Dibenzylbenzene, ar-methyl derivative

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

There is one reliable OECD 421 study performed according to GLP (CIT, 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 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 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

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.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological 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

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

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.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

80 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Critical effects observed:

no

Key result

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

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Reproduction toxicity screening study (OECD 421, CIT 2020):

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.

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 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.

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.

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.

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).

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 (CIT, 2020) both performed according to OECD 414.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Limit test:

no

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

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

Remarks:

(maternal toxicity)

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

Abnormalities:

no effects observed

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)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

no effects observed

Developmental effects observed:

no

Conclusions:

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 1000 mg/kg/d for fetal 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 1000 mg/kg/d for fetal toxicity.

Reference 2

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

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

Dose / conc.:

10 mg/kg bw/day

Dose / conc.:

30 mg/kg bw/day

Dose / conc.:

75 mg/kg bw/day

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:

See 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

Endocrine 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.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

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.

Remarks on result:

not determinable due to absence of adverse toxic effects

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.

Anogenital distance of all rodent fetuses:

not examined

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.

Sex:

male/female

Basis for effect level:

visceral malformations

Abnormalities:

effects observed, treatment-related

Localisation:

visceral/soft tissue: urinary

visceral/soft tissue: cardiovascular

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 5^thmedian 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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

10 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

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 (CIT, 2020b)

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).

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).

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.

 

3/Dose range finding toxicity study in rabbit (CIT 2020a)

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.

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.

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).

In summary, 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.

Toxicity to reproduction: other studies

Description of key information

A Uterotrophic Bioassay in Rodents was conducted on the registered substance in accordance with the OECD Testing Guideline 440 (CRL, 2021). The objective of the study was to evaluate the estrogenic and anti-estrogenic activities of the test item (dibenzylbenzene, ar-methyl derivative) following daily oral administration (gavage) to juvenile, sexually immature female rats for 3 consecutive days. The results were compared to those of a reference estrogen (17-α-Ethynylestradiol). The investigations performed at the end of the treatment period focused on uterus weight changes and microscopic examination of the uterus and vagina.
Under the experimental condition of this study at dose levels ≥ 250 mg/kg/day, the registered substance was associated with microscopic changes suggestive of an estrogenic effect in the female reproductive organs of immature rats, although uterine weights were not increased, most likely because of effects on the terminal body weights. Given the high mortality at the intermediate- and high-doses of the combination of the test item and the reference item, definite conclusion on the anti-estrogenic potential of the test item could not be drawn.

Justification for classification or non-classification

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

In the rabbit developmental study (CIT, 2020b), 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 1B, H360D.

 

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

In the screening reproduction toxicity study OECD 421 (CIT, 2020c), 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 1B (H360F)