Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
extended one-generation reproductive toxicity - with both developmental neuro- and immunotoxicity (Cohorts 1A, 1B without extension, 2A, 2B, and 3)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 17 January 2017 to 24 July 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Deviations:
yes
Remarks:
Deviations were considered not to have compromised the validity or integrity of the study
GLP compliance:
yes (incl. certificate)
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:

- Premating exposure duration for parental (P0) animals: Considering that the extension of Cohort 1B was included in the study design, and that this extension involved a 10-week premating exposure (covering the complete spermatogenesis and folliculogenesis before mating), a 2-week premating exposure for the P generation was deemed adequate.
- Basis for dose level selection: Doses were selected based on previous repeated-dose toxicity and toxicity to reproduction studies performed on the same grade of the registered substance. 540 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. 60 and 180 mg/kg/day). After 6 days of treatment, 3 males from the high dose-group (540 mg/kg/day) had severe clinical signs and body weight losses. Therefore it was decided to lower the high-dose level to 360 mg/kg/day (2-fold interval between mid- and high-dose groups).
- Inclusion/exclusion of extension of Cohort 1B: It was considered necessary to include the extension of Cohort 1B as it allows to evaluate the potential effects on reproductive performance of the F1 animals which are exposed already during critical life stages in utero and early postnatal periods.
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B: Some observations performed during a previous toxicity to reproduction study performed on Grade 5 of the registered substance suggested potential neurodevelopmental toxicity, which supported further elucidating testing. Therefore Cohorts 2A and 2B were included.
- Inclusion/exclusion of developmental immunotoxicity Cohort 3: Findings from previous toxicity to reproduction study performed on Grade 5 suggested a potential mode of action for the registered substance that could induce developmental immunotoxicity. Therefore Cohort 3 was included.
- Route of administration: Oral was considered to be the relevant route of exposure.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
RjHan: SD
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) males: approximately 13 weeks old. (P) females: approximately 12 weeks old.
- Weight at study initiation: (P) males: 459 g to 565 g. (P) females: 251 g to 329 g.
- Fasting period before study: No
- Housing: The P and F1 adult animals were group-housed (except 2 weeks before mating and during mating, gestation and lactation) in polycarbonate cages with stainless steel lids (in Tecniplast 2000P cages: up to 4/sex per cage (2065 cm2) for Cohort 1A animals and up to 5/sex per cage for Cohort 2A and Cohort 3 animals; in Tecniplast 2154 cages (954 cm2): up to 2/sex per cage before mating for Cohort 1B animals) containing autoclaved sawdust (SICSA, Alfortville, France) (see § Study plan adherence). Toward the end of gestation and during lactation, autoclaved wood shavings (SICSA, Alfortville, France) were provided to females and their litter as nesting material. Each cage contained two objects (rat hut and nylabone) for the environmental enrichment of the animals. The cages were placed in numerical order on the racks.
- Diet: SSNIFF R/M-H pelleted maintenance diet, batch Nos 68211320 and 89416605 (SSNIFF Spezialdiäten GmbH, Soest, Germany) ad libitum.
- Water: Tap water (filtered with a 0.22 µm filter) ad libitum.
- Acclimation period: Males were acclimated to the study conditions for a period of 17 days before treatment. 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.


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): 12h/12h

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Preparation procedure (homogeneity and stability testing) for a range of concentrations covering the lowest and highest used in this study was investigated in a separate study.

VEHICLE
- Justification for use and choice of vehicle: The test substance is poorly soluble in water (<0.1 g/L) but proved to be soluble in olive oil. This vehicle was previously used during the screening for toxicity to reproduction and was therefore preferred for consistency’s sake.
- Concentration in vehicle: 0, 12, 36, 72 and 108 mg/mL
- Amount of vehicle: A constant dosage-volume of 5 mL/kg/day was used.
- Lot/batch no. BCBS0084V and BCBT7822
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation:
- Proof of pregnancy: vaginal plug or for sperm in a vaginal lavage referred to as Day 0 p.c.
- Each female was placed with the same male until mating occurred or 14 days had elapsed. When mating had not occurred after 2 weeks, the animals were separated without further opportunity for mating.
- After successful mating each pregnant female was caged: Individual housing was chosen since it is preferable for pregnant animals, littering and lactating females in order to not jeopardize gestation, littering and lactation phases, and to avoid aggressive behaviour around mating in males.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical technique: High Performance Liquid Chromatography with UV detection (HPLC/UV)

Principle and validation of the method: Analytical method developed and validated during a dedicated study prior to dose formulation analysis

Determination of test item concentrations in dose formulations:
- Eight dosages: once in each P-premating, P-mating, P gestation, P-lactation, F1-postweaning, F1-premating, F1 gestation and F1-lactation periods
- A sample was taken from control and test item dose formulations and analyzed using the validated method
- Acceptance criterion: measured concentration = nominal concentration ± 15%
Duration of treatment / exposure:
P Generation
- in the males (at least 10 weeks of treatment): 2 weeks before mating, during the mating period (up to 2 weeks), until euthanasia (after weaning of the pups),
- in the females (at least 8 to 10 weeks of treatment): 2 weeks before mating, during the mating period (up to 2 weeks), during gestation, during lactation until Day 21 p.p. (or Day 22 p.p. sacrifice after hematology, blood chemistry and urinalysis on Day 23 p.c. for the selected females), until euthanasia for females with no evidence of mating or no delivery (26 days after the last day of the mating period).

Cohort 1A
- in the males and females: from weaning (Day 22 p.p.), until euthanasia (from Days 98 to 101 p.p.).

Cohort 1B
- in the males: from weaning (Day 22 p.p.) for at least 10 weeks before mating, during the mating period (up to 2 weeks), after sacrifice of F2 pups (on Day 4 p.p.),
- in the females: from weaning (Day 22 p.p.) for at least 10 weeks before mating, during the mating period (up to 2 weeks), during gestation, during lactation until Day 4 p.p. inclusive, until euthanasia for females with no evidence of mating or no delivery (24-26 days after the last day of the mating period).

Cohort 2A
- in the males and females: from weaning (Day 22 p.p.), until euthanasia (after completion of behavioral testing: Days 75 to 78 p.p.).

Cohort 2B
There was no direct dosing in Cohort 2B animals (sacrificed on Day 22 p.p.).

Cohort 3
- in the males and females: from weaning (Day 22 p.p.), until 5 days after KLH injection on Day 56 (± 3) p.p. (sacrifice on Day 61 (± 3) p.p.).
Frequency of treatment:
Daily
Details on study schedule:
- F1 parental animals not mated until 10 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 22 days of age.
- Age at mating of the mated animals in the study: 13 or 14 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
60 mg/kg bw/day (nominal)
Dose / conc.:
180 mg/kg bw/day (nominal)
Dose / conc.:
360 mg/kg bw/day (nominal)
Remarks:
In the first instance 540 mg/kg bw/day was selected as the high dose level. After 6 days of treatment, 3 males from the high dose-group had severe clinical signs and body weight losses. Therefore it was decided to:
- lower the high-dose level to 360 mg/kg bw/day (2-fold interval between mid- and high-dose groups),
- not treat the males on Day 7,
- treat the females at 540 mg/kg/day on Day 7,
- treat all high-dose group animals at 360 mg/kg bw/day from Day 8.
No. of animals per sex per dose:
P: 24 animals/sex/dose
Cohort 1A: 20 animals/sex/dose
Cohort 1B: 20 animals/sex/dose
Cohort 2A: 10 animals/sex/dose
Cohort 2B: 10 animals/sex/dose
Cohort 3: 10 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on previous repeated-dose toxicity and toxicity to reproduction studies performed on the test substance via the oral route.
Positive control:
Not included

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: From arrival, each animal was observed at least once a day as part as routine examinations. From the start of the treatment period each animal was observed at least twice a day (before and after treatment) at approximately the same time of day, for the recording of clinical signs.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical examinations were performed on all animals once a week until the end of the study. Observations included (but were not limited to) changes in the skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behavior (e.g. self mutilation, walking backwards) were also recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each male was recorded on the first day of treatment (Study Day 1), then at least once a week until euthanasia.
The body weight of each female was recorded on the first day of treatment (Study Day 1), then once a week until mated (or until euthanasia for females with no evidence of mating), on Days 0, 4, 7, 10, 14, 17 and 20 p.c. and, on Days 1, 4, 7, 14 and 21 p.p.
In addition, to the scheduled period of body weight measurements, males from high-dose group (540/360 mg/kg/day) were weighed on Day 7 (see § Study plan adherence).
Animals were weighed at euthanasia as normal procedure before pathology examination.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- The quantity of food consumed by each male was measured once a week from the first day of treatment until the start of the mating period and after the mating period until euthanasia.
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 0-4, 4-7, 7-10, 10-14, 14-17 and 17-20 p.c. and during lactation for the intervals: Days 1-4, 4-7, 7-14, and 14 21 p.p.
During the mating period, food consumption was not measured for males or females.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
Oestrous cyclicity (parental animals):
The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning:
- during 12 days of the pre-treatment period,
- during the 2 weeks of the premating period,
- during the mating period, until the females were mated or the mating period had ended,
- on the day of sacrifice (premature or scheduled)
Sperm parameters (parental animals):
Parameters examined in P and F1 male parental generations: testis weight, epididymis weight, sperm count in testes, sperm count in epididymides, sperm motility, sperm morphology.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups.
The following parameters were examined in F2 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies.

GROSS EXAMINATION OF DEAD PUPS: Yes
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and euthanized by exsanguination after weaning of the F1 progeny.
- Maternal animals: All surviving animals were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and euthanized by exsanguination on Days Days 22-23 p.p, or on Days 26 or 27 p.c. for P females which did not deliver and 26 days after the end of the mating period when no delivery occurred.

GROSS NECROPSY
- Gross necropsy consisted of examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, 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 implantation sites were recorded for females euthanized on Days 22-23 p.p. The numbers of corpora lutea and implantation sites were recorded for the female euthanized 26 days after the end of the mating period with no evidence of mating and for females euthanized on Day 26 p.c. due to no delivery.


HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at [#?] days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, 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 implantation sites were recorded for females euthanized on Days 22-23 p.p. The numbers of corpora lutea and implantation sites were recorded for the female euthanized 26 days after the end of the mating period with no evidence of mating and for females euthanized on Day 26 p.c. due to no delivery.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.
Statistics:
Body weight, food consumption and reproductive data: Data were compared by one-way variance analysis and Dunnett's test, (mean values being considered as normally distributed, variances being considered as homogeneous) or by Fisher’s exact probability test (proportions).

Organ weights: PathData software was used to perform the statistical analysis of organ weight data (level of significance of 0.05 or 0.01).

Auditory startle reflex test, splenic lymphocyte immunophenotyping and number of primary follicles
Statistical analysis was performed using the SAS Enterprise Guide software. To compare the auditory startle reflex response (categorical data using positive or negative values) between treatment groups, a Fisher's exact test was used. If a global statistically significant difference is observed between treatment groups, multiple Fisher's exact tests was performed to compare the auditory startle reflex responses of each test item-treated group with that of the control group. The latency and amplitude of auditory startle reflex responses (by sex for each of the sound frequencies used) and the mean number per section of primordial follicles of P and F1 females were analyzed using the following decision tree. Animals for which auditory startle reflex data was not interpretable are excluded from statistical analyses.

Ano-genital distance, locomotor activity, number of nipples and areolae, time of preputial separation/vaginal opening, time to first estrous after vaginal opening/patency, seminology, hematology, blood biochemistry, urinalysis, thyroid hormones, anti KLH IgM and post-implantation loss: Citox software was used to perform the statistical analyses of data.
Reproductive indices:
Female mating index
Female fertility index
Lactation index
Offspring viability indices:
Live birth index
Viability index on Day 4 p.p.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Males: Ptyalism was observed in all groups (including controls) but at increased incidence in test item treated groups. This finding was considered to be test item treatment-related but not adverse.
At 540/360 mg/mg/kg/day, there were test item treatment-related effects which were considered to be adverse based on findings (thin appearance, hunched posture, piloerection, hypotonia , dyspnea and/or soiled anus/soft feces), incidence (up to 16 males affected vs. one in controls) and duration (from Day 6 up to 59).
At 180 mg/kg/day, piloerection was recorded. Taking into account the limited incidence (2 males vs. 1 male in controls) and duration (from Day 10 to 12), this findings was considered to be non-adverse.
At 60 mg/kg/day, there were no adverse findings.
Other findings observed at low and/or comparable incidence across groups (including controls) are commonly observed in this species/strain of animals and therefore not considered to be test item treatment-related.

Females: Ptyalism was observed in all groups (including controls) but at increased incidence in test item treated groups. This finding was considered to be test item treatment-related but not adverse.
At 540/360 mg/mg/kg/day, there were test item treatment-related effects which were considered to be adverse based on findings (hunched posture and piloerection), incidence (up to 3 females affected vs. none in controls) and duration (up to 5 days).
At 180 mg/kg/day, there were no adverse findings.
At 60 mg/kg/day, one female had an episode of hunched posture/piloerection on study Day 19. As this finding was limited to one animal, to one day and not observed at the mid-dose, a test item treatment-related effect was considered unlikely.
Other findings observed at low and/or comparable incidences across groups (including controls) are commonly observed in this species/strain of animals or in pregnant animals, and therefore not considered to be test item treatment-related.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
In males, there were no unscheduled deaths.

In the control group:
- One female was sacrificed for difficulties to deliver on Day 23 p.c.
- One female was sacrificed on Day 26 p.c. (study Day 43) for no delivery.
- One female was sacrificed on Day 6 p.c. (study Day 25) due to the presence of a mass close to a mammary gland.
In the 60 mg/kg/day group:
- One female was found dead on Day 24 p.c. (study Day 43).
In the 180 mg/kg/day group:
- One female was sacrificed on Day 26 p.c. (study Day 43) for no delivery.
- One female was sacrificed on study Day 57 for no mating.
In the 540/360 mg/kg/day group:
- One female was sacrificed for difficulties to deliver on Day 24 p.c. (study Day 44).
- One female was sacrificed for difficulties to deliver on Day 23 p.c. (study Day 41).
- One female was sacrificed on Day 26 p.c. (study Day 45) for no delivery.
- One female was sacrificed on Day 26 p.c. (study Day 46) for no delivery.

Overall, there were no tendencies toward any test item treatment-related deaths in females. The only potential concern could be the difficulty to deliver observed in two high-dose females. However, this is a common observation in animals of this species/strain (also observed in one control female) for which a test item treatment relationship was therefore excluded.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weight: when compared with controls, there were statistically significant differences at 180 mg/kg/day in males and, at 540/360 mg/kg/day in both sexes. While treatment-related, none of these differences raised a toxicological concern (less than 10% differences when compared with controls). There were no effects at 60 mg/kg/day.

Mean body weight change: in the 540/360 mg/kg/day group, there was a body weight loss in males (-28g vs. +22 g in controls) on Days 1 to 8. Mean body weight gain returned toward control values when the high dose level was lower to 360 mg/kg/day. In the 180 or 60 mg/kg/day group there were no adverse effects on mean body weight gains, despite a few statistically significant differences of low amplitudes which were considered to test item treatment-related.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
In the 540/360 mg/kg/day and when compared with controls, males had a lower mean food consumption (-30% vs. controls, p<0.001) on Days 1 to 8. Mean food consumption returned toward control values when the high dose level was lower to 360 mg/kg/day. In females, there were no dose related differences.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
When compared with control or Historical Control Data, there were no relevant differences in hematology and coagulation parameters. In females and when compared with Historical Control Data, there were incidental higher mean neutrophils counts in all groups, including controls.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In males:
- at 60 mg/kg/day when compared with controls and Historical Control Data, there were no relevant differences,
- from 180 mg/kg/day, there was an increased cholesterol concentration (+89% vs. controls, p<0.01) which exceeds the upper limit of the Historical Control Data (3.77 vs. 2.69 mmol/L),
- at 540/360 mg/kg/day, there were higher concentration of calcium (+11% vs. controls, p<0.01) which exceeds the upper limit of the Historical Control Data (2.87 vs. 2.83 mmol/L), higher urea and creatinine concentration (+15% and +12% vs. controls, p<0.05) which exceeds the upper limit of the Historical Control Data (42.62 vs. 40.22 µmol/L) and higher alanine aminotransferase activity (+38% vs. controls, p<0.01) which exceeds the upper limit of the Historical Control Data (65 vs. 52 U/L).

In females:
- at 60 and 180 mg/kg/day when compared with controls and Historical Control Data there were no relevant differences,
- at 540/360 mg/kg/day, there was an increased cholesterol concentration (+52% vs. controls, p<0.01) which marginally exceeds the upper limit of the Historical Control Data (4.10 vs. 4.08 mmol/L).

Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no effects on urinalysis parameters.

At 540/360 mg/kg/day and when compared with controls, there was a statistically significant higher urinary volume (12 vs. 7 mL, p<0.5) in males. However the value remained lower than the maximal range of Historical Control Data and this increase was therefore considered fortuitous.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Lower final body weights were noted in males treated at 180 or 540/360 mg/kg/day, respectively at -5 and 7% (p<0.05 and 0.01).
Increased absolute and relative-to-body liver weights were recorded in males and females treated at 540/360 mg/kg/day (up to +31% in males in relative-to-body weights; p<0.01). These differences were related to the test item administration and correlated with the microscopic hepatocellular hypertrophy.
Increased absolute and relative-to-body adrenal gland weights were recorded in males and females treated at 540/360 mg/kg/day (up to +22% in females in absolute and relative-to-body weights; p<0.01). These differences were related to the test item administration and correlated with the microscopic increased cortical vacuolation.
Increased absolute and relative-to-body thyroid gland weights were recorded in males treated at 540/360 mg/kg/day (up to +24% in males in relative-to-body weights; p<0.01). These differences were related to the test item administration and correlated with the microscopic follicular cell hypertrophy.
The other organ weight changes were not considered to be test item related because they were of insufficient magnitude, were not dose-related and/or did not correlate to microscopic findings.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Slightly increased incidence of dilated pelvis was noted in the kidneys from males treated at 540/360 mg/kg/day. This change correlated with the microscopic dilation of pelvis.
Although poorly dose-related, slightly increased black/brown/red discolorations were noted in the stomach from males and females treated at 60, 180 or 540/360 mg/kg/day. This correlated with erosion/ulcer seen at minimally higher incidence in test item animals. A relationship to test item administration could not be excluded.

The few other isolated gross observations were considered to be consistent with spontaneous findings encountered in the rats of these strain and age.
This included the testes and epididymides that were small in 2 males treated at 540/360 mg/kg/day. In addition, small testes were noted in 1 male treated at 180 mg/kg/day. Although seen only in mid- and high-dose males, these findings were considered to be in the range of reference data and thus were considered to be of spontaneous origin.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The following test item-related findings were noted:
- kidney: minimal to moderate lesions including mineralization of tubules; mineralization of pelvis; scar in cortex; increased basophilia of tubules; increased infiltrates of mononuclear cells; dilation of tubule; increased dilation of pelvis in males treated at 540/360 mg/kg/day. The relationship to test item of tubular mineralization in 3/24 males treated at 180 mg/kg/day was considered to be equivocal in view of the low incidence and the absence of other renal test item-related findings,
- thyroid glands: minimal to slight follicular cell hypertrophy in males and females treated at 540/360 mg/kg/day,
- lungs: minimal foamy alveolar macrophages in males treated at 540/360 mg/kg/day,
- liver: increased incidence of minimal altered cell foci (clear cells) in males treated at 540/360 mg/kg/day that was considered to be adverse; minimal single cell necrosis/apoptosis located in centrilobular areas in males treated at 540/360 mg/kg/day and that was considered to be adverse; minimal to slight hepatocellular hypertrophy (centrilobular or centrilobular/midzonal distribution) in males and females treated at 180 or 540/360 mg/kg/day,
- adrenal glands: minimal increased vacuolation in the cortex from males treated at 540/360 mg/kg/day,
- jejunum: minimal to moderate ectasia of lymphatics in males and females treated at 180 or 540/360 mg/kg/day and considered to be related in part to the oily nature of the vehicle (olive oil),
- sternum: minimal increased bone mass in trabeculae in males treated at 540/360 mg/kg/day,
- bone marrow: minimal increased myeloid/erythroid ratio; minimal decreased cellularity in males treated at 540/360 mg/kg/day,
- skin: slight ulcer and minimal to slight serocellular crust in 2 males treated at 540/360 mg/kg/day with cutaneous macroscopic lesions,
- stomach: minimal to slight erosion/ulcer in 5/22 females treated at 540/360 mg/kg/day. In females treated at 60 or 180 mg/kg/day, the presence of isolated erosion/ulcer was considered to be unrelated to test item administration in view of their low incidence.

These findings were considered not to be adverse except when specified (i.e. altered cell foci and single cell necrosis/apoptosis in the liver from males treated at 540/360 mg/kg/day).

In addition, focal mineralization was noted in the urinary bladder from one male treated at 540/360 mg/kg/day, in association with reactive urothelium hyperplasia. Although isolated, this finding was considered to be probably related to test item administration in view of the other renal findings including mineralization.
The stomach erosion/ulcer in high-dose females was associated occasionally with degeneration/necrosis of glandular cells or infiltrate of giant cells around mineralization. These findings were considered to be related to the test item administration, although seen at low incidences.

The remaining microscopic findings were not considered to be associated with the test item administration because these findings were consistent with spontaneous and background findings described in the literature, the findings were distributed randomly among groups, or their appearance was similar to changes found in controls.

There were no test item-related changes in the male or female reproductive organs nor in nervous system.

There was a good correspondence between the vaginal smears and the histopathological examination of estrus cycle.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Thyroid hormones: At 540/360 and 180 mg/kg/day, when compared with controls, there were dose related decreases in T4 concentrations (26.4 and 30.5 vs. 35.3 ng/mL, respectively with p<0.01 or not statistically significant) associated with increased concentrations in TSH (5736 and 4777 vs. 1934 pg/mL, respectively with p <0.01). These findings were considered to be test item treatment-related and adverse at 540/360 mg/kg/day (TSH value exceeding the upper limit of Historical Control Data). At 60 mg/kg/day there were no effects,

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
In the 540/360 mg/kg/day group and when compared with controls, there was an increased number of females in diestrus (7.3 days vs. 5.0 days, p<0.001), resulting in a prolongation of mean oestrous cycle duration (5.4 days vs. 3.9 days, p<0.001). This finding was considered to be test item treatment-related and adverse.

In the 180 and 60 mg/kg/day groups and when compared with controls, there were no effects on mean oestrous parameters.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
There were no test item treatment-related effects in sperm analysis data
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no effects on mean mating and fertility data of the P generation.

In the 540/360 mg/kg/day group and when compared with controls or Historical Control Data, there was a lower fertility index (91.7% vs. 100% in controls) close to the lower value recorded in Historical Control Data (91.7% vs. 92.0% in HCD). Therefore a test-item treatment relationship was considered unlikely.

Details on results (P0)

There was no test item-related mortality.

Clinical signs:
- ptyalism was observed in all groups (including controls) but at increased incidences in test item treated groups. This finding was considered to be test item treatment-related but not adverse,
- after 6 days of treatment, 3 out 24 males from the high dose-group (540 mg/kg/day) had severe clinical signs and body weight losses which resulted in the decision to decrease the high dose level to 360 mg/kg/day. At 540/360 mg/mg/kg/day, there were test item treatment-related effects which were considered to be adverse based on findings (thin appearance, hunched posture, piloerection, hypotonia, dyspnea and/or soiled anus/soft feces), incidence (up to 16 males affected vs. none in controls) and duration (from Day 6 up to 59),
- in females, at 540/360 mg/mg/kg/day, there were test item treatment-related effects which were considered to be adverse based on findings (hunched posture and piloerection), incidence (up to 3 females affected vs. none in controls) and duration (up to 5 days),
- there were no test item treatment-related adverse clinical signs at 180 or 60 mg/kg/day.

Mean body weight and mean body weight changes: When compared with controls, there were a few statistically significant differences at 180 and 540/360 mg/kg/day in males or at 540/360 mg/kg/day in females. While treatment-related, none of these differences raised a toxicological concern (less than 10% differences when compared with controls). There were no effects at 60 mg/kg/day.

Food consumption: In the 540/360 mg/kg/day and when compared with controls, males,had a lower mean food consumption (-30% vs. controls, p<0.001) on Days 1 to 8. Mean food consumption returned toward control values when the high dose level was lower to 360 mg/kg/day. In females, there were no dose related differences.

Estrous cycle: In the 540/360 mg/kg/day group and when compared with controls, there was an increased mean number of diestrus (7.3 days vs. 5.0 days, p<0.001), resulting in a prolonged duration of mean estrous cycle (5.4 days vs. 3.9 days, p<0.001). This finding was considered to be test item treatment related and adverse. In the 180 and 60 mg/kg/day groups and when compared with controls, there were no effects on mean estrous parameters.

Mating and fertility: There were no effects on mean mating and fertility data of the P generation.

Delivery data: There were no relevant effects on mean delivery data of the P generation females.

Increased liver and adrenal gland weights were recorded in males and females treated at 540/360 mg/kg/day and increased thyroid gland weights were recorded in males treated at 540/360 mg/kg/day.
Slightly increased incidence of dilated pelvis was grossly noted in the kidneys from males treated at 540/360 mg/kg/day.
The following test item-related microscopic findings were noted:
- kidney: mineralization of tubules, mineralization of pelvis, scar in cortex, increased basophilia of tubules, increased infiltrates of mononuclear cells, dilation of tubule and/or increased dilation of pelvis in males treated at 540/360 mg/kg/day,
- thyroid glands: follicular cell hypertrophy in males and females treated at 540/360 mg/kg/day,
- lungs: foamy alveolar macrophages in males treated at 540/360 mg/kg/day,
- liver: increased incidence of altered cell foci (clear cells) and single cell necrosis/apoptosis that were considered to be adverse, in males treated at 540/360 mg/kg/day, hepatocellular hypertrophy in males and females treated at 180 or 540/360 mg/kg/day,
- adrenal glands: increased vacuolation in the cortex from males treated at 540/360 mg/kg/day,
- jejunum: ectasia of lymphatics in males and females treated at 180 or 540/360 mg/kg/day,
- sternum: increased bone mass in trabeculae in males treated at 540/360 mg/kg/day,
- bone marrow: increased myeloid/erythroid ratio and decreased cellularity in males treated at 540/360 mg/kg/day,
- skin: ulcer and minimal to slight serocellular crust in 2 males treated at 540/360 mg/kg/day,
- stomach: erosion/ulcer in 5/22 females treated at 540/360 mg/kg/day.

Quantitative evaluation of primordial follicles on PCNA-stained slides: There were no differences between the high-dose and control groups, with respectively a mean total number of 14.50 and 11.42 primordial follicles per section.

Hematology and coagulation: When compared with control or Historical Control Data, there were no relevant differences in hematology and coagulation parameters.

Blood biochemistry: In males, at 60 mg/kg/day there were no relevant differences; from 180 mg/kg/day, there was an increased cholesterol concentration (+89% vs. controls, p<0.01) and at 540/360 mg/kg/day, there were higher concentrations of calcium (+11% vs. controls, p<0.01) and creatinine (+12% vs. controls, p<0.05) along with higher alanine aminotransferase activity (+38% vs. controls, p<0.01). In females, at 60 and 180 mg/kg/day there were no relevant differences; at 540/360 mg/kg/day, there was an increased cholesterol concentration (+52% vs. controls, p<0.01).

Urinalysis: There were no test item treatment-related findings on urinalysis parameters.

Thyroid hormones: At 540/360 and 180 mg/kg/day, when compared with controls, there were dose related decreases in T4 concentrations (26.4 and 30.5 vs. 35.3 ng/mL, respectively with p<0.01 or not statistically significant) associated with increased concentrations in TSH (5736 and 4777 vs. 1934 pg/mL, respectively with p <0.01). These findings were considered to be test item treatment-related and adverse at 540/360 mg/kg/day (TSH value exceeding the upper limit of Historical Control Data). At 60 mg/kg/day there were no effects.

Sperm analysis: There were no test item treatment-related effects in sperm analysis data.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
180 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic
reproductive function (oestrous cycle)
other: Thyroid hormones

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Ptyalism was observed in all treated groups at dose level related increased incidences. This finding was considered to be test item treatment-related but not adverse. There were no other test item treatment-related clinical signs.
Mortality / viability:
no mortality observed
Description (incidence and severity):
There were no test item treatment-related deaths.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no adverse effects.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no effects on mean food consumption.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
In males and females, despite a few statistically significant differences when compared with controls, there were no relevant findings when compared with Historical Control Data.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In males:
- at 60 mg/kg/day when compared with controls and Historical Control Data there were no relevant differences,
- at 180 and 540/360 mg/kg/day, there were increased cholesterol concentrations (+69% and +137% vs. controls, respectively with p<0.01) which exceeds the upper limit of the Historical Control Data (2.69 mmol/L).

In females:
- at 60 and 180 mg/kg/day when compared with controls and Historical Control Data there were no relevant differences,
- at 540/360 mg/kg/day, there was an increased cholesterol concentration (+100% vs. controls, p<0.01) which marginally exceeds the upper limit of the Historical Control Data (4.28 vs. 4.08 mmol/L).
Urinalysis findings:
no effects observed
Description (incidence and severity):
In males and females, despite a statistically significant difference in mean urinary volume in females when compared with controls, there were no relevant findings when compared with Historical Control Data.
Sexual maturation:
no effects observed
Description (incidence and severity):
When compared with controls, there were no statistically significant differences in the mean age of balanopreputial separation in males, vaginal opening in females or mean time to first oestrous after vaginal opening in females.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A: Increased absolute and relative-to-body kidney weights were recorded in males and females treated at 360 mg/kg/day (up to +14% in males in relative-to-body weights or in females in absolute weights; p<0.01). These differences were related to the test item administration and correlated with the microscopic renal lesions, mainly the pelvis dilatation.
Increased absolute and relative-to-body liver weights were recorded in males and females treated at 180 or 360 mg/kg/day (up to +34% in males in relative-to-body weights; p<0.01). These differences were related to the test item administration and correlated with the microscopic hepatocellular hypertrophy.
Increased absolute and relative-to-body adrenal gland weights were recorded in males and females treated at 360 mg/kg/day (up to +24% in males in relative-to-body weights or in females in absolute weights; p<0.01). These differences were related to the test item administration and correlated with the microscopic increased cortical vacuolation.
The other organ weight changes were not considered to be test item related because they were of insufficient magnitude, were not dose-related and/or did not correlate to microscopic findings.

Cohort 1B: Increased absolute and relative-to-body liver weights were recorded in males treated at 60 or 180 mg/kg/day, and in males and females treated at 360 mg/kg/day (up to +34% in males in relative to body weights; p<0.01). These differences were related to the test item administration and correlated with the microscopic hepatocellular hypertrophy.
Decreased absolute and relative-to-body pituitary gland weights were recorded in females treated at 360 mg/kg/day (up to -15% in relative-to-body weights; p<0.05). In the absence of microscopic examination of this Cohort, in view of the low magnitude of this observation and since there were no similar findings in Cohort 1A, these differences were considered to be probably not related to the test item administration.
Decreased absolute prostate weights were recorded in males treated at 360 mg/kg/day (-17%; p<0.01). In the absence of microscopic examination of this Cohort, in view of the low magnitude of this observation and since there were no similar findings in Cohort 1A, these differences were considered to be probably not related to the test item administration.
The other organ weight changes were not considered to be test item related because they were of insufficient magnitude and/or were not dose-related.

Cohort 2A: Increased absolute and relative-to-body liver weights were recorded in males and females treated at 360 mg/kg/day (up to +32% in males in absolute weights; p<0.01). These differences were related to the test item administration and correlated probably with the microscopic hepatocellular hypertrophy, although only partial microscopic examination was performed.
The other organ weight changes were not considered to be test item related because they were of insufficient magnitude, were not dose-related and/or did not correlate to microscopic findings.

Cohort 2B: There were no test item-related organ weight changes. The few organ weight changes were not considered to be test item related because they were of insufficient magnitude and/or were not dose-related.

Cohort 3: Increased absolute and relative-to-body liver weights were recorded in males and females treated at 360 mg/kg/day (up to +30% in males in relative-to-body weights; p<0.01). These differences were related to the test item administration.
Increased absolute and relative-to-body adrenal gland weights were recorded in females treated at 360 mg/kg/day (up to +25% in females in relative-to-body weights; p<0.01). These differences were related to the test item administration.
Increased absolute and relative-to-body ovaries weights were recorded in females treated at 360 mg/kg/day (up to +21% in relative-to-body weights; p<0.01). In the absence of similar change in the other cohorts and since the ovaries were not examined microscopically, the relationship to test item administration was considered to be equivocal.
The other organ weight changes were not considered to be test item related because they were of insufficient magnitude, were not dose-related and/or did not correlate to microscopic findings.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A: Dose-related test item-related dilated pelvis was noted in the kidneys from several males treated at 60 or 180 mg/kg/day, and in males and females treated at 360 mg/kg/day. This change correlated to the microscopic dilation of pelvis.

Cohort 1B: Dose-related dilated pelvis was noted in the kidneys from a few males treated at 60, 180 or 360 mg/kg/day.
Enlarged liver was recorded in one male treated at 360 mg/kg/day.
Cutaneous scabs were noted in the dorsal region from a few males treated at 60, 180 or 360 mg/kg/day and in one female treated at 180 mg/kg/day. Although seen also in one control female, a relationship to test item administration could not be excluded in males.
The few other isolated gross observations were considered to be consistent with spontaneous findings encountered in the rats of these strain and age.

Cohort 2A: Dilated pelvis was noted in the kidneys from 9/10 males treated at 360 mg/kg/day. This change correlated to the microscopic dilation of pelvis and was related to the test item administration. The dilated pelvis seen in isolated female treated at 60 mg/kg/day and males treated at 180 mg/kg/day were not related to the test item administration in view of the minimal incidence and the non-dose-relationship in females.
Enlarged liver was recorded in one male treated at 360 mg/kg/day and correlated with hepatocellular hypertrophy. The enlarged liver seen in one female treated at 60 mg/kg/day was not observed microscopically and thus a relationship to test item could not be ruled out.
Black discoloration was seen in the stomach from 2/10 females treated at 180 mg/kg/day and in 2/10 females 360 mg/kg/day. Although seen also in 2/10 control males, a relationship to test item administration could not be excluded.
The few other isolated gross observations were considered to be consistent with spontaneous findings encountered in the rats of these strain and age.

Cohort 2B: There were no test item-related gross changes. The few isolated gross observations were considered to be consistent with spontaneous findings encountered in the rats of these strain and age.

Cohort 3: Black discoloration was seen in the stomach from 1/10 control males and in 1/10 females treated at 360 mg/kg/day and brown discoloration was recorded in the stomach from 1/10 females treated at 360 mg/kg/day. Although seen at low incidences and also in control males, a relationship to test item administration of these changes could not be excluded in females.
The few other isolated gross observations were considered to be consistent with spontaneous findings encountered in the rats of these strain and age.
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A: The following test item-related findings were noted:
- kidney: minimal to marked lesions including dystrophic mineralization in interstitium; interstitium fibrosis; dilation of tubules; pigment; cyst; dilation of pelvis; chronic inflammation in interstitium that was considered to be adverse in males and females treated at 360 mg/kg/day because the grade was noted up to marked; inflammation in pelvis. All these findings were considered to be related to test item administration in males and females treated at 360 mg/kg/day. In the other groups treated at 60 or 180 mg/kg/day, the incidence and severity were considered to be similar to those encountered in untreated rats. The isolated degeneration/necrosis of tubules seen in one male treated at 60 mg/kg/day and in one female treated at 360 mg/kg/day was considered not to be related to the test item administration in view of the low number of affected animals and the absence of dose-relationship in males,
- thyroid glands: minimal to slight follicular cell hypertrophy in males or females treated at 360 mg/kg/day,
- lungs: minimal foamy alveolar macrophages in males treated at 180 or 360 mg/kg/day and considered to be of low toxicological importance in view of the low incidence (3/20 or 2/20) and severity,
- liver: minimal single cell necrosis/apoptosis in males treated at 180 or 360 mg/kg/day that was considered to be adverse; minimal to slight hepatocellular hypertrophy in males treated at 180 mg/kg/day and in males and females treated at 360 mg/kg/day,
- jejunum: minimal to slight ectasia of lymphatics considered to be related in part to the oily nature of the vehicle (olive oil) in males and females treated at 180 or 360 mg/kg/day,
- sternum: minimal increased bone mass in trabeculae in males and females treated at 180 or 360 mg/kg/day. The relationship to test item of the minimal increased bone mass in trabeculae seen an isolated female treated at 60 mg/kg/day was considered to be equivocal in view of the low incidence of this change,
- stomach: minimal erosion/ulcer in 2/20 males treated at 360 mg/kg/day.
These findings were considered not to be adverse except when specified i.e. renal chronic inflammation in interstitium at 360 mg/kg/day and single cell necrosis/apoptosis in the liver from males treated at 180 or 360 mg/kg/day. No test item related findings were seen in the urinary bladder.
The remaining microscopic findings were not considered to be associated with the test item administration because these findings were consistent with spontaneous and background findings described in the literature, the findings were distributed randomly among groups, or their appearance was similar to changes found in controls.
There were no test item-related changes in the male or female reproductive organs nor in nervous system.
There was a good correspondence between the vaginal smears and the histopathological examination of estrus cycle.

Cohort 2A: The following test item-related findings were noted:
- kidney: minimal to moderate lesions including dystrophic mineralization in interstitium; interstitium fibrosis; mononuclear inflammatory cell infiltrate; dilation of tubules; cyst; dilation of pelvis; chronic inflammation in interstitium that was considered to be adverse at 360 mg/kg/day,
- liver: minimal single cell necrosis/apoptosis that was considered to be adverse; minimal hepatocellular hypertrophy,
- stomach: minimal to slight erosion/ulcer seen with higher incidence and/or severity in high-dose males and females,
- ureters: dilated lumen in high-dose males,
- skin: slight ulcer; moderate sero-cellular crust; minimal hyperkeratosis.
These findings were considered not to be adverse except when specified (i.e chronic inflammation in the kidneys).
The examination by the pathologist of the HE-stained multiple brain sections allowed examination of olfactory bulbs, cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalamus, mid-brain (thecum, tegmentum, and cerebral peduncles), brain-stem and cerebellum. There were no test item-related findings in these areas.
There were no effects in the eyes (retina and optic nerve), peripheral nerve, muscle or spinal cord.


Cohort 2B: There were no test item-related microscopic findings.
The few microscopic findings were not considered to be associated with the test item administration because these findings were consistent with spontaneous and background findings.
The examination by the pathologist of the HE-stained multiple brain sections allowed examination of olfactory bulbs, cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalamus, mid-brain (thecum, tegmentum, and cerebral peduncles), brain-stem and cerebellum. There were no test item-related findings in these areas.
There were no effects in the eyes (retina and optic nerve), peripheral nerve, muscle or spinal cord.

Cohort 3: Slight erosion/ulcer was noted in the stomach from 2/10 females treated at 540/360 mg/kg/day and was considered to be related to the test item administration.
The few other microscopic findings were not considered to be associated with the test item administration because these findings were consistent with spontaneous and background findings.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Thyroid hormones:
In non-selected F1 offspring: there were no effects on T4 or TSH plasma samples.
In Cohort 1A males and females: In males at 540/360 and 180 mg/kg/day, when compared with controls, there were increased concentrations in TSH (2937 and 3051 vs. 1421pg/mL, respectively with no statistical significance and p<0.05). These increases were not associated with a similar trend for T4 levels. Therefore a test-item treatment relationship was considered dubious. At 60 mg/kg/day there were no effects. In females, at 540/360 and 180 mg/kg/day, when compared with controls, there were increased concentrations in TSH (996 and 1085 vs. 544 pg/mL, respectively with p <0.01 and p<0.05) associated with increases in T4 levels (+12 and +13% vs. controls, respectively not statistically significant). These findings were considered to be test item treatment-related but not adverse (within the range of the Historical Control Data). At 60 mg/kg/day there were no effects.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Cohort 2A animals were tested once between Day 64 and 66 p.p.. There was no abnormal reactivity to manipulation or to different stimuli.

Detailed clinical examination
- "touch escape" or "ease of removal from the cage" (reactivity to handing) were normal,
- fur appearance (except for 1 or 2 animals/sex in the 180 or 540/360 mg/kg/day group, respectively), gait, posture, behavior and breathing were normal,
- there were no excessive grooming, defecation, urination in treated groups when compared with the control group,
- there were no salivation, no lacrimation, no piloerection and no palpebral closure,
- all pups had a normal pupil size (myosis) at examination and there were no exophthalmos,
- there were no tremors, no twitches, no clonic/tonic convulsions, no ataxia, no hypoactivity, no hyperactivity, no hypotonia and no stereotypies.

Reactivity to stimuli
- visual stimulus response and papillary reflex were normal in all groups,
- auditory startle reflex was normal in all groups,
- forelimb strength was normal in all groups.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
no effects observed
Description (incidence and severity):
Cohort 1A: At 540/360 mg/kg/day and when compared with controls, significant variations consisted in a moderate decrease in relative cytotoxic T cells count (-20% vs. controls) in males. In females, a moderate decrease in relative NK cells count (-32% vs. controls) and a moderate increase in relative T cells count (+28% vs. controls) was observed. Other observed changes in the studied parameters were considered to be either non-significant or non-related to the treatment.
At 180 or 60 mg/kg/day and when compared with controls, there were no effect.

Cohort 3: Before KLH immunization, all samples displayed quantifiable concentrations of anti-KLH IgM. This result can be explained by the fact that rat serum is expected to contain IgMs directed against some glycosylated moieties similar to those of KLH.
After KLH immunization and for all groups (including controls), a slight increase of anti KLH IgM concentration was observed when compared to levels before immunization (around two times more).
For all treated groups and when compared with controls, there were no statistically significant differences.

Details on results (F1)

Cohort 1A
There was no test item-related mortality.
Clinical signs: Ptyalism was observed in all treated groups at dose level related increased incidences. This finding was considered to be test item treatment-related but not adverse. There were no other test item treatment-related clinical signs.
Body weight, body weight change and food consumption: There were no adverse effects.
Pathology: Increased kidney and adrenal gland weights were recorded in males and females treated at 360 mg/kg/day. Increased liver weights were recorded in males and females treated at 180 or 360 mg/kg/day.
Dose-related test item-related dilated pelvis was grossly noted in the kidneys from several males treated at 60 or 180 mg/kg/day, and in males and females treated at 360 mg/kg/day.
The following test item-related microscopic findings were noted:
- Kidney: chronic interstitial inflammation that was considered to be adverse in males and females treated at 360 mg/kg/day because of the up to moderate grade, interstitial dystrophic mineralization and fibrosis, dilation of tubules, pigment, cyst, dilation of pelvis and inflammation in pelvis in males and females treated at 360 mg/kg/day.
- Thyroid glands: follicular cell hypertrophy in males or females treated at 360 mg/kg/day.
- Lungs: foamy alveolar macrophages in males treated at 180 or 360 mg/kg/day.
- Liver: single cell necrosis/apoptosis in males treated at 180 or 360 mg/kg/day that was considered to be adverse; hepatocellular hypertrophy in males treated at 180 mg/kg/day and in males and females treated at 360 mg/kg/day.
- Jejunum: ectasia of lymphatics in males and females treated at 180 or 360 mg/kg/day.
- Sternum: increased bone mass in trabeculae in males and females treated at 180 or 360 mg/kg/day.
- Stomach: erosion/ulcer in 2/20 males treated at 360 mg/kg/day.
Quantitative evaluation of primordial follicles on PCNA-stained slides: There was a slight difference between the high-dose and control groups, with a total number of 9.63 and 15.57 primordial follicles per section respectively. A relationship to test item administration was considered to be unlikely in view of the low magnitude of the difference, of the lack of dose-relationship, of the high inter-individual variability and of the lack of reproduction abnormalities.
Sexual development: When compared with controls, there were no statistically significant differences in the mean age of balanopreputial separation in males, vaginal opening in females or mean time to first oestrous after vaginal opening in females.
Estrous cyles: There were no effects.
Mating, fertility and delivery data: There were no effects.
Blood chemistry: In males, at 60 mg/kg/day there were no relevant differences; from 180 mg/kg/day, there was an increased cholesterol concentration (+69% vs. controls, p<0.01). In females, at 60 and 180 mg/kg/day there were no relevant differences; at 540/360 mg/kg/day, there was an increased cholesterol concentration (+100% vs. controls, p<0.01).
Urinalysis: No test item treatment-related findings on urinalysis parameters.
Thyroid hormones: In males at 540/360 and 180 mg/kg/day, when compared with controls, there were increased concentrations in TSH (2937 and 3051 vs. 1421pg/mL, respectively with no statistical significance and p<0.05). These increases were not associated with a similar trend for T4 levels. Therefore a test-item treatment relationship was considered dubious. At 60 mg/kg/day there were no effects. In females, at 540/360 and 180 mg/kg/day, when compared with controls, there were increased concentrations in TSH (996 and 1085 vs. 544 pg/mL, respectively with p <0.01 and p<0.05) associated with increases in T4 levels (+12 and +13% vs. controls, respectively not statistically significant). These findings were considered to be test item treatment-related but not adverse (within the range of the Historical Control Data). At 60 mg/kg/day there were no effects.
Sperm analysis: There were no test item treatment-related effects in sperm analysis data.
Lymphocyte subtyping: At 540/360 mg/kg/day and when compared with controls, significant variations consisted in a moderate decrease in relative cytotoxic T cells count (-20% vs. controls) in males. In females, a moderate decrease in relative NK cells count (-32% vs. controls) and a moderate increase in relative T cells count (+28% vs. controls) was observed. Other observed changes in the studied parameters were considered to be either non-significant or non-related to the treatment. At 180 or 60 mg/kg/day and when compared with controls, there were no effect.


Cohort 1B
There was no test item-related mortality.
Clinical signs: Ptyalism was observed in all treated groups at dose level related increased incidences. This finding was considered to be test item treatment-related but not adverse. There were no other test item treatment-related clinical signs.
Body weight, body weight change and food consumption: There were no adverse effects.
Pathology: Increased liver weights were recorded in males treated at 60 or 180 mg/kg/day, and in males and females treated at 360 mg/kg/day.
Decreased absolute and relative-to-body pituitary gland weights were recorded in females treated at 360 mg/kg/day.
Dose-related dilated pelvis grossly was noted in the kidneys from a few males treated at 60, 180 or 360 mg/kg/day.
Enlarged liver was recorded in one male treated at 360 mg/kg/day.
No microscopic examination was performed.
Sexual development: When compared with controls, there were no statistically significant differences in the mean age of balanopreputial separation in males, vaginal opening in females or mean time to first oestrous after vaginal opening in females.
Estrous cyles: There were no effects.
Mating, fertility and delivery data: There were no effects.

Cohort 2A
There was no test item-related mortality.
Clinical signs: Ptyalism was observed with increased incidence in test item treated groups. This finding was considered to be test item treatment-related but not adverse.
Body weight, body weight change and food consumption: There were no adverse effects.
Pathology: Increased liver weights were recorded in males and females treated at 360 mg/kg/day.
Dilated pelvis was grossly noted in the kidneys from 9/10 males treated at 360 mg/kg/day.
Enlarged liver was recorded in one male treated at 360 mg/kg/day.
Black discoloration was seen in the stomach from 2/10 females treated at 180, and in 2/10 females treated at 360 mg/kg/day.
The following test item-related microscopic findings were noted:
- Kidney : chronic interstitial inflammation that was considered to be adverse; interstitial dystrophic mineralization and fibrosis; mononuclear inflammatory cell infiltrate; tubular dilation; cyst; pelvic dilation
- Liver: minimal single cell necrosis/apoptosis that was considered to be adverse; hepatocellular hypertrophy
- Stomach: erosion/ulcer
- Ureters: dilated lumen
- Skin: ulcer; sero-cellular crust; hyperkeratosis
Sexual development: When compared with controls, there were no statistically significant differences in the mean age of balanopreputial separation in males or vaginal opening in females.
Neurobehavioral testing: There were no findings at auditory startle testing, reactivity to manipulation or on different stimuli (Functional Observation Battery) and, motor activity (horizontal movements and rearing) testing.
Neurohistopathology: There were no noteworthy findings with the exception of higher cornu ammonis thickness in high dose males than in controls with ambiguous evidence.

Cohort 2B
There was no mortality during the course of the study.
Clinical signs: Ptyalism was observed with increased incidence in test item treated groups. This finding was considered to be test item treatment-related but not adverse.
Body weight, body weight change and food consumption: There were no adverse effects.
There were no test item-related organ weight changes.
There were no test item-related gross changes.
There were no test item-related microscopic findings.
Neurohistopathology: There were no differences between controls and high-dose groups for the measurements performed except for the striatum thickness that was lower in high dose females than in controls with ambiguous evidence.

Cohort 3
There was no test item-related mortality.
Clinical signs: Ptyalism was observed in all test item treated animals and at increased incidence. This finding was considered to be test item treatment-related but not adverse.
Body weight, body weight change and food consumption: There were no adverse effects.
Pathology: Increased liver weights were recorded in males and females treated at 360 mg/kg/day.
Increased adrenal gland weights were recorded in females treated at 360 mg/kg/day.
Black discoloration was seen in the stomach from 1/10 females treated at 360 mg/kg/day and brown discoloration in the stomach from 1/10 females treated at 360 mg/kg/day.
Slight erosion/ulcer was noted in the stomach from 2/10 females treated at 360 mg/kg/day.
Sexual development: When compared with controls, there were no statistically significant differences in the mean age of balanopreputial separation in males or vaginal opening in females.
Quantification of anti-KLM IgM response: For all treated groups and when compared with controls, there were no statistically significant differences.

Non selected pups
There was no mortality during the course of the study.
Decreased thymus weights were recorded in females treated at 360 mg/kg/day. In the absence of microscopic examination, no correlation could be performed.
There were no test item-related gross changes.
There were no test item-related findings.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
180 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic

Results: F2 generation

General toxicity (F2)

Clinical signs:
no effects observed
Description (incidence and severity):
When compared to controls, there no relevant clinical signs in test item treated groups (both in terms of finding and associated severity) which could have reveal any impairment of maternal behaviour (nursing, care to pups).

All findings observed in lactating pups are commonly observed in this species/strain of animals at this age.
Mortality / viability:
no mortality observed
Description (incidence and severity):
In the test item treated groups and when compared with controls or Historical Control Data, there were no significant differences in the repartition of found dead or cannibalized pups and in viability index on Day 4 p.p.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
In test item treated group and when compared with controls, there was no statistically significant differences in mean body weight and mean body weight change.
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:
not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Details on results (F2)

- There were no significant differences in the repartition of found dead or cannibalized pups,
- There were no effects on viability index on Day 4 p.p. in F2 lactating pups,
- There were no test item treatment-related clinical signs in F2 lactating pups,
- There were no test item treatment-related effects on mean F2 body weight and body weight gain.

Effect levels (F2)

Dose descriptor:
NOAEL
Generation:
F2 (cohort 1B)
Effect level:
360 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
180 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
NOAEL for reproductive/developmental toxicity = 180 mg/kg/day
NOAEL for developmental neurotoxicity = 360 mg/kg/day
NOAEL for developmental immunotoxicity = 360 mg/kg/day
NOAEL for systemic toxicity (male) = 60 mg/kg bw/d
NOAEL for systemic toxicity (female) = 180 mg/kg bw/d
Executive summary:

The test item, 4,4’Isopropylidenediphenol, propoxylated, Grade 4PO, was administered daily by oral gavage, at dose levels of 0, 60, 180 or 540/360 mg/kg bw/d, sexually-mature male and female rats (parental (P) generation) starting 2 weeks before mating and continuously through mating, gestation and weaning of their pups (F1 generation). At weaning, F1 generation was also exposed to graduated doses of the test item while being assigned to Cohorts of animals for reproductive/developmental toxicity, developmental neurotoxicity or developmental immunotoxicity testing.

 

Systemic toxicity evaluation:

The No Observed Adverse Effect Levels (NOAELs) for systemic toxicity (excluded reproductive and developmental toxicity endpoints) were considered to be:

- 60 mg/kg bw/d in males based on adverse pathology findings (single cell necrosis) from 180 mg/kg bw/d in Cohort 1A males;

- 180 mg/kg bw/d in females based on adverse pathology findings (single cell necrosis) at 540/360 mg/kg bw/d in Cohort 1A females.

 

Reproductive/developmental toxicity testing:

- in P generation animals, there was an increased number of females in diestrus at 540/360 mg/kg bw/d, resulting in a prolonged duration of mean estrous cycle associated with a lower fertility index at this dose level,

- in Cohorts 1A or 1B animals there were no adverse effects on reproductive function.

Therefore the No Observed Adverse Effect Level (NOAEL) for reproductive/developmental toxicity was considered to be 180 mg/kg bw/d.

 

Developmental neurotoxicity testing:

There were no adverse effects at neurobehavioral testing but ambiguous evidence of developmental neurotoxicity in high dose males from cohort 2A (corpus callosum increased thickness) and high dose females from cohort 2B (striatum decreased width).

Therefore the No Observed Adverse Effect Level (NOAEL) for developmental neurotoxicity was considered to be 360 mg/kg bw/d.

 

Developmental immunotoxicity testing:

in Cohort 1A:

-          moderate alterations in spleen immunophenotyping were observed differently in males (decrease of relative cytotoxic T cells count) and females (decrease of relative NK cells count and increase of T cell relative count) at 360 mg/kg bw/d,

in Cohort 3:

-          after KLH immunization and for all groups (including controls), there were no statistically significant differences.

Therefore the No Observed Adverse Effect Level (NOAEL) for developmental immunotoxicity was considered to be 360 mg/kg bw/d.