Dibenzylbenzene, ar-methyl derivative

Toxicity to reproduction

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2020

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Dibenzylbenzene, ar-methyl derivative

EC Number:

258-649-2

EC Name:

Dibenzylbenzene, ar-methyl derivative

Cas Number:

53585-53-8

Molecular formula:

C21H20

IUPAC Name:

dibenzylbenzene, ar-methyl derivative

Test material form:

liquid

Test animals

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.

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

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)

Examinations

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))

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Effect levels (P0)

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

Target system / organ toxicity (P0)

1

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

2

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

Results: F1 generation

General toxicity (F1)

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.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

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

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

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

Any other information on results incl. tables

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.

 

Applicant's summary and conclusion

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).