Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information
Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 November 2017 - 26 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
exception: on Days 34 (males) and 51-54 (females), following urobilinogen QC deviations at the beginning of the series on the positive control, the samples were analyzed with an invalid QC (urobilinogen data non GLP): no impact on the study conclusions
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 males were approximately 10 weeks old and the females were approximately 11 weeks old
- Weight: on the first day of treatment, the males had a mean body weight of 411 g (range: 389 g to 426 g) and the females had a mean body weight of 274 g (range: 247 g to 312 g)
- Housing: F0 animals were individually housed, except during mating (males + females) and lactation (females + pups), in polycarbonate cages (Tecniplast 2154, 940 cm2) with stainless steel lids and containing autoclaved sawdust
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: the males were acclimated to the study conditions for a period of 7 days before treatment and the females were acclimated to the study conditions for a period of 6 days before the beginning of estrous cycle monitoring during the pre-treatment period.

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: 03 January 2018 to 26 February 2018.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING FORMULATIONS:
- Suspension in the vehicle (solution in the vehicle at 3 mg/mL)
- Concentration in vehicle: 3, 10 and 30 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation (mating period): until mating occurred
- Proof of pregnancy: vaginal plug or sperm in the morning vaginal lavage referred to as day 0 post-coitum
- After successful mating each pregnant female was caged individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Type of method: High Performance Liquid Chromatography with UV detection (HPLC/UV)
Test item concentrations: remained within an acceptable range of variations (-4.1% to +0.6%) when compared to the nominal values (criterion: ± 15% of the nominal concentrations).
Homogeneity/Stability: the dose formulations containing the test item at 3 mg/mL or at 10 and 200 mg/mL in corn oil were found to be homogeneous and stable after 24 hours or 2 days at room temperature and protected from light, respectively.
Duration of treatment / exposure:
In the males:
- 2 weeks before mating,
- during the mating period (up to 2 weeks),
- until sacrifice (at least 4 weeks in total),

In the females:
- 2 weeks before mating,
- during the mating period (up to 2 weeks),
- during gestation,
- during lactation until Day 13 post-partum inclusive,
- until euthanasia for females with no delivery.
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for dose level selection:
The dose levels were selected in agreement with the Sponsor, on the basis of the results of a previous study performed in the same species, in which the test item was administered daily by gavage to five males and five females at dose levels of 100, 300 or 1000 mg/kg/day for 2 weeks.
The dose level of 1000 mg/kg/day was associated, in both sexes, with mortality, premature euthanasia of the surviving animals and signs of poor clinical condition (i.e. thin appearance, hunched posture, piloerection, pallor of extremities, half-closed eyes, hypoactivity, hypotonia, staggering gait, decreased grasping reflex and/or dyspnea), body weight loss leading to a lower body weight, markedly reduced food consumption and macroscopic changes in the stomach (white, black or red discoloration, gas distension), forestomach (white or red discoloration), intestine (distension with feces or with gas and/or thick or liquid brown content), spleen (enlargement), ureters (dilatation), kidneys (enlargement, pelvis dilatation, gelatinous hilus and perirenal adipose tissue and/or yellow discoloration), pancreatic lymph nodes (enlargement) and liver (enlargement and/or yellow discoloration). No microscopic observation was performed.
The dose level of 300 mg/kg/day was associated with poor clinical condition (i.e. and hunched posture and piloerection in one male and one female), body weight loss in males at the beginning of the treatment period, moderate reduced food consumption in both sexes during the first week of the treatment period, organ weight changes in the liver (absolute and relative liver weights moderately increased day in both sexes), the spleen (marked increases in the mean absolute and relative spleen weights in both sexes), and in the kidneys and the heart (mean absolute and relative kidney or heart weights minimally increased in females reaching statistical significance for the absolute weight), macroscopic changes in the spleen (enlargement in both sexes) and ureters (dilatation in males) and microscopic changes in the liver (slight to moderate hepatocellular hypertrophy and minimal hematopoiesis in both sexes), the spleen (increased severity of hematopoiesis and congestion along with increased hemosiderin in both sexes suggestive of hemolysis) and the kidneys (eosinophilic granules in renal tubules in males suggestive of hemolysis).
The dose level of 100 mg/kg/day was associated with slight reduced food consumption in females during the study, macroscopic changes in the spleen (enlargement in both sexes) and microscopic changes in the liver (minimal hepatocellular hypertrophy in males and minimal hematopoiesis in both sexes), and the spleen (increased severity of hematopoiesis in both sexes and congestion along with increased hemosiderin in females suggestive of hemolysis).

As the doses of 300 and 1000 mg/kg/day are considered to be higher than the Maximum Tolerable Dose (MTD), 150 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. 15 and 50 mg/kg/day).

- Rationale for animal assignment: stratification procedure
Positive control:
no (not required)
Parental animals: Observations and examinations:
MORTALITY/MORBIDITY:
- Time schedule: Each animal was checked for mortality or signs of morbidity once a day before the treatment period and at least twice a day during the treatment period, including weekends and public holidays.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: detailed clinical examinations were performed on all animals, once before the beginning of the treatment period and then once a week until the end of the study.

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

BODY WEIGHT:
- Time schedule: the body weight of each male was recorded on the first day of treatment (Day 1), then once a week until euthanasia.
The body weight of each female was recorded on the first day of treatment (Day 1), then once a week until mated, on Days 0, 7, 14 and 20 p.c. (post-coitum) (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 period, 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.

WATER CONSUMPTION:
- Time schedule: the quantity of water 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 water consumed by each female was measured once a week from the first day of treatment until the start of the mating period, 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, water consumption was not measured for males or females.

NEUROBEHAVIOURAL EXAMINATION:
Functional observation battery
The first five males and the first five surviving females euthanized on Day 14 p.p., from each group, were evaluated with a functional observation battery once at the end of the treatment period. For females, this was performed on Day 13 p.p. after euthanasia of the pups.

This included a detailed clinical examination, the assessment of reactivity to manipulation and to different stimuli and motor activity.
All animals were observed in the cage, in the hand and in the standard arena.

Detailed clinical examination
The following parameters were assessed and graded:
- in the cage: “touch escape” or ease of removal from the cage,
- in the hand: fur appearance, salivation, lacrimation, piloerection, exophthalmos, reactivity to handling, pupil size (presence of myosis or mydriasis),
- in the standard arena (2-minute recording): grooming, palpebral closure, defecation, urination, tremors, twitches, tonic and clonic convulsions, gait, arousal (hypo- and hyper-activity), posture, stereotypy, behavior, breathing, ataxia and hypotonia.

Reactivity to manipulation and different stimuli
The following measurements, reflexes and responses were recorded:
- touch response,
- forelimb grip strength,
- pupillary reflex,
- visual stimulus response,
- auditory startle reflex,
- tail pinch response,
- righting reflex,
- landing foot splay,
- at the end of observation: rectal temperature.

Motor activity
Motor activity was measured once by automated infra-red sensor equipment over a 60-minute period.

HAEMATOLOGY:
The parameters were determined on the day of euthanasia for the first five males and the first five females from each group euthanized on Day 14 p.p.

CLINICAL CHEMISTRY:
The parameters were determined on the day of euthanasia for the first five males and the first five females from each group euthanized on Day 14 p.p.

URINALYSIS:
The parameters were determined on the day of euthanasia for the first five males and the first five females from each group euthanized on Day 14 p.p.

THYROID HORMONES:
Blood samples were taken, in the first half of the morning (between 7.5 and 10 am), as follows:
- at termination on Day 14 p.p. from all F0 females,
- at termination from all F0 males.
REPRODUCTION (apart from indices):
- Pre-coital time and duration of gestation were recorded.
Oestrous cyclicity (parental animals):
Monitoring of estrous cycle
The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning (between 7:5 and 10 am):
- during the 2 weeks of the pre-treatment period (including for the 2 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 Day 14 p.p. before euthanasia, to allow correlation with reproductive organs histopathology.

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.
Sperm parameters (parental animals):
Parameters examined in males of parental generation:
- weighing and microscopic examination: see Tissue Procedure Table below
- special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.
Litter observations:
STANDARDISATION OF LITTERS: Yes, performed on Day 4 p.p.

PARAMETERS EXAMINED:
- number and sex of pups,
- number of live, dead and cannibalized pups,
- presence of gross anomalies, weight gain, clinical signs,
- anogenital distance,
- presence of nipples and of areolae in male pups on Day 12p.p.

GROSS EXAMINATION OF DEAD:
- external abnormalities

THYROID HORMONES:
Blood samples were taken, in the first half of the morning (between 7.5 and 10 am), as follows:
- at termination on Day 4 p.p. from at least 2 pups/litter culled,
- at termination on Day 13 p.p. from at least 2 pups/litter.

The levels of the thyroid hormone (T4) and thyroid stimulating hormone (TSH) were determined respectively by LC-MS/MS or Luminex MAP® technology for samples from pups collected on Day 13 p.p. and from F0 males collected at termination.

Plasma samples obtained on Day 4 p.p. from pups and on Day 14 p.p. from F0 females were put into storage at -80°C pending possible analysis.
Postmortem examinations (parental animals):
SACRIFICE
On completion of the treatment period, after at least 14 hours fasting, all surviving F0 animals were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and euthanized by exsanguination:
- males: after the end of the mating period (at least 4 weeks of treatment in total),
- females: on Day 14 p.p.
The following F0 females were euthanized by the same way without overnight fasting:
- females that did not deliver: on Day 25 or 26 p.c. (after the recording of body weight to check for a possible un noticed delivery).

ORGAN WEIGHTS: see table below
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.

GROSS PATHOLOGY:
A complete macroscopic post-mortem examination was performed on all F0 animals including females that died during the study or were euthanized prematurely. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. Special attention was paid to the reproductive organs.
The numbers of corpora lutea and implantation sites were recorded for females euthanized as scheduled on Day 14 p.p. and for females euthanized on Day 25 or 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.

HISTOPATHOLOGY:
A microscopic examination was performed on:
- all tissues listed in the Tissue Procedure Table from the first five euthanized as scheduled males and the first five females euthanized on Day 14 p.p., of the control and high-dose groups (groups 1 and 4),
- all macroscopic lesions of all groups,
- reproductive organs and thyroids with parathyroids from from any animal that did not mate or conceive, or from pregnant females that did not deliver, to investigate possible causes: i.e. one male and one female (group 2), one male and one female (group 3) and one male and one female (group 4).
Special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

In agreement with the Sponsor and based upon the results of the microscopic examination of the high-dose group, other tissues of the low- and intermediate-dose groups were examined in the first five euthanized as scheduled males and the first five females euthanized on Day 14 p.p. as follows:
-heart (females only),
-kidneys (both sexes),
-liver (both sexes),
-sternum with bone marrow (females only),
-spleen (both sexes),
-urinary bladder (both sexes).
In agreement with the Sponsor and based upon the results of the microscopic examination of the high-dose group, microscopic examination of histological slides stained with Perl’s procedures were performed as described in the previous section.

Postmortem examinations (offspring):
SACRIFICE: on Day 13 post-partum
Pups were euthanized by an intraperitoneal injection of sodium pentobarbital (or by decapitation under isoflurane anesthesia on Day 4 p.p. when blood samples were collected), followed by exsanguination when the thyroids were collected:
-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.

ORGAN WEIGTHS:
The body weight of one selected pup/sex/litter (or of 2 pups from the same sex when there was only one sex in the litter) euthanized on Day 13 p.p. was recorded before euthanasia.

HISTOPATHOLOGY: 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 Fisher’s exact probability test (proportions).

Hematology, blood biochemistry, urinalysis, hormones, motor activity, anogenital distance, nipples/aerolae, post-implantation loss and sex ratio
CITOX software was used to perform the statistical analyses.
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 implantations
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 / Number of delivered pups)
Viability index on day 4 p.p. = 100 * (Number of surviving pups on day 4 p.p. / Number of live born pups)
Lactation index on Day 13 p.p.: 100 * Number of surviving pups on Day 13 p.p. / Number of surviving pups on Day 4 p.p. (after culling)
AGD/cube root of body weight ratio (calculated with Excel) = AGD / ³vBody weight
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
See table 1.
Non-adverse test item-related clinical signs, namely round back (from Day 29 to Day 33) and piloerection (Day 29) were noted in Week 5 in 1/10 males given 150 mg/kg/day.
Ptyalism was observed with a dose-related incidence in test item-treated males and females during the premating, pregnancy and lactation periods. This sign, commonly noted when a test item is administered by gavage, was not considered to represent an adverse effect.

At 50 mg/kg/day, piloerection or emaciated appearance were noted in 2/8 females for 4 or 2 days, respectively, during the lactation period. As these clinical signs were not dose-related and/or were most probably due to a technical problem of water access (emaciated appearance), a test item relationship was considered to be unlikely.

All the other clinical signs (i.e. areas of hair loss, cutaneous lesions, scabs, chromodacryorrhea, short tail, reflux at dosing and/or reddish vaginal discharge) were considered to be unrelated to the test item as they were present in control animals, were not dose-related, were reported sporadically in only a few animals, are commonly observed findings in this species and strain and/or resulted from the gavage procedure.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
See table 2.
There were no unscheduled deaths in males.

. At 0 mg/kg/day
One female was found dead on Day 24 p.c. Five dead pups (two males and three females) were found in the litter. No remarkable clinical signs were observed prior to death. At necropsy, there were 11 dead fetuses and 5 placentae without fetuses in the uterus, which suggested that death was related to parturition issues. The thymus was red, which correlated with agonal congestion/hemorrhage. In the thymus, in addition to agonal congestion/hemorrhage, there was moderately increased apoptosis in the cortex and slightly decreased lymphocyte cellularity in the cortex/medulla, which were both suggestive of stress associated with poor health.

. At 15 mg/kg/day
Two females were euthanized on Days 26 and 25 p.c., respectively, for no delivery. At necropsy, these females were found to be non-pregnant. Ptyalism was transiently noted during the premating and/or gestation period together with chromodacryorrhea and cutaneous lesions on the forelimbs in one female. At necropsy, a vaginal transverse septum was observed in one female, which is associated with the absence of pregnancy.
No necropsy findings were observed in one female. However, follicular cysts and absence of corpora lutea in the ovaries, increased thickness of the squamous epithelium with keratinization in the vagina were suggestive of persistent estrus. This absence of normal estrous cycling explained the absence of pregnancy, and was considered to be incidental.

. At 50 mg/kg/day
One female was euthanized on Day 26 p.c. for no delivery. Ptyalism was transiently noted during the premating and gestation periods. At necropsy, no macroscopic post-mortem findings were observed. This female was found to be non-pregnant and no relevant microscopic changes were observed in the reproductive organs.
One female was prematurely euthanized on Day 13 p.c. due to a subcutaneous mass on the urogenital area (3.0 x 3.0 cm in diameter), which resulted from a nodosity that appeared during the mating period (i.e. on Day 17 of the study). Ptyalism was transiently noted during the premating and gestation periods. At necropsy, there was a large perigenital white mass (3.5x3 cm approximatively), which correlated with an adenocarcinoma of the mammary gland. This isolated finding in a mid-dose individual was considered to be incidental.
This female was found to be pregnant with 10 live concepti and 2 early resorptions.

. At 150 mg/kg/day
Two females were euthanized on Days 26 and 25 p.c., respectively, for no delivery. Ptyalism was transiently noted during the premating period and throughout the gestation period.
One female was found to be pregnant. At necropsy, the spleen was enlarged and 2 implantation sites were observed in uterine horns.
One female was found to be non-pregnant. At necropsy, no macroscopic post-mortem findings were observed and no relevant microscopic changes were noted in the reproductive organs.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See table 5.
Differences between control and test item-treated animals consisted of lower mean body weight gain in males given 150 mg/kg/day between Days 1 and 8 (+30 g vs. +39 g in controls, p<0.05) and between Days 22 and 29 (+18 g vs. +24 g in controls), and lower mean body weight gain in females given 150 mg/kg/day between Days 1 and 4 p.p. (+1 g vs. +9 g in controls). These variations resulted in lower mean body weights in males on Days 8 and 29 (-3% and -4% vs. controls, respectively, p<0.05).
These findings were considered to be of minor toxicological importance as they were of slight magnitude, isolated and/or with no effects on the mean body weight.

There were no effects on mean body weight or mean body weight change at 15 and 50 mg/kg/day in males and females.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
See table 6.
Differences between control and test item-treated animals consisted of higher mean food consumption between Days 8 and 15 of the premating period (+13% vs. controls, p<0.05) in females given 50 or 150 mg/kg/day. This trend persisted in females given 150 mg/kg/day over the first week of the gestation period (+16% vs. controls, p<0.01). These test item-related findings were considered to be of minor toxicological importance as they were of slight magnitude, isolated and/or with no effects on the mean body weight.
Other differences from controls were observed during the lactation period (i.e. higher mean food intake in females given 15 mg/kg/day). A relationship with the test item was considered to be unlikely as these variations were not dose-related.

There were no effects on mean food consumption in males at any dose level or in females at 15 mg/kg/day.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
See table 7.
Differences between control and test item-treated animals consisted of higher mean water consumption:
- in males given 15, 50 or 150 mg/kg/day over the second week of the premating period (+19, +16 and +24% vs. controls, respectively, statistically significant),
- in females given 15 mg/kg/day over the first week of the gestation period (+17% vs. controls),
- in females given 50 mg/kg/day or 150 mg/kg/day over the premating period (+13 to +19% and +20 to +22% vs. controls, respectively), throughout the gestation period (+17 to +28% and +31 to +38% vs. controls, respectively) with statistical significance, and on some occasions during the lactation period (+15% between Days 8 and 13 p.p. at 50 mg/kg/day and +16% between Days 4 and 8 p.p. at 150 mg/kg/day) but to a lesser extent. These differences were statistically significant during the premating and gestation periods and more marked between Days 0 and 7 p.c.
These test item-related findings correlated with slightly higher mean urine volumes recorded in females given 15, 50 or 150 mg/kg/day at the end of the study. They were considered to be of minor toxicological importance as they were of slight magnitude and were not associated with other adverse findings.

Other differences from controls were observed during the lactation period (i.e. higher mean water intake in females at all dose levels from Day 4 p.p. without statistically significance). A relationship with the test item was considered to be unlikely as these variations were poorly dose-related and mainly due to the low water consumption of one control female.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
See details in 7.5 Repeated dose toxicity (OECD 422).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
See details in 7.5 Repeated dose toxicity (OECD 422).
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
See details in 7.5 Repeated dose toxicity (OECD 422).
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
See tables 3 and 4.
See details in 7.5 Repeated dose toxicity (OECD 422).
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See details in 7.5 Repeated dose toxicity (OECD 422).

Unscheduled deaths
One female (50 mg/kg/day) prematurely sacrificed on Day 31 (i.e. on Day 13 p.c.), had an adenocarcinoma of the mammary gland in the perigenital region i.e. a malignant tumor. This isolated finding in a mid-dose individual was considered to be incidental.

In one control female found dead on Day 40 (i.e. on Day 24 p.c.), there were 11 dead fetuses in the uterus, which suggested that death was related to delivery issues. In the thymus, in addition to agonal congestion/hemorrhage, there was moderately increased apoptosis in the cortex and slightly decreased lymphocyte cellularity in the cortex/medulla, which were both suggestive of stress associated with the poor health status.

There were no relevant microscopic changes in the male and female reproductive organs.
In one low-dose female which was not pregnant, follicular cysts and absence of corpora lutea in the ovaries, increased thickness of the squamous epithelium with keratinization in the vagina were suggestive of persistent estrus. This absence of normal estrous cycling explained the absence of pregnancy, and was considered to be incidental. Other females (lone mid-dose and one high-dose) which were not pregnant did not show any abnormalities in the estrous cycle or reproductive organs. There were no relevant changes in the reproductive organs in the corresponding mating males.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Thyroid hormones
See table 11.
The thyroid hormone levels were considered to be unaffected by the test item treatment in F0 males.

In males, in all treated groups and when compared with controls, there were lower mean T4 concentrations reaching statistical significance at 15 and 150 mg/kg/day but remaining in the range of the Historical Control Data. These variations were considered fortuitous as they were not dose-related, did not correlate with a dose-related response in mean TSH levels and also resulted from higher mean control values (above mean Historical Control Data).
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
See table 9.
There were no effects on mean length of the estrous cycle or mean number of cycles.
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
Pairing, mating and fertility data
See table 8.
There were no relevant effects on mating, fertility or gestation index.

Low percentages in the fertility index of test item-treated groups were not dose-related or were within the range of Historical Control Data (fertility index: 90-100%, Historical Control Data OCDE 421-422). They were therefore considered not to be test item-related.

When compared with controls, slightly higher pre-coital time was observed in females 15, 50 or 150 mg/kg/day. As these differences were poorly dose-related, were not statistically significant, were within the range of the Reference Data and mainly resulted from the low pre-coital time recorded in controls, they were considered to be of no toxicological importance.

Delivery data
See table 10.
There were no effects on the mean duration of gestation, numbers of corpora lutea and implantation sites or percentages of pre /post implantation losses.

The low mean number of implantation sites and high mean post-implantation loss recorded in control females were mainly due to one control female (5 implantation sites and 80% post-implantation loss).
Dose descriptor:
NOAEL
Remarks:
for parental systemic toxicity
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Remarks:
for reproductive performance
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
yes
Lowest effective dose / conc.:
150 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
bladder
heart
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
See table 13.
There were no test-item treatment related behavioral changes or external abnormalities at pup examination.

At 50 and 150 mg/kg/day, clinical signs of poor clinical condition (i.e. generalized palor, bluish appearance, coldness to the touch, dyspnea, emaciated appearance, absence of milk in the stomach and/or hematoma on the abdomen) were noted in 3/8 litters and 5/8 litters vs. 0/9 control litters, respectively. These findings were considered to be test item related in view of the litter incidence (they were also observed in all found dead pups).

The other clinical signs were not attributed to the test item as they were not dose-related, were reported in isolated animals and/or are routinely observed in rat pups of this strain and age.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
See table 12.
At 50 and 150 mg/kg/day and when compared with controls, there were increased numbers of found dead or cannibalized pups (10 and 17 pups vs. 1 pup in controls, respectively) and increased incidences of litters affected (4/8 and 5/8 vs. 1/9 in controls, respectively). This was associated with low live birth and viability indexes which were below the lower limit of the Reference Data. Therefore, these findings were considered to be test-item treatment related.
At 15 mg/kg/day and when compared with controls, there were no effects on pup viability.

There were no effects on the lactation index.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
See table 14.
There were no effects on mean body weight or mean body weight change during the lactation period.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Anogenital distance
See table 16.
At 150 mg/kg/day and when compared with controls, males had a statistically significant, lower mean AGD/BW1/3 ratio (-6%) for which a test item relationship could not be excluded.
Most of the pups from groups 2, 3 and 4 are in the range of AGD/BW1/3 of the male control pups [1.99 – 3.29]. Indeed, 2 pups at 15 mg/kg/day (1.93; 1.98), 1 pup at 50 mg/kg/day (1.79) and 2 pups at 150 mg/kg/day (1.87; 1.93) showed lower values, closed to the control range and within the range of the reference data.
In view of the slight magnitude of the difference and as this was not associated with any other findings, this variation was considered to be of minor toxicological importance.
There were no test item-related effects on AGD/BW1/3 on female pups.

Nipples and areolae numbers
No nipples or areolae were observed in any male pups.

Thyroid hormones
See table 17.
The thyroid hormone levels were considered to be unaffected by the test item treatment in pups.

For pups in the 15 mg/kg/day group and when compared with controls, there was lower mean TSH concentration (not statistically significant). This difference was considered as fortuitous in the absence of a dose-response relationship.

Sex ratio
See table 15.
There were no test item-related effects on sex ratios (% of male pups).
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
clinical signs
Critical effects observed:
no
Reproductive effects observed:
yes
Lowest effective dose / conc.:
50 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
not specified
Dose response relationship:
yes
Relevant for humans:
not specified

P0 tables

Table 1: Clinical signs

 

Sex

Male

Female

Dose level (mg/kg/day)

0

15

50

150

0

15

50

150

Pre-mating (females) or whole study (males)

Ptyalism

0

4

10

10

0

2

10

10

Round back

0

0

0

1

0

0

0

0

Piloerection

0

0

0

1

0

0

0

0

Total affected animals

0/10

4/10

10/10

10/10

0/10

2/10

10/10

10/10

Gestation

 

 

 

 

 

 

 

 

Ptyalism

-

-

-

-

0

3

8

8

Total affected animals

-

-

-

-

0/9

3/8

8/8

8/8

Lactation

 

 

 

 

 

 

 

 

Ptyalism

-

-

-

-

0

1

8

8

Piloerection

-

-

-

-

0

0

1

0

Emaciated appearance

-

-

-

-

0

0

1

0

Total affected animals

-

-

-

-

0/9

1/8

8/8

8/8

-: not applicable.

Table 2: Mortality

In females: 

Dose level (mg/kg/day)

0

15

50

150

Humane reasons (mass)

0

0

1(pg)

0

No delivery

0

2(npg)

1(npg)

2(1npg 1pg)

Found dead

1(pg)

0

0

0

Total surviving animals

9/10

8/10

8/10

8/10

pg: pregnant; npg: not pregnant.

Table 3: Motor activity

 

Sex

Male

Female

Dose level (mg/kg/day)

0

15

50

150

0

15

50

150

Horizontal movements

692

689

569

652

529

460

341

339

SD

204.5

113.6

109.6

279.0

250.0

150.8

153.1

163.1

% from controls

-

0

-18

-6

-

-13

-36

-36

Rearing

166

172

178

190

128

135

107

87

SD

26.1

21.8

50.1

48.1

64.2

37.1

50.6

43.7

% from controls

-

+4

+7

+14

-

+5

-16

-32

-: not applicable.

Statistical analysis: no significance.

Table 4: Functional Observation Battery

 

Sex

Male

Female

Dose level (mg/kg/day)

0

15

50

150

0

15

50

150

Urination

 

 

 

 

 

 

 

 

. absence

4

2

3

2

4

3

5

4

. presence

1

3

2

3

1

2

0

1

Landing foot splay (mm)

81

77

82

95

73

82

90

96

SD

23

38

29

25

19

22

19

28

% from controls

-

-5

+1

+17

-

+12

+17

+32

-: not applicable.

Table 5: Body weight and body weight change

 

Sex

Male

Female

Dose level

(mg/kg/day)

0

15

50

150

0

15

50

150

Pre-mating period (females) and whole study (males)

. Day 1

412

410

412

408

276

272

273

276

% from controls

-

0

0

-1

-

-1

-1

0

. Day 8

452

452

450

438*

279

271

277

280

% from controls

-

0

0

-3

-

-3

-1

0

. Day 15

476

481

479

464

284

275

286

288

% from controls

-

+1

+1

-3

-

-3

+1

+1

. Day 29

516

521

522

497*

-

-

-

-

% from controls

-

+1

+1

-4

-

-

-

-

. Days 1 - 8

+39

+41

+38

+30*

+3

-1

+4

+4

. Days 1 - 15

+64

+70

+68

+56

+8

+4

+14

+12

. Days 22 - 29

+24

+26

+28

+18

-

-

-

-

. Days 15 - 29

+40

+41

+43

+33

-

-

-

-

. Days 1 - 29

+104

+111

+111

+89

-

-

-

-

Gestation

 

 

 

 

 

 

 

 

. Day 0p.c.

-

-

-

-

285

284

286

293

% from controls

-

-

-

-

-

0

0

+3

. Day 20p.c.

-

-

-

-

424

432

443

446

% from controls

-

-

-

-

-

+2

+4

+5

. Days 0 - 20p.c.

-

-

-

-

+140

+149

+156

+153

Lactation

 

 

 

 

 

 

 

 

. Day 1p.p.

-

-

-

-

334

339

340

351

% from controls

-

-

-

-

-

+1

-2

+5

. Day 13p.p.

-

-

-

-

358

367

375

383

% from controls

-

-

-

-

-

+3

+5

+7

. Days 1 - 4p.p.

-

-

-

-

+9

+7

+6

+1

. Days 4 - 8p.p.

-

-

-

-

+10

+18

+12

+17

. Days 8 - 13p.p.

 

 

 

 

+5

+4

+16

+15

. Days 1 - 13p.p.

-

-

-

-

+24

+28

+34

+32

-: not applicable.

Statistical significance *: p<0.05.


 

Table 6: Food consumption

 

Sex

Male

Female

Dose level

(mg/kg/day)

0

15

50

150

0

15

50

150

Pre-mating period (females) and whole study (males)

Days 1 - 8

29

30

29

28

17

17

17

18

% from controls

-

+3

0

-3

-

0

0

+6

Days 8 -15

27

28

29

29

16

17

18*

18*

% from controls

-

+4

+7

+7

-

+6

+13

+13

Gestation

 

 

 

 

 

 

 

 

Days 0 - 7p.c.

-

-

-

-

19

20

20

22**

% from controls

 

 

 

 

-

+5

+5

+16

Days 7 - 14p.c.

-

-

-

-

22

23

23

24

% from controls

 

 

 

 

-

+5

+5

+9

Days 14 - 20p.c.

-

-

-

-

27

27

28

29

% from controls

 

 

 

 

-

0

+4

+7

Lactation

 

 

 

 

 

 

 

 

Days 1- 4p.p.

-

-

-

-

33

37

32

33

% from controls

 

 

 

 

-

+12

-3

0

Days 4- 8p.p.

-

-

-

-

45

52

48

49

% from controls

 

 

 

 

-

+16

+7

+9

Days 8- 13p.p.

-

-

-

-

57

63

62

59

% from controls

 

 

 

 

-

+11

+9

+4

-: not applicable.

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


 

Table 7: Water consumption

 

Sex

Male

Female

Dose level

(mg/kg/day)

0

15

50

150

0

15

50

150

Pre-mating period (females) and whole study (males)

Days 1 - 8

38

42

40

42

27

27

32**

33#

% from controls

-

+11

+5

+11

-

0

+19

+22

Days 8 -15

37

44**

43*

46#

30

31

34*

36**

% from controls

-

+19

+16

+24

-

+3

+13

+20

Gestation

 

 

 

 

 

 

 

 

Days 0 - 7p.c.

-

-

-

-

29

34

37*

40#

% from controls

 

 

 

 

-

+17

+28

+38

Days 7 - 14p.c.

-

-

-

-

38

42

45*

51#

% from controls

 

 

 

 

-

+11

+18

+34

Days 14 - 20p.c.

-

-

-

-

52

55

61*

68#

% from controls

 

 

 

 

-

+6

+17

+31

Lactation

 

 

 

 

 

 

 

 

Days 1- 4p.p.

-

-

-

-

57

60

58

60

% from controls

 

 

 

 

-

+5

+2

+5

Days 4- 8p.p.

-

-

-

-

69

77

75

80

% from controls

 

 

 

 

-

+12

+9

+16

Days 8- 13p.p.

-

-

-

-

84

92

97

94

% from controls

 

 

 

 

-

+10

+15

+12

-: not applicable.

Statistical significance *: p<0.05, **: p<0.01 and #: p<0.001.


 

Table 8: Pairing, mating and fertility data

 

Dose level (mg/kg/day)

0

15

50

150

Ref. Data

Number of animals paired (M + F)

10 + 10

10 + 10

10 + 10

10 + 10

59 + 60

Number of males mated

10

10

10

10

59

Number of females mated

10

10

10

10

60

Mean number of days taken to mate
(pre-coital time) ± SD

1.4 ± 0.8

2.3 ± 1.1

2.4 ± 1.1

2.5 ± 1.4

[1.8;4.3]

Number of pregnant females

10

8

9

9

60

Number of females with live born pups

9

8

8

8

58

Mating index (%)

 

 

 

 

 

. male

100

100

100

100

100

. female

100

100

100

100

100

Fertility index (%)

 

 

 

 

 

. male

100

80

90

90

100

. female

100

80

90

90

100

Gestation index (%)

90.0

100.0

88.9

88.9

[80;100]

M: male; F: female.

Statistical analysis: no significance.

Ref. Data: reference data collected from six GLP OECD 422 studies covering a period ranging from August 2016 to October 2017, [min.; max.]

 

Table 9: Estrous cycle data

 

Dose level (mg/kg/day)

0

15

50

150

Mean number of cycles ± SD

3.0 ± 0.0

3.0 ± 0.0

2.8 ± 0.6

3.0 ± 0.0

Mean cycle length (days) ± SD

4.0 ± 0.0

3.9 ± 0.2

4.0 ± 0.1

4.0 ± 0.2

Number of females having a mean average cycle of 4-5 days

10

7

9

8

Statistical analysis: no significance.

Table 10: Delivery data

 

Dose level (mg/kg/day)

0

15

50

150

Ref. Data

Number of pregnant females

10

8

9

9

60

Number of females which delivered

9

8

8

8

58

Mean duration of gestation (days) ± SD

22.0 ± 0.5

22.1 ± 0.4

22.1 ± 0.4

22.3 ± 0.5

[22.0;22.1]

Mean number of corpora lutea ± SD

15.4 ± 4.2

15.5 ± 2.2

17.1 ± 1.7

15.5 ± 1.6

[13.9;17.5]

% from controls

/

+1

+11

+1

/

Mean number of implantation sites ± SD

12.7 ± 3.9

14.3 ± 1.6

16.0 ± 2.9

14.6 ± 0.9

[13.9;14.9]

% from controls

/

+13

+26

+15

/

Mean pre-implantation loss (%) ± SD

17.6 ±14.1

7.2 ± 10.5

6.3 ± 15.6

5.2 ± 5.8

[0.0;14.8]

% from controls

/

-59

-64

-70

/

Mean number of pups delivered ± SD

11.0 ±4.7

13.1 ±2.5

12.9 ±3.5

12.3 ±1.6

[11.0;12.8]

Mean post-implantation loss (%) ± SD

17.8± 25.8

8.5 ± 9.1

20.9±13.8

16.1±11.0

[13.2;24.0]

% from controls

 

-52

+17

-10

/

/:             not applicable.

Statistical analysis: no significance.

Ref. Data: reference data collected from six GLP OECD 422 studies covering a period ranging from August 2016 to October 2017, [min.; max.].

Table 11: Thyroid  hormones

 

Thyroid hormones

Sex

F0 males

Dose level (mg/kg/day)

0

15

50

150

T4 (ng/mL)

39.32

34.65*

36.10

32.53**

HCD

36.6 Minimum: 26.9 Maximum: 46.9

Standard deviation

6.183

2.746

2.668

3.664

n

10

10

10

10

% from controls

/

-12

-8

-17

TSH (pg/mL)

1435

1974

2193

1622

HCD

1830 Minimum: 424 Maximum: 6935

Standard deviation

699.8

1292.0

1032.1

476.3

n

10

10

10

10

% from controls

/

+38

+53

+13

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

HCD: Historical Control Data.

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

 

F1 generation tables

Table 12: Mortality

 

Dose level (mg/kg/day)

0

15

50

150

Ref. Data

Number of pups (litters)

99 (9)

105 (8)

103 (8)

98 (8)

704 (58)

Mean live birth index (%) ± SD

100 ± 0.0

99 ± 2.4

92 ± 12.8

81 ±26.1

[98;100]

Number of pups found dead

or cannibalized (litters affected)

1 (1)

3 (2)

10 (4)

17 (5)

 

30 (14)

Mean viability index ± SD (Day 4p.p.%)

99 ± 2.2

98 ± 4.9

90 ± 12.7

80 ± 26.1

[91;98]

Mean lactation index ± SD(Day 13p.p.%)

100 ± 0.0

100 ± 0.0

100 ± 0.0

100 ± 0.0

[99;100]

Statistical analysis: no significance.

Ref. Data: reference data collected from six GLP OECD 422 studies covering a period ranging from August 2016 to October 2017, [min.; max.]. Values are rounded.

Table 13: Clinical signs

 

Dose level (mg/kg/day)

0

15

50

150

Abnormal color, n (L)

 

1 (1)

 

 

Generalized palor

 

 

 

1 (1)

Bluish appearance, n (L)

 

 

1 (1)

 

Cold to the touch, n (L)

 

1 (1)

13 (3)

8 (1)

Dyspnea, n (L)

 

 

1 (1)

 

Blackish abdomen, n (L)

 

1 (1)

 

 

Emaciated appearance, n (L)

 

 

1 (1)

1 (1)

Absence of milk in stomach, n (L)

 

 

2 (2)

11 (5)

Hematoma abdomen, n (L)

 

 

1 (1)

 

Scab, n (L)

 

 

 

1 (1)

Total pups affected, n (L)

0 (0)

1 (1)

16 (3)

20 (6)

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

Table 14: Pup body weight

 

Sex

Male

Female

Dose level (mg/kg/day)

0

15

50

150

0

15

50

150

Body weight(g)

 

 

 

 

 

 

 

 

. Day 1p.p.

7.8

7.9

7.6

7.7

7.5

7.6

7.4

7.2

. Day 4p.p.post-culling

11.6

11.7

11.8

11.7

11.0

11.4

11.3

11.4

. Day 8p.p.

21.2

21.7

21.8

22.0

19.7

21.2

20.9

20.9

. Day 13p.p.

35.0

35.8

36.6

35.7

32.8

35.5

35.3

34.1

Pup body weight change(g)

. Days 1 to 4p.p.

+3.8

+3.8

+4.0

+4.0

+3.6

+3.8

+3.9

+4.0

. Days 4to 8p.p.

+9.6

+9.9

+10.0

+10.3

+8.7

+9.8

+9.5

+9.6

. Days 4 to 13p.p.

+23.4

+24.1

+24.8

+24.0

+21.8

+24.1

+23.9

+22.8

. Days 8 to 13p.p.

+13.8

+14.2

+14.8

+13.7

+13.1

+14.3

+14.4

+13.2

Statistical analysis: no significance.

Table 15: Sex ratio on Day 1 p.p. (% of males)

 

Dose level (mg/kg/day)

0

15

50

150

. Day 1p.p.(%)

41.8± 18.68

51.0± 12.00

45.4± 15.99

38.1± 18.82

. Day 13p.p.(%)

44.4± 17.80

48.4± 4.42

46.6± 10.60

42.2± 17.60

Statistical analysis: no significance.

Table 16: Pup development

 

Sex

Males

Females

Dose level (mg/kg/day)

0

15

50

150

0

15

50

150

Number of pups

45

53

43

34

54

51

52

48

AGD

5.20

5.12

4.94

4.85

2.92

3.03

2.81

2.84

Standard deviation

0.699

0.581

0.538

0.453

0.397

0.489

0.405

0.406

BW on Day 1p.p.

7.8

7.9

7.6

7.7

7.5

7.6

7.4

7.2

AGD/BW1/3

2.63

2.58

2.51

2.46*

1.51

1.55

1.45

1.47

Standard deviation

0.310

0.275

0.276

0.221

0.201

0.256

0.214

0.215

Ref. Data

[1.68-3.18]

 

 

[0.92-2.17]

 

 

Statistical significance: *: p<0.05

Ref. Data: reference data collected from three GLP OECD 422 studies covering a period ranging from June 2017 to October 2017; [min.; max.] for individual values.

Table 17: Thyroid hormones

 

 

Thyroid hormones

Sex

Pups, Day 13p.p.

Dose level (mg/kg/day)

0

15

50

150

T4 (ng/mL)

34.42

37.23

38.19

35.31

Standard deviation

3.615

2.074

4.959

5.110

n

9

8

8

8

% from controls

/

+8

+11

+3

TSH (pg/mL)

1745

1454

1751

1854

Standard deviation

355.4

589.6

728.6

660.5

n

8

6

8

8

% from controls

/

-17

0

6

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

 

Conclusions:
The test item was administered daily by oral gavage to male and female Sprague Dawley rats for 2 weeks before mating, during mating and, for females, throughout gestation and until Day 13 p.p. at the dose levels of 15, 50 or 150 mg/kg/day.

Based on the experimental conditions of this study:
- the No Observed Adverse Effect Level (NOAEL) for parental systemic toxicity was considered to be 50 mg/kg/day based on the microscopic findings observed at 150 mg/kg/day in the urinary bladder (i.e. hyperplasia and single cell necrosis of the urothelium) of some males and females and in heart (i.e. cardiac atrial thrombosis and hypertrophy) of one female,
- the No Observed Adverse Effect Level (NOAEL) for reproductive performance (mating, fertility and delivery) was considered to be 150 mg/kg/day in the absence of adverse findings at this high-dose level,
- the No Observed Adverse Effect Level (NOAEL) for toxic effects on progeny was considered to be 15 mg/kg/day (based on clinical signs and live birth and viability indexes at 50 and 150 mg/kg/day).
Executive summary:

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

This study provides information on:

. the possible health hazards likely to arise from repeated exposure over a relatively limited period of time,

.male and female reproductive performance, such as gonadal function, mating behavior, conception, conceptus development, and parturition.

The study was performed according to OECD guideline No. 422 (29 July 2016) and in compliance with GLP regulations.

 

Methods

Three groups of ten male and ten female Sprague-Dawley rats received the test item.

The test item was administered daily by the oral route (gavage) at dose levels of 15, 50 or 150 mg/kg/day, under a constant dosage volume of 5 mL/kg/day.

Males were treated for an overall period of 4 weeks: two weeks before mating, during the mating period (up to 1 week) and until the day before sacrifice. Females were treated for an overall period of 7 to 8 weeks: two weeks before mating, throughout mating (up to one week) and gestation (3 weeks) until Day 13 post-partum (p.p.) inclusive. A control group of ten males and ten females received the vehicle only (corn oil) under the same experimental conditions.

The actual test item concentrations in the dose formulations were determined in Weeks 1 and 6 using a validated HPLC/UV detection analytical method.

The F0 animals were checked daily during the treatment period for mortality, morbidity and clinical signs. Detailed clinical observations were conducted weekly. Body weight, and food and water consumption were recorded weekly during the premating, mating (with the exception of food and water consumption), gestation and lactation periods. Estrous cycle stage was determined each morning from 2 weeks before the treatment period until the females had mated and on Day 14 p.p. before euthanasia.

The F0 animals were paired for mating after 2 weeks of treatment and the dams were allowed to litter 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. Pups not selected on Day 4 p.p. were euthanized and discarded without further examination.

Pups were weighed on Days 1, 4, 8 and 13 p.p. and observed daily for clinical signs, abnormal behaviour and external abnormalities. The physical development of pups was assessed by measuring the anogenital distance on Day 1 p.p. (all pups before culling) and by counting the number of nipples and areolae in male pups on Day 12 p.p.

A Functional Observation Battery (FOB) was performed on five F0 animals per sex and group at the end of the treatment period.

Laboratory investigations (hematology, blood biochemistry and urinalysis) were carried out on at least five F0 males and five F0 females from each group at the end of the study. Plasma thyroid hormone levels (TSH and T4) were determined at terminationin all F0 males and in pups euthanized on Day 13 p.p.

The F0 males were euthanized after completion of the mating period and F0 females were euthanized on Day 14 p.p.


A full macroscopic post-mortem examination was performed, with a particular attention to the reproductive organs. Designated organs were weighed and selected tissue specimens were preserved. A microscopic examination was performed on selected tissues from five males and lactating females in the control and high-dose groups, on all macroscopic lesions (all groups) and on the heart (females only), kidneys, liver, sternum with bone marrow (females only), spleen and urinary bladder from five males and lactating females at the low- and intermediate dose levels. Reproductive organs and thyroids with parathyroids from one pair at each test item dose level were also microscopically observed as the females were found to be non-pregnant. Perl’s staining was performed on the spleen and kidneys of the five males and/or five lactating females.

 

Pups were euthanized on Day 13 p.p. and a detailed external examination was performed, with a particular attention to the external genital organs. Thyroids with parathyroids from one pup/sex/litter were preserved.

 

Results

 

Chemical analysis:

The actual test item concentrations in the analyzed dose formulations (i.e. in Weeks 1 and 6) remained within an acceptable range of variation (-4.1% to +0.6% when compared with the nominal values; nominal concentration ± 15% required).

 

F0 animals:

Mortality: there were no test item-related unscheduled deaths.

 

Clinical signs: round back and piloerection were transiently noted in 1/10 males given 150 mg/kg/day. These findingswere considered to be related to the test item but of minor toxicological importance. Ptyalism occurred in both sexes and in all test item treated groups with dose-related increased incidences. This sign, commonly observed when a test item is administered by gavage, was not considered as an adverse effect.

 

Functional Observation Battery and motor activity: slightly lower incidences of horizontal and rearing movements were recorded in females given 50 or 150 mg/kg/day, along with higher landing foot splay values at the same dose levels in females. These effects were considered to be test item-related but of minor toxicological importance.

 

Body weight and body weight change: at 150 mg/kg/day and when compared with controls, a few variations of body weight gain were noted in males during the whole treatment period and in females on the first 4 days of the lactation period. These differences resulted in slightly transient lower mean body weights in males.They were considered to be related to the test item but of minor toxicological importance in view of their magnitude and/or the absence of effects on the body weight.

 

Food consumption: at 50 and 150 mg/kg/day and when compared with controls, a few episodes of slightly higher food consumption were noted in females in the premating period and/or at the beginning of the gestation period. These findings were considered to be related to the test item but of minor toxicological importance in view of their magnitude and the absence of effects on the body weight.

There were no effects on food consumption at any dose level in males.


Water consumption: at 15, 50 and 150 mg/kg/day and when compared with controls, higher water consumption was recorded in males and/or females over the premating period. High water intake was also observed in all dose females throughout the gestation period and then on a few occasions during the lactation. These effects were more marked at the beginning of the pregnancy period. These findings were considered to be related to the test item but of minor toxicological importance in view of their magnitude and in absence of any correlates with adverse findings.

 

Hematology: at 150 mg/kg/day and when compared with controls, there was a decrease in red blood cell count in females (-13%, p<0.05) along with increased mean cell volume and mean cell hemoglobin (+13% and +14%, respectively, p<0.01). These changes were associated with increased reticulocyte count (+38%), which was also observed in males (+59%, p<0.01).These hematological variations correlated with histopathological findings suggestive of hemolysis (increased extramedullary hematopoiesis and pigment consistent with hemosiderin in the spleen of both sexes and increased erythroid cell numbers in the bone marrow of females). Marginally higher mean neutrophil count (+37% vs. controls, p<0.05) was also observed in females. In view of their magnitude and as mean values remained within or close to the range of the Historical Control Data, hematological changes were considered to be non-adverse.

 

Blood biochemistry: when compared with controls, blood biochemistry changes included a dose-related decreased glucose level in males at all dose levels (-14 to -20%, p<0.05 to p<0.01) and the opposite trend in females (+21% to +29%, not statistically significant) in females, an increase in the inorganic phosphorus level in females (+30%, p<0.01) and an increase in the bile acids level in males and females from 50 mg/kg/day. In view of their magnitude, as mean values remained within the range of the Historical Control Data and in absence of histopathological findings, these changes were considered to be non-adverse.

 

Urinalysis: urinalysis changes consisted of higher volume in females at all dose levels (+64% vs. controls) corresponding to non-adverse higher mean water consumption, increased bilirubin levels in both sexes from 50 mg/kg/day, colored urine in males from 50 mg/kg/day, presence of nitrites in all males at all dose levels and in all females from 50 mg/kg/day, and of moderate to marked proteinuria in 3/5 males at 50 mg/kg/day and in 2/5 males and 1/5 females at 150 mg/kg/day.Bilirubinuria correlated with variations observed in the red blood cell mass of males and females given 150 mg/kg/day and with histopathological findings suggestive of increased red blood cell turnover and proteinuria could be related to adverse microscopic changes observed in the kidneys and/or in the urinary bladder at 150 mg/kg/day. As mean values remained within the range of the Historical Control Data, were poorly dose-related and without statistically significance, in absence of any other correlates and/or as findings could be consecutive from histopathological findings, these changes were considered to be non-adverse.

 

Thyroid hormones: there were no test item-related effects on the T4 and TSH levels in F0 males or pups at any dose level.

 

Pathology: At 150 mg/kg/day, at microscopic examination, adverse hyperplasia and single cell necrosis of the urothelium was observed in the urinary bladder in both sexes. Non-adverse microscopic findings were noted in the spleen (increased congestion and pigment consistent with hemosiderin) and kidneys (brown pigment), with compensatory hematopoiesis (erythropoiesis) in the spleen, correlated with increased spleen weights (both sexes), and bone marrow (females). Minimal and non-adverse tubular vacuolation was noted in two females in the kidneys at 150 mg/kg/day. In addition in one female at this dose-level, there were adverse cardiac atrial thrombosis and hypertrophy, glomerulopathy and renal tubular hyaline casts, potentially related to the test item administration. Non-adverse slight hepatocellular hypertrophy correlated with increased liver weights was noted in the liver at 150 mg/kg/day (both sexes).

 

Estrous cycle: there were notest item-relatedeffects on the mean length of the estrous cycle or the mean number of cycles at any dose level.

 

Pairing, mating and fertility data: there were no relevant effects on the mating, fertility or gestation index at any dose level.

 

Delivery data: there were no effects on mean duration of gestation, numbers of corpora lutea and implantation sites or percentages of pre-/post-implantation losses at any dose level.

 

Pups:

Mortality and viability:at 50 and 150 mg/kg/day and when compared with controls, there were reductions in the live birth (92% and 81% vs.100%, respectively) and viability (90% and 80% vs. 99%, respectively) indexes associated with increased number of pups found dead and cannibalized (10 and 17 pups vs. 1 pup, respectively) and in the incidence of litters affected (4/8 and 5/8 vs.1/9, respectively).These findings were dose-related and the indexes were below the lower limit of the Reference Data. They were therefore considered to be adverse.

 

Clinical signs and external abnormalities: there were no external abnormalities at any dose level.At 50 and 150 mg/kg/day and when compared with controls, increased clinical signs (generalized palor, bluish appearance, coldness to the touch, dyspnea, emaciated appearance, absence of milk in the stomach and/or hematoma on the abdomen) was observed in pups, along with increased number of affected litters (3/8 litters with 16/103 pups and 5/8 litter with 19/98 pups vs. 0/9 litters in controls, respectively).These findings, also observed in all found dead pups, were considered to be adverse.

 

Body weight: there were no effects on mean body weight or mean body weight change in pups at any dose level.

 

Sex ratio: there were no effects on the mean percentage of male fetuses (sex ratio) at any dose level.

 

Development: there were no nipples or areolae in male pups at any dose level. At 150 mg/kg/day and when compared with controls, males had a slightly statistically significant, lower mean AGD/BW1/3ratio (-6%) for which a test item relationship could not be excluded although individual values were within the range of the reference data. This finding was considered to be non-adverse in view of the slight magnitude.

 

Conclusion

The test item was administered daily by oral gavage to male and female Sprague-Dawley rats for 2 weeks before mating, during mating and, for females, throughout gestation and until Day 13 p.p. at the dose levels of 15, 50 or 150 mg/kg/day.

 

Based on the experimental conditions of this study:

. the No Observed Adverse Effect Level (NOAEL) for parental systemic toxicity was considered to be 50 mg/kg/day based on the microscopic findings observed at 150 mg/kg/day in the urinary bladder (i.e. hyperplasia and single cell necrosis of the urothelium) of some males and females and in heart (i.e.cardiac atrial thrombosis and hypertrophy)of one female,

the No Observed Adverse Effect Level (NOAEL) for reproductive performance (mating, fertility and delivery) was considered to be 150 mg/kg/day in the absence of adverse findings at this high-dose level,

.the No Observed Adverse Effect Level (NOAEL) for toxic effects on progeny was considered to be 15 mg/kg/day (based on clinical signs and live birth and viability indexes at 50 and 150 mg/kg/day).

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The key study is considered to be reliable, as conducted with GLP and OECD guidelines.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

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

This study provides information on:

. the possible health hazards likely to arise from repeated exposure over a relatively limited period of time,

.male and female reproductive performance, such as gonadal function, mating behavior, conception, conceptus development, and parturition.

 

Methods

Three groups of ten male and ten female Sprague-Dawley rats received the test item.

The test item was administered daily by the oral route (gavage) at dose levels of 15, 50 or 150 mg/kg/day, under a constant dosage volume of 5 mL/kg/day.

Males were treated for an overall period of 4 weeks: two weeks before mating, during the mating period (up to 1 week) and until the day before sacrifice. Females were treated for an overall period of 7 to 8 weeks: two weeks before mating, throughout mating (up to one week) and gestation (3 weeks) until Day 13post-partum(p.p.) inclusive. A control group of ten males and ten females received the vehicle only (corn oil) under the same experimental conditions.

The actual test item concentrations in the dose formulations were determined in Weeks 1 and 6 using a validated HPLC/UV detection analytical method.

The F0 animals were checked daily during the treatment period for mortality, morbidity and clinical signs. Detailed clinical observations were conducted weekly. Body weight, and food and water consumption were recorded weekly during the premating, mating (with the exception of food and water consumption), gestation and lactation periods. Estrous cycle stage was determined each morning from 2 weeks before the treatment period until the females had mated and on Day 14p.p.before euthanasia.

The F0 animals were paired for mating after 2 weeks of treatment and the dams were allowed to litter and rear their progeny until Day 13p.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 4p.p.by culling extra pups to obtain as nearly as possible four males and four females per litter. Pups not selected on Day 4p.p.were euthanized and discarded without further examination.

Pups were weighed on Days 1, 4, 8 and 13p.p.and observed daily for clinical signs, abnormal behaviour and external abnormalities. The physical development of pups was assessed by measuring the anogenital distance on Day 1p.p.(all pups before culling) and by counting the number of nipples and areolae in male pups on Day 12p.p.

A Functional Observation Battery (FOB) was performed on five F0 animals per sex and group at the end of the treatment period.

Laboratory investigations (hematology, blood biochemistry and urinalysis) were carried out on at least five F0 males and five F0 females from each group at the end of the study. Plasma thyroid hormone levels (TSH and T4) were determined at terminationin all F0 males and in pups euthanized on Day 13 p.p.

The F0 males were euthanized after completion of the mating period and F0 females were euthanized on Day 14p.p.


A full macroscopicpost-mortemexamination was performed, with a particular attention to the reproductive organs. Designated organs were weighed and selected tissue specimens were preserved. A microscopic examination was performed on selected tissues from five males and lactating females in the control and high-dose groups, on all macroscopic lesions (all groups) and on the heart (females only), kidneys, liver, sternum with bone marrow (females only), spleen and urinary bladder from five males and lactating females at the low- and intermediate dose levels. Reproductive organs and thyroids with parathyroids from one pair at each test item dose level were also microscopically observed as the females were found to be non-pregnant. Perl’s staining was performed on the spleen and kidneys of the five males and/or five lactating females.

 

Pups were euthanized on Day 13p.p.and a detailed external examination was performed, with a particular attention to the external genital organs. Thyroids with parathyroids from one pup/sex/litter were preserved.

 

Results

 

Chemical analysis:

The actual test item concentrations in the analyzed dose formulations (i.e.in Weeks 1 and 6) remained within an acceptable range of variation (-4.1% to +0.6% when compared with the nominal values; nominal concentration ± 15% required).

 

F0 animals:

Mortality: there were no test item-related unscheduled deaths.

 

Clinical signs: round back and piloerection were transiently noted in 1/10 males given 150 mg/kg/day. These findingswere considered to be related to the test item but of minor toxicological importance. Ptyalism occurred in both sexes and in all test item treated groups with dose-related increased incidences. This sign, commonly observed when a test item is administered by gavage, was not considered as an adverse effect.

 

Functional Observation Battery and motor activity: slightly lower incidences of horizontal and rearing movements were recorded in females given 50 or 150 mg/kg/day, along with higher landing foot splay values at the same dose levels in females. These effects were considered to be test item-related but of minor toxicological importance.

 

Body weight and body weight change: at 150 mg/kg/day and when compared with controls, a few variations of body weight gain were noted in males during the whole treatment period and in females on the first 4 days of the lactation period. These differences resulted in slightly transient lower mean body weights in males.They were considered to be related to the test item but of minor toxicological importance in view of their magnitude and/or the absence of effects on the body weight.

 

Food consumption: at 50 and 150 mg/kg/day and when compared with controls, a few episodes of slightly higher food consumption were noted in females in the premating period and/or at the beginning of the gestation period. These findings were considered to be related to the test item but of minor toxicological importance in view of their magnitude and the absence of effects on the body weight.

There were no effects on food consumption at any dose level in males.


Water consumption: at 15, 50 and 150 mg/kg/day and when compared with controls, higher water consumption was recorded in males and/or females over the premating period. High water intake was also observed in all dose females throughout the gestation period and then on a few occasions during the lactation. These effects were more marked at the beginning of the pregnancy period. These findings were considered to be related to the test item but of minor toxicological importance in view of their magnitude and in absence of any correlates with adverse findings.

 

Hematology:at 150 mg/kg/day and when compared with controls, there was a decrease in red blood cell count in females (-13%, p<0.05) along with increased mean cell volume and mean cell hemoglobin (+13% and +14%, respectively, p<0.01). These changes were associated with increased reticulocyte count (+38%), which was also observed in males (+59%, p<0.01).These hematological variations correlated with histopathological findings suggestive of hemolysis (increased extramedullary hematopoiesis and pigment consistent with hemosiderin in the spleen of both sexes and increased erythroid cell numbers in the bone marrow of females). Marginally higher mean neutrophil count (+37%vs. controls, p<0.05) was also observed in females. In view of their magnitude and as mean values remained within or close to the range of the Historical Control Data, hematological changes were considered to be non-adverse.

 

Blood biochemistry:when compared with controls, blood biochemistry changes included a dose-related decreased glucose level in males at all dose levels (-14 to -20%, p<0.05 to p<0.01) and the opposite trend in females (+21% to +29%, not statistically significant) in females, an increase in the inorganic phosphorus level in females (+30%, p<0.01) and an increase in the bile acids level in males and females from 50 mg/kg/day. In view of their magnitude, as mean values remained within the range of the Historical Control Data and in absence of histopathological findings, these changes were considered to be non-adverse.

 

Urinalysis: urinalysis changes consisted of higher volume in females at all dose levels (+64%vs.controls) corresponding to non-adverse higher mean water consumption, increased bilirubin levels in both sexes from 50 mg/kg/day, colored urine in males from 50 mg/kg/day, presence of nitrites in all males at all dose levels and in all females from 50 mg/kg/day, and of moderate to marked proteinuria in 3/5 males at 50 mg/kg/day and in 2/5 males and 1/5 females at 150 mg/kg/day.Bilirubinuria correlated with variations observed in the red blood cell mass of males and females given 150 mg/kg/day and with histopathological findings suggestive of increased red blood cell turnover and proteinuria could be related to adverse microscopic changes observed in the kidneys and/or in the urinary bladder at 150 mg/kg/day. As mean values remained within the range of the Historical Control Data, were poorly dose-related and without statistically significance, in absence of any other correlates and/or as findings could be consecutive from histopathological findings, these changes were considered to be non-adverse.

 

Thyroid hormones: there were no test item-related effects on the T4 and TSH levels in F0 males or pups at any dose level.

 

Pathology: At 150 mg/kg/day, at microscopic examination, adverse hyperplasia and single cell necrosis of the urothelium was observed in the urinary bladder in both sexes. Non-adverse microscopic findings were noted in the spleen (increased congestion and pigment consistent with hemosiderin) and kidneys (brown pigment), with compensatory hematopoiesis (erythropoiesis) in the spleen, correlated with increased spleen weights (both sexes), and bone marrow (females). Minimal and non-adverse tubular vacuolation was noted in two females in the kidneys at 150 mg/kg/day. In addition in one female at this dose-level, there were adverse cardiac atrial thrombosis and hypertrophy, glomerulopathy and renal tubular hyaline casts, potentially related to the test item administration. Non-adverse slight hepatocellular hypertrophy correlated with increased liver weights was noted in the liver at 150 mg/kg/day (both sexes).

 

Estrous cycle: there were notest item-relatedeffects on the mean length of the estrous cycle or the mean number of cycles at any dose level.

 

Pairing, mating and fertility data: there were no relevant effects on the mating, fertility or gestation index at any dose level.

 

Delivery data:there were no effects on mean duration of gestation, numbers ofcorpora luteaand implantation sites or percentages of pre-/post-implantation losses at any dose level.

 

Pups:

Mortality and viability:at 50 and 150 mg/kg/day and when compared with controls, there were reductions in the live birth (92% and 81%vs.100%, respectively) and viability (90% and 80%vs.99%, respectively) indexes associated with increased number of pups found dead and cannibalized (10 and 17 pupsvs.1 pup, respectively) and in the incidence of litters affected (4/8 and 5/8vs.1/9, respectively).These findings were dose-related and the indexes were below the lower limit of the Reference Data. They were therefore considered to be adverse.

 

Clinical signs and external abnormalities: there were no external abnormalities at any dose level.At 50 and 150 mg/kg/day and when compared with controls, increased clinical signs (generalized palor, bluish appearance, coldness to the touch, dyspnea, emaciated appearance, absence of milk in the stomach and/or hematoma on the abdomen) was observed in pups, along with increased number of affected litters (3/8 litters with 16/103 pups and 5/8 litter with 19/98 pupsvs.0/9 litters in controls, respectively).These findings, also observed in all found dead pups, were considered to be adverse.

 

Body weight: there were no effects on mean body weight or mean body weight change in pups at any dose level.

 

Sex ratio: there were no effects on the mean percentage of male fetuses (sex ratio) at any dose level.

 

Development: there were no nipples or areolae in male pups at any dose level. At 150 mg/kg/day and when compared with controls, males had a slightly statistically significant, lower mean AGD/BW1/3ratio (-6%) for which a test item relationship could not be excluded although individual values were within the range of the reference data. This finding was considered to be non-adverse in view of the slight magnitude.

 

Conclusion

The test item was administered daily by oral gavage to male and female Sprague-Dawley rats for 2 weeks before mating, during mating and, for females, throughout gestation and until Day 13p.p.at the dose levels of 15, 50 or 150 mg/kg/day.

 

Based on the experimental conditions of this study:

. the No Observed Adverse Effect Level (NOAEL) for parental systemic toxicity was considered to be 50 mg/kg/day based on the microscopic findings observed at 150 mg/kg/day in the urinary bladder (i.e. hyperplasia and single cell necrosis of the urothelium) of some males and females and in heart (i.e.cardiac atrial thrombosis and hypertrophy)of one female,

the No Observed Adverse Effect Level (NOAEL) for reproductive performance (mating, fertility and delivery) was considered to be 150 mg/kg/day in the absence of adverse findings at this high-dose level,

.the No Observed Adverse Effect Level (NOAEL) for toxic effects on progeny was considered to be 15 mg/kg/day (based on clinical signs and live birth and viability indexes at 50 and 150 mg/kg/day).

Effects on developmental toxicity

Description of key information

No specific developmental toxicity study was performed on nitrosodiphenylamine.

However, some adverse effets on the development were observed in theCombined repeated dose toxicity study with reproduction/developmental toxicity study (OECD422) as explained above.

The no observed adverse effect Level (NOAEL) for toxic effects on progeny was considered to be 15 mg/kg/day (based on clinical signs and live birth and

viability indexes at 50 and 150 mg/kg/day).

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
15 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The OECD 422 study is considered to be reliable.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available data (OECD 422), no classification for fertility is required for Nitrosodiphenylamine according to the Regulation EC 1272/2008.

Justification: no adverse effects on fertility were observed in the OECD 422 study.

Based on the available data (OECD 422), a classification for foetal development is required according to the Regulation EC 1272/2008: Repro 2 (H361d).

Justification: Adverse effects were observed in the pups in absence of maternal toxicity. Indeed, clinical signs and pups mortalities are observed at 50 and 150 mg/kg/day during the first day of lactation period.

Additional information


Route: .live2