n = 50 female pups).

AGD: anogenital distance on Day 1p.p.

(BW)1/3: cube root of body weight recorded on Day 1p.p.

(): in brackets, percentage (%) differencevs.controls.

No statistical significancevs.controls.

Table 14: Areolae and nipples

Dose level (mg/kg/day)

0

80

250

800

Mean number of areolae

0.2

0.0

0.0

0.3

Mean number of nipples

0.0

0.0

0.0

0.0

No statistical significancevs.controls.

 

Conclusions:
The test item was administered daily by oral gavage to male and female Sprague-Dawley rats for 15 days before mating, during mating, and (for females) throughout gestation and until Day 13 p.p. at dose levels of 0, 80, 250 or 800 mg/kg/day.

Under the experimental conditions of the study:
- the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 250 mg/kg/day, based on thyroid follicular hypertrophy associated with low T4 concentration and decreased mean number of corpora lutea/implantation sites in females at 800 mg/kg/day,
- the No Observed Adverse Effect Level (NOAEL) for reproductive performance (mating, fertility and delivery) was considered to be 250 mg/kg/day based on decreased fertility index at 800 mg/kg/day,
- the No Observed Adverse Effect Level (NOAEL) for F1 development was considered to be 80 mg/kg/day based on marked decreases in body weight on Days 8-13 p.p. (males and females).
Executive summary:

Objectives

The objective of the present study was to evaluate the potential toxicity effects of the test item following daily oral administration (gavage) to male and female rats from before mating, through mating and, for females, through gestation until Day 13 post-partum (p.p.).

This study provided initial information on male and female reproductive performance, such as gonadal function, mating behaviour, conception, development of the conceptus and parturition.

 

Materials and methods

Three groups of 10 male and 10 female Sprague-Dawley rats received the test item daily by oral route (gavage) at dose levels of 80, 250 and 800 mg/kg/day. Males were treated for 2 weeks before mating, throughout mating and then until the day before euthanasia (i.e. after a minimum treatment period of 4 consecutive weeks). Females were treated for 2 weeks before pairing, throughout mating and gestation periods, until Day 13 p.p. inclusive.

Another group of 10 males and 10 females received the vehicle only [0.5% (w/v) carboxymethylcellulose 400-800cPs + 0.5% (w/v) Tween 80 in drinking water] under the same experimental conditions and acted as a control group. A constant dosage volume of 5 mL/kg/day was used.

The actual test item concentrations in the dose formulations prepared for use in Weeks 1, 3, 6 and 7 were determined using a validated Gas Chromatography with Flame Ionization Detection (GC-FID) analytical method.

Animals were checked daily for clinical signs and mortality. Body weight and food consumption were recorded weekly until mating and then at designated intervals throughout gestation and lactation.

The dams were allowed to deliver and rear their progeny until Day 13 p.p. The total litter sizes and numbers 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. The pups were observed daily for clinical signs and abnormal behavior and were weighed on Days 1, 4, 8 and 13 p.p. The physical development of pups was assessed by measuring the anogenital distance on Day 1 p.p. and by counting the number of nipples and areolae in male pups on Day 12 p.p.

Thyroid hormone (TSH and T4) levels were determined in all F0 males and females at sacrifice and in pups sacrificed on Day 4 p.p. and on Day 13 p.p.

Males were euthanized after completion of the mating period. Dams were euthanized on Day 14 p.p.

A full macroscopic post-mortem examination was performed, with attention accorded to the reproductive organs. Designated organs were weighed, and selected tissue specimens were preserved. A microscopic examination was performed on epididymides, ovaries and testes from the control and high-dose groups and on all macroscopic lesions.

A macroscopic post-mortem examination was performed on pups, including those found dead before study termination.


Results

The test item concentrations in the administered dose formulations analyzed in Weeks 1, 3, 6 and 7 remained within an acceptable range of variation (-9.1% to +10.9%) when compared to the nominal values (± 15% of the nominal concentrations). No test item was observed in the control dose formulation.

 

At all dose-levels, there were no test item-related deaths and no adverse clinical signs.

 

At all dose-levels, there were no test item-related effects on mean body weight and mean body weight change during the premating (males and female), gestation/lactation (females) and post-mating (males) periods. There were no test item-related adverse effects on mean food consumption during the pre-mating period (males and females) and gestation/lactation (females) periods.

 

At all dose-levels, there were no test item-related effects on the estrous cycle during the pre-mating period. There were no effects on mating index and all groups had comparable pre-coital time intervals (number of days taken to mate).

 

At 80 and 250 mg/kg/day, there were no effects on fertility index. In all test item-treated groups and when compared with controls, there were no effects on mean duration of gestation.

At 250 and 80 mg/kg/day, there were no effects on reproductive and litter data.

 

At 800 mg/kg/day and when compared with controls, there was a lower fertility index (77.8% vs. 100%) as a consequence of two non-pregnant females at this dose level for which a test item treatment relationship cannot be excluded.

 

At 80 and 250 mg/kg/day, there were no effects on reproductive and litter data.

At 800 mg/kg/day and when compared with controls, there was a lower mean number of pups delivered (9.1 vs. 14.9, p<0.001) because of the lower mean numbers of corpora lutea (13.3 vs. 17.4, p<0.001) and implantation sites (11.9 vs. 17.2, p<0.001). When compared with Historical Control Data, the values were below the lower limits of the ranges. Therefore, these findings were considered to be test item treatment-related and adverse.

At 800 mg/kg/day, in pups from both sexes and when compared with controls, there were lower mean body weight after culling (down to -27% on Day 13 p.p., p<0.001) as a consequence of lower mean body weight gain (+9.6 g vs. +14.6 g). These effects were also observed from 250 mg/kg/day in females with a decrease in mean body weight (-11% on Day 13 p.p.) as a consequence of lower mean body weight gain (+12.7g vs. +14.5 g, p<0.05).

Taking into account the amplitudes of the effects these findings were considered to be adverse at 800 mg/kg/day.

 

At all dose-levels, there were no test-item treatment effects on mean anogenital distance (and normalized AGD). There were no test-item related areolae or nipples in male pups on Day 12 p.p.

 

There were no effects on the incidences of pups found dead or cannibalized andno test item-related effects on pup viability at all dose-levels.

 

At 80 mg/kg/day, there were no effects on mean TSH or T4 plasma concentration levels.

At 250 and 800 mg/kg/day, in F0 parent males, there were increases in mean TSH concentrations (up to +38% at 800 mg/kg/day, not statistically significant) associated with lower mean T4 concentrations at 800 mg/kg/day (-22%, p<0.001). When compared with Historical Control Data, all values remained within the ranges. Therefore, while a test-item treatment relationship cannot be excluded, this finding was considered not to be adverse.

At 250 and 800 mg/kg/day, in Day 13 p.p. pups and when compared with controls, there were no effects on mean TSH concentrations while lower T4 concentrations were observed from 250 mg/kg/day (down to -44% at 800 mg/kg/day and outside the range of Historical Control Data). Therefore, this finding was considered to be adverse at 800 mg/kg/day.

 

Pathology

The premature sacrifice of two test item-treated females was most probably unrelated to the test item administration.

There were test item-related decreased thyroid gland weight in males at 800 mg/kg/day and microscopic thyroid gland hypertrophy in males treated at = 80 mg/kg/day (parents).

In view of the correlation with the decreased T4 concentration and in spite of its low severity, the follicular hypertrophy was considered to be adverse at 800 mg/kg/day.

The thyroids pups were unaffected at microscopic examination.

 

Conclusion

 The test item was administered daily by oral gavage to male and female Sprague-Dawley rats for 15 days before mating, during mating, and (for females) throughout gestation and until Day 13 p.p. at dose levels of 0, 80, 250 or 800 mg/kg/day.

 

Under the experimental conditions of the study:

. the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 250 mg/kg/day, based on thyroid follicular hypertrophy associated with low T4 concentration and decreased mean number of corpora lutea/implantation sites in females at 800 mg/kg/day,

. the No Observed Adverse Effect Level (NOAEL) for reproductive performance (mating, fertility and delivery) was considered to be 250 mg/kg/day based on decreased fertility index at 800 mg/kg/day,

. the No Observed Adverse Effect Level (NOAEL) for F1 development was considered to be 80 mg/kg/day based on marked decreases in body weight on Days 8-13 p.p. (males and females).

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subacute
Species:
rat

Effects on developmental toxicity

Description of key information

The effects of Dibenzylbenzene, ar-methyl derivative on the development was evaluated in one developmental toxicity study in rats (Kurosaki, 1988) and one develomental toxicity in rabbits (Haag, 2020) both performed according to OECD 414..

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
some information such as the purity or batch number of the test substance or concerning the exposure conditions are lacking.
GLP compliance:
not specified
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: no data
- Age at study initiation: 14 weeks for males and 12-13 weeks for females
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: individual aluminium cages for the gravid females
- Diet (e.g. ad libitum): solid diet (aliment MF, oriental yeast Co., Ltd.)
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period:


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 25 +/- 1
- Humidity (%): 55 +/- 5
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12 (6:00 to 18:00)
Route of administration:
oral: gavage
Vehicle:
other: Sesame oil (japanese pharmacopea)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: no data

VEHICLE
- Justification for use and choice of vehicle (if other than water): dibenzyltoluene is not soluble in water
- Concentration in vehicle: 50%
- Amount of vehicle (if gavage): 2, 06 and 0.2 ml/kg, respectively for the dose-levels of 1000, 300 and 100 mg/kg/d
- Lot/batch no. (if required): no data
- Purity: no data
Details on analytical verification of doses or concentrations:
No data
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: no data
- Length of cohabitation: one night
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
from day 7 to day 17 of gestation
Frequency of treatment:
once a day
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
20 dams
Control animals:
other: sesame oil
Details on study design:
- Dose selection rationale: The high-dose level was fixed according to preliminary assays showing a decrease in the body weight gain of gravid females given 1000 mg/kg/d of dibenzyltoluene.

Maternal examinations:
CLINICAL OBSERVATION: daily

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION : Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/rat/day

WATER CONSUMPTION: No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on 21 day of gestation
- Organs examined: no data
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of : Yes
- Number of dead and liv conceptuses : Yes
Fetal examinations:
- External examinations: Yes (third per litter )
- Soft tissue examinations: Yes (third per litter ) after fixation for 2 weeks in Bouin's mixture according to the wilson's method
- Skeletal examinations: Yes (third per litter )
- Head examinations: Yes (third per litter )
- fetal weight: Yes
Statistics:
- Implantation rate: Chi-square
- Weight of dams, food consumption, number of corpora lutea, number of implants, number of fetuses, fetal body weight: Student's t test (if homogeneous variance), Aspin-Welch t test (in other cases)
- Fetal mortality, sex ratio, abnormalities frequency: Wilcoxon test.
Indices:
No data
Historical control data:
No data
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
CLINICAL FINDINGS:
Piloerection was observed in the highest dose group (1000 mg/kg/day).

MORTALITY:
Maternal mortality was not reported.

BODY WEIGHT:
A slight decrease in body weight gain was noted in dams exposed to 1000 mg/kg/day when compared to that of control.

FOOD CONSUMPTION:
A visible decrease in food intake was observed at 1000 mg/kg/day but no dose-related effect was found.

NECROPSY:
- Fertility parameters:
. All dams had live fetuses.
. A slight but statistically significant decrease in the number of corpora lutea was observed at 1000 mg/kg/day (274 corpora lutea in 20 pregnant females) when compared to control (299 corpora lutea) however since dibenzyltoluene was administered after the implantation should be performed (day 6), this effect was not considered to be treatment-related .
. Number of implants were unaffected by treatment and implant ratio was similar in all groups.

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No teratogenic effects were found.
- Mortality:
. Fetal mortality was similar in treated and control groups.
- Litters examination:
. Litter sizes in all groups were similar.
. No significant difference in sex ratio was found.
. Pups body weight was nonetheless statistically reduced in the highest dose group when compared to control (3.8 vs 4.1 g in male, 3.6 vs 3.9 g in females).
. No toxicologically significant malformations were observed.
Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
Under these experimental conditions, the No-Observed-Adverse-Effect-Level (NOAEL) was 300 mg/kg/d for maternal and foetal toxicity.
Executive summary:

The potential of Dibenzylbenzene, ar-methyl derivative to induce developmental toxicity after maternal exposure during the critical period of organogenesis was evaluated in rat according to a study design comparable to OECD Guideline N° 414.

Dibenzylbenzene, ar-methyl derivative was administered orally by gavage to three groups of 20 bred female Sprague-Dawley rats once daily from gestation days 7 through 17. Dosage levels were 0, 100, 300 and 1000 mg/kg/d.

Animals were observed daily for mortality and morbidity. Clinical observations, body weights and food consumption were recorded at appropriate intervals.

On gestation day 20, a hysterectomy was performed on each female. The uteri, placenta and ovaries were examined, and the number of foetuses, resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded. The foetuses were weighted, sexed and examined for external, visceral and skeletal malformations and developmental variations.

All animals survived to the scheduled necropsy. Piloerection was observed in females given 1000 mg/kg/d as well as a slight decrease in body weight gain and food consumption.

The slight increase in the resorption of corpora lutea noted in the highest dose group was not considered as relevant since the administration of Dibenzylbenzene, ar-methyl derivative occurred after the implantation. The survival of the pups was unaffected by Dibenzylbenzene, ar-methyl derivative administration at all dose levels. However, pups body weight was reduced in the highest dose group when compared to controls but this effect was related to the maternal toxicity observed. Foetal external, soft tissue and skeletal malformations observed in control and treated pups were considered to be spontaneous in origin. The developmental variations expressed in the treated groups were generally similar to those present in the control group or occurred in a manner that was not dose related.

Under these experimental conditions, the No-Observed-Adverse-Effect-Level (NOAEL) was 300 mg/kg/d for maternal toxicity and the No-Observed-Adverse-Effect-Level (NOAEL) was 300 mg/kg/d for fetal toxicity.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 August 2019 - 17 October 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
25 June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
Name: dibenzylbenzene, ar methyl derivative
Synonyms:
JARYTHERM DBT
Jarytherm® DBT
DIBENZYLBENZENE, AR-METHYL DERIVATIVE
Dibenzyltoluene
CAS No.: 53585-53-8
Batch No.: B529183511
Descriptions: liquid, colorless to yellow, slightly aromatic
Storage condition: at room temperature in a dry and well-ventilated place
Purity: 98.3% (according to CoA)
Considered as 100% (UVCB)
Correction factor: none
Expiry date: 18 December 2020
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: breeder: Charles River Laboratories France (Châtillon-sur-Chalaronne, France)
- Age at the beginning of the treatment period: 18-21 weeks old
- Mean body weight at the beginning of the treatment period: 3520 g (range: 2840 g to 4115 g).
- Fasting period before study: no
- Housing: individually
- Diet: breeding pelleted diet “type 110C”. Due to low food consumption, carrots were also given to all females on some occasions.
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: for at least 5 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 ± 3°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): about 5 to 15 cycles/hour of filtered, non-recycled air.
- Photoperiod (hrs dark / hrs light): 8 h/16 h

IN-LIFE DATES: 02 September 2019 to 17 October 2019
Route of administration:
oral: gavage
Vehicle:
other: 0.5% (w/v) carboxymethylcellulose 400-800cPs + 0.5% (w/v) Tween 80 in drinking water treated by reverse osmosis
Details on exposure:
PREPARATION OF DOSING FORMULATIONS:
- Emulsion in the vehicle
- Justification for use and choice of vehicle (if other than water): suitable formulation in the selected vehicle
- Concentration in vehicle: 2, 6 and 15 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Type of method: Gas Chromatography with FID detection (GC-FID)
Test item concentrations: were within an acceptable range of variation compared to nominal values (-7.3 to +3.1% vs. +/- 15%) on Days 6 and 28 p.c.
Homogeneity: the dose formulations containing the test item and prepared at 0.5 mg/mL and 200 mg/mL in 0.5% (w/v) carboxymethylcellulose 400-800cPs + 0.5% (w/v) Tween 80 in drinking water treated by reverse osmosis were found to be homogeneous after preparation
Stability: diluted analytical samples prepared from 0.16 mg/mL and 240 mg/mL dose formulations in 0.5% (w/v) carboxymethylcellulose 400-800cPs + 0.5% (w/v) Tween 80 in drinking water treated by reverse osmosis were found to be stable for 6 days when they were stored at room temperature.
Details on mating procedure:
- Impregnation procedure: purchased time pregnant
- Proof of pregnancy: vaginal plug (at the breeder's facility); referred to as Day 0 post coitum
Duration of treatment / exposure:
Days 6 to 28 post coitum inclusive
Frequency of treatment:
Daily
No. of animals per sex per dose:
24 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for dose selection:
The dose levels were selected in agreement with the Sponsor, based on the results from a preliminary prenatal development study in which New Zealand White female rabbits (5 to 8 mated females per group) received the test item at 0, 30, 75, 150, 450 or 800 mg/kg/day, from Day 6 to Day 28 p.c., inclusive.
At 150, 450 and 800 mg/kg/day, excessive maternal toxicity was noted, defined by signs of poor clinical condition, severely reduced food consumption and body weight loss. All females at 450 or 800 mg/kg/day, and 4/8 females at 150 mg/kg/day died or were prematurely euthanized between Day 11 and 14 p.c.
At 30 and 75 mg/kg/day, no clinical signs or effects on body weight or food consumption attributed to the test item were noted. No macroscopic post-mortem findings were observed in the dams. However, at 75 mg/kg/day and when compared with controls, there was a significantly higher post implantation loss rate which was outside HCD (mainly late resorptions) resulting in a significant lower number of live fetuses.
Based on the results of this study, 75 mg/kg/day was selected as the high dose level. This dose was expected to induce significant embryo-fetal toxicity without compromising the group size of live fetuses for a comprehensive evaluation of external, visceral and skeletal development.
The low dose and mid dose were selected using a ratio representing approximately a 2.5 to 3.0 fold interval (i.e. 10 and 30 mg/kg/day).

- Rationale for animal assignment:stratified procedure.
Maternal examinations:
MORTALITY/MORBIDITY:
- Time schedule: each animal was checked for mortality and morbidity once a day before the treatment period and twice a day during treatment period, except for one day, including weekends.

CLINICAL OBSERVATIONS:
- Time schedule: from arrival, each animal was observed once a day as part of routine examinations. From the start of the treatment period, each animal was observed once a day, at approximately the same time for the recording of clinical signs (including evidence of abortion).

BODY WEIGHT:
- Time schedule: the body weight of each female was recorded on Days 2, 4, 5, 6, 9, 12, 15, 19, 24 and 29 p.c.

FOOD CONSUMPTION:
- Time schedule: the quantity of food consumed by each female was recorded for the following intervals: Days 2-4, 4-5, 5-6, 6-9, 9-12, 12-15, 15-19, 19-24 and 24-29 p.c.

POST-MORTEM EXAMINATION:
- Sacrifice on Day 29 post coitum
- Examined: principal thoracic and abdominal organs
Ovaries and uterine content:
The ovaries and uterine content were examined after termination, including:
- gravid uterus weight,
- number of corpora lutea,
- number and distribution of dead and live fetuses,
- number and distribution of early and late resorptions,
- number and distribution of uterine scars,
- number and distribution of implantation sites.

Fetal examinations:
- External examinations: Yes: all fetuses per litter
- Soft tissue examinations: Yes: all fetuses per litter
- Skeletal examinations: Yes: all fetuses per litter
- Head examinations: Yes: half fetuses per litter
- Other: fetal weight, fetal sex
Statistics:
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).
Indices:
% Pre-implantation loss = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea
% Post-implantation loss = 100 * (Number of implantation sites - Number of live fetuses) / Number of implantation sites
Historical control data:
Cf attached document
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
See table 2.
No test item-related clinical signs were observed during the study at any dose level.
All clinical signs recorded in surviving females (all pregnant except one female at 30 mg/kg/day) during the study were considered to be unrelated to the test item treatment as they were present before treatment initiation, and/or both in control and test item-treated animals and/or were observed in isolated animals.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female given 75 mg/kg/day was prematurely euthanized on Day 24 p.c. due to signs of poor clinical condition: emaciated appearance on the day of sacrifice and absence of feces from Day 19 p.c., associated with body weight loss on Days 9-24 p.c. (-15%) and almost no food intake from Day 12 p.c.
At necropsy, the gall bladder was dilated. Ten live fetuses and two late resorptions were found in the uterine horns. Although the cause of poor clinical condition was not identified, a relationship to the test item treatment was considered to be unlikely as there were no signs of marked toxicity in the surviving females at this dose level.
One female given 30 mg/kg/day was prematurely euthanized on Day 29 p.c. due to delivery. Seven fetuses and three placentas were found in the bedding. Blood in the bedding was observed on the day of euthanasia. At necropsy, reddish color of lungs was noted. Eight uterine scars were observed. This isolated premature delivery was considered to be incidental.
No unscheduled deaths occurred in females given 0 or 10 mg/kg/day.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See table 3.
At 30 and 75 mg/kg/day, when compared to controls, moderately lower mean body weight gain was recorded between Days 6 and 29 p.c. (+272 and +253 g, respectively, vs. +356 g in controls, not statistically significant). These differences were especially due to continuous lower mean body weight gain at 30 mg/kg/day and transient mean body weight losses at 75 mg/kg/day.
While a relationship to the test item treatment cannot be excluded, the differences in mean body weight changes were not statistically significant and did not impact the Day 29 p.c. body weight (-3 and -2%, respectively). They were therefore considered to be non-adverse.
At 10 mg/kg/day, no effects on mean body weight or mean body weight change were noted.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
See table 4.
There were no effects on mean food consumption.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
See table 6.
None of the macroscopic findings observed were considered to be related to the test item administration.
In the stomach, the mucosa was reddish in 1/24 females at 30 mg/kg/day and 1/24 females at 75 mg/kg/day. In the uterus of 1/24 females at 75 mg/kg/day, the wall of one uterine horn was thickened and whitish content was present. The kidneys were enlarged in 1/24 females at 75 mg/kg/day. Given the low incidence/isolated occurrence of these findings in the stomach, uterus and kidneys, any relationship with the test item administration was considered to be unlikely.
The other macroscopic findings noted in test item-treated animals were considered incidental changes, as they also occurred in controls, were isolated or of low incidence, had no dose relationship in incidence, and/or are common background findings for the rabbit.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
See table 5.
Uterus weight, carcass weight
At 75 mg/kg/day, when compared to controls, mean gravid uterus weight was lower (-11%). This finding was not attributed to the test item treatment, but was rather related to the higher percentage of pre-implantation loss recorded at this dose level.
The more pronounced mean net body weight loss from Day 6 p.c. correlated with the lower body weight gain at this dose level, with no impact on the mean carcass weight.
At 10 or 30 mg/kg/day, no relevant effects were noted on mean gravid uterus weight, carcass weight or net body weight changes.
Number of abortions:
no effects observed
Description (incidence and severity):
See table 7.
No dams with abortions.


Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
See table 7.
At 75 mg/kg/day, when compared to controls, mean percentage of pre-implantation loss per female was higher (17.5 vs. 12.6%). This was mainly due to the contribution of two females, for which the percentage of pre-implantation loss was 62.5 and 53.3%, respectively.
At 10 mg/kg/day, the higher mean percentage of pre-implantation loss per female (19.4%) was mainly due to one female (80%).
These changes were associated at both dose levels with lower mean number of implantation sites per female and lower mean number of live fetuses per animal. As the differences from controls were of low magnitude, not dose-related and not statistically significant, these changes were considered to be unrelated to the test item treatment.
No relevant effects were noted on the other parameters and no effects were reported at 30 mg/kg/day.

Number of dams with pre-implantation loss = 12, 16, 11, 16, at 0, 10, 30 or 75 mg/kg/day, respectively
Number of dams with post-implantation loss = 8, 8, 7, 9, at 0, 10, 30 or 75 mg/kg/day, respectively
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
See table 7.
No dams with total resorption.

Mean number of resorptions per female: 0.7; 0.6; 0.8 and 0.8 respectively at 0, 10, 30 and 75 mg/kg/day.
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
See table 7.
Mean number of early resorptions: 0.2; 0.4; 0.5 and 0.3 at 0, 10, 30 or 75 mg/kg/day, respectively.
Mean number of late resorptions: 0.5; 0.2; 0.3 and 0.5 at 0, 10, 30, or 75 mg/kg/day, respectively.
Dead fetuses:
no effects observed
Description (incidence and severity):
See table 7.
No dead fetuses.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
See table 7.

At hysterectomy on Day 29 p.c., 21/24, 21/24, 20/24 and 21/24 females were pregnant with live fetuses in the groups treated at 0, 10, 30 and 75 mg/kg/day, respectively.
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: no effects in maternal animals
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
See table 8.
Mean fetal body were unaffected by the test item treatment at any dose level.

Fetal body weight of males: 38.9 g, 41.7 g, 37.1 g and 37.8 g at 0, 10, 30 or 75 mg/kg/day, respectively.
Fetal body weight of females: 37.0 g, 39.8 g, 36.9 g and 36.7 g at 0, 10, 30 or 75 mg/kg/day, respectively.
Reduction in number of live offspring:
not examined
Changes in sex ratio:
effects observed, non-treatment-related
Description (incidence and severity):
Mean fetal sex ratio were unaffected by the test item treatment at any dose level.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
See table 10.
No test item-related external malformations were observed at any dose level.
Agnathia, astomia and gastroschisis were observed in one fetus at 75 mg/kg/day.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
See table 15.
No test item-related skeletal malformations were observed at any dose level.
The findings recorded were considered to be incidental and not related to the test item treatment as they were noted both in control and test item-treated animals and/or in isolated fetuses and/or with no dose-relationship.
Fused mandible was observed in the teras. This fetus also had abnormal color of amniotic fluid, agnathia, astomia, gastroschisis, abnormal liver lobation, short and fused mandible, several skeletal variations associated with lower body weight (35.3 g vs. 45.3 to 51.6 g for the three other fetuses of the same litter). A test item effect was considered to be unlikely as these observations were only seen in this fetus (except for abnormal color of amniotic fluid).

Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
See table 12.
At 75 mg/kg/day, three dams had fetuses with malpositionned right kidney.One fetus also had enlarged kidney (and adhesion between kidney and intestines).
At 30 mg/kg/day, one dam had one fetus with absence of right kidney and ureter.
At 10 mg/kg/day, one dam had one fetus, with malpositionned right kidney and another dam had one fetus, with malpositionned bilateral kidneys, associated with marked dilated renal pelvis and ureters.
The malposition of kidney was only recorded at 10 and 75 mg/kg/day (2 and 5 fetuses were affected, respectively, statistically significant at the high dose level, vs. 0 in controls ) and the incidences were outside the range of the HCD (this was recorded at most in one fetus per study).
This finding was attributed to the test item, and as this malformation is a major abnormality, it was considered as adverse.
The other findings in upper urinary tract, i.e. marked dilation of renal pelvis/ureters at 10 mg/kg/day and absence of kidney/ureter at 30 mg/kg/day, were also recorded with an higher incidence when compared to the HCD, and were therefore attributed to the test item and considered as adverse.

The other findings recorded were considered to be incidental and not related to the test item treatment as they were noted both in control and test item-treated animals and/or with no dose-relationship.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
See table 9.
External variations:
At 75 mg/kg/day, two dams had fetuses with abnormal color of amniotic fluid and one dam had one fetus with malrotated paw.
At 30 mg/kg/day, one dam had one fetus with polyhydramnios and one dam had one fetus with paw hyperflexion.
At 10 mg/kg/day, one dam had one fetus with paw hyperflexion.
Although the above-mentioned findings were noted in a few animals and/or with no dose-relationship, they were not reported among control animals and were outside the range of the HCD. Therefore a relationship to the test item treatment cannot be excluded, but these variations are minor abnormalities considered as non-adverse.

Skeletal variations:
See table 14.
At 75 mg/kg/day, when compared to controls, the higher incidence of incomplete ossification of the hyoid centrum and unossification/incomplete ossification of the 5th forepaw median phalanx was considered to be test item-related and correlated with the cartilage findings. These variations are minor abnormalities considered as non-adverse.

The other findings recorded were considered to be incidental and not related to the test item treatment as they were noted both in control and test item-treated animals and/or in isolated fetuses and/or with no dose-relationship.
Short mandible and other skeletal variations were observed in the teras.

Cartilages
See table 13.
At 75 mg/kg/day, when compared to controls, the higher incidence of non-ossified hyoid centrum and forepaw median phalanxes (only the respective cartilages were present) was considered to be test item-related. These variations are minor abnormalities considered as non-adverse.
The other findings recorded were considered to be incidental and not related to the test item treatment as they were noted both in control and test item-treated animals and/or in isolated fetuses and/or with no dose-relationship.


Visceral variations:
See table 11.
At 75 mg/kg/day, two dams had fetuses with kidneys variations with dilated renal pelvis, with enlarged kidney/adhesion between kidney and intestines, and three dams had fetuses with a heart great vessel variation (absence of innominate artery).
At 30 mg/kg/day, one dam had one fetus with absence of innominate artery.
At 10 mg/kg/day, one dam had one fetus with dilated renal pelvis.

The absence of innominate artery was only recorded at the two higher dose levels of the test item (statistically significant at 75 mg/kg/day) and the incidences were outside the range of the HCD. This finding was attributed to the test item, but this variation is a minor abnormality considered as non-adverse.

Although the variations in upper urinary tract were noted in a few animals and/or with no dose-relationship, they were not reported among control animals and the incidences were outside the range of the HCD. Therefore a relationship to the test item treatment cannot be excluded, but these variations are minor abnormalities considered as non-adverse.

The other findings recorded were considered to be incidental and not related to the test item treatment as they were noted both in control and test item-treated animals and/or with no dose-relationship and/or were isolated findings.

Dose descriptor:
LOAEL
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
visceral malformations
Developmental effects observed:
yes
Lowest effective dose / conc.:
10 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Table 1: Pregnancy status

Dose level (mg/kg/day)

0

10

30

75

Number of females

24

24

24

24

Pregnant females

21

21

21

22

Females with live fetuses at termination

21

21

20

21

Female prematurely euthanized (pregnant)

0

0

1

1

Non-pregnant females

3

3

3

2

 

Table 2: Clinical signs

Dose level (mg/kg/day)

0

10

30

75

Noremarkable clinicalsigns

18/24

22/24

19/23

18/23

Emaciated appearance

-

-

1

2

Aggressive behaviour

-

1

-

-

Absence of urine

4

1

1

3

Absence of feces

1

-

1

2

Cutaneous lesion on hindlimb

1

-

-

-

Difficulty during gavage administration

1

-

1

-

-: no findings.

 

Table 3:Body Weight and Body Weight Change

Dose level (mg/kg/day)

0

10

30

75

Body weight (g)

 

 

 

 

Day 6p.c.

3522

3506

3498

3588

Day 9p.c.

3562

3557

3520

3576

Day 12p.c.

3590

3589

3548

3602

Day 15p.c.

3660

3655

3598

3629

Day 19p.c.

3695

3678

3618

3619

Day 24p.c.

3752

3756

3687

3723

Day 29p.c.

3878

3846

3770

3816

% from controls

-

-1

-3

-2

Body weight change (g)

 

 

 

 

Days 6 - 9p.c.

+40

+50

+22

-12

Days 9 - 12p.c.

+29

+32

+28

+27

Days 12 - 15p.c.

+70

+66

+51

+27

Days 15 - 19p.c.

+34

+24

+19

-10

Days 19 - 24p.c.

+57

+78

+70

+104

Days 24 - 29p.c.

+126

+90

+83

+84

Days 6 - 29p.c.

+356

+340

+272

+253

% from controls

-

-4

-24

-29

-: not applicable.

No statistically significant differences versus controls.


 

Table 4: Food consumtion

Dose level (mg/kg/day)

0

10

30

75

Days 6 - 9p.c.

135

147

119

124

Days 9 - 12p.c.

134

148

115

116

Days 12 - 15p.c.

114

118

91

95

Days 15 - 19p.c.

121

135

115

94

Days 19 - 24p.c.

119

123

116

125

Days 24 - 29p.c.

95

91

87

105

Days 6 to 29p.c.

118

128

105

112

No statistically significant differences versus controls.

 

Table 5: Gravid Uterus and Carcass Weights

Dose level (mg/kg/day)

0

10

30

75

Gravid uterus weight

549.9

515.3

513.0

488.7

% from controls

-

-6

-7

-11

Carcass weight

3328.2

3330.9

3285.2

3327.0

Net weight change from Day 6p.c.

-193.9

-175.5

-211.8

-235.9

-: not applicable.

No statistically significant differencesversus controls.

 

Table 6: Macroscopic post-mortem examination

Dose level (mg/kg/day)

0

10

30

75

Mass (lung or adipose tissue)

-

-

2

-

Lungs

Irregular color

-

-

 

1

-

Spleen

Colored nodules

-

-

 

1

-

Liver

Colored nodules

-

-

 

1

-

Stomach

Colored areas

-

-

 

1

 

1

Kidney

Enlarged

-

-

-

 

1

Female gonads

Serous cyst(s)

(connective tissue, periovarian region)

3

1

3

5

Uterus

Thickened wall of uterine horn

Colored contents in uterine horn

-

-

-

1

1

Gall bladder

Dilatation

Reduced in size

-

-

1

2

-

-: not recorded.

 


 

Table 7: Hysterectomy data

Dose level (mg/kg/day)

0

10

30

75

HCD

Number of females with live fetuses at termination

21

21

20

21

282

Mean number ofcorpora luteaper animal

11.7

11.2

11.6

11.4

9.0-12.1

Mean number of implantation sites per animal

10.6

9.2

10.3

9.5

8.0-10.9

Mean percentage of pre-implantation loss (%)

12.6

19.4

11.6

17.5

2.5-13.0

Mean number of live fetuses per animal

9.9

8.6

9.5

8.7

/

Dead fetuses (%)

0.0

0.0

0.0

0.0

/

Mean number of resorptions per female

0.7

0.6

0.8

0.8

/

Mean number of implantation scars

0.0

0.0

0.0

0.0

/

Mean number of early resorptions

0.2

0.4

0.5

0.3

0.0-1.8

Mean number of late resorptions

0.5

0.2

0.3

0.5

0.0-0.6

Mean percentage of post-implantation loss (%)

7.3

6.2

7.5

8.6

0.0-20.9

HCD: Historical Control Data- minimum-maximum incidence (New Zealand White Rabbits, 2018) supplied by Charles River Laboratories Lyon.

/: not recorded in HCD.

 

Table 8: Fetal body and sex ratio

Dose level (mg/kg/day)

0

10

30

75

HCD

Mean fetal body weight (g)

37.8

40.8

37.1

37.4

33.5-45.0

Mean percentage of male fetuses (%)

42.6

47.1

55.9*

57.1*

39.3-57.5

Statistically significant vs.controls: *: p<0.05.

HCD: Historical Control Data- minimum-maximum incidence (New Zealand White Rabbits, 2018) supplied by Charles River Laboratories Lyon.

/: not recorded in HCD.

 

Table 9: External variations

Dose level (mg/kg/day)

0

10

30

75

HCD

Lyon 2016-18

HCD Lyon 2019

Reference data Evreux 2019

Number of litters

21

21

20

21

 

 

 

Number of fetuses

208

181

190

183

5480

 

 

General

 

 

 

 

 

 

 

. abnormal color of amniotic fluid, F (L)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

1.1 (9.5)

0.0

0.0

0.5 (1.6)

. polyhydramnios, F (L)

0.0 (0.0)

0.0 (0.0)

0.5 (5.0)

0.0 (0.0)

0.0

0.0

0.2 (1.6)

Paw and digit

 

 

 

 

 

 

 

. malrotated paw F (L)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.5 (4.8)

0.02

0.01 (0.16)

0.2 (1.6)

. paw hyperflexion, F (L)

0.0 (0.0)

0.6 (4.8)

0.5 (5.0)

0.0 (0.0)

0.13

0.09 (0.49)

0.2 (1.6)

Trunk

 

 

 

 

 

 

 

. abdomen, enlarged F (L)

0.5 (4.8)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.0

 

 

. abdomen, blackish color, F (L)

0.5 (4.8)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.0

 

 

Litters affected, n (%)

1 (4.8)

1 (4.8)

2 (10.0)

3 (14.3)

 

 

 

Fetus affected, n (%)

1 (0.5)

1 (0.6)

2 (1.1)

3 (1.6)

 

 

 

HCD: Historical Control Data-mean fetal incidence (2016-2018) or mean fetal (litter) incidence (2019) supplied by Charles River Laboratories Lyon (New Zealand White rabbits).

For HCD 2019 from Charles River Laboratories Lyon and reference data 2019 from Charles River Laboratories

Evreux, only the data for the findings discussed below are shown.

 

Table 10: External malformations

Dose level (mg/kg/day)

0

10

30

75

HCD

Number of litters

21

21

20

21

 

Number of fetuses

208

181

190

183

5480

Mouth, jaw, palate

 

 

 

 

 

. Agnathia, F (L)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.5 (4.8)

0.0

. Astomia, F (L)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.5 (4.8)

0.0

Eyes

 

 

 

 

 

. Open eye, F (L)

0.5 (4.8)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.05

Trunk

 

 

 

 

 

. Gastroschisis, F (L)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.5 (4.8)

0.02

Cranium

 

 

 

 

 

. Anencephaly, F (L)

0.5 (4.8)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.0

Litters affected, n (%)

1 (4.8)

0 (0.0)

0 (0.0)

1 (4.8)

 

Fetus affected, n (%)

1 (0.5)

0 (0.0)

0 (0.0)

1 (0.5)

 

HCD: Historical Control Data-mean fetal incidence (New Zealand White rabbits, 2016 2018) supplied by Charles River Laboratories Lyon.

n: number, No statistically significant differences vs.controls.

 

Table 11: Soft tissue variations

Dose level (mg/kg/day)

0

10

30

75

HCD Lyon 2016-2018

HCD Lyon 2019

Reference

Data

Evreux 2019

Number of litters

21

21

20

21

 

 

 

Number of fetuses

208

181

190

183

4635

 

 

Gall bladder

 

 

 

 

 

 

 

. bilobed, F (L)

0.5 (4.8)

0.0 (0.0)

0.5 (5.0)

1.1 (9.5)

0.06

 

 

. small, F (L)

1.0 (9.5)

2.8 (9.5)

0.5 (5.0)

0.5 (4.8)

0.41

 

 

. enlarged, F (L)

0.0 (0.0)

0.0 (0.0)

1.1 (10.0)

0.5 (4.8)

0.04

 

 

Liver

 

 

 

 

 

 

 

. colored nodule, F (L)

1.0 (9.5)

1.1 (9.5)

0.0 (0.0)

0.5 (4.8)

0.0

 

 

. enlarged, F (L)

0.5 (4.8)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.0

 

 

Kidneys

 

 

 

 

 

 

 

. dilated renal pelvis, F (L)

0.0 (0.0)

0.6 (4.8)

0.0 (0.0)

0.5 (4.8)

0.15

0.13 (1.04)

0.7 (6.6)

. enlarged kidney, F (L)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.5 (4.8)

0.0

0.0 (0.0)

0.0 (0.0)

. adhesion, F (L)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.5 (4.8)

0.0

0.0 (0.0)

0.0 (0.0)

Gonads

 

 

 

 

 

 

 

. hemorrhagic ovary, F (L)

1.0 (4.8)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.0

 

 

Vessels

 

 

 

 

 

 

 

. absent brachiocephalic trunk,

F (L)

31.7 (81.0)

37.0 (76.2)

25.3 (75.0)

20.2 (57.1)

0.0

0.0

31.5 (78.7)

. short innominate artery, F (L)

3.8 (23.8)

1.7 (14.3)

2.6 (15.0)

1.6 (9.5)

0.0

0.0

0.5 (4.9)

. absent innominate artery, F (L)

0.0 (0.0)

0.0 (0.0)

0.5 (5.0)

2.2* (14.3)

0.35

0.15 (1.12)

0.5 (1.6)

Thymus

 

 

 

 

 

 

 

. reddish color, F (L)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.5 (4.8)

0.04

 

 

. reddish focus, F (L

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.5 (4.8)

0.0

 

 

General

 

 

 

 

 

 

 

. fluid-filled abdomen, F (L)

0.5 (4.8)

0.0 (0.0)

0.5 (5.0)

0.0 (0.0)

0.02

 

 

. edema, F (L)

0.5 (4.8)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.0

 

 

. fluid-filled thoracic cavity, F (L)

0.5 (4.8)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.02

 

 

Miscellaneous

 

 

 

 

 

 

 

. thickening, F (L)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.5 (4.8)

0.0

 

 

Litters affected, n (%)

19 (90.5)

17 (81.0)

18 (90.0)

17 (81.0)

 

 

 

Fetus affected, n (%)

73 (35.1)

75 (41.4)

54 (28.4)

47 (25.7)

 

 

 

HCD: Historical Control Data-mean fetal incidence (2016-2018) or mean fetal (litter) incidence (2019) supplied by Charles River Laboratories Lyon (New Zealand White rabbits).

For HCD 2019 from Charles River Laboratories Lyon and reference data 2019 from Charles River Laboratories

Evreux, only the data for the findings discussed below are shown.

n: number, /: not recorded in HCD.

Statistically significant vs.controls: *: p<0.05.

 

Table 12: Soft tissue malformations

Dose level (mg/kg/day)

0

10

30

75

HCD Lyon 2016-18

HCD Lyon mean 2019

HCD Lyon max 2019

Number of litters

21

21

20

21

 

 

 

Number of fetuses

208

181

190

183

4635

 

 

Brain

 

 

 

 

 

 

 

. small cerebrum, F (L)

0.5 (4.8)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.0

 

 

. misshapen cerebrum, F (L)

0.5 (4.8)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.05

 

 

Heart

 

 

 

 

 

 

 

. ventricular septum defect, F (L)

0.5 (4.8)

0.0 (0.0)

0.5 (5.0)

0.0 (0.0)

0.04

 

 

. hydropericardium, F (L)

0.5 (4.8)

0.6 (4.8)

0.5 (5.0)

0.5 (4.8)

0.0

 

 

. cor triloculare, F (L)

0.5 (4.8)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.0

 

 

. cardiomegaly, F (L)

0.5 (4.8)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.0

 

 

Gall bladder

 

 

 

 

 

 

 

. absent, F (L)

0.0 (0.0)

0.6 (4.8)

0.0 (0.0)

0.0 (0.0)

0.09

 

 

Liver

 

 

 

 

 

 

 

. abnormal liver lobation, F (L)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.5 (4.8)

0.19

 

 

Kidneys

 

 

 

 

 

 

 

. marked dilated renal pelvis, F (L)

0.0 (0.0)

0.6 (4.8)

0.0 (0.0)

0.0 (0.0)

0.02

0.02 (0.15)

0.56 (5.00)

. malpositioned kidney, F (L)

0.0 (0.0)

1.1 (9.5)

0.0 (0.0)

2.7*(14.3)

0.04

0.13 (0.93)

1.67 (5.56)

. absent kidney, F (L)

0.0 (0.0)

0.0 (0.0)

0.5 (5.0)

0.0 (0.0)

0.02

0.02 (0.16)

0.53 (5.26)

Vessels

 

 

 

 

 

 

 

. narrowed aortic arch, F (L)

0.0 (0.0)

0.0 (0.0)

0.5 (5.0)

0.0 (0.0)

0.02

 

 

. dilated pulmonary trunk, F (L)

0.5 (4.8)

0.0 (0.0)

0.5 (5.0)

0.0 (0.0)

/

 

 

. dilated aortic arch, F (L)

0.5 (4.8)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.00

 

 

Ureter

 

 

 

 

 

 

 

. marked dilated ureter, F (L)

0.0 (0.0)

0.6 (4.8)

0.0 (0.0)

0.5 (4.8)

0.00

 

 

. absent ureter, F (L)

0.0 (0.0)

0.0 (0.0)

0.5 (5.0)

0.0 (0.0)

0.0

 

 

Litters affected, n (%)

3 (14.3)

4 (19.0)

2 (10.0)

5 (23.8)

 

 

 

Fetus affected, n (%)

3 (1.4)

4 (2.2)

3 (1.6)

8 (4.4)

 

 

 

HCD: Historical Control Data-mean fetal incidence (2016-2018) or mean fetal (litter) incidence (2019) supplied by Charles River Laboratories Lyon (New Zealand White rabbits).

For HCD 2019, only the data for the findings discussed below are shown.

n: number, /: not recorded in HCD.

Statistically significant vs.controls: *: p<0.05.

 

Table 13: Cartilages

Dose level (mg/kg/day)

0

10

30

75

Number of litters

21

21

20

21

Number of fetuses

208

181

190

183

Head-others

 

 

 

 

. cartilage of hyoid centrum: present, F (L)

5.3 (33.3)

4.4 (28.6)

5.3 (35.0)

11.5* (38.1)

Caudal vertebrae

 

 

 

 

. cartilage of caudal vertebra(e): present , F (L)

0.0 (0.0)

1.7 (9.5)

5.3#(30.0**)

1.6 (14.3)

Forepaw phalanx

 

 

 

 

. cartilage of median phalanx present , F (L)

19.7 (57.1)

12.7 (57.1)

23.2 (70.0)

32.2** (66.7)

Litters affected, n (%)

21 (100)

21 (100)

20 (100)

21 (100)

Fetus affected, n (%)

176 (84.6)

148 (81.8)

160 (84.2)

158 (86.3)

n: number.

Statistically significant vs.controls:*: p<0.05, **: p<0.01,#: p<0.001.

 

Table 14: Skeletal variations

Dose level (mg/kg/day)

0

10

30

75

Number of litters

21

21

20

21

Number of fetuses

208

181

190

183

Head-others

 

 

 

 

. hyoid: incomplete ossification of centrum, F (L)

4.3 (23.8)

4.4 (28.6)

3.7 (30.0)

11.5* (38.1)

. Mandible: short

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.5 (4.8)

Forepaw phalanx

 

 

 

 

. unossified 5thmedian phalanx,

F (L)

13.5 (52.4)

7.2 (47.6)

14.7 (60.0)

25.1** (57.1)

. incomplete ossification median phalanx, F (L)

0.5 (4.8)

0.0 (0.0)

0.5 (5.0)

3.8* (14.3)

 

 

 

 

 

 

 

 

 

 

Litters affected, n (%)

21 (100)

21 (100)

20 (100)

21 (100)

Fetus affected, n (%)

201 (96.6)

171 (94.5)

185 (97.4)

177 (96.7)

Statistically significant vs.controls:*: p<0.05, **: p<0.01.

Table 15: Skeletal malformations

Dose level (mg/kg/day)

0

10

30

75

HCD

Number of litters

21

21

20

21

 

Number of fetuses

208

181

190

183

4561

Head-skull

 

 

 

 

 

. parietal, split F (L)

0.0 (0.0)

0.0 (0.0)

0.5 (5.0)

0.0 (0.0)

0.0

. interparietal: absent, F (L)

0.5 (4.8)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.0

. parietal: absent, F (L)

0.5 (4.8)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.0 

. skullcap: hole, F (L)

0.5 (4.8)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.0

. supraoccipital:absent, F (L)

0.5 (4.8)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.0

Head-others

 

 

 

 

 

. mandible: fused, F (L)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.5 (4.8)

0.0 /

Cervical vertebrae

 

 

 

 

0.0

. cervical rib, F (L)

0.0 (0.0)

0.0 (0.0)

0.5 (5.0)

0.0 (0.0)

0.0

Thoracic vertebrae

 

 

 

 

 

. absent, F (L)

0.0 (0.0)

0.6 (4.8)

0.0 (0.0)

0.0 (0.0)

0.0

Lumbar vertebrae

 

 

 

 

 

. absent, F (L)

0.0 (0.0)

0.0 (0.0)

0.5 (5.0)

0.0 (0.0)

0.0

Sternebrae

 

 

 

 

 

. fused sternebrae , F (L)

1.4 (14.3)

0.0 (0.0)

0.5 (5.0)

0.5 (4.8)

0.26

Rib

 

 

 

 

 

. branched rib(s), F (L)

0.0 (0.0)

0.6 (4.8)

0.5 (5.0)

0.0 (0.0)

0.02

. fused ribs, F (L)

0.0 (0.0)

0.6 (4.8)

0.0 (0.0)

0.0 (0.0)

0.0

Litters affected, n (%)

5 (23.8)

1 (4.8)

4 (20.0)

2 (9.5)

 

Fetus affected, n (%)

5 (2.4)

1 (0.6)

4 (2.1)

2 (1.1)

 

HCD: Historical Control Data-mean fetal incidence (New Zealand White rabbits, 2016 2018) supplied by Charles River Laboratories Lyon.

n: number.

No statistically significant differences vs.controls.

Conclusions:
The test item was administered daily to time-mated female NZW rabbits by the oral route (gavage) at dose levels of 10, 30 or 75 mg/kg/day from Day 6 to Day 28 p.c., inclusive.
Based on the results obtained in this study:
• the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 75 mg/kg/day based on the absence of adverse findings at this dose level,
• no No Observed Effect Level (NOEL) or No Observed Adverse Effect Level (NOAEL) for embryo-fetal development could be established based on the presence of malformations in upper urinary tract (kidneys and ureters) at all dose levels of the test item. The Low Observed Adverse Effect Level (LOAEL) is considered to be 10 mg/kg/day.
Executive summary:

The objective of this study was to evaluate the potential toxic effects of the test item on the pregnant female and on embryonic and fetal development following daily oral administration (gavage) to pregnant female rabbits from implantation to the day prior to the scheduled hysterectomy (Day 6 to Day 28 post-coitum (p.c.) inclusive).

Methods

Three groups of 24 time-mated female New-Zealand White rabbits received the test item, once daily by the oral route (gavage) at dose levels of 10, 30 or 75 mg/kg/day from Day 6 to Day 28 p.c. inclusive. A control group of 24 time-mated females received the vehicle (0.5% carboxymethylcellulose with 0.5% Tween 80), under the same experimental conditions. A constant dosage volume of 5 mL/kg/day was used.

The actual test item concentrations were determined in the dose formulations on two occasions, using a validated GC-FID analytical method.

The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded at designated intervals.

On Day 29 p.c., the females were euthanized and a macroscopic post-mortem examination was performed. Hysterectomies were performed and the numbers of corpora lutea, implantation sites, early and late resorptions, and live and dead fetuses were recorded. Placentas were observed. The fetuses were weighed and sexed by internal examination of the gonads. The fetuses were subjected to detailed external, soft tissue and skeletal (bones + cartilages) examinations.

Results

Actual concentrations of the test item in the dose formulations analyzed during the study were within an acceptable range of variations (-7.3% to +3.1%) when compared with the nominal concentrations.

At hysterectomy on Day 29 p.c., 21/24, 21/24, 20/24 and 21/24 females were pregnant with live fetuses in the groups treated at 0, 10, 30 and 75 mg/kg/day, respectively.

No test item-related deaths or clinical signs were recorded.

Lower body weight gain was recorded between Days 6 and 29 p.c. at 30 and 75 mg/kg/day (+272 g and +253 g, respectively, vs. +356 g in controls). These differences, especially due to continuous lower body weight gain at 30 mg/kg/day and transient body weight losses at 75 mg/kg/day, did not impact the body weight on Day 29 p.c. (-3 and -2%, respectively) and were considered to be non-adverse. This was associated with a net body weight loss from Day 6 p.c.more pronounced at 75 mg/kg/day, vs. controls, but with no impact on the carcass weight.

Food consumption was unaffected by the test item treatment.

At necropsy of the dams, none of the macroscopic findings observed were considered to be related to the test item administration. Gravid uterus weight was considered to be not impacted by the test item treatment. No effects on the hysterectomy parameters were noted.

The fetal body weight and the sex ratio were unaffected at any dose level.

At fetal examination, the major anomaly consisted of malposition of the kidney(s), recorded with higher incidence at 10 and 75 mg/kg/day, associated with marked dilation of renal pelvis in one fetus at 10 mg/kg/day. Absence of kidney and ureter was noted in one fetus at 30 mg/kg/day. These malformations were accompanied at 75 mg/kg/day by perturbation of vessel development (absence of innominate artery) and delay in skeletal ossification (unossified hyoid centrum and forepaw median phalanxes). External and visceral variations of uncertain relationship to the test item were noted at all dose levels (malrotation/hyperflexion of paw, polyhydramnios/abnormal color of amniotic fluid, dilated renal pelvis, enlarged kidneys and/or adhesion kidney-intestine).

Conclusion

The test item was administered daily to time-mated female NZW rabbits by the oral route (gavage) at dose levels of 10, 30 or 75 mg/kg/day from Day 6 to Day 28 p.c., inclusive.

Based on the results obtained in this study:

· the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 75 mg/kg/day based on the absence of adverse findings at this dose level,

· no No Observed Effect Level (NOEL) or No Observed Adverse Effect Level (NOAEL) for embryo-fetal development could be established based on the presence of malformations in upper urinary tract (kidneys and ureters) at all dose levels of the test item. The Low Observed Adverse Effect Level (LOAEL) is considered to be 10 mg/kg/day.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 June 2019 - 07 August 2019
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
Preliminary range-finding study
GLP compliance:
no
Remarks:
not required for a range-finding study
Limit test:
no
Specific details on test material used for the study:
Name: dibenzylbenzene, ar-methyl derivative
Synonyms: JARYTHERM DBT; Jarytherm® DBT; DIBENZYLBENZENE, AR-METHYL DERIVATIVE; Dibenzylbenzene, ar-methyl derivative; Dibenzyltoluene
CAS No.: 53585-53-8
Batch No.: B529183511
Description: liquid; colorless to yellow; slightly aromatic
Storage conditions: at room temperature; in a dry and well-ventilated place
Purity: 98.3% (according to CoA); considered as 100% (UVCB)
Correction factor: None
Expiry date: 18 December 2020.
Species:
rabbit
Strain:
other:
Remarks:
New Zealand White, INRA, A 1077 from Centre LAGO (for first receipt) KBL New Zealand White from Charles River Laboratories France (for second receipt)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source:
first receipt: New Zealand White, INRA, A 1077 from Centre LAGO (Vonnas, France),
second receipt: KBL New Zealand White from Charles River Laboratories France, Châtillon-sur-Chalaronne, France
- Age at the beginning of the treatment period: the animals were 18-20 weeks old
- Mean body weight at the beginning of the treatment period: the animals had a mean body weight of 3670 g (range: 3175 g to 4370 g) (first receipt) or 3599 g (range: 3085 g to 4105 g) (second receipt)
- Fasting period before study: no
- Housing: individually
- Diet: breeding pelleted diet “type 110C” (free access) (SAFE)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: for at least 5 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 ± 3°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): about 5 to 15 cycles/hour of filtered, non-recycled air.
- Photoperiod (hrs dark / hrs light): 8 h/16 h.

IN-LIFE DATES: 11 June 2019 to 07 August 2019.
Route of administration:
oral: gavage
Vehicle:
other: 0.5% (w/v) carboxymethylcellulose 400-800cPs + 0.5% (w/v) Tween 80 in drinking water treated by reverse osmosis
Details on exposure:
PREPARATION OF DOSING FORMULATIONS:
- Justification for use and choice: suitable formulations in the selected vehicle
- Emulsion in the vehicle
- Concentration in vehicle: 30, 90 and 160 mg/mL (first receipt) ; 6 and 15 mg/mL (second receipt)
- Amount of vehicle: 5 mL/kg/day
Analytical verification of doses or concentrations:
no
Details on mating procedure:
Females were mated at the breeder's facilities. The day of confirmed mating (visual assessment) was designated as Day 0 p.c.
Duration of treatment / exposure:
Days 6 to 28 post coitum inclusive
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
first receipt
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
first receipt
Dose / conc.:
450 mg/kg bw/day (nominal)
Remarks:
first receipt
Dose / conc.:
800 mg/kg bw/day (nominal)
Remarks:
first receipt
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
second receipt
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
second receipt
Dose / conc.:
75 mg/kg bw/day (nominal)
Remarks:
second receipt
No. of animals per sex per dose:
8 females per dose (first receipt)
5 females per dose (second receipt)
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for dose selection:
The dose levels were selected in agreement with the Sponsor, on the basis of the results of a previous dose range-finding study performed in non pregnant rabbits.
In this study, three females received daily by gavage the test item, at the dose level of 300 mg/kg/day for 7 days, then at the dose level of 1000 mg/kg/day for another 7-day treatment period.
At 300 mg/kg/day, no test item-related effects were noted on any parameter.
At 1000 mg/kg/day, moderately to severely reduced food consumption was noted in the two surviving females associated with a 12% body weight loss in one of them (the third female was prematurely sacrificed, no relationship to the test item treatment was established).
No test item-related macroscopic findings were observed at final sacrifice.
Therefore, 800 mg/kg/day was selected as the high-dose level. The low-dose and mid dose levels were selected using a ratio representing approximately a 2- to 3-fold interval (i.e. 150 and 450 mg/kg/day).
As severe maternal toxic effects (signs of poor clinical condition associated with body weight loss and drop in food consumption) were observed at 450 and 800 mg/kg/day, the decision was taken to test two lower dose levels (30 and 75 mg/kg/day).

- Rationale for animal assignment: stratified procedure.
Maternal examinations:
MORTALITY/MORBIDITY:
- Time schedule: each animal was checked for mortality or morbidity once a day before the treatment period and three times a day during treatment period, including weekends and public holidays.

CLINICAL SIGNS;:
- Time schedule: from arrival, each animal was observed once a day as part of routine examinations.
From the start of the treatment period, each animal was observed once a day, at approximately the same time of day, for the recording of clinical signs (including evidence of abortion).

BODY WEIGHT:
- Time schedule: the body weight of each female was recorded on Days 2, 4, 5, 6, 9, 12, 15, 19, 24 and 29 p.c. and prior to premature euthanasia.

FOOD CONSUMPTION:
- Time schedule: The quantity of food consumed by each animal was recorded for the following intervals: Days 2-4, 4-5, 5-6, 6-9, 9-12, 12-15, 15-19, 19-24 and 24-29 p.c.

POST-MORTEM EXAMINATION:
- Sacrifice on Day 29 post coitum
- Examined: principal thoracic and abdominal organs
Ovaries and uterine content:
The ovaries and uterine content were examined after termination, including:
- gravid uterus weight,
- number of corpora lutea,
- number and distribution of dead and live fetuses,
- number and distribution of early and late resorptions,
- number and distribution of uterine scars,
- number and distribution of implantation sites.

A gross evaluation of placentas was also undertaken.
Fetal examinations:
- External examinations: Yes: all fetuses per litter
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
- Other: fetal weight.
Statistics:
Data were compared by one-way analysis of variance and the Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous) or by the Fisher’s exact probability test (proportions).
Indices:
% Pre-implantation loss = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea
% Post-implantation loss = 100 * (Number of implantation sites - Number of live fetuses) / Number of implantation sites
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
See table 2.
At 450 or 800 mg/kg/day, the major finding was emaciated appearance, recorded generally from Day 11 or 12 p.c., accompanied by hypotonia, absence of feces or soft feces, soiled urogenital area and only at 800 mg/kg/day by tremors and cold to the touch. These signs of poor clinical condition, recorded in a dose related manner, were attributed to the test item treatment and were consistent with those noted in found dead animals at the same dose levels.
Emaciated appearance and/or absence of feces/urine were also reported among some control females or females at 30 or 75 mg/kg/day. As these signs were transient and sometimes recorded before treatment initiation, they were considered to be unrelated to the test item treatment.
The other findings, i.e. blood in the bedding in one control female and one female given 75 mg/kg/day, cutaneous lesions/scabs in one control female or one female at 75 mg/kg/day and difficulty during gavage administration in two females at 30 mg/kg/day, are commonly recorded in pregnant females of this strain and age and were therefore not considered to be test item-related.
Mortality:
mortality observed, treatment-related
Description (incidence):
At 450 and 800 mg/kg/day, before the premature euthanasia on Days 12-14 p.c., the following deaths were recorded:

At 450 mg/kg/day
. one female was found dead on Day 11 p.c.
Prior to death, signs of poor clinical condition were observed (tremors on Day 9 p.c. and emaciated appearance and absence of feces from Day 10 p.c.), associated with body weight loss (-415 g i.e. -11%) and almost no food intake (10 g/day) between Days 6 and 9 p.c.
At necropsy, the esophagus was perforated. Nine implantation sites were recorded.
. one female was found dead on Day 13 p.c.
Absence of feces was noted from Day 10 p.c., associated with body weight loss (-665 g i.e. -19%) and little or no food intake (0 to 22 g/day) between Days 6 and 9 p.c.
At necropsy, white discoloration of the stomach mucosa with depressed blackish area was noted, associated with dilatation of the gall bladder. Nine implantation sites were recorded in the uterine horns.

At 800 mg/kg/day
. one female was found dead on Day 12 p.c.
Prior to death, signs of poor clinical condition were observed (soft feces from Day 10 p.c., hypotonia and emaciated appearance on the day of death), associated with body weight loss (-590 g i.e. -16%) and almost no food intake between Days 6 and 12 p.c.
At necropsy, black foci were observed on the mucosa of the stomach, associated with dilatation of the gall bladder. No implantation site was noted in the uterine horns.

. one female was found dead on Day 13 p.c.
Prior to death, signs of poor clinical condition were observed (absence of feces from Day 10 p.c., hypotonia and emaciated appearance from Day 12 p.c.), associated with body weight loss (-780 g i.e. -22%) and little or no food intake (0 to 32 g/day) between Days 6 and 12 p.c.
At necropsy, red foci were observed on the mucosa of the stomach. Eleven implantation sites were recorded in the uterine horns.

These premature deaths led to the euthanasia of all surviving animals in these two groups on Days 12-14 p.c.
The details are included in the corresponding sections.

At 150 mg/kg/day, four females were prematurely euthanized due to poor clinical condition:
. one female was prematurely euthanized on Day 13 p.c.
Emaciated appearance was observed from Day 12 p.c., associated with body weight loss (-460 g i.e. -14%) from Day 6 p.c. and reduced food intake (7 to 72 g/day) between Days 6 and 12 p.c. At necropsy, the pyloric mucosa was thickened.
Four implantation sites were recorded in the uterine horns.

. one female was prematurely euthanized on Day 14 p.c.
Emaciated appearance was observed from Day 13 p.c., associated with body weight loss (-635 g i.e. -18%) from Day 6 p.c. and reduced food intake (3 to 58 g/day) on Days 6-12 p.c. At necropsy, red foci were observed on the stomach and the pyloric mucosa was thickened.
Nine implantation sites were recorded in the uterine horns.

. one female was prematurely euthanized on Day 14 p.c.
Emaciated appearance was observed from Day 13 p.c., associated with body weight loss (-580 g i.e. -16%) from Day 6 p.c. and little or no food intake (0 to 28 g/day) on Days 6-12 p.c. No necropsy findings were observed.
No implantation site was recorded in the uterine horns.

. one female was prematurely euthanized on Day 18 p.c.
Emaciated appearance was observed from Day 17 p.c., associated with body weight loss (-600 g i.e. -17%) from Day 9 p.c. and no food intake (3 g/day) between Days 12 and 15 p.c. At necropsy, white deposits were observed in the stomach and the spleen was reduced in size. Ten implantation sites were recorded in the uterine horns.

For all females mentioned above at 150, 450 or 800 mg/kg/day, the death or the poor clinical condition was considered to have resulted from the test item treatment. For one female, it is more difficult to differentiate the effects secondary to the esophagus perforation from those directly attributable to the test item.

At 75 mg/kg/day, one female was prematurely euthanized on Day 13 p.c. due to poor clinical conditions (absence of feces and urine on a few occasions, emaciated appearance from Day 8 p.c. and blood in the bedding from Day 12 p.c.), associated with body weight loss (-830 g i.e. -23%) and no food intake from Day 2 p.c.
No necropsy findings were noted. Ten implantation sites were recorded in the uterine horns.
This death was not attributed to the test item treatment as the major factor contributing to the poor clinical condition of the animal, i.e. the absence of food intake, was already noted before treatment initiation.

At 30 mg/kg/day, no unscheduled deaths occurred.

At 0 mg/kg/day, the following deaths occurred:
. one female (group 1) was prematurely euthanized following abortion on Day 21 p.c. (blood and one fetus and one placenta in the bedding). No clinical observations and no necropsy findings were reported. One implantation site was recorded in the uterine horns. This isolated case was considered to be incidental.
. one female (group 5) was prematurely euthanized on Day 15 p.c. due to poor clinical conditions (absence of feces and urine on several occasions and emaciated appearance from Day 8 p.c.), associated with body weight loss (-385 g i.e. -10%) and no food intake from Day 2 p.c.
No necropsy findings were noted. Eleven implantation sites were recorded in the uterine horns.
The major factor contributing to the poor clinical condition of the animal was attributed to the absence of food intake, already noted before treatment initiation.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See table 3.
At 450 and 800 mg/kg/day, when compared to controls, a dose-related, statistically significant mean body weight loss was recorded between Days 6 and 12 p.c. (-523 and -670 g, respectively, vs. +88 g), leading to lower mean body weight on Day 12 p.c. before sacrifice (-14 and -19%, respectively). These effects on body weight and body weight change were attributed to the test item treatment.

At 150 mg/kg/day, when compared to controls, a mean body weight loss was recorded between Days 6 and 15 p.c. (-158 g vs. +150 g). This effect on body weight change was due to the contribution of the three pregnant females prematurely euthanized. The individual body weight gain of the three surviving pregnant females between Day 6 and 24 p.c. was similar to those recorded in controls.
On Days 24-29 p.c., a statistically significant mean body weight loss was recorded (-188 g vs. +56 g in controls). This effect on body weight change was attributed to the test item treatment, but it did not impact the Day 29 p.c. body weight (-3% vs. controls).
At 30 and 75 mg/kg/day, no effects attributed to the test item were noted on body weight or body weight change. The mean body weight loss recorded in females given 75 mg/kg/day on Days 6-12 p.c. was due to the contribution of one female, prematurely euthanized on Day 13 p.c. following absence of food intake from Day 2 p.c.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
See table 4.
At 450 and 800 mg/kg/day, when compared to controls, dose-related, statistically significant reduced mean food intake was recorded between Days 6 and 9 p.c. (less than 50 g/day in the majority of animals) and Days 9 and 12 p.c. (almost zero food intake, except one female at 450 mg/kg/day). From Days 9/10 p.c., hay was distributed daily to the animals. This effect on food intake was attributed to the test item treatment and correlated with body weight loss during the same period.

At 150 mg/kg/day, when compared to controls, slightly lower mean food intake was recorded between Days 6 and 15 p.c. (-30%, only statistically significant on interval Days 6-9 p.c.). This effect on body weight change was due to the contribution of the three pregnant females prematurely euthanized (see § Mortality). The individual food intake of the three surviving pregnant females between Day 6 and 24 p.c. was similar to those recorded in controls.
On Days 24-29 p.c., lower mean food intake was recorded (-50%). This effect on food intake was attributed to the test item treatment and correlated with body weight loss during the same period.

At 30 and 75 mg/kg/day, no relevant effects were noted on food intake.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
See table 6.
On Days 12-14 p.c., at 450 and 800 mg/kg/day, the findings recorded in the stomach (colored foci, thickened mucosa and depressed areas) and in the intestines (liquid content) were consistent with those noted in found dead animals at the same dose levels. These gastrointestinal changes were considered to have contributed to the poor clinical condition of the animals. Dilatation of the gall bladder is a non-specific finding, secondary to the absence of food intake.
On Day 29 p.c., no test item-related findings were observed in animals given 30, 75 or 150 mg/kg/day.
The only findings, i.e. colored foci on the lungs in 1/7 control female (group 1), colored stomach mucosa in 1/4 control female (group 5), colored foci on the stomach in 1/5 females given 30 mg/kg/day and periovarian serous cysts in 1/5 female given 30 mg/kg/day, were considered to be incidental as these findings were reported in isolated animals and/or were noted with no dose relationship.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Gravid uterus weight
See table 5.
At 150 mg/kg/day, when compared to controls, the mean gravid uterus weight was lower (-32%). The mean carcass weight was unaffected by the test item treatment.
At 30 and 75 mg/kg/day, no effects on mean gravid uterus weight or carcass weight were noted.
Number of abortions:
no effects observed
Description (incidence and severity):
No dams with abortions.
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
See table 7.
Number of dams with pre-implantation loss:
4, 3 at 0 or 150 mg/kg/day and 5, 4 and 2 at 0, 30 or 75 mg/kg/day.

Number of dams with post-implantation loss:
4, 5 at 0 or 150 mg/kg/day and 1, 0 and 4 at 0, 30 or 75 mg/kg/day.

At 150 mg/kg/day, when compared to controls, mean pre- and post-implantation loss rate per female was higher, associated with lower mean number of live fetuses per female (7.7 vs. 8.7). This was mainly due to one out of the three females for which 42.9 and 25% of pre-and post-implantation loss rate, respectively, was recorded.
At 75 mg/kg/day, when compared to controls, mean post-implantation loss rate per female was higher (21.1 vs. 0%), mainly corresponding to higher mean number of early and late resorptions per female. This resulted in a lower mean number of live fetuses per female (8.3 vs. 10.0).
Although these differences were poorly dose-related, a relationship to the test item treatment could not be excluded.
At 30 mg/kg/day, no effects on hysterectomy data were noted.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No dams with total resorption.
Early or late resorptions:
effects observed, treatment-related
Description (incidence and severity):
At 150 mg/kg/day, when compared to controls, mean pre- and post-implantation loss rate per female was higher, associated with lower mean number of live fetuses per female (7.7 vs. 8.7). This was mainly due to one out of the three females for which 42.9 and 25% of pre-and post-implantation loss rate, respectively, was recorded.
At 75 mg/kg/day, when compared to controls, mean post-implantation loss rate per female was higher (21.1 vs. 0%), mainly corresponding to higher mean number of early and late resorptions per female. This resulted in a lower mean number of live fetuses per female (8.3 vs. 10.0).
Although these differences were poorly dose-related, a relationship to the test item treatment could not be excluded.

Number of dams with resorptions:
3 and 2 at 0 or 150 mg/kg/day, respectively
1, 0 and 3 at 0, 30 or 75 mg/kg/day, respectively.
Dead fetuses:
no effects observed
Description (incidence and severity):
See table 7.
No dead fetuses.
Changes in number of pregnant:
effects observed, treatment-related
Description (incidence and severity):
Table 1.
At hysterectomy, on Day 29 p.c., 6/8, 3/8, 4/5, 5/5 and 4/5 females were pregnant with live fetuses in the groups treated at 0 (1st receipt), 150, 0 (2nd receipt), 30 and 75 mg/kg/day, respectively.
At dose levels of 450 and 800 mg/kg/day, all surviving females were prematurely euthanized on Day 13 or 14 and Day 12 or 13 p.c., respectively.
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (nominal)
Basis for effect level:
pre and post implantation loss
Remarks on result:
other: High post-implantation loss at 75 mg/kg:day
Abnormalities:
not examined
Description (incidence and severity):
Excessive reduced food consumption leading to body weight loss and poor clinical condition. Macroscopic post-mortem lesions were observed in the stomach and intestines.
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
See table 8.
At 150 mg/kg/day, when compared to controls, mean fetal body weight was lower (individual values of 22.0, 28.1 and 51.3 g vs. at least 33.8 g in controls). This finding was attributed to the test item treatment and correlated with the lower mean gravid uterus weight recorded at this dose level.
At 30 and 75 mg/kg/day, no effect on mean fetal body weight was noted.
Reduction in number of live offspring:
not examined
Changes in sex ratio:
not examined
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
Malformations
No external malformations were observed at any dose level.
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Variations
No external variations were observed at any dose level.
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (nominal)
Basis for effect level:
reduction in number of live offspring
Remarks on result:
other: reduced numer of fetuses at 75 mg/kg/day
Abnormalities:
no effects observed
Developmental effects observed:
yes
Lowest effective dose / conc.:
75 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
not specified
Dose response relationship:
yes
Relevant for humans:
not specified

Table 1: Pregnancy status

Dose level (mg/kg/day)

0

150

450

800

0

30

75

Group

1

2

3

4

5

6

7

Number of females

8

8

8

8

5

5

5

Pregnant females

7

6

7

7

5

5

5

Females with live fetuses at termination

6

3

0

0

4

5

4

Females dead (pregnant)

0

0

2

1

0

0

0

Females prematurely
sacrificed (pregnant)

1

3

5

6

1

0

1

 

Table 2: Clinical signs

Dose level (mg/kg/day)

0

150

450

800

0

30

75

Group

1

2

3

4

5

6

7

Number of females

7

4

6

6

4

5

4

Tremors

-

-

-

1

-

-

-

Emaciated appearance

-

1

5

6

-

1

-

Hypotonia

-

-

1

6

-

-

-

Cold to the touch

-

-

-

1

-

-

-

Absence of urine

-

-

-

-

1

3

1

Absence of feces

-

-

1

2

-

1

1

Soft feces

-

-

1

4

-

-

-

Soiled urogenital area

-

-

1

4

-

-

-

Cutaneous lesion on neck ventral area

-

-

-

-

-

-

1

Scab on hindlimb

-

-

-

-

1

-

-

Blood in the bedding

1

-

-

-

-

-

1

Difficulty during gavage administration

-

-

-

-

-

2

-

-: no findings

Table 3: Body weight and body weight change

Dose level (mg/kg/day)

0

150

450

800

0

30

75

Body weight (g)

Day 6 p.c.

3609

3599

3676

3659

 

3574

 

 

3538

 

3686

Day 9 p.c.

3661

3546

3327

3260*

3587

3544

3672

% from controls

-

-3

-9

-11

 

-1

+2

Day 12 p.c.

3698

3501

3169*

2989**

3638

3559

3666

% from controls

-

-5

-14

-19

 

-2

+1

Day 15 p.c.

3760

3726

-

-

3714

3562

3896

% from controls

-

-1

 

 

 

-4

+5

Day 19 p.c.

3815

4003

-

-

3768

3711

3983

% from controls

-

+5

 

 

 

-2

+6

Day 24 p.c.

3933

4072

-

-

3788

3675

4046

% from controls

-

+4

 

 

 

-3

+7

Day 29 p.c.

3988

3883

-

-

3818

3787

4130

% from controls

-

-3

 

 

 

-1

+8

Body weight change (g)

 

 

 

 

Days 6-9 p.c.

52

-53

-349#

-399#

13

6

-14

Days 9-12 p.c.

36

-45

-174**

-271#

51

15

-6

Days 12-15 p.c.

62

-60

-

-

76

3

49

Days 15-19 p.c.

55

103

-

-

-26

149*

86

Days 19-24p.c.

64

68

-

-

20

-36

64

Days 24-29 p.c.

56

-188*

-

-

30

112

84

Days 6-2 9 p.c.

334

83

-

-

219

249

363

Statistically significant vs. controls: *: p<0.05; ** : p<0.01, # : p<0.001.

-: not applicabl

 

Table 4: Food consumption

Dose level (mg/kg/day)

0

150

450

800

0

30

75

Days 6-9 p.c.

195

140*

36#

31#

126

102

119

Days 9-12 p.c.

183

120

25#

1#

114

91

108

Days 12-15 p.c.

171

122

-

-

97

60

125

Days 15-19 p.c.

179

188

-

-

140

108*

140

Days 19-24 p.c.

289

281

-

-

116

100

139

Days 24-29 p.c.

111

56

 

 

73

97

114

Statistically significant vs. controls: *: p<0.05; **: p<0.01; # : p<0.001.

-: not applicable.

 

Table 5: Gravid uterus weight, carcass weight and net body weight change

Dose level (mg/kg/day)

0

150

0

30

75

Gravid uterus weight (g)

490.5

333.9

523.8

538.8

547.4

Carcass weight (g)

3497.8

3549.4

3293.7

3248.2

3582.7

Net body weight change
from Day 6
p.c. (g)

-156.3

-250.6

-305.0

-289.8

-184.8

No statistically significant differences from controls.

 

Table 6: Macroscopic post-mortem examination

Dose level (mg/kg/day)

0

150

450

800

0

30

75

Group

1

2

3

4

5

6

7

Number of females

7

4

6

6

4

5

4

Lung : colored focus

1

-

 

 

-

 

-

Stomach : colored foci

-

-

-

4

-

1

-

Stomach : colored mucosa

-

-

-

-

1

-

-

Stomach : depressed area

-

-

-

1

-

-

-

Stomach: thickened mucosa

-

-

4

3

-

-

-

Intestines : liquid content (greenish/reddish)

-

-

4

4

-

-

-

Gall bladder : dilatation

-

-

2

1

-

-

-

Periovarian region : serous cyst

-

-

-

-

-

1

-

-: no findings.

 

Table 7: Hysterectomy data

Dose level (mg/kg/day)

0

150

0

30

75

Number of females with live fetuses at termination

6

3

4

5

4

Mean number ofcorpora luteaper animal

10.5

10.3

11.3

13.0

11.8

Mean number of implantation sites per animal

9.3

8.7

10.0

11.4

10.8

Mean pre-implantation loss (%)

11.4

19.8

11.3

12.4

8.7

Mean number of live fetuses per animal

8.7

7.7

10.0

11.4

8.3**

Dead fetuses (%)

0.0

0.0

0.0

0.0

0.0

Mean number of resorptions per female

0.7

1.0

0.0

0.0

2.5*

Mean number of early resorptions

0.7

0.3

0.0

0.0

0.3

Mean number of late resorptions

0.0

0.7

0.0

0.0

2.3*

Mean post-implantation loss (%)

6.0

14.4

0.0

0.0

21.1*

Statistically significant vs. controls: *: p<0.05; **: p<0.01.

 

Table 8: Fetal body weight

Dose level (mg/kg/day)

0

150

0

30

75

Mean fetal body weight (g)

38.3

33.8

36.4

33.3

39.0

% from controls

-

-12

 

-9

+7

No statistically significant differences versus controls.

Conclusions:
The test item was administered daily to time-mated female New Zealand White rabbits by the oral route (gavage) at 30, 75 or 150 mg/kg/day from Day 6 to Day 18 p.c. or at 450 or 800 mg/kg/day from Day 6 to Day 12, 13 or 14 p.c.
At 150, 450 and 800 mg/kg/day, there was overt maternal toxicity, excessive reduced food consumption leading to body weight loss and poor clinical condition. All females at 450 and 800 mg/kg/day and half of the females at 150 mg/kg/day, were prematurely sacrificed between Days 12 and 14 p.c.
Macroscopic post-mortem lesions were observed in the stomach and intestines.
At 30 and 75 mg/kg/day, no maternal toxicity was noted. The only finding consisted of a significant higher post implantation loss rate with a significant lower number of fetuses at 75 mg/kg/day.
Based on the results of this study, the high dose level in the further main study should not exceed 75 mg/kg/day.
Executive summary:

The objective of this study was to evaluate the potential toxic effects of the test item, on the pregnant female and on embryonic and fetal development following daily oral administration (gavage) to pregnant female rabbits from implantation to the day prior to the scheduled hysterectomy (Day 6 to Day 28 post-coitum (p.c.) inclusive), in order to select dose levels for a further main study.

 

Methods

Four groups of eight timed-mated female New-Zealand White rabbits received the test item, suspended in 0.5% carboxymethylcellulose and 0.5% Tween 80, once daily by the oral route (gavage), at dose levels of 0, 150, 450 or 800 mg/kg/day (group 1, 2, 3 or 4, respectively). Due to severe toxic effects at 150, 450 and 800 mg/kg/day, leading to the premature sacrifice of half of females at 150 mg/kg/day and all surviving females at 450 or 800 mg/kg/day, three groups of five timed-mated females were added and treated at dose levels of 0, 30 or 75 mg/kg/day (groups 5, 6 and 7, respectively).

The females were treated between Day 6 and 18 p.c. inclusive or until Day 12, 13 or 14 p.c. if case of premature sacrifice at 150, 450 or 800 mg/kg/day.

The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recordedat designated intervals. On Day 29 p.c., the surviving females were euthanized and a macroscopic post-mortem examination was performed. Hysterectomies were performed and the numbers of corpora lutea, implantation sites, early and late resorptions, and live and dead fetuses were recorded. The fetuses were weighed and examined for external abnormalities.

 

Results

At hysterectomy, on Day 29 p.c., 6/8, 3/8, 4/5, 5/5 and 4/5 females were pregnant with live fetuses in the groups treated at 0 (1st receipt), 150, 0 (2nd receipt), 30 and 75 mg/kg/day, respectively.

At dose levels of 450 and 800 mg/kg/day, 2/8 females in each group were found dead between Days 11 and 13 p.c. preceded by signs of poor clinical condition (such as emaciated appearance, hypotonia, absence of feces), body weight loss and almost no food intake. The surviving females were euthanized between Days 12 and 14 p.c., together with 4/5 females given 150 mg/kg/day as the same life effects were observed.

No test item-related clinical signs were reported in the 4/8 surviving females at 150 mg/kg/day or in females at 30 or 75 mg/kg/day.

No effects on body weight, body weight change or food intake were noted at 30 or 75 mg/kg/day. At 150 mg/kg/day, lower food intake correlated with body weight loss was reported between Days 24 and 29 p.c., but these effects did not impact the terminal body weight.

 

At necropsy of the dams, no test item-related macroscopic post-mortem findings were observed at 30 or 75 mg/kg/day or in the 4/8 surviving females at 150 mg/kg/day. At 450 and 800 mg/kg/day and in the 4/8 prematurely euthanized females at 150 mg/kg/day, findings in the stomach and intestines were recorded.

 

At hysterectomy, higher post-implantation loss rates were noted at 75 and 150 mg/kg/day, corresponding to higher number of resorptions. These resulted in lower numbers of live fetuses per female. This latter finding, associated with the lower fetal body weight at 150 mg/kg/day (-12% vs. controls), accounted for the lower gravid uterus weight at this dose level (-32% vs. controls).


Conclusion

The test item was administered daily to time-mated female New-Zealand White rabbits by the oral route (gavage) at 30, 75 or 150 mg/kg/day from Day 6 to Day 18 p.c. or at 450 or 800 mg/kg/day from Day 6 to Day 12, 13 or 14 p.c.

 

At 150, 450 and 800 mg/kg/day, there was overt maternal toxicity, excessive reduced food consumption leading to body weight loss and poor clinical condition. All females at 450 and 800 mg/kg/day and half of the females at 150 mg/kg/day, were prematurely sacrificed between Days 12 and 14 p.c.

Macroscopic post-mortem lesions were observed in the stomach and intestines. 

At 30 and 75 mg/kg/day, no maternal toxicity was noted. The only finding consisted of a significant higher post-implantation loss rate with a significant lower number of fetuses at 75 mg/kg/day.

 

Based on the results of this study, the high dose level in the further main study should not exceed 75 mg/kg/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
10 mg/kg bw/day
Study duration:
subacute
Experimental exposure time per week (hours/week):
7
Species:
rabbit
Additional information

1/Rat developmental study (Kurosaki, 1988)

The potential ofDibenzylbenzene, ar-methyl derivativeto induce developmental toxicity after maternal exposure during the critical period of organogenesis was evaluated in rat according to a study design comparable to OECD Guideline N° 414.

Dibenzylbenzene, ar-methyl derivativewas administered orally by gavage to three groups of 20 bred female Sprague-Dawley rats once daily from gestation days 7 through 17. Dosage levels were 0, 100, 300 and 1000 mg/kg/d.

.

All animals survived to the scheduled necropsy. Piloerection was observed in females given 1000 mg/kg/d as well as a slight decrease in body weight gain and food consumption.

The slight increase in the resorption of corpora lutea noted in the highest dose group was not considered as relevant since the administration ofDibenzylbenzene, ar-methyl derivativeoccurred after the implantation. The survival of the pups was unaffected byDibenzylbenzene, ar-methyl derivativeadministration at all dose levels. However, pups body weight was reduced in the highest dose group when compared to controls but this effect was related to the maternal toxicity observed. Foetal external, soft tissue and skeletal malformations observed in control and treated pups were considered to be spontaneous in origin. The developmental variations expressed in the treated groups were generally similar to those present in the control group or occurred in a manner that was not dose related.

Under these experimental conditions, the No-Observed-Adverse-Effect-Level (NOAEL) was 300 mg/kg/d for maternal toxicityandthe No-Observed-Adverse-Effect-Level (NOAEL) was 300 mg/kg/d for fetal toxicity.

2/Rabbit developmental toxicity study (Haag, 2020)

The objective of this study was to evaluate the potential toxic effects of the test item on the pregnant female and on embryonic and fetal development following daily oral administration (gavage) to pregnant female rabbits from implantation to the day prior to the scheduled hysterectomy (Day 6 to Day 28post-coitum (p.c.) inclusive).

Methods

Three groups of 24 time-mated female New-Zealand White rabbits received the test item, once daily by the oral route (gavage) at dose levels of 10, 30 or 75 mg/kg/day from Day 6 to Day 28p.c.inclusive. A control group of 24 time-mated females received the vehicle (0.5% carboxymethylcellulose with 0.5% Tween 80), under the same experimental conditions.

No test item-related deaths or clinical signs were recorded.

Lower body weight gain was recorded between Days 6 and 29p.c. at 30 and 75 mg/kg/day (+272 g and +253 g, respectively,vs.+356 g in controls). These differences, especially due to continuous lower body weight gain at 30 mg/kg/day and transient body weight losses at 75 mg/kg/day, did not impact the body weight on Day 29p.c.(-3 and -2%, respectively) and were considered to be non-adverse. This was associated with a net body weight loss from Day 6p.c.more pronounced at 75 mg/kg/day,vs.controls, but with no impact on the carcass weight.

Food consumption was unaffected by the test item treatment.

At necropsy of the dams, none of the macroscopic findings observed were considered to be related to the test item administration. Gravid uterus weight was considered to be not impacted by the test item treatment. No effects on the hysterectomy parameters were noted.

The fetal body weight and the sex ratio were unaffected at any dose level.

At fetal examination, the major anomaly consisted of malposition of the kidney(s), recorded with higher incidence at 10 and 75 mg/kg/day, associated with marked dilation of renal pelvis in one fetus at 10 mg/kg/day. Absence of kidney and ureter was noted in one fetus at 30 mg/kg/day. These malformations were accompanied at 75 mg/kg/day by perturbation of vessel development (absence of innominate artery) and delay in skeletal ossification (unossified hyoid centrum and forepaw median phalanxes). External and visceral variations of uncertain relationship to the test item were noted at all dose levels (malrotation/hyperflexion of paw, polyhydramnios/abnormal color of amniotic fluid, dilated renal pelvis, enlarged kidneys and/or adhesion kidney-intestine).

Conclusion

The test item was administered daily to time-mated female NZW rabbits by the oral route (gavage) at dose levels of 10, 30 or 75 mg/kg/day from Day 6 to Day 28p.c., inclusive.

Based on the results obtained in this study:

· the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 75 mg/kg/day based on the absence of adverse findings at this dose level,

· no No Observed Effect Level (NOEL) or No Observed Adverse Effect Level (NOAEL) for embryo-fetal development could be established based on the presence of malformations in upper urinary tract (kidneys and ureters) at all dose levels of the test item. The Low Observed Adverse Effect Level (LOAEL) is considered to be 10 mg/kg/day.

Justification for classification or non-classification

Classification for Reproductive Toxicity Hazard related to unborn child according to CLP criteria:

In the rabbit developmental study (Haag, 2020), the presence of a clear evidence of fetal malformations incidence of the upper urinary tract (kidneys and ureters) at all dose levels associated with absence of significant maternal toxicity or any other toxic effects has lead to the impossibility to derive a NOEL for developmental toxicity and instead to derive a Low Observed Adverse Effect Level (LOAEL) of 10 mg/kg/day. Based on these adverse fetal findings, the following hazard statement is warranted (Repro 1 B, H360D).

Classification for Reproductive Toxicity related to Fertility according to CLP Criteria:

In the screening reproduction toxicity study OECD 421 (Spézia, 2020), evidence of fertility adverse effects were noted in the absence of significant maternal toxicity, they were susbtantiated by :

- Thyroid follicular hypertrophy associated with low T4 concentration and decreased mean number of corpora lutea/implantation sites/litter size and consequently a significant decrease in female fertility index at 800 mg/kg/day,

. the No Observed Adverse Effect Level (NOAEL) for reproductive performance (mating, fertility and delivery) was considered to be the mid dose-level 250 mg/kg/day based on decreased fertility index at 800 mg/kg/day,

. the No Observed Adverse Effect Level (NOAEL) for F1 development was considered to be 80 mg/kg/day based on marked decreases in body weight on Days 8-13p.p. (males and females).

Based on the evidence of fertility adverse effects, the following hazard statement is warranted: (Repro 1 B, H360F)

Additional information