Registration Dossier

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Diss Factsheets

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
extended one-generation reproductive toxicity - with developmental neurotoxicity (Cohorts 1A, 1B without extension, 2A and 2B)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 3 December 2020 to 13 August 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022
Report date:
2022

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
28 July 2011 (Figure 1 corrected 2 October 2012)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
SPECIFICATIONS OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:
Based on ECHA decision No.: TPE-D-2114498472-38-01/F
- 10 weeks Pre-mating exposure duration for parental (P0) animals as according to ECHA:
1) There is no substance-specific information in the dossier supporting shorter premating exposure duration.
2) Lipophilicity of the test item (Log Pow 7.3) ensures that steady state in parental animals has been reached before mating.

- Exclusion of extension of Cohort 1B:
The conditions to include the extension of Cohort 1B has not been met. The substance has no uses leading to significant exposure of consumers or professionals.

- Inclusion of developmental neurotoxicity Cohorts 2A and 2B:
According to ECHA, there is a particular concern on (developmental) neurotoxicity based on the effects on thyroid observed at 1000 mg/kg bw/day group during the OECD 408 and 422 studies. Signs of thyroid toxicity rise a particular concern on developmental neurotoxicity.

- Exclusion of developmental immunotoxicity Cohort 3:
No triggers for the inclusion of Cohort 3 have been identified. No effects on the lymphoid tissues have been reported in the OECD 408 and 422 studies.

- Route of administration: oral
The oral route was selected as it is the recommended route of administration for this type of study with an industrial chemical.

Test material

Constituent 1
Chemical structure
Reference substance name:
[1,3(or 1,4)-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide
EC Number:
246-678-3
EC Name:
[1,3(or 1,4)-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide
Cas Number:
25155-25-3
Molecular formula:
C20H34O4
IUPAC Name:
Reaction mass of 1,3-bis[2-(terbutylperoxy)propan-2-yl]benzene and 1,4-bis[2-(terbutylperoxy)propan-2-yl]benzene
Test material form:
solid: flakes

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl CD (SD) IGS BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Italia, Calco, Italy
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) males 6 wks and females 5 weeks
- Weight at study initiation: (P) Males: 185-225 g; Females: 132-172 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. They were housed in polycarbonate cages with stainless steel lids (4/sex per cage for P generation and Cohort 1A animals and 5/sex per cage for Cohort 2A animals; up to 4/sex per cage for Cohort 1B animals) containing autoclaved sawdust. 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 behavior around mating in males.
- Use of restrainers for preventing ingestion (if dermal): not applicable
- Diet (e.g. ad libitum): SSNIFF rat/mouse pelleted maintenance diet (SSNIFF Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water (e.g. ad libitum): tap water (filtered with a 0.22 um filter) in bottles, ad libitum
- Acclimation period: 8 days
No contaminants were present in the diet, drinking water, sawdust or wood shavings at levels which may be expected to interfere with or prejudice the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C (target)
- Humidity (%): 50 ± 20% (target)
- 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: From: 18 December 2020 To: 9 July 2021

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item dosing solutions were prepared as solutions in the vehicle corn oil (based on visual observation) according to CRL Study No. 48344 VAS (2021) over a dose formulation concentration range of 1 to 200 mg/mL for determination of homogeneity and stability.
Frequency of preparation: 11 days at room temperature followed by approximately a 4-hour magnetic stirring in a water bath at 33±2°C for dose formulations from 1 to 200 mg/mL and based on vehicle expiry.
Control dose formulations were prepared in line with the frequency of test item dose formulations.

At receipt in the study room, the dose formulations (groups 1 to 4) were placed under magnetic stirring in a water bath at 33°C±2°C for at least 15 minutes before administration. These conditions were maintained throughout the administration procedure, which was performed within 4 hours after the 15 minutes stirring for re homogenization.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Selected based on previous experimental work.
- Concentration in vehicle: 1 to 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw/day
- Lot/batch no. (if required): MKCN9742, MKCM9808, MKCK6411and MKCM3364.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 14 days or until evidence of mating had been obtained
- Proof of pregnancy: presence of a vaginal plug or sperm in a vaginal lavage in the morning referred to as Day 0 of pregnancy
- After successful mating each pregnant female was caged (how): individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Validated analytical method (covering a 1 to 200 mg/mL range) validated at Charles River Laboratories France (Study No. 48344 VAS, 2021) prior to dose formulation analysis.
Checked parameters, acceptance criteria and obtained results are detailed in the validation report
Analytical verification was performed on 9 occasions in the study (i.e. on Study Days -2, 7, 15, 30 and then once a month, including at least the first and last week of Cohort 2A treatment period).
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) Males:
at least 10 weeks before mating, up to 2 weeks during the mating period, until euthanasia after weaning of their F1 offspring.

(P) Females:
at least 10 weeks before mating, up to 2 weeks during the mating period, 3 weeks during gestation.
Part of the females until Postnatal Day 21 (weaning of their offspring).
Part of the females until the day prior to euthanasia on Postnatal Day 23 weeks during lactation.
Females with no evidence of mating or no delivery were treated until 24-26 days after the last day of the mating period.

(F1) Males:
Cohort 1A from weaning until euthanasia (Postnatal Days 22 until 90-93).
Cohort 1B from weaning (Postnatal Day 22), for at least 10 weeks, before euthanasia (after necropsy of Cohort 1A pending no alteration of estrous cycle or sperm parameters, and no macroscopic findings after reproductive organs examination were observed).
Cohort 2A from weaning until euthanasia, after completion of behavioral testing (Postnatal Days 22 until 77-80).
Cohort 2B: there was no direct dosing in Cohort 2B animals (euthanized on Postnatal Day 22).

(F1) Females:
Cohort 1A from weaning until euthanasia (postnatal days 22 until 90-93)
Cohort 1B from weaning (Postnatal day 22), for at least 10 weeks before euthanasia (after necropsy of Cohort 1A pending no alteration of estrous cycle or sperm parameters, and no macroscopic findings after reproductive organs examination were observed).
Cohort 2A from weaning until euthanasia, after completion of behavioral testing (Postnatal Days 22 until 77-80).
Cohort 2B: there was no direct dosing in Cohort 2B animals (euthanized on Postnatal Day 22 ).
Frequency of treatment:
The treatment was performed once daily at approximately the same time each day (7 days a week) and on the days of developmental landmark testing, when animals were treated after the end of the test.
Details on study schedule:
- Age at mating of the mated animals in the study: males: 16 weeks; females: 15 weeks old
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Group 1 (Concentration 0 mg/mL)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Group 2 (Concentration: 20 mg/mL)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Group 3 (Concentration: 60 mg/mL)
Dose / conc.:
800 mg/kg bw/day (actual dose received)
Remarks:
Group 4 (Concentration: 160 mg/mL)
No. of animals per sex per dose:
24 animals/sex/dose (P0 generation)
20 animals/sex/dose (Cohort 1A and 1B)
10 animals/sex/dose (Cohort 2A and 2B)
Control animals:
yes, concurrent vehicle
Details on study design:
- Basis for dose level selection: Selected based on 4 previous studies.
An OECD 422 GLP study was performed in which Sprague Dawley rats of both sexes were treated orally with BIS PEROXIDE (administered as VULCUP®R) at 0, 100, 300, 1000 mg/kg bw/day by gavage. At 1000 mg/kg bw/day, decreased numbers of pregnant dams, with decreased numbers of corpora lutea, implantation sites, live embryos at first litter check, and higher postnatal loss were observed. At 300 mg/kg bw/day, a slightly higher postnatal loss was also observed, but without statistical significance. Living pups at 1000 and 300 mg/kg bw/day had lower birth weight gain until post-natal day 4. The NOAEL for systemic toxicity in the parental generation was considered to be 300 mg/kg bw/day, based on decreased body weight gain and food consumption in males and females and microscopic changes in the kidneys of females at 1000 mg/kg/day. The NOAEL for fertility was 1000 mg/kg bw/day in males and 300 mg/kg/day in females. The NOAEL for fetal development was 100 mg/kg bw/day, based on the lower body weight gain at 300 and 1000 mg/kg bw/day.
An OECD 408 GLP study was performed in which BIS PEROXIDE (administered as Luperox® F Flakes) was administered daily by oral gavage to Wistar rats of both sexes at dose levels of 0, 50, 200 and 800 mg/kg bw/day for a period of 91/92 days. At the end of the treatment period, some animals from control and high dose groups were kept for a 28-day treatment-free recovery period. Salivation was observed at all dose levels after administration. The incidence and frequency of salivation increased with the dose level. At 800 mg/kg bw/day, slightly lower absolute food consumption was recorded in males, while slightly increased food consumption was observed in females of this group. In group 4, decreased body weight and lower body weight gain were recorded in males from treatment week 2 onwards (-15 and - 40%, respectively compared to controls). This change was considered as adverse. At 800 and 200 mg/kg bw/day, kidney weights were increased in males, while hyaline droplets immunostaining with an anti-alpha 2µ-globulin antibody were recorded in the proximal tubules of males at all dose levels with a dose-related increase in the mean severity. Karyomegaly, tubular basophilia and pigmented vacuoles in the proximal tubules of the kidneys of males and females were recorded at 800 mg/kg bw/day, with higher mean severity in males. Changes in kidneys were considered as adverse. Based on the results of this study, the NOAEL in male and female rats was established at 200 mg/kg bw/day, considering the adverse effects observed at 800 mg/kg/day on the body weight gain in males and on the kidneys in males and females.
An OECD 414 GLP study was performed in which BIS PEROXIDE (administered as Luperox® F Flakes) was administered once daily by gavage at dose levels of 0, 100, 300 and 1000 mg/kg bw/day to four groups of 24 mated RccHanTM: WIST(SPF) female rats. All females were sacrificed on Day 21 p.c. and the fetuses were removed by cesarean section. Treatment with BIS PEROXIDE caused a reversible reduction in food consumption at 1000 mg/kg/day, not considered adverse. Treatment with BIS PEROXIDE caused a statistically significant reduction in body weight, body weight gain (41 vs. 48% for controls) and corrected body weight gain (4.8 vs. 10.8% for controls) (body weight gain corrected for the gravid uterus weight) at 1000 mg/kg bw/day. This effect was considered adverse. A reversible reduction in body weight gain in the absence of significant effects on absolute body weight or corrected body weight gain was recorded at 300 mg/kg bw/day. This effect was not considered to be adverse. Relevant reproduction data were not affected by treatment with BIS PEROXIDE (post-implantation loss and number of fetuses per dam). No treatment-related findings were recorded on any of the offspring parameters, except for a light increase in the incidence of fused zygomatic arch and malpositioned pelvic girdle at 1000 mg/kg bw/day. This finding was not considere adverse. Based on these results, the NOAEL (No Observed Adverse Effect Level) for maternal toxicity was considered to be 300 mg/kg/day whereas the NOEL (No Observed Effect Level) was 100 mg/kg bw/day. For prenatal developmental toxicity, the NOEL was considered to be 300 mg/kg bw/day and the NOAEL was considered to be 1000 mg/kg bw/day.
A preliminary (not GLP) study for effects on pre- and post-natal development (PPND study) by oral gavage in Sprague Dawley rats was performed in which BIS PEROXIDE (Luperox® F Flakes) was administered daily at 0, 100, 300 or 1000 mg/kg bw/day to time-mated and pregnant female Sprague-Dawley rats throughout gestation (from Gestation Day 6 until and including Postnatal Day 21. Then, after weaning, male and female pups (F1 generation) received the test item from Day 22 until and including Day to 35.
Under the experimental conditions of this study, 1000 mg/kg bw/day was considered a dose level exceeding the Maximal Tolerated Dose, reflected in mortality and absence of body weight gain at the beginning of the gestation period in F0 females, adverse effects on early pup survival and adverse findings in F1 generation (clinical signs, body weight/body weight change and food consumption). At this high-dose level, there was also a marked increase in the number of pups and litters with test item-related clinical signs (yellowish area(s) on the body, soiled urogenital region, abnormal reddish color of anus, dehydration and/or emaciated appearance).
In conclusion, 1000 mg/kg bw/day was not used as the high dose level based on the following:
• the probable test item related mortality and/or premature euthanasia of three females observed at 1000 mg/kg bw/day in the PPND study;
• the absence of body weight gain observed over the Gestation Days 6-9 interval (0 g vs. +10 g; p<0.05) during the gestation period at 1000 mg/kg bw/day in the preliminary PPND study;
• statistically significant reduction in body weight, body weight gain and corrected body weight gain (body weight gain corrected for the gravid uterus weight) at 1000 mg/kg bw/day considered adverse in the OECD 414 study in rats;
• the effects on fertility observed at 1000 mg/kg bw/day in the OECD 422 study substantiated by decreased mean number of corporal lutea, implantation sites and pups body weight on Postnatal Day 4;
• the increase in the number of pups found dead at 1000 mg/kg bw/day in the preliminary PPND study as well as their small weight from Postnatal Days 1 to 17 considered adverse,
• the marked increase in the number of pups and litters with test item-related clinical signs at 1000 mg/kg bw/day in the preliminary PPND study. These findings were considered to represent abnormal maternal care and were considered to be adverse.
To be note that, the decreased body weight gain in pups in the preliminary PPND study was less marked than the one observed in the OECD 422 study at the same dose-level. This is likely due to the absence of premating exposure to the test substance of the P generation animals during the preliminary PPND study, although a similar trend was observed. Consequently, it can be expected the effects observed at 300 and 1000 mg/kg bw/day in regard to the decreased body weight to be accentuated by a 10-week pre-treatment period, thus compromising the survival of the pups in case of use of a too high dose level.
In addition, Cohorts 2A and 2B were requested by ECHA based on the effects on thyroid observed in pups from the 1000 mg/kg bw/day group during the OECD 422 Study. This effect was deemed a consequence of an enhanced liver cell metabolism due to the hepatocellular hypertrophy and not to represent a direct effect of the test item. Consequently, effects on thyroid were not investigated in the low and intermediate groups, and it cannot be confirmed if pups from the 100 and 300 mg/kg bw/day displayed the same effect on thyroid. While the selection of doses should aim at replicating this effect during the OECD 443 Study, it is considered that a dose of 1000 mg/kg bw/day will most likely not allow to obtain a sufficient number of live pups for the F1 Generation, due to the adverse effects observed at this dose-level in the various studies performed with the test-item.
Based on the findings detailed above the dose of 800 mg/kg bw/day was thus selected as the
high-dose level and considered to represent a good compromise in order preventing from an excessive mortality in the parental and pups groups but allowing the investigation of the effects observed in the previous studies at the high dose levels. In addition, effects observed at 800 mg/kg bw/day on body weight gain in males and kidneys in males and females in the OECD 408 study were considered as adverse. The low-dose and mid-dose were selected using a ratio representing approximately a 2.5 to 3-fold interval (i.e. 100 and 300 mg/kg bw/day).
- Rationale for animal assignment (if not random): during the acclimation period, the required number of animals (96 males and 96 females) were selected according to body weight and clinical condition and allocated to the groups (by sex), according to a computerized stratification procedure, so that the average body weight of each group was similar (i.e. ± 20% of the global mean values among the groups).
- Fasting period before blood sampling for clinical biochemistry: overnight period of at least 14 hours

Examinations

Parental animals: Observations and examinations:
GENERAL OBSERVATIONS, MORTALITY, MORBIDITY: Yes
- Time schedule: Each animal was checked for mortality, morbidity and general clinical observations once a day during the acclimation period and at least twice a day during the treatment period.
- 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 evaluated.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a week until the end of the study

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each male was recorded for group allocation and 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 for group allocation and 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 Gestation Days 0, 4, 7, 10, 14, 17 and 20 and on Postnatal Days 1, 4, 7, 14 and 21.
Animals were weighed at euthanasia as normal procedure before pathology examination.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The quantity of food consumed by each male was measured at least 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: Gestation Days 0-4, 4-7, 7-10, 10-14, 14-17 and 17-20 and during lactation for the intervals: Postnatal Days 1-4, 4-7, 7-14, and 14-21.
During the mating period, food consumption was not measured for males or females.

Oestrous cyclicity (parental animals):
The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning:
• for 2 weeks during the premating period,
• during the mating period, until the females were mated or the mating period had ended,
• on the day of euthanasia.
Sperm parameters (parental animals):
Full seminology investigations were performed on all surviving P and Cohort 1A males from all groups.

Parameters examined:
-testis weight;
-epididymis weight;
-sperm motility and morphology: sperm from the cauda of the left epididymis was sampled for motility and morphology investigations. The number of motile and non-motile spermatozoa was evaluated under a microscope using a 40-fold magnification from a sample of 200 spermatozoa. Results were expressed as the percentage of motile and non-motile spermatozoa. Sperm morphology was determined from a smear, after eosin staining, counting 200 spermatozoa per slide. Results were expressed as the percentage of spermatozoa in each of the following categories: normal, normally shaped head separated from flagellum, abnormal head separated from flagellum, abnormal head with normal flagellum, abnormal head with abnormal flagellum, normally shaped head with abnormal flagellum.
-enumeration of cauda epididymal sperm reserve: cauda of the left epididymis (sampled after anesthesia and before euthanasia) was separated from the corpus using a scalpel and subsequently frozen at -20°C for further investigation. After thawing, the left cauda epididymis was weighed, minced and homogenized. An aliquot of the suspension was sampled and the number of spermatozoa was counted in a Malassez cell. Results were expressed as the number of spermatozoa per cauda and per gram of cauda.
-sperm count in testis: After thawing, the left testis was weighed and ground. The resulting preparation was diluted and sperm heads resistant to homogenization (i.e. elongated spermatids and mature spermatozoa) were counted in a Neubauer cell. Results were expressed as the number of sperm heads per gram of testis and the daily sperm production rate was calculated (using a time divisor of 6.10; Blazak et al., 1993).
- Testicular staging (Control and high dose groups): a detailed examination of the testes was performed, using a thorough understanding of tubule development through the different stages of the spermatogenic cycle, and allowed detection of retained spermatids, missing germ cell layers or types, multinucleated giant cells or sloughing of spermatogenic cells into the lumen, etc.
- Epididymis (Control and high dose groups): examination included the caput, corpus and cauda and allowed detection of leukocyte infiltration, change in prevalence of cell types, aberrant cell types, phagocytosis of sperm, etc.
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:
The total litter size and sex of each pup were recorded on Postnatal Day 1. The litters were then observed daily in order to note the number of live, dead and cannibalized pups.
The pups were observed daily for clinical signs, abnormal behavior and external abnormalities (including oral cavity and orifices). The first clinical examination (on Postnatal Day 1) included: gross external examination (e.g. external visible abnormalities, cleft palate, subcutaneous hemorrhages, abnormal skin color or texture; presence of umbilical cord, lack of milk in the stomach, presence of dried secretion) and qualitative assessment of body temperature, activity and reaction to handling.
The body weight of each live pup was recorded on Postnatal Days 1, 4, 7, 14 and 21.
The following physical development measurements were performed in pups of each litter:
• anogenital distance (AGD) on Postnatal Day 1 (all pups before culling). The AGD was normalized to the cube root of body weight recorded on Postnatal Day 1 (Normalized Ano-Genital Distance (Norm. AGD) = AGD / ³vBody weight);
• number of nipples and of areolae in male pups: on Postnatal Day 12.

GROSS EXAMINATION OF DEAD PUPS:
A macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed on all found dead (except cannibalized) pups. Special attention was paid to the reproductive organs and to whether the pup had fed (e.g. presence of milk in the stomach). Macroscopic lesions were preserved in appropriate fixative.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY :
Cohorts 2A and 2B:
Cohort 2A: On Postnatal Day 22, 20 pups/group (10 males and 10 females/group; one male or one female/litter; all litters represented by at least 1 pup; randomly selected) were selected for neurohistopathology assessment at weaning.
Cohort 2B: On Postnatal Day 22, 20 pups/group (10 males and 10 females/group; one male or one female/litter; all litters represented by at least 1 pup; randomly selected) were selected for neurobehavioral testing followed by neurohistopathology assessment as adults.

Neurobehavioural testing:
Auditory Startle Test: Cohort 2A animals were subjected to an auditory startle test on Postnatal Day 23 using a computerized shuttle box system to determine the amplitude and time to response.
Functional Observation Battery: Cohort 2A animals were observed once on Postnatal Days 63-65, in the cage, in the hand and in the standard arena. Where possible, the same observer evaluated the animals in a given test. 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, exophthalmia, reactivity to handling, pupil size (presence of myosis or mydriasis),
• in the standard arena (two-minute recording): grooming, palpebral closure, defecation, and urination counts, tremors, twitches, convulsions (clonic and tonic), gait, arousal (hypo- and hyperactivity), posture, stereotypy, behaviour and breathing, ataxia, hypotonia.
In addition, the following parameters, reflexes and responses were recorded:
• pupil reflex,
• auditory startle reflex,
• forelimb grip strength,
• visual stimulus.
Motor activity: Cohort 2A animals were subjected to motor activity testing at the same time as the clinical examination for reactivity to manipulation or to different stimuli using an automated infra-red sensor equipment recording individual animal activity over a 60-minute period.
The following parameters were reported:
• horizontal movements,
• vertical movements.

Neuropathology assessment:
-At weaning (Postnatal Day 22) in Cohort 2B animals neurohistopathology assessment.
-Adult age: Cohort 2A animals were terminated after behavioral testing (between Postnatal Days 77 and 80), with brain weight recorded and full neurohistopathology for purposes of neurotoxicity assessment.

Neurohistopathology:
Neurohistopathology was performed for all high-dose and control animals of each sex following completion of neurobehavioral testing (between Postnatal Days 77 and 80 for Cohort 2A animals and on Postnatal Day 22 for Cohort 2B animals). Cohort 2A were perfusion-fixed (the vascular system was flushed by saline, followed by a perfusion with 10% neutral buffered formalin).
Multiple sections were examined from the brain to allow examination of olfactory bulbs, cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalamus, mid-brain (thecum, tegmentum, and cerebral peduncles), brainstem and cerebellum. The eyes (retina and optic nerve) and samples of peripheral nerve, muscle and spinal cord were also examined.
The brain and the pituitary gland of all animals were fixed in formalin after opening the brain cavity, but with the brain supported by the skull base; after fixation, the brain and the pituitary gland were removed and weighed after study termination. Weighing was at least 48 hours after necropsy to prevent physical damage of the soft tissue during weighing.
All tissue samples were processed to paraffin wax blocks embedded. The brain tissue from all dose-groups was processed to blocks at the same time to avoid shrinkage artefacts associated with prolonged storage in fixative.
Control and high-dose groups were processed to slides and examined.
Morphometric (quantitative) evaluations were performed on representative areas of the brain (homologous sections carefully selected based on reliable microscopic landmarks) and included linear and/or areal measurements of specific brain regions. For neurohistopathology, as relevant changes from controls were found in the high-dose group, morphometric analyses from mid-and low-dose groups were also performed on:
• L3 L2+6 sensory cortex thickness, L4-1 dendate gyrus thickness and L4-3 entire hippocampus thickness from Cohort 2A males,
• L4-2 cornu ammonis thickness and L4-3 entire hippocampus thickness from Cohort 2A females.
At least three consecutive sections were taken at each level in order to select the most homologous and representative section for the specific brain area to be evaluated. Non homologous sections were not used for evaluation. A minimum of 6 (up to 10) brains/sex was evaluated for the control and high-dose group, except for the L3 4+8 and L7 measurements in control males (n=5 for both) and for the L7 measurement in high-dose males (n=5).
The comparisons for these parameters were considered to be of lower reliability than for the other measurements. However, the achieved number of adequate brain sections was close to the target and this was considered to have no negative impact on the definitive conclusions.
The appropriate relative dimensions of the cerebral cortex, hippocampus and cerebellum were assessed in each examined brain.
All aspects of the preparation of tissue samples, from tissue fixation, through the dissection of tissue samples, tissue processing, and staining of slides, were employed a counterbalanced design, such that each batch contained representative samples from each dose group where possible.

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY (Cohort 1A):
For the investigation of pre- and post-natal induced immunotoxic effects, 10 males and 10 females from each group in Cohort 1A animals (all litters represented by at least one pup, as much as possible; randomly selected) were subjected to a splenic lymphocyte subpopulation analysis [T lymphocytes, CD4+ and CD8+ T lymphocytes, B lymphocytes, Natural Killer (NK) cells and NKT cells] using one half of the spleen (weight determined at necropsy), the other half of the spleen was preserved for histopathological evaluation.
Analysis of splenic lymphocyte subpopulations in non-immunised (Cohort 1A) animals was for the determination as to exposure-related shifts in the immunological steady-state distribution of "helper" (CD4+) or "cytotoxic" (CD8+) thymus-derived lymphocytes or Natural Killer (NK) cells (rapid responses to neoplastic cells and pathogens); when compared with concurrent control or reference ranges.
The lymphocyte subtyping by flow cytometry followed the method validated in Study No. 44564 RDR and the internal procedures. Samples were prepared from half of the spleen. After collection, spleen samples were dissociated to obtain single cells suspensions that were stained and finally acquired on the MACSQuant Analyzer 10 (Miltenyi Biotec). All individual results in relative and absolute counts for each splenocyte subset as well as summary tables composed of mean and SD per group and per sex with statistical results (for relative and absolute counts) were reported.
Postmortem examinations (parental animals):
SACRIFICE
- On completion of the treatment period, all surviving P animals were euthanized by an intraperitoneal injection of sodium pentobarbital followed by exsanguination.
- Male animals: All surviving animals after weaning of the F1 progeny.
- Maternal animals: All surviving animals on Postnatal Days 23-24 after weaning of the F1 progeny. P females which did not deliver: on Days 24-26 post-coitum (after body weight recording to check for a possible unnoticed delivery. P females with no evidence of mating: 24-26 days after the end of the mating period when no delivery occurred. One high dose female with total litter loss was euthanized on Postnatal Day 3.

GROSS NECROPSY
- A complete macroscopic post-mortem examination was performed on all P animals. This included 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 corpora lutea and implantation sites were recorded for females euthanized on Postnatal Days 23-24.

Two mid-dose males were found dead and submitted for a complete macroscopic post-mortem examination. Macroscopic lesions were preserved. The numbers of corpora lutea and implantation sites were recorded for one mid-dose female euthanized on Day 25 post-coitum due to no delivery.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 (Section Additional Information) were prepared for microscopic examination and weighed, respectively.

Histology:
The tissues specified in Tissue Procedure Table (Table 1; Section Additional Information) were preserved in 10% buffered formalin (except for the eyes with optic nerves, testes and epididymides which were preserved in Modified Davidson's Fixative). Ovaries (all groups from P generation and Cohorts 1A and 1B) were fixed for at most 96 hours in formalin before being embedded in paraffin wax.
All tissues required for microscopic examination were trimmed based on the RITA guidelines, when applicable, embedded in paraffin wax, sectioned at a thickness of approximately 4 microns and stained with hematoxylin-eosin (except for the testes and epididymides which were stained with hematoxylin/PAS and for the right ovaries which were stained by PCNA immunohistochemistry). Ovaries and kidneys were fixed for at most 96 hours in formalin.
Five step-sections (100 µm apart in the middle third) were made for the right ovary.
One additional kidney slide of control and high-dose males (Control and high dose groups) was immunostained with an antibody for a2µ globulin.

Microscopic examination
A microscopic examination was performed on:
• all tissues listed in the Tissue Procedure Table (Table 1; Section "Additional information on methods") from animals of the control and high-dose groups euthanized at the end of the treatment period,
• reproductive organs from animals that did not mate or conceive, from pregnant females that did not deliver, from females with abnormal estrous cycles and from males with abnormalities at sperm analysis, to investigate possible causes,
• all macroscopic lesions of all groups, including those from found dead and prematurely euthanized animals,
• kidneys of males, and liver, thyroid glands and thymus of males and females from
of the low- and mid-dose groups euthanized at the end of the treatment period,

Right ovary (Control and high dose groups): a detailed and careful microscopic examination was made of five step-sections of the right ovary of each female, with enumeration of the total number of primordial follicles on PCNA-stained slides.

The uteri of all P females was examined for the presence and number of implantation sites (visual examination only).
A vaginal smear was made on the day of necropsy to allow correlation with histopathology in reproductive organs. The smears were stained with Harris Shorr.
Postmortem examinations (offspring):
SACRIFICE
- F1 pups were euthanized by an intraperitoneal injection of sodium pentobarbital followed by exsanguination.
- F1 males (Cohort 1A): on Posnatal Days 90-92;
- F1 females (Cohort 1A): on Postnatal Days 91-93;
- F1 males and females (Cohort 1B): after euthanasia of the Cohort 1A animals (i.e. on Postnatal Days 98-100;
- F1 males (Cohort 2A): on Postnatal Days 77-79;
- F1 females (Cohort 2A): on Postnatal Days 77-80;
- The F1 offspring not selected at weaning were sacrificed on Postnatal Day 22.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- A complete macroscopic post-mortem examination was performed on all F1 animals, including F1 pups culled on Postnatal Day 4 and not selected F1 pups on Postnatal Day 22 . This included 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.

Unscheduled deaths:
Animals found death or euthanized for humane reaons were submitted for a complete macroscopic post mortem examination. Macroscopic lesions were preserved.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table 1 (Section Additional Information were prepared for microscopic examination and weighed, respectively.

Histology
The tissues specified in Tissue Procedure Table (Table 1; Section Additional Information) were preserved in 10% buffered formalin (except for the eyes with optic nerves, testes and epididymides which were preserved in Modified Davidson's Fixative).
Ovaries (all groups from P Cohorts 1A and 1B) were fixed for at most 96 hours in formalin before being embedded in paraffin wax.
All tissues required for microscopic examination were trimmed based on the RITA guidelines, when applicable, embedded in paraffin wax, sectioned at a thickness of approximately 4 microns and stained with hematoxylin-eosin (except for the testes and epididymides which were stained with hematoxylin/PAS and for the right ovaries which were stained by PCNA immunohistochemistry). Ovaries and kidneys were fixed for at most 96 hours in formalin.
Five step-sections (100 µm apart in the middle third) were made for the right ovary.
One additional kidney slide of control and high-dose males (Control and high dose groups from Cohort 1A) was immunostained with an antibody for a2µ globulin.
Tissues of Cohort 1B were processed to the block stage. If microscopy is requested for animals that did not mate or conceive, of pregnant females that did not deliver, from females with abnormal estrous cycles, the reproductive organ blocks will be processed to histological slides.

Microscopic evaluation
A microscopic examination was performed on:
• all tissues listed in the Tissue Procedure Table (Table 1; Section Additional Information) from animals of the control and high-dose groups euthanized at the end of the treatment period,
• reproductive organs from animals that did not mate or conceive, from pregnant females that did not deliver, from females with abnormal estrous cycles and from males with abnormalities at sperm analysis, to investigate possible causes (Cohorts 1A and 1B),
• all macroscopic lesions of all groups (from F1 cohort), including those from found dead and prematurely euthanized animals,
• kidneys of males, and liver and thyroid glands of males and females from Cohort 1A males of low- and mid-dose groups euthanized at the end of the treatment period,
• thyroid glands from all Cohort 2B females euthanized at the end of the treatment period (Control and high dose groups).

Right ovary (groups 1 and 4): a detailed and careful microscopic examination was made of five step-sections of the right ovary of each Cohort 1A female, with enumeration of the total number of primordial follicles on PCNA-stained slides.
A vaginal smear was made on the day of necropsy to allow correlation with histopathology in reproductive organs. The smears were stained with Harris Shorr.
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
The statistical analysis of organ weight data (level of significance of 0.05 or 0.01) according to the sequence given in Figure 1 of the attached file.

Splenic Lymphocyte Immunophenotyping
Statistical analysis was performed using the sequence given in Figure 2 of the attached file.

Number of Primary Follicles/Corpora Lutea, Ano-genital Distance, Number of Nipples and Areolaes, Time of Preputial Separation/Vaginal Opening, Time to First Estrous After Vaginal Opening/Patency, Seminology, Hematology, Coagulation, Blood Biochemistry, Urinalysis, Thyroid Hormones, Anti KLH IgM, Auditory Startle Reflex Test, Motor Activity, Post-implantation Loss, Sex Ratio, Live Birth, Viability and Lactation Indexes
Statistical analysis was performed using the sequence given in Figure 3 of the attached file.
Reproductive indices:
Post-implantation loss = [(Number of implantation sites - Number of live pups)/ Number of implantation sites] x 100

Mating index = (Number of mated animals / Number of paired animals) x 100

Fertility index = (Number of pregnant female partners / Number of mated pairs) x 100

Gestation index = (Number of females with live born pups / Number of pregnant females) x 100
Offspring viability indices:
Live birth index = (Number of live pups on Postnatal Day 1 / Number of delivered pups) x 100

Viability index on Postnatal Day 4 = (Number of surviving pups on Postnatal Day 4 (before culling) / Number of delivered pups) x 100

Lactation index = [(Number of surviving pups on Postnatal Day 21 / Number of surviving pups on Postnatal Day 4 (after culling)] x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Males: There were no adverse clinical signs in test-item treated male rats at any dose level.
Ptyalism (hypersalivation), associated with reflux at dosing at 800 mg/kg bw/day, was recorded from 100 mg/kg bw/day with dose-related increased incidence and onset. These findings were considered to be test item-related but not adverse. Other findings observed with a low and/or similar incidence across the groups were those commonly observed in this species/strain of animals, and were therefore not considered to be test item treatment-related.

Females: At 800 mg/kg bw/day, close or at the end of the premating period, there was a series of clinical signs in 1 to 5 females (loud breathing, exophthalmos, hunched posture and/or piloerection) which were considered to be adverse based on their severity and/or duration.
At 300 and 100 mg/kg bw/day, there were no adverse clinical signs in test item-treated female rats. In female rats, ptyalism (hypersalivation), associated with reflux at dosing at 800 mg/kg bw/day, was recorded from 100 mg/kg bw/day with a dose-related increased incidence and onset. These findings were considered to be test item-related but not adverse. Other findings observed with a low and/or similar incidence across the groups were those commonly observed in this species/ strain of animals, and were therefore not considered to be test item treatment-related.

Details are given in Table 1 (males) and Table 2 (females) of the Section "Any other information on results".
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were no test-item related deaths.
In the 300 mg/kg bw/day group, two males were found dead on study days 102 and 116, respectively. At necropsy, no observations were noted (no microscopic examination performed). Occasionally, ptyalism was observed, accompanied in one of the males by alopecia and scabs. Both deaths were considered not related to test item.
In the 800 mg/kg bw/day group, one female was euthanised on Postnatal Day 3 (Study day 110) because of the death of her litter. This female had thinning of hair and ptyalism before euthanasia. At necropsy, no findings were observed. This death was not considered to be test item-related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males:
Premating and postmating: • at 800 mg/kg/day, when compared to controls, there was a lower mean body weight throughout the treatment (down to -26% on Days 127 to 134, p<0.001) and a low mean body weight gain (+307 g vs. +475 g in controls on Days 1 to 140, p<0.001). These findings were considered to be test item treatment-related and adverse.
At 300 mg/kg bw/day, when compared to controls, there was a lower mean body weight throughout the treatment (down to -15% on Day 140, p<0.001) and a low mean body weight gain (+377 g vs. +475 g in controls on Days 1 to 140, p<0.001). These findings were considered to be test item treatment-related and adverse.
At 100 mg/kg bw/day, when compared to controls, there was a lower mean body weight throughout the treatment (down to -9% on Days 120 to 134, p<0.01) and a low mean body weight gain (down to +9 g vs. +17 g in controls on Days 99 to 106, p<0.01). These findings were considered to be test item treatment-related and but not adverse.

Females:
Premating and postmating: At 800, 300 and 100 mg/kg bw/day, when compared to control animals, there were no effects on mean body weight and no test item treatment-related effects on mean body weight change during the premating period.
Gestation:
At 800 mg/kg bw/day and when compared to controls, mean body weight was lower from Gestation Day 7 (down to -10% vs. controls on Gestation Day 20 , p<0.001) resulting in low mean body weight gain over the pregnancy period (+111 g vs. +140 g in controls on Gestation Days 0 to 20, p<0.001). A test item-relationship cannot be ruled out, but taking into account the amplitude of the changes, these findings were considered to be non-adverse.
At 300 and 100 mg/kg bw/day, when compared to controls, there were no test item
treatment-related effects on mean body weight or mean body weight gain during the pregnancy period.
Lactation:
At 800 mg/kg/day and when compared to controls, there was a lower mean body weight during the lactation period (-11 to -9 % vs. controls on Days 1 to 21 p.p., p<0.001). This was consider to be the result of the lower body weight gain observed during the pregnancy period. However, a return towards control values can be observed from Day 4 p.p. (less than 10% differences vs. controls, despite the statistically significant differences) resulting in a higher body weight gain over the lactation period (+42 g vs. +27 g on Days 1 to 21 p.p., p<0.01).
At 300 and 100 mg bw/kg /day, when compared to controls, there were no test item treatment-related effects on mean body weight or mean body weight gain during the lactation period.

Details are given in Table 3 (males and females; absolute body weights), Table 4 (males and females; body weight change), Table 5 (females; gestation) and Table 6 (females; lactation) of the Section "Any other information on results".
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males:
Pre-mating and post-mating: At 800 mg/kg/day and when compared to controls, mean food consumption was mainly lower overall the treatment period, most pronounced from Days 1 to 8 (-13% vs. controls, p<0.01). A test item-relationship cannot be ruled out but taking into account the amplitude of the changes, this finding was considered to be non-adverse. At 300 and 100 mg/kg bw/day, when compared to controls, there were no adverse effects on mean food consumption.


Females:
Premating and post mating:
At 800, 300 and 100 mg/kg bw/day, when compared to controls, there were no adverse effects on mean food consumption. In test-item treated females and when compared to controls; there was higher mean food consumption (up to +27% vs. controls on Days 36-43 and 57-64 at 800 mg/kg bw/day). This finding was considered to be test item-related but non-adverse.
Gestation:
At 800, 300 and 100 mg/kg bw/day, when compared to controls, there were no adverse effects on mean food consumption (the few statistically significant differences were minimal and/or not dose-related).
Lactation:
At 800 mg/kg bw/day and when compared to controls, mean food consumption was lower over the Postnatal Day 4 - 21 period (down to -18% vs. controls on Postnatal Days 4 to 7, p<0.001); a test item-relationship cannot be ruled out, but taking into account the amplitude of the changes, this finding was considered to be non-adverse. At 300 and 100 mg/kg bw/day, when compared to controls, there were no adverse effects on mean food consumption.

Details are given in Table 7 (males and females (premating and postmating), Table 8 (females; gestation) and Table 9 (females; lactation).
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item treatment-related effects on hematology and/or coagulation in P generation animals. When compared to controls, the few statistically significant differences were of limited amplitude, were not dose-related, were observed in one sex only and/or were within the range of the Historical Control Data. Therefore a link with the test item-treatment was considered to be unlikely.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item treatment-related effects on blood biochemistry in P generation animals.
When compared to controls, the few statistically significant differences reported were of limited amplitude, not dose-related, observed in one sex only and/or were within the range of the Historical Control Data. Therefore a link with the test item-treatment was considered to be unlikely.
Endocrine findings:
effects observed, treatment-related
Description (incidence and severity):
In males, from 100 mg/kg bw/day and when compared to controls, there were dose-related decreases in mean T4 levels (down to -36% vs. controls at 800 mg/kg bw/day, p<0.01) associated with dose related increases in mean TSH levels (up to +130% vs. controls at 800 mg/kg bw/day, p<0.01). These findings, considered to be test item-related, correlated with thyroid follicular hypertrophy at microscopic examination and in view of hepatocellular hypertrophy could be considered as compensatory to the increased hepatic metabolism of thyroid hormones following induction of hepatic microsomal enzymes. These thyroid hormonal findings were not considered as adverse, but rather adaptive changes.
Details are given in Table 10 of the Section "Any other information on results".

In females, when compared to control or Historical Control Data (OECD443 - 2015-2021, n = 8 studies), there were no effects on mean T4 or TSH level.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item treatment-related effects on urinalysis parameters in P generation animals. When compared to controls, the few statistically significant differences reported were of limited amplitude, were not dose-related, were observed in one sex only and/or were within the range of the Historical Control Data. Therefore a link with the test item-treatment was considered to be unlikely.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related changes were noted in the kidneys, liver, thyroid glands and thymus.
Kidneys: Minimal to slight degeneration/necrosis was noted in the tubules of males treated at 800 mg/kg bw/day, associated with an increased incidence of pigment. In addition, minimal to moderate hyaline droplet accumulation and tubule dilatation were seen in the kidneys of males treated at = 100 mg/kg bw/day with increased severity and incidence when compared to controls.
It is noteworthy that there were no differences in the staining of Alpha-2µ-globulin immuno-histochemical slides between controls and high-dose males. However, the hyaline droplets were positively stained.
No test item-related changes were noted in the kidneys of females.

Liver: Minimal to moderate dose-related hepatocellular hypertrophy (centrilobular or diffuse) was noted in males and females treated at = 100 mg/kg bw/day.

Thyroid glands: Minimal dose-related follicular cell hypertrophy was noted in males and females treated at =100 mg/kg bw/day.

Thymus: Dose-related increased severity and incidence of thymic lymphoid decreased cellularity were seen in males treated at = 100 mg/kg bw/day and in females treated at 800 mg/kg bw/day when compared to controls.

Details are given in Table 13 of the Section "Any additional information on results".

The remaining microscopic findings were not considered to be associated with the test item administration because these findings were consistent with spontaneous background findings described in the literature, the findings were distributed randomly among the groups and/or their appearance was similar to changes found in controls. These included minimal alveolar macrophages or osseous metaplasia in the lungs, retinal atrophy, pigment in the spleen, forestomach hyperkeratosis, jejunal intussusception in isolated high-dose males, and pituitary gland cyst, necrosis/hemorrhage in the adrenal gland and tail fracture in isolated high-dose females. These changes were considered to be part of the spontaneous background findings.

Reproductive organs: There were no test item-related changes in the male or female reproductive organs. There was a good correlation between the vaginal smears and the histopathological examination of estrus cycle in all examined females.
Enumeration of Corpora Lutea in HE - Stained Slides: At enumeration of corpora lutea in the ovaries of high-dose and control females, there were no statistical differences in the mean number of corpora lutea: 8.68 (±5.00) and 8.50 (±3.30) per animal respectively.
Enumeration of the Number of Primordial Follicles on PCNA-Stained Slides: There were no statistically significant differences between the high-dose and control groups, with mean numbers of 13.41 (±8.90) and 12.46 (±8.89) primordial follicles per animal, respectively, on PCNA-stained slides.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
At 800 mg/kg bw/day and when compared to controls, there was a lower mean number of cycles
(1.6 vs. 2.4 in controls, p<0.01) as a consequence of an increased mean duration of cycles
(7.3 vs. 5.1 days in controls, not statistically significant) resulting in a lower percentage of females cycling normally (33.3% vs. 65.2% in controls, not statistically significant). A test item-relationship cannot be ruled out, but this finding was considered to be non-adverse based on the absence of impact on mating and fertility data and absence of microscopic correlates in female reproductive organs.
At 300 and 100 mg/kg bw/day, when compared to controls, there were no effects on mean estrous cycle parameters.

A comparison with Historical Control Data, collected in Sprague Dawley rats over a 15-day period, indicates that in the present study there were longer period of diestrus in all groups, including controls. Seasonal variations may have impacted the estrous cyclicity, even in a controlled environment.

Details are given in Table 14 of the Section "Any other information on results".
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
In test item-treated groups and when compared to controls, there were no effects on sperm parameters (motility, morphology, sperm/testicular numerations or daily sperm production rate).
Reproductive performance:
no effects observed
Description (incidence and severity):
At 800, 300 and 100 mg/kg bw/day, when compared to controls, there were no effects on mating or fertility data, reflected in the number of females mated, the female mating index, the mean number of days taken to mate and the female fertility index.
In addition, there were no effects on mean duration of gestation, delivery, number of implantation sites, number of live pups, post-implantation loss and live birth index.

Details on results (P0)

PATHOLOGY DISCUSSION
KIDNEY
In the P0 generation, adverse degeneration/necrosis was noted in the renal tubules of males treated at 800 mg/kg bw/day, together with pigment and dilatation of tubules. The other findings were considered to be non-adverse in view of their morphology, their limited distribution and their low magnitude and incidence. They included hyaline droplet accumulation that correlated with increased kidney weights in males treated at = 100 mg/kg bw/day and kidney enlargement at necropsy. Although there was no increase in the Alpha-2-u-globulin immuno-histochemical staining when compared to controls in the current study, this renal finding, positively stained and seen in males only, was highly suggestive of hyaline droplet nephropathy, a male rat specific change of no relevance to human. This could correlate with the increased urinary volume in males treated at 800 mg/kg bw/day.

LIVER AND THYROID
In the liver, hepatocellular hypertrophy was seen at =100 mg/kg bw/day in males and females and correlated with enlargement and accentuated lobular pattern at necropsy. This could correlate with increased proteinemia and albuminemia in males and females treated at = 300 mg/kg bw/day. In the thyroid glands, follicular hypertrophy correlated with increased weights at
= 100 mg/kg bw/day. In view of the low magnitude of the liver hypertrophy and in the absence of a degenerative associated process, it was not considered as adverse under the conditions of this study, but rather an adaptive change (Greaves, 2012).
This was consistent with microsomal enzyme induction that resulted in increased turnover of T4 (thyroxine) and secondary thyroid gland hypertrophy due to stimulation of the pituitary-thyroid-endocrine axis (Hall et al, 2012). This was confirmed by the increase in TSH and decrease in T4 noted in P generation males.

THYMUS
The decreased lymphoid cellularity seen in males at = 100 mg/kg bw/day and in females at 800 mg/kg bw/day correlated with macroscopic small thymus in males. A relationship to stress associated with the decreased body weights and food consumption could not be excluded.

REPRODUCTION AND FERTILITY
At quantitative evaluation of primordial follicles or corpora lutea, there were no differences between the high-dose and control groups. There were no test item-related macroscopic or microscopic lesions in male or female reproductive organs and the vaginal smears correlated with the estrous cycle in females. This was consistent with the estrous cycle, mating and fertility results in spite there were non-adverse lower mean number of cycles as a consequence of an increased mean duration of cycles which resulted in a lower percentage of females cycling normally at 800 mg/kg bw/day.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
P0 Systemic toxicity
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Remarks:
P0 Systemic toxicity
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical signs
Key result
Dose descriptor:
NOAEL
Remarks:
reproduction and fertility
Effect level:
> 800 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

Target system / organ toxicity (P0)

open allclose all
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
thyroid gland
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
During lactation:
There were no test item-related findings in F1 pups on Postnatal Day 1 (external examination, qualitative assessment of body temperature and/or activity/reaction to handling). From 300 mg/kg bw/day, there were increased incidences of pups with an abnormal yellowish urogenital region and skin dryness. A test item-relationship cannot be ruled out but in the absence of impact on pup viability, these findings were considered to be non adverse.
Details are given in Table 15 of the Section "Any other information on results".
The other findings were observed with low incidences, no dose level relationship and/or are common findings in this species and strain maintained in the experimental conditions of this study.

Cohort 1A:
At all dose levels, there were no adverse clinical signs in test item-treated male rats.
At 800 mg/kg bw/day, 9/19 males had an increased anus size during the Day 9-36 period. This finding was considered to be test item-related but not adverse based on its reversibility and/or an absence of associated effects.
There were no adverse clinical signs in test item-treated female rats at any dose level.
From 300 mg/kg/day, there was a dose-related increased incidence and onset of females with increased anus size. This finding was considered to be test item-related but not adverse based on its reversibility and/or an absence of associated findings.

Ptyalism (hypersalivation) was recorded in all groups (including controls) with a dose-related increased incidence and onset from 100 mg/kg bw/day. This finding was considered to be test
item-related but not adverse.
Other findings observed with a low and/or similar incidence across the groups were those commonly observed in this species/strain of animals, and were therefore not considered to be test item-related.

Cohort 1B:
There were no adverse clinical signs in test item-treated male rats at any dose level.
Ptyalism (hypersalivation) was recorded in all groups with a dose-related increase. This finding was considered to be test item-related but not adverse.
There were no adverse clinical signs in test-item treated female rats at any dose level.
At 800 mg/kg/day, 14/20 females had an increased anus size over the Day 21-78 period. This finding was considered to be test item-related but not adverse based on reversibility and/or an absence of associated effects.

Other findings observed with a low and/or similar incidence across the groups were those commonly observed in this species/strain of animals, and were therefore not considered to be test item-related.

Cohort 2A:
There were no adverse clinical signs in test-item treated male rats at any dose level.
At 800 mg/kg bw/day, 3/9 males had an increased anus size during the Day 9-42 period. This finding was considered to be test item-related but not adverse based on its reversibility and/or an absence of associated effects.
There were no adverse clinical signs in test item-treated female rats at any dose level.
From 300 mg/kg/day, females had an increased anus size. This finding was considered to be test item-related but not adverse based on its reversibility and/or an absence of associated effects.

Ptyalism (hypersalivation) was recorded in all groups with a dose-related increase. This finding was considered to be test item-related but not adverse.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Birth: At 800, 300 and 100 mg/kg bw/day, when compared to controls, there were no effects on mean number of live pups.

Lactation: There were no effects on the repartition of deaths in F1 lactating pups. At 800 mg/kg bw/day, one female was prematurely euthanized on Postnatal Day 3 because of the death of her litter. This isolated finding is a common observation in rats that was therefore not considered to be test item treatment-related. There was no test item-related effect on F1 pup viability index.
Before culling on Postnatal Day (PND) 4 and when compared to controls or Historical Control Data, there was a lower PND 4 viability index (85.7% vs. 94.0% in controls), mainly related to afore-mentioned female with a dead litter.

Cohort 1A:
No test item-related mortality was noted.
In the 300 mg/kg bw/day group, one male was prematurely euthanized on Day 12. Prior to euthanasia, thin appearance, pallor of extremities, increase abdomen size and soft feces were observed. At necropsy, histopathological examination revealed the following dosing-related changes: thymic adhesions to sternum (correlating with fibrosis in adjacent fat), adhesion and yellow mass in the lungs (correlating with moderate granulomatous inflammation), yellow discoloration in skeletal muscle (not submitted for microscopic examination), red discoloration in cecum (correlating with chronic transparietal inflammation and necrosis), red discoloration in the mesentery (not submitted for microscopic examination) and yellow mass in the diaphragm (correlating with chronic inflammation of the pulmonary serosa).
In the 800 mg/kg bw/day group, one male was found dead on Day 4. No clinical signs were observed before death and there were no findings at necropsy. The cause of this death could not be established, but it was considered to be unrelated to the test item administration, in view of the early occurrence, and incidental. This animal was replaced by another male.
In addition, one female was prematurely euthanized on Day 2 (PND 23) due to its poor health (soiled urogenital region, wound/scab on anus, pallor of extremities, cold to the touch, and prostration) observed before euthanasia.There was red discoloration of the cranium (correlating with focal hemorrhage), red pituitary gland (not submitted for microscopic examination), red lungs (correlating with congestion and atelectasia) and thickened wall of the rectum (correlating with chronic transparietal inflammation). The cause of moribundity was considered to be an accident, in view of the hemorrhage in the cranium and the early occurrence of this sacrifice.
A second female at 800 mg/kg bw/day found dead in Week 1 had dosing-related changes: esophageal perforation (correlating with myofiber degeneration/regeneration) and red content in the stomach and thoracic cavity (not submitted for microscopic examination).

Cohort 1B:
There were no unscheduled deaths in Cohort 1B males or females.

Cohort 2A:
In Cohort 2A males, one male in the 800 mg/kg bw/day group was found dead on Study Day 20. Ptyalism was observed on Days 13, 16 and 18. At necropsy, no macroscopic lesions were noted. This was an accidental death and thus was considered to be unrelated to the test item administration.
There were no unscheduled deaths in Cohort 2A females.

COHORT 2B
The evaluation of mortality was not applicable in this cohort.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
During lactation:
At 800 mg/kg bw/day and when compared with controls, there were lower mean body weights in males and females from PND4 (down to -27% and -26% vs. controls on PND 7 in males and females with p<0.001, respectively) and lower mean body weight gains throughout the lactation period. These findings were considered to be adverse.
At 300 and 100 mg/kg bw/day, there were no effects.
Details are given in Table 16 of the Section "Any additional information on results".

Cohort 1A:
Males: at 800 mg/kg bw/day, when compared to controls, there was a lower mean body weight throughout the treatment period (almost -20%, p<0.01) and a low mean body weight gain (+358 g vs. +448 g in controls on Days 1 to 64, p<0.01). These findings were considered to be test item treatment-related and adverse (no recovery in terms of body weight or body weight gain). At 300 mg/kg bw/day, when compared to controls, there was a lower mean body weight (down to -10% on Day 64, p<0.01) and a low mean body weight gain (+400 g vs. +448 g in controls on Days 1 to 64, p<0.01). These findings were considered to be test item treatment-related but not adverse (no more than 10% difference in terms of body weight and a return towards control values in terms of body weight gain). at 100 mg/kg bw/day, when compared to controls, there were no test item-related differences.
Females: at 800 mg/kg bw/day, when compared to controls, there was a lower mean body weight during the first 3 weeks of the treatment period (down to -21% on Day 8, p<0.01) and a lower mean body weight gain during the first week of the treatment period (+28 g vs. +38 g in controls, p<0.01). Thereafter, mean body weight and mean body weight gain returned towards controls values. Therefore, these findings were considered to be test item-related but not adverse based on their reversibility. At 300 and 100 mg/kg bw/day, when compared to controls, there were no test item-related differences.
Details are given in Table 17 of the Section "Any other information on results".

Cohort 1B:
Males: at 800 mg/kg/day, when compared to controls, there was a lower mean body weight throughout the treatment period (-19% to -22%, p<0.001) and a low mean body weight gain (+372 g vs. +469 g in controls on Days 1 to 71, p<0.001). These findings were considered to be test item treatment-related and adverse (no recovery in terms of body weight or body weight gain). At 300 mg/kg bw/day, when compared to controls, there was a lower mean body weight and a low mean body weight gain. These findings were considered to be test item treatment-related but not adverse (no more than 10% difference in terms of body weight and a return towards control values in terms of body weight gain). At 100 mg/kg/day, when compared with controls, there were no test item-related differences.
Females: At 800 mg/kg bw/day, when compared to controls, there was a lower mean body weight during the first 4 weeks of the treatment period (down to -21% on Day 8, p<0.001) and a lower mean body weight gain during the first week of the treatment period (+29 g vs. +37 g in controls, p<0.001). Thereafter, mean body weight and mean body weight gain returned towards controls values. Therefore, these findings were considered to be test item-related but not adverse based on their reversibility. At 300 and 100 mg/kg bw/day, when compared to controls, there were no test item-related differences.
Details are given in Table 18 of the Section "Any other information on results".

Cohort 2A:
Males: at 800 mg/kg bw/day, when compared to controls, there was a lower mean body weight throughout the treatment period (-17% to -25%, p<0.01) and a low mean body weight gain (+326 g vs. +397 g in controls on Days 1 to 50, p<0.01). These findings were considered to be test item treatment-related and adverse (no recovery in terms of body weight or body weight gain). At 300 mg/kg bw/day, when compared to controls, there was a lower mean body weight and a low mean body weight gain. These findings were considered to be test item treatment-related but not adverse (no more than 10% difference in terms of body weight and a return towards control values in terms of body weight gain). At 100 mg/kg bw/day, when compared to controls, there were no test item-related differences.
Females: At 800 mg/kg bw/day, when compared to controls, there was a lower mean body weight during the first 4 weeks of the treatment period (down to -23% on Days 1 and 8, p<0.01) and a lower mean body weight gain during the first week of the treatment period (+28 g vs. +38 g in controls, p<0.01). Thereafter, mean body weight and mean body weight gain returned towards controls values. Therefore, these findings were considered to be test item-related but not adverse based on their reversibility. At 300 and 100 mg/kg bw/day, when compared to controls, there were no test item-related differences.
Details are given in Table 19 of the Section "Any other information on results".
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A:
Males: At 800 mg/kg bw/day and when compared to controls, mean food consumption was lower on Days 1 to 15 (down to -24% vs. controls on Days 1 to 8, p<0.01), with a tendency towards a return to control values thereafter. A test item-relationship cannot be ruled out but taking into account the amplitude of the changes, this finding was considered to be non-adverse. At 300 and 100 mg/kg bw/day, when compared to controls, there were no adverse effects on mean food consumption.
Females: At 800, 300 and 100 mg/kg bw/day, when compared to controls, there were no adverse effects on mean food consumption. In test item-treated females and when compared to controls, mean food consumption was lower on Days 1 to 8 (-21% vs. controls, p<0.01), with a return to control values thereafter with higher mean food consumption (up to +27% vs. controls on Days 36-43 at 800 mg/kg bw/day). This finding was considered to be test item-related but non-adverse.
Details are given in Table 20 of the Section "Any other information on results".

Cohort 1B:
Males: At 800 mg/kg bw/day and when compared to controls, mean food consumption was lower on Days 1 to 22 (down to -22% vs. controls on Days 1 to 8, p<0.01), with a tendency towards a return to control values thereafter. A test item-relationship cannot be ruled out but taking into account the amplitude of the changes, this finding was considered to be non-adverse.
At 300 and 100 mg/kg bw/day, when compared to controls, there were no adverse effects on mean food consumption.
Females: At 800, 300 and 100 mg/kg bw/day, when compared to controls and despite a few statistically significant differences, there were no adverse effects on mean food consumption.
Details are given in Table 21 of the Section "Any other information on results".

Cohort 2A:
There were no effects on mean food consumption in Cohort 2A animals.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1A:
There were no test item treatment-related effects on hematology and/or coagulation in Cohort 1A animals.
When compared to controls, the few statistically significant differences reported were of limited amplitude, were not dose-related, were observed in one sex only and/or were within the range of the Historical Control Data. Therefore a link with the test item-treatment was considered to be unlikely.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1A:
There were no test item treatment-related effects on blood biochemistry in Cohort 1A animals.
When compared to controls, the few statistically significant differences reported were of limited amplitude, were not dose-related, were observed in one sex only and/or were within the range of the Historical Control Data. Therefore a link with the test item-treatment was considered to be unlikely.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1A:
There were no test item treatment-related effects on urinalysis parameters in Cohort 1A animals.
When compared to controls, the few statistically significant differences reported were of limited amplitude, were not dose-related, were observed in one sex only and/or were within the range of the Historical Control Data. Therefore a link with the test item-treatment was considered to be unlikely.
Sexual maturation:
effects observed, treatment-related
Description (incidence and severity):
Balano-preputial separation
At 800 mg/kg bw/day and when compared to controls (or Historical Control Data), there was an increase in the mean age at balanopreputial separation (51.0 vs. 48.2 days, p<0.01). At this age, the mean body weight of the pups was lower (-10% vs. controls, p<0.01). While a test-item relationship cannot be excluded, the mean values remained within the range of the Historical Control Data. Therefore, in the absence of adverse findings at sperm analysis (see section Sperm Analysis) and in the absence of macro-/microscopic observations in male reproductive organs (see section Pathology), these delays were considered to be non-adverse.
At 300 and 100 mg/kg bw/day, when compared to controls or Historical Control Data, there were no effects.
Details are given in Table 24 of the Section "Any additional information on results".

Vaginal opening
At 800 mg/kg bw/day and when compared to controls (or Historical Control Data), there was an increase in the mean age at vaginal opening (36.4 vs. 33.4 days, p<0.01). While a test-item relationship cannot be excluded, the mean value remained within the range of the Historical Control Data. Therefore, in the absence of adverse findings on estrous cycle (see Cohort 1A, section Estrous Cycle) and in the absence of macro-/microscopic observations in female reproductive organs (see section Pathology), this delay was considered to be non-adverse.
At 300 and 100 mg/kg bw/day, when compared to controls or Historical Control Data, there were no effects.
Details are given in Table 25 of the Section "Any additional information on results".
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
There were no effects on postnatal Day 1 mean anogenital distance (AGD) or normalized AGD, in males or females.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
There were no nipples or areolae in male pups examined on Postnatal Day 12.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
NON-SELECTED PUPS
There were no test item-related organ weight differences.
No organ weight changes were considered to be test item-related because they were of insufficient magnitude and/or were not dose-related. These included various decreases in absolute weights and/or increases in relative-to-body weights seen in brain, spleen and thymus that were related to the decreases in body weights in males and females treated at 800 mg/kg bw/day (up to -27%; p<0.01).

COHORT 1A
There were test item-related organ weight differences in kidneys, liver and thyroid glands.
Increased absolute and/or relative-to-body kidney weights were noted in males treated at
= 100 mg/kg bw/day and in females treated at = 300 mg/kg bw/day, and correlated with tan discoloration at necropsy and hyaline droplet accumulation at microscopic examination.
Increased absolute and/or relative-to-body liver weights were noted in males treated at
= 300 mg/kg bw/day and in females treated at =100 mg/kg bw/day, and correlated with hepatocellular hypertrophy at microscopic examination.
Increased absolute and/or relative-to-body thyroid gland weights were noted in males and females treated at = 300 mg/kg bw/day, and correlated with follicular hypertrophy at microscopic examination.
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 with microscopic findings. These included the various decreases in absolute weights and/or increases in relative-to-body weights seen in adrenal glands, brain, epididymides, heart, pituitary gland, prostate, spleen, testes and thymus were related to the decreases in body weights in males treated at = 300 mg/kg bw/day (down to -22%; p<0.01).
Details are given in Table 27 of Section "Any other information on results".

COHORT 1B
There were no test item-related organ weight differences.
No organ weight changes were considered to be test item-related because they were of insufficient magnitude, were not dose-related and/or did not correlate with microscopic findings. These included the increased absolute and relative-to-body pituitary gland weight in males treated at 100 mg/kg bw/day (up to +21% in relative-to-body weight; p<0.01). This difference had no macroscopic correlates and was not dose-related. Therefore it was considered to be unrelated to the test item administration. In addition, various decreases in absolute weights and/or increases in relative-to-body weights were seen in brain, epididymides, prostate, seminal vesicles and testes were related to the decreases in body weights in males treated at = 300 mg/kg bw/day (up to -22%; p<0.01).

COHORT 2A
There were test item-related organ weight differences in kidneys, liver and t= hyroid glands.
Increased relative-to-body kidney weights were noted in males treated at = 300 mg/kg bw/day.
Increased absolute and/or relative-to-body liver weights were noted in males treated at
= 300 mg/kg bw/day and in females treated at = 100 mg/kg bw/day, and correlated with enlargement at necropsy.
Increased absolute and relative-to-body thyroid gland weights were noted in males treated at 800 mg/kg bw/day.
No other organ weight changes were considered to be test item related because they were of insufficient magnitude, were not dose-related and/or did not correlate with microscopic findings. These included various decreases in absolute weights and/or increases in relative-to-body weights seen in brain, heart, pituitary gland, seminal vesicles, epididymides and testes that were related to the decreases in body weights in males treated at 800 mg/kg bw/day (-20%; p<0.01).
Details are given in Table 28 of Section "Any other information on results".

COHORT 2B
There were test item-related organ weight differences in thyroid glands.
Increased relative-to-body thyroid gland weights were noted in females treated at = 300 mg/kg bw/day (up to +62%; p<0.01). This correlated with microscopic follicular hypertrophy.
No other organ weight changes were considered to be test item related because they were of insufficient magnitude, were not dose-related and/or did not correlate to microscopic findings. These included various decreases in absolute weights and/or increases in relative-to-body weights seen in adrenal gland, brain, epididymides, heart, kidneys, liver, testes, thymus, prostate and spleen were related to the decreases in body weights in males and females treated at 800 mg/kg bw/day (down to -27%; p<0.01). In addition, increased absolute and relative-to-body weights of uterus were observed in females treated at 800 mg/kg bw/day. This was related to an outlier that had a green mass correlating with transparietal necrosis. In view of the isolated occurrence of this change and its absence in the other cohorts, it was considered to be unrelated to the test item administration.
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.
Details are given in Table 29 of the Section "Any other information on results".
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
DEAD PUPS OF PARENTAL GENERATION
There were no test item-related findings. The few isolated gross observations were considered to be consistent with spontaneous findings encountered in rats of this strain and age.

NON-SELECTED PUPS
There were no test item-related gross changes. The findings in the tail (black discoloration and shortening) were seen in control females.

CULLED PUPS
There were no test item-related gross changes. There was a dilated pelvis in 1/46 pups at 800 mg/kg bw/day and a dilated ureter in 1/82 pups at 300 mg/kg bw/day and 1/46 pups at 800 mg/kg bw/day. These changes were considered to be unrelated to the test administration as no similar lesions were seen in the kidneys and ureters of examined animals from the other cohorts of the study and as the incidences of these findings were very low.

PUPS EUTHANIZED AFTER WEANING
There were no test item-related findings in any group. The few findings were observed with low incidences, no dose level relationship and/or are common observations in this species and strain maintained in the experimental conditions of this study.

COHORT 1A
There were test item-related findings in kidneys and liver.
In the kidneys, tan discoloration was noted in 1/19 males treated at 300 mg/kg bw/day and 2/20 males treated at 800 mg/kg bw/day. This correlated with hyaline droplet accumulation at microscopic examination.
In the liver, enlargement was noted in 1/19 males treated at 300 mg/kg bw/day. Accentuated lobular pattern was noted in 1/20 males and 1/20 females treated at 100 mg/kg bw/day, 4/19 males and 1/20 females treated at 300 mg/kg bw/day and 2/20 males and 2/19 females treated at 800 mg/kg bw/day. These changes correlated with hepatocellular hypertrophy at microscopic examination.
The other isolated gross observations were considered to be consistent with spontaneous findings encountered in rats of this strain and age. These included the isolated ileal intussusception in one high-dose male and the isolated exophtalmia and red discoloration of the eyes, small thymus, pulmonary yellow mass (correlating with congestion and atelectasis) and adhesion to the ribs (correlating with granulomatous inflammation) in the lungs, enlarged mandibular lymph nodes, red skin and black discoloration of harderian glands in occasional high-dose females that correlated with common microscopic findings and were considered to be part of the spontaneous background findings.
Details are give in Table 26 of the Section "Any other information on results".

COHORT 1B
There were no test item-related findings.
The isolated gross observations were considered to be consistent with spontaneous findings encountered in rats of this strain and age. They included the cysts with translucent content in the ovaries of 1 mid-dose and 1 high-dose female. There was also accentuated lobular pattern in 1/20 low-dose males. In view of the low incidence of this change and the absence of a dose- relationship, it was considered to be unrelated to the test item administration.

COHORT 2A
There were no test item-related findings except enlarged liver in 1/10 females treated at 800 mg/kg bw/day.
The other isolated gross observations were considered to be consistent with spontaneous findings encountered in rats of this strain and age. These included black discoloration in the stomach (correlating with erosion), white discoloration in the forestomach (no correlates) and scabs (correlatingd with crusts) in 1/10 low and 1/10 high-dose females.

COHORT 2B
There were no test item-related findings.
The isolated gross observations were considered to be consistent with spontaneous findings encountered in rats of this strain and age (including the renal cysts and deformation, scabs and thickening in the skin of 1/10 females treated at 800 mg/kg BW/day). In addition, there was a green mass in the uterus of one high-dose female that correlated with transparietal necrosis at microscopic examination and with macroscopic adhesion in the spleen (considered to be contiguous inflammation).
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
NON-SELECTED PUPS
No examination was performed.

COHORT 1A
Test item-related changes were noted in the kidneys, liver and thyroid glands.

Kidneys: Minimal to moderate hyaline droplet accumulation was seen in the kidneys of males treated at = 300 mg/kg bw/day with increased severity and/or incidence when compared to controls. A relationship with the test item administration for the minimal to slight dilatation of tubules in 3/20 males treated at 100 mg/kg bw/day, 3/20 males treated at 800 mg/kg bw/day and 2/19 females treated at 800 mg/kg bw/day was considered to be equivocal in view of the low incidence of this change.

Liver: Minimal to slight hepatocellular hypertrophy (centrilobular) was noted in males and females treated at = 100 mg/kg bw/day. Of note, slight degeneration/necrosis was seen in 1/19 males treated at 800 mg/kg bw/day, and minimal bile duct hyperplasia was seen in another male treated at 800 mg/kg bw/day. A relationship with the test item administration was considered to be equivocal in view of the low incidence of these changes.

Thyroid glands: Minimal to slight follicular cell hypertrophy was noted in males and females treated at = 100 mg/kg bw/day. One female treated at 800 mg/kg bw/day had focal unilateral hyperplasia of the follicular cells. A relationship with the test item administration was considered to be equivocal in view of the low incidence of this change.

The remaining microscopic findings were not considered to be associated with the test item administration because these findings were consistent with spontaneous background findings described in the literature, the findings were distributed randomly among the groups, and/or their appearance was similar to changes found in controls. These findings included dilatation of deferent duct, decreased lymphoid cellularity of the thymus, mononuclear cell infiltrates in the urinary bladder or lungs, intussusception in the ileum, intrasinusoidal erythrocytes and pigment in the renal lymph node in isolated high-dose males, and hemorrhage with erosion/ulcer in the cornea, decreased lymphoid cellularity of the thymus, intrasinusoidal erythrocytes with increased germinal centers in the mandibular lymph node, intimal thickening in the heart, dilatation of the pelvis, fibrosis in the kidney, edema in the submucosa of the stomach and pigment in the harderian gland in isolated high-dose females. These changes were considered to be part of the spontaneous background findings.

The malignant lymphoma seen in the mesenteric lymph node of one male at 300 mg/kg bw/day was considered to be spontaneous because it was seen in only one male in the mid-dose group and because this tumor may occur as early as 19 weeks old (Matsushima et al, 2010).

Reproductive organs:
There were no test item-related changes in the male or female reproductive organs. There was a good correlation between the vaginal smears and the histopathological examination of estrous cycle in all examined females.
Enumeration of corpora lutea (HE-stained slides): There were no differences at the enumeration of corpora lutea in the ovaries of high-dose and control females. The mean numbers of corpora lutea were 17.16 (± 6.57) and 16.90 (± 8.05) per animal, respectively, reaching no statistical significance.
Enumeration of the Number of Primordial Follicles on PCNA-Stained Slides: There were no differences at the enumeration of primordial follicles in the ovaries of high-dose and control females. The mean numbers of primordial follicles were 12.76 (± 5.15) and 13.17 (± 6.92) per animal, respectively, reaching no statistical significance.

Details are given in Table 30 of the Section "Any other information on results".

COHORT 1B
Microscopic examination of macroscopic lesions: There were no test item-related findings.
No microscopic findings were considered to be associated with the test item administration because the findings were consistent with spontaneous background findings described in the literature, were distributed randomly among groups, and/or their appearance was similar to changes found in controls.

COHORT 2A
Microscopic examination of macroscopic lesions: There were no test item-related findings.
No microscopic findings were considered to be associated with the test item administration because they were consistent with spontaneous background findings described in the literature, were distributed randomly among groups, and/or their appearance was similar to changes found in controls.

COHORT 2B
Microscopic examination of macroscopic lesions and of thyroid glands of all females: There was test item-related minimal follicular cell hypertrophy in the thyroid glands of 2/10 females treated at 300 mg/kg bw/day and 5/10 females treated at 800 mg/kg bw/day. This correlated with the increased weights.
No other microscopic findings were considered to be associated with the test item administration because they were consistent with spontaneous background findings described in the literature, were distributed randomly among groups, and/or their appearance was similar to changes found in controls. The findings included focal transparietal necrosis seen in the uterus of 1/10 females treated at 800 mg/kg bw/day, associated with spleen and peritoneal inflammation.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
THYROID HORMONES COHORT 1A
Males: At 800 mg/kg bw/day and when compared to controls, there was a decrease in mean T4 level (-22% vs. controls, p<0.05) associated with an increase in mean TSH level (+118% vs. controls, p<0.05). These findings, considered to be test item-related, correlated with thyroid follicular hypertrophy at microscopic examination and in view of hepatocellular hypertrophy could be considered as compensatory to the increased hepatic metabolism of thyroid hormones following induction of hepatic microsomal enzymes. These thyroid hormonal findings were not considered as adverse, but rather adaptive changes.
Details are given in Table 22 of Section "Any additional information on results".

Females: from 100 mg/kg bw/day and when compared to controls, there were dose-related decreases in mean T4 levels (down to -20% vs. controls at 800 mg/kg bw/day) associated with dose related increases in mean TSH levels (up to +109% vs. controls at 800 mg/kg/day, p<0.01). These findings, considered to be test item-related, correlated with thyroid follicular hypertrophy at microscopic examination and in view of hepatocellular hypertrophy could be considered as compensatory to the increased hepatic metabolism of thyroid hormones following induction of hepatic microsomal enzymes. These thyroid hormonal findings were not considered as adverse, but rather adaptive changes.
Details are given in Table 23 of Section "Any additional information on results".

SPERM ANALYSIS COHORT 1A
There were no test item-related effects on sperm parameters (motility, morphology, sperm/testicular numerations or daily sperm production rate).
When compared to controls, the few statistically significant differences reported in the above table were of limited amplitude, were not dose-related, had no microscopic correlates at histopathological examination, and/or were within the range of the Historical Control Data. Therefore a relationship with the test item-treatment was considered to be unlikely.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
AUDITORY STARTLE TEST
There were no test item-related effects on the mean latency time. At 100 mg/kg bw/day and when compared to controls, the isolated statistically significant difference in males (25 vs. 24 ms, p<0.05) was considered to be fortuitous based on its minimal difference and an absence of any dose level relationship.
From 300 mg/kg/day and when compared to controls, there were dose-related decreases in the magnitude of the responses (down to -39% vs. controls in males on Trials 41 to 50 with p<0.01 and -44% vs. controls in females on Trials 31 to 40 with p<0.01 at 800 mg/kg/day). Taking into account the dose level relationship, the amplitudes of the differences and the persistence of this finding over the trials, these changes were considered to be test item-related but non adverse in the absence of findings at auditory reflex evaluation (see section Reactivity to Manipulation or to Different Stimuli).
At 100 mg/kg bw/day and when compared to controls, the isolated statistically significant differences in females were considered to be fortuitous based on the absence of a dose level relationship.
Details are given in Table 31 of the Section "Any other information on results".
REACTIVITY TO MANIPULATION OR TO DIFFERENT STIMULI (FUNCTIONAL OBSERVATION BATTERY)
Cohort 2A animals were tested once between Postnatal Days 63 to 65. 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) was normal,
• fur appearance was normal when compared to controls,
• there was no excessive grooming, defecation or urination in treated groups when compared to the control group,
• there was no salivation, no lacrimation, no piloerection and no palpebral closure,
• all pups had a normal pupil size (myosis) at examination and there was no exophthalmos,
• all pups had normal gait, posture, behavior and breathing,
• 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 pupillary reflex were normal in all groups,
• auditory startle reflex was normal in all groups,
• forelimb grip strength was normal in all groups.
MOTOR ACTIVITY
The motor activity test addressed horizontal movements and rearing. There were no effects on the mean number of horizontal movements or rearing.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
effects observed, non-treatment-related
Description (incidence and severity):
LYMPHOCYTE SUBTYPING (COHORT 1A)
Relative counts: No significant differences were observed in terms of relative count in test item-treated animals when compared to controls (excepted for B cell subset, which was found to be increased in females treated at high dose: 45.7% compared to 39.3% in control females).

Absolute counts: In terms of absolute count, no major differences were evidenced in animals treated at the low or high dose. In contrast, all female subsets treated at the mid dose (test item at 300 mg/kg bw/day) were found to have significantly decreased absolute counts when compared to the control group. The same trend towards a decrease was found in males at 300 mg/kg bw/day when compared to the control group, even though the results were not statistically significant.
Regarding the different evaluated spleen-derived lymphocyte subpopulations, the main observation was based on statistically significant decreases found in the different studied cell subsets in both sexes, but only in the mid dose group (animals treated at 300 mg/kg/day) and only in term of absolute count. Consequently, there was no dose-related treatment effect.

Details on results (F1)

ESTROUS CYCLES (COHORT 1A)
When compared to controls, there was no significant difference in the mean time of the
first estrous after the onset of vaginal patency. There were no effects on mean estrous cycle parameters (mean number of days in diestrus, proestrus, estrus, metestrus, mean number of cycles, mean duration of cycles, mean percent of females cycling normally and mean percent of females with all stages).
At quantitative evaluation of primordial follicles or corpora lutea, there were no differences between the high-dose and control groups. There were no test item-related macroscopic or microscopic lesions in male or female reproductive organs and the vaginal smears correlated with the estrous cycle in females. This was consistent with estrous cycle results.

NEUROHISTOPATHOLOGY
COHORT 2A
Brain weights:
There were no test item-related brain weight differences. The lower absolute weight (-9%; p<0.01) and increased relative to body weight (+12%; p<0.01) in males treated at 800 mg/kg bw/day were considered to be related to the decreased in body weight (-20%; p<0.01). The other observed differences were minimal and/or not dose-related. These included lower absolute brain weights in males treated at 300 mg/kg bw/day (-7%; p<0.05).

Qualitative Microscopic Examination:
The examination 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), brainstem 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, skeletal muscle or spinal cord.

Morphometric Measurements of Regions of Interest in Cerebral Cortex (Level 3), Hippocampus (Level 4) and Cerebellum (Level 7)
A minimum of 6 (up to 10) brains/group/sex that were considered as appropriate were obtained for all cortex, hippocampus and cerebellum measurements in control and high-dose males and females. Males treated at 800 mg/kg bw/day had lower cortex (L3 L2+6: -6%; p<0.05) and hippocampus (L4 1: -13%; p<0.01 and L4 3: -9% p<0.05) thickness measurements. These differences were considered to be ambiguous as they were of low magnitude and were most probably secondary to the absolute brain weight decrease (-9%; p<0.01). Other changes in the thickness of these areas in males at 100 and 300 mg/kg bw/day were not considered to be toxicologically significant in view of their direction, low magnitude, and/or lack of a dose-relationship. Females treated at 800 mg/kg bw/day had lower hippocampus thickness measurements (L4 3: -6% p<0.01) when compared to controls. This difference was considered to be ambiguous as it was seen in a single brain region, and was of low magnitude.
The other measurements were similar in test item-treated animals and controls.
Details are given in Table 31 of the Section "Any other information on results".

COHORT 2B
Brain Weights
There were no test item-related brain weight differences. The lower absolute weight (down to
-9%; p<0.01 or 0.05) and increased relative to body weight (up to +28%; p<0.01) in males and females treated at 800 mg/kg bw/day were considered to be related to the decreased in body weight (down to -27%; p<0.01). The other observed differences were minimal and/or not dose-related.

Qualitative Microscopic Examination
The examination 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), brainstem 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, skeletal muscle or spinal cord.

Morphometric Measurements of Regions of Interest in Cerebral Cortex (Level 3), Hippocampus (Level 4) and Cerebellum (Level 7)
A minimum of 6 (up to 10) brains/group/sex, considered as appropriate, were obtained for all cortex, hippocampus and cerebellum measurements in control and high-dose males and females, except for the L3 4+8 and L7 measurements in control males (n=5 for both) and for the L7 measurement in high-dose males (n=5). The comparisons for these parameters were considered to be of lower reliability than for the other measurements. However, the achieved number of adequate brain sections was close to the target and this was considered to have no negative impact on the definitive conclusions. There were no statistically significant differences between controls and animals treated at 800 mg/kg bw/day for the measurements performed.

Neuropathology discussion: At neurohistopathology, there were ambiguous test item-related changes.
In cohort 2B (PND 22), there were no test item-related changes. In cohort 2A (PND 75-90), there were no differences between controls and the high-dose groups for the measurements performed except in males at = 100 mg/kg/day, which had lower cortex measurements (1 measurement out of 4) and hippocampus (2 measurements out of 3) and in females treated at 800 mg/kg/day that had lower measurements in hippocampus (1 measurement out of 3). In males, the relationship to test item of these differences were considered to be ambiguous based on the integrative weight-of-evidence approach of Garman et al. (2016) as they were not dose-related, were of low magnitude and were most probably secondary to the absolute brain weight decrease. In males at 800 mg/kg bw/day, there was a -20% decreased body weight, similar to the -9% decreased absolute brain weights; a relationship between low body weight and smaller brain is likely (Carney et al, 2004). These statistically significant differences appeared to be inconsistent: a similar difference was not seen in 2A females for the cortex, or in the males and females from cohort 2B. There was an absence of test item treatment-related gross abnormalities (brain size reduction readily visible as a gross lesion) or microscopic alteration in the central nervous system, either in parent or 1A, 2A or 2B F1 cohorts, either in males or females. No microscopic degenerative abnormalities, such as dysplasia, hypoplasia, hypo/-dysmyelination or ectopia, or neuronal necrosis, myelinic edema or vacuolation were present in these brains. Finally there was no evidence of test item treatment-related nervous behavioral effects when compared to controls, except dose-related decreases in the magnitude of the responses in the auditory startle test in males treated at = 300 mg/kg bw/day.
Similarly, Cohort 2A females treated at 800 mg/kg bw/day had lower hippocampus measurements when compared to controls. The relationship to test item of this difference was considered to be ambiguous as it was isolated (seen in a single brain region), not dose-related, of low magnitude and did not correlate with any macroscopic/microscopic lesions or clinical signs except decreases in the magnitude of the responses in the auditory startle test in females treated at 800 mg/kg bw/day.
In cohort 2B, there was no test item-related mortality. Increased relative-to-body thyroid gland weights were noted in females treated at = 300 mg/kg bw/day. These correlated with non-adverse microscopic follicular cell hypertrophy. There were no gross test item-related findings.

PATHOLOGY DISCUSSION
KIDNEY, LIVER AND THYMUS
In cohort 1A, similar non-adverse lesions were noted as in the P0 generation, but with lower severity and fewer affected tissues: there were test item-related organ weight increases in the kidneys at = 100 mg/kg bw/day that correlated with tan discoloration at necropsy, hyaline droplet accumulation at microscopic examination and increased phosphoremia at clinical pathology in males treated at 800 mg/kg bw/day, in the liver at =100 mg/kg bw/day that correlated with hepatocellular hypertrophy at microscopic examination and with increased proteinemia and albuminemia in males treated at 800 mg/kg bw/day and females treated at = 100 mg/kg bw/day, and in the thyroid glands at = 300 mg/kg bw/day that correlated with follicular hypertrophy at microscopic examination.
In cohort 1B, no test item-related mortality, no organ weight differences and no gross or microscopic findings were noted.
In cohort 2A, there were test item-related organ weight increases in the kidneys of males at
= 300 mg/kg bw/day, in the liver at =100 mg/kg bw/day that correlated with enlargement at necropsy, and in the thyroid glands of males treated at 800 mg/kg bw/day. There were no gross test item-related findings except the enlarged liver in 1/10 females at 800 mg/kg bw/day. There were no microscopic test item-related lesions (microscopic examination performed on gross lesions only).

In line with the findings of similar nature in the P0 generation, the renal findings were suggestive of hyaline droplet nephropathy, a male rat specific change of no relevance to human. The liver and thyroid findings were evaluated as non-adverse and rather adaptive changes.

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
F1 Reproduction toxicity (Sexual development postweaning)
Generation:
F1 (cohort 1A)
Effect level:
> 800 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
No effect on time to first estrous and estrous cycles.
Key result
Dose descriptor:
NOAEL
Remarks:
F1 Reproduction toxicity (Sexual development post weaning)
Generation:
F1 (cohort 1A)
Effect level:
> 800 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
No effect on sperm parameters
Key result
Dose descriptor:
NOAEL
Remarks:
Developmental immunotoxicity (limited evaluation)
Generation:
F1 (cohort 1A)
Effect level:
> 800 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
Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Remarks:
F1 systemic toxicity (post weaning)
Generation:
F1 (cohort 2A)
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat. (total fraction)
Sex:
male
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Remarks:
F1 systemic toxicity (from weaning onwards)
Generation:
F1
Effect level:
> 800 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOEL
Remarks:
Developmental neurotoxicity
Generation:
F1 (cohort 2A)
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
developmental neurotoxicity
Remarks on result:
other: At neurohistopathology (Cohort 2B), there were ambiguous test item-related changes (lower cortex thickness in males and lower hippocampus thickness in females at 800 mg/kg bw/day.

Target system / organ toxicity (F1)

open allclose all
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
thyroid gland
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

CHEMICAL ANALYSIS OF THE DOSE FORMULATIONS


The concentrations of the dose formulations prepared for treatments were all found to be within an acceptable range of variations (measured concentrations at ± 15% of the theoretical concentrations). No test item was found in the control dose formulation.


(TO BE COMPLETED WHEN APPENDIX 3 IS AVAILABLE: Actual measured dose concentrations need to be included)


 


CLINICAL SIGNS P0 ANIMALS


Table 1: Clinical Signs in P Generation Terminated as Scheduled Males (Number of Animals Affected per Group)

































































































Dose level
(mg/kg bw/day)



0



100



300



800



Number terminated as scheduled males



24



24



22



24



Ptyalism (before treatment)



 



 



7


(Days 25-60)



8


(Days 26-59)



Ptyalism (after treatment)



2


(Days 87-139)



9


(Days 31-143)



22


(Days 18-143)



24


(Days 9-143)



Reflux at dosing



 



 



 



1


(Day 57)



Soiled (head, nose, neck, forelimbs)



2
(Days 56-122)



2
(Days 27-141)



 



1


(Day 27)



Alopecia and/or Thinning of hair



8


(Days 41-144)



3


(Days 71-138)



4


(Days 62-144)



 



Chromodacryorrhea and/or Chromorhynorrhea



3


(Days 25-93)



1


(Day 93)



2


(Days 54-77)



 



Shortened tail



 



 



1
(From Day 106)



 



Short broken teeth



1
(Days 115-122)



 



1
(Days 69-71)



 



Hematoma (nose and tongue)



1
(Day 114)



 



 



 



Increase in size (ear)



 



1
(Day 54)



 



 



Scabs and/or Nodosities (eye, ear, neck, forelimb, head, thorax and/or tail)



12


(Days 7-144)



6


(Days 16-138)



7


(Days 10-96)



3


(Days 56-92)



In bold: clinical signs considered relevant.


In italics: clinical signs considered to be unrelated to the test item treatment.


(): period of occurrence.


 


Table 2: Clinical Signs in Terminated as Scheduled P Generation Females (Number of Animals Affected per Group)















































































































Dose level
(mg/kg bw/day)



0



100



300



800



Number terminated as scheduled females



24



24



24



23



Loud breathing



 



 



 



1


(Day 68)



Exophthalmos (right and left)



 



 



 



5
(Days 56-83)



Hunched posture



 



 



 



1


(Days 79-97)



Piloerection



 



 



 



4


(Days 68-101)



Ptyalism (before treatment)



 



 



2


(Days 58-59)



5


(Days 28-59)



Ptyalism (after treatment)



1
(Day 87)



8


(Days 56-121)



24


(Days 10-128)



23


(Days 10-128)



Reflux at dosing (after treatment)



 



 



 



1


(Day 72)



Alopecia and/or Thinning of hair



4


(Days 19-128)



9


(Days 65-138)



10


(Days 19-130)



9


(Days 10-125)



Chromodacryorrhea and/or Chromorhynorrhea



2


(Days 54-69)



 



2
(Days 54-73)



3


(Days 32-95)



Short broken teeth



 



 



1
(Day 69)



 



Nodosities and/or Scab(s) (ear, neck, limbs, flank, cheek, interscapular region and/or tail)



5


(Days 16-126)



7


(Days 21-124)



5


(Days 13-105)



3


(Days 16-129)



Soiled (head, abdomen, neck, nose and/or urogenital region)



 



1


(Days 54-56)



1
(Days 98-104)



3
(Days 56-110)



Reddish vaginal discharge



 



1


(Day 79)



 



1


(Day 81)



In bold: clinical signs considered relevant.


In italics: clinical signs considered to be unrelated to the test item treatment.


(): period of occurrence.


 


BODY WEIGHTS P0 ANIMALS


Table 3 Mean Body Weight (g) in P Generation Animals (premating and postmating)
































































































































































































































































Sex



Male



Female



Dose level
(mg/kg/day)



0



100



300



800



0



100



300



800



Mean body weight (g)



. Day 1



206



202


(-2)



202


(-2)



203


(-1)



152



154


(+1)



151


(-1)



152


(0)



. Day 8



275



267


(-3)



267


(-3)



260#


(-5)



184



183


(-1)



177


(-4)



180


(-2)



. Day 15



340



328


(-4)



325*


(-4)



310#


(-9)



212



212


(0)



207


(-2)



202


(-5)



. Day 22



390



373*


(-4)



366**


(-6)



351#


(-10)



230



232


(+1)



224


(-3)



220


(-4)



. Day 29



429



408*


(-5)



396#


(-8)



380#


(-11)



242



249


(+3)



238


(-2)



236


(-2)



. Day 36



462



433**


(-6)



422#


(-9)



405#


(-12)



254



261


(+3)



248


(-2)



246


(-3)



. Day 43



486



456*


(-6)



442#


(-9)



419#


(-14)



264



272


(+3)



257


(-3)



254


(-4)



. Day 50



508



474**


(-7)



462#


(-9)



431#


(-15)



270



278


(+3)



266


(-1)



261


(-3)



. Day 57



529



492**


(-7)



478#


(-10)



443#


(-16)



274



285


(+4)



275


(0)



265


(-3)



. Day 64



549



506**


(-8)



489#


(-11)



445#


(-19)



277



287


(+4)



276


(0)



265


(-4)



. Day 71



568



523**


(-8)



505#


(-11)



452#


(-20)



290



304


(+5)



292


(+1)



277


(-4)



. Day 78



581



539**


(-7)



519#


(-11)



462#


(-20)



295



316*


(+7)



299


(+1)



282


(-4)



. Day 85



588



548*


(-7)



522#


(-11)



462#


(-21)



/



/



/



/



. Day 92



600



559*


(-7)



535#


(-11)



469#


(-22)



/



/



/



/



. Day 99



614



569*


(-7)



547#


(-11)



478#


(-22)



/



/



/



/



. Day 106



630



579**


(-8)



550#


(-13)



486#


(-23)



/



/



/



/



. Day 113



646



592**


(-8)



556#


(-14)



490#


(-24)



/



/



/



/



. Day 120



659



601**


(-9)



566#


(-14)



495#


(-25)



/



/



/



/



. Day 127



669



608**


(-9)



573#


(-14)



498#


(-25)



/



/



/



/



. Day 134



676



615**


(-9)



579#


(-14)



501#


(-26)



/



/



/



/



. Day 140



679



623*


(-8)



576#


(-15)



510#


(-25)



/



/



/



/



Statistical significance: *: p<0.05; **: p<0.01; #: p< 0.001.


(): in brackets, percentage difference (%) vs. controls.


/: not applicable.


 


Table 4 Mean Body Weight Change (g) in P Generation Animals (premating and postmating)
































































































































































































































































Sex



Male



Female



Dose level
(mg/kg/day)



0



100



300



800



0



100



300



800



Mean body weight (g)



. Day 1



206



202


(-2)



202


(-2)



203


(-1)



152



154


(+1)



151


(-1)



152


(0)



. Day 8



275



267


(-3)



267


(-3)



260#


(-5)



184



183


(-1)



177


(-4)



180


(-2)



. Day 15



340



328


(-4)



325*


(-4)



310#


(-9)



212



212


(0)



207


(-2)



202


(-5)



. Day 22



390



373*


(-4)



366**


(-6)



351#


(-10)



230



232


(+1)



224


(-3)



220


(-4)



. Day 29



429



408*


(-5)



396#


(-8)



380#


(-11)



242



249


(+3)



238


(-2)



236


(-2)



. Day 36



462



433**


(-6)



422#


(-9)



405#


(-12)



254



261


(+3)



248


(-2)



246


(-3)



. Day 43



486



456*


(-6)



442#


(-9)



419#


(-14)



264



272


(+3)



257


(-3)



254


(-4)



. Day 50



508



474**


(-7)



462#


(-9)



431#


(-15)



270



278


(+3)



266


(-1)



261


(-3)



. Day 57



529



492**


(-7)



478#


(-10)



443#


(-16)



274



285


(+4)



275


(0)



265


(-3)



. Day 64



549



506**


(-8)



489#


(-11)



445#


(-19)



277



287


(+4)



276


(0)



265


(-4)



. Day 71



568



523**


(-8)



505#


(-11)



452#


(-20)



290



304


(+5)



292


(+1)



277


(-4)



. Day 78



581



539**


(-7)



519#


(-11)



462#


(-20)



295



316*


(+7)



299


(+1)



282


(-4)



. Day 85



588



548*


(-7)



522#


(-11)



462#


(-21)



/



/



/



/



. Day 92



600



559*


(-7)



535#


(-11)



469#


(-22)



/



/



/



/



. Day 99



614



569*


(-7)



547#


(-11)



478#


(-22)



/



/



/



/



. Day 106



630



579**


(-8)



550#


(-13)



486#


(-23)



/



/



/



/



. Day 113



646



592**


(-8)



556#


(-14)



490#


(-24)



/



/



/



/



. Day 120



659



601**


(-9)



566#


(-14)



495#


(-25)



/



/



/



/



. Day 127



669



608**


(-9)



573#


(-14)



498#


(-25)



/



/



/



/



. Day 134



676



615**


(-9)



579#


(-14)



501#


(-26)



/



/



/



/



. Day 140



679



623*


(-8)



576#


(-15)



510#


(-25)



/



/



/



/



Statistical significance: *: p<0.05; **: p<0.01; #: p< 0.001.


(): in brackets, percentage difference (%) vs. controls.


/: not applicable.


 


Table 5 Mean Body Weight (g) and Mean Body Weight Change (g) in P Generation Females (Gestation)





















































































































Dose level
(mg/kg/day)



0



100



300



800



Mean body weight (g)



. Gestation Day (GD) 0 



294



312


(+6)



297


(+1)



282


(-4)



. GD 4 



309



330*


(+7)



317


(+3)



294


(-5)



. GD 7 



320



339*


(+6)



325


(+2)



298*


(-7)



. GD 10



331



348


(-5)



334


(+1)



305**


(-8)



. GD 14 



348



368*


(+6)



355


(+2)



319#


(-8)



. GD 17 



380



401*


(+6)



389


(+2)



344#


(-9)



. GD 20 



434



456


(+5)



444


(+2)



392#


(-10)



Mean body weight change (g)



. GD 0 to 4 



+15



+18



+21



+13



. GD 4 to 7 



+11



+9



+7



+4**



. GD 7 to 10 



+11



+9



+10



+7**



. GD 10 to 14 



+16



+20



+20



+14



. GD 14 to 17 



+32



+33



+34



+26*



. GD 17 to 20 



+54



+55



+55



+48



. GD 0 to 20 



+140



+144



+147



+111#



Statistical significance: *: p<0.05; **: p<0.01; #: p< 0.001.


(): in brackets, percentage difference (%) vs. controls


 


Table 6 Mean Body Weight (g) and Mean Body Weight Change (g) in P Generation Females (Lactation)

























































































Dose level
(mg/kg/day)



0



100



300



800



Mean body weight (g)



. Postnatal Day (PND) 1 



331



353*


(+7)



340


(+3)



295#


(-11)



. PND 4 



336



362**


(+8)



351


(+4)



307**


(-9)



. PND 7 



352



376**


(+7)



363


(+3)



321#


(-9)



. PND 14 



373



393*


(+5)



378


(+1)



347**


(-7)



. PND 21 



358



369


(+3)



358


(0)



338*


(-6)



Mean body weight change (g)



. PND 1 to 4 



+5



+9



+11



+11



. PND 4 to 7 



+16



+14



+12



+14



. PND 7 to 14 



+20



+17



+15



+26



. PND 14 to 21 



-15



-24



-20



-9



. PND 1 to 21 



+27



+16



+18



+42**



Statistical significance: *: p<0.05; **: p<0.01; #: p< 0.001.


(): in brackets, percentage difference (%) vs. controls


 


FOOD CONSUMPTION P0 ANIMALS


Table 7 Mean Food Consumption (g/animal/day) in P Generation Animals (Premating and postmating)




























































































































































































































Sex



Male (a)



Female



Dose level
(mg/kg bw/day)



0



100



300



800



0



100



300



800



. Days 1 to 8



26.5



25.0**


(-6)



25.3*


(-5)



23.1**


(-13)



17.0



16.9


(-1)



17.6


(+4)



15.9


(-6)



. Days 8 to 15



28.8



27.1*


(-6)



27.1*


(-6)



25.6**


(-11)



18.1



17.8


(-2)



19.6


(+8)



17.0


(-6)



. Days 15 to 22



28.8



27.1*


(-6)



27.7


(-4)



26.1**


(-9)



19.9



18.1


(-9)



20.3


(+2)



17.4


(-13)



. Days 22 to 29



27.8



26.3


(-5)



25.6


(-8)



24.9**


(-10)



17.3



17.5


(+1)



19.4


(+12)



19.0


(+10)



. Days 29 to 36



26.5



24.2**


(-9)



24.2**


(-9)



24.6*


(-7)



17.2



17.9


(+4)



19.9


(+16)



19.2


(+12)



. Days 36 to 43



25.1



23.8


(-5)



24.2


(-4)



23.6


(-6)



16.0



17.4


(+9)



18.9*


(+18)



20.3**


(+27)



. Days 43 to 50



25.0



23.0*


(-8)



23.9


(-4)



22.7*


(-9)



16.2



17.1


(+6)



18.1*


(+12)



19.8**


(+22)



. Days 50 to 57



25.2



24.0


(-5)



24.9


(-1)



23.7


(-6)



16.0



17.4


(+9)



19.1**


(+19)



19.4**


(+21)



. Days 57 to 64 (b)



27



25*


(-7)



24#


(-11)



24#


(-11)



15



18**


(+20)



17**


(+13)



19#


(+27)



. Days 64 to 71



25



24


(-4)



25


(0)



24


(-4)



17



19


(+12)



20*


(+18)



21#


(+24)



. Days 71 to 78



26



25


(-4)



25


(-4)



26


(0)



18



20*


(+11)



20*


(+11)



20


(+11)



. Days 95 to 99



25



23


(-8)



23


(-8)



23


(-8)



/



/



/



/



. Days 99 to 106



25



24


(-4)



24


(-4)



26


(+4)



/



/



/



/



. Days 106 to 113



27



25


(-7)



24*


(-11)



26


(-4)



/



/



/



/



. Days 113 to 120



25



24


(-4)



24


(-4)



22**


(-12)



/



/



/



/



. Days 120 to 127



25



23*


(-8)



24


(-4)



25


(0)



/



/



/



/



. Days 127 to 134



24



24


(0)



24


(0)



24


(0)



/



/



/



/



. Days 134 to 140



24



24


(0)



24


(0)



22


(-8)



/



/



/



/



 


(a): food consumption not recorded during the pairing period. /: not applicable.


(b): differences in terms of rounded values are related to the use of different computer systems over the
Day 57-64 period.


( ): percentage (%) difference vs. controls. Statistical significance: *: p<0.05; **: p<0.01; #: p<0.001.


 


Table 8 Mean Food Consumption (g/animal/day) in P Generation Females during the Gestation Period























































Dose level
(mg/kg bw/day)



0



100



300



800



. Gestation Days (GD) 0 to 4 



18



21


(+17)



23**


(+28)



22*


(+22)



. GD 4 to 7 



20



22


(+10)



22*


(+10)



20


(0)



. GD 7 to 10 



21



23


(+10)



23


(+10)



20


(-5)



. GD 10 to 14 



21



23*


(+10)



24**


(+14)



21


(0)



. GD 14 to 17 



22



24


(+9)



26*


(+18)



22


(0)



. GD 17 to 20



25



27*


(+8)



29#


(+16)



25


(0)



( ): percentage (%) difference vs. controls. Statistical significance: *: p<0.05; **: p<0.01; #: p<0.001.


 


Table 9 Mean Food Consumption (g/animal/day) in P Generation Females during the Lactation Period





































































Dose level
(mg/kg bw/day)



0



100



300



800



. Postnatal Days (PND) 1 to 4 p.p.



31



36



37*



30



 



 



(+16)



(+19)



(-3)



. PND 4 to 7 



55



57



55



45#



 



 



(+4)



(0)



(-18)



. PND 7 to 14



70



71



69



58#



 



 



(+1)



(-1)



(-17)



. PND 14 to 21 



84



83



82



70#



 



 



(-1)



(-2)



(-17)



( ): percentage (%) difference vs. controls. Statistical significance: *: p<0.05; #: p<0.001.


 


THYROID HORMONES P0 ANIMALS


Table 10 Thyroid Hormones in P Generation Males






























Dose level
(mg/kg/day)



0



100



300



800



HCD


[min. – max.]



T4 (ng/mL ± SD)



46.7 ± 5.4



40.2* ± 4.5


(-14)



35.6** ± 4.7


(-24)



30.1** ± 5.6


(-36)



[24.6 - 58.0]



TSH (pg/mL ± SD)



1589 ± 503



3052** ± 1248


(+92)



2075 ± 569


(+31)



3654** ± 1670


(+130)



[353 - 3987]



HCD: Historical Control Data (OECD443 - 2015-2021, n = 8 studies).


( ): percentage (%) difference vs. controls. Statistical significance vs. controls: *: p<0.05; **: p<0.01.


 


ORGAN WEIGHTS P0 ANIMALS


Table 11 Relevant Changes in Mean Final Body Weights and Organ Weights in Treated Groups
(% Changes from Controls)
































































































































Sex



Male



Female



Group



2



3



4



2



3



4



Dose-level (mg/kg/day)



100



300



800



100



300



800



Examined animals / Number of animals



24/24



22/24



24/24



24/24



24/24



23/24



- Final body weight



-9**



-15**



-27**



+4



+1



-6*



- Kidneys


      

.absolute



+6



+11**



+2



+7*



+6



+2



.relative



+16**



+31**



+39**



+3



+6



+9**



- Liver


      

.absolute



+13*



+16**



+24**



+11



+25**



+36**



.relative



+24**



+37**



+70**



+7



+25**



+45**



- Thyroid glands


      

.absolute



+11



+16**



+3



+7



+15**



+18**



.relative



+21*



+36**



+41**



+3



+15*



+25**



Statistically significant from controls: *: p<0.05, **: p<0.01. The significance concerned the organ weights values and not the percentages.


MACROSCOPICAL FINDINGS P0 ANIMALS


Table 12 Test Item-Related Macroscopic Findings in P0 animals


 
























































































Sex



Male



Female



Group



1



2



3



4



1



2



3



4



Dose-level (mg/kg/day)



0



100



300



800



0



100



300



800



Number of animals



24



24



22



24



24



24



24



23



KIDNEYS; enlargement



-



-



-



1



-



-



-



-



LIVER; enlargement



-



-



1



5



-



-



1



1



LIVER; accentuated lobular pattern



-



1



-



1



-



-



-



-



THYMUS; reduced in size



-



3



1



4



3



1



-



3



-: not observed in the group.


HISTOPATHOLOGY P0 ANIMALS


Table 13 Main Microscopic Lesions (Incidence and Severity) - P0 Animals

















































































































































































































































Sex



Male



Female



Group



1



2



3



4



1



2



3



4



Dose-level (mg/kg/day)



0



100



300



800



0



100



300



800



Number of animals per group



24



24



22



24



24



24



24



23



KIDNEYS; degeneration/necrosis



grade 1



-



-



-



10



-



na



na



-



grade 2



-



-



-



1



-



na



na



-



KIDNEYS; hyaline droplet accumulation



grade 1



2



1



4



6



-



na



na



-



grade 2



-



2



2



14



-



na



na



-



grade 3



-



-



-



3



-



na



na



-



KIDNEYS; pigment



grade 1



-



2



2



16



-



na



na



-



grade 2



1



-



-



-



-



na



na



-



KIDNEYS; dilated tubules



grade 1



1



1



4



5



1



na



na



-



grade 2



2



-



-



4



-



na



na



-



LIVER; hypertrophy; hepatocellular



grade 1



-



7



14



14



-



13



15



10



grade 2



-



-



3



7



-



-



-



9



grade 3



-



-



-



1



-



-



-



-



THYROID GLAND; hypertrophy, follicular cell



grade 1



-



14



11



17



-



3



9



18



THYMUS; lymphoid decreased cellularity



grade 1



6



12



10



14



10



9



7



12



grade 2



-



1



1



4



-



-



-



2



grade 3



-



-



1



-



-



-



-



-



-: not observed; na: not applicable.


 


ESTROUS CYCLE P0 ANIMALS


Table 14 Mean Estrous Cycle Parameters (treatment period) in P Generation Females






















































































Dose level
(mg/kg bw/day)



0



100



300



800



HCD


[Min.,; Max.]



Number of females examined



24



24



24



24



142



Mean number of days of diestrus (± SD)



7.2 ± 2.7



8.6 ± 3.4



8.2 ± 3.1



9.4 ± 2.9



[3.8; 5.9]



Mean number of days of proestrus (± SD)



2.5 ± 1.4



1.8 ± 1.1



2.4 ± 1.1



1.9 ± 2.7



[1.7; 3.6]



Mean number of days of estrus (± SD)



2.8 ± 1.2



2.6 ± 2.3



2.3 ± 1.8



1.9 ± 1.3



[3.5; 4.5]



Mean number of days of metestrus (± SD)



2.5 ± 1.1



1.9 ± 1.3



2.1 ± 1.2



1.8 ± 1.0



[3.0; 4.9]



Mean number of cycles (± SD)



2.4 ± 0.7



1.9 ± 0.9



2.3 ± 0.8



1.6** ± 0.9



[2.8; 3.2]



Mean duration of cycles (days)
(± SD)



5.1 ± 2.6



5.7 ± 3.1



5.9 ± 3.1



7.3 ± 4.1



[3.8;4.5]



Mean percent of females cycling normally (%)



65.2



65.0



61.1



33.3



[67.0; 100]



Mean percent of females with all stages (%)



87.5



87.5



79.2



75.0



[87.0; 100]



HCD: Historical Control Data (OECD443 - P generation, 2017-2020, n = 6 studies). Statistical significance: **: p<0.01.


CLINICAL OBSERVATIONS F1 GENERATION


Table 15 Clinical Signs in F1 Pups During Lactation











































































































































Dose level
(mg/kg BW/day)



0



100



300



800



Number of litters evaluated



22



20



20



20



Number of pups evaluated



317



295



286



247



- abnormal yellowish color (urogenital area), P/L



 



 



10/2



60/13



- skin dryness tail or hindlimb P/L



 



 



 



85/15



- cold to the touch, P/L



16/1



 



16/1



 



- emaciated appearance, P/L



1/1



3/2



10/3



 



- hematoma/scab (limbs, back, neck, abdominal or urogenital region, head and/or tail), P/L



2/2



3/3



6/4



3/3



- malformed and necrosed tail, P/L



1/1



 



 



 



- necrosed forelimb, P/L



 



 



 



1/1



- nodosities (mouth or forelimb), P/L



 



 



1/1



1/1



- pallor (generalized), P/L



1/1



 



 



 



- shortened tail, P/L



1/1



 



2/2



 



- stiffness, P/L



 



 



16/1



 



- swollen/wound (forelimb, urogenital region and/or anus), P/L



1/1



 



1/1



2/2



- thinning of hair P/L



 



 



10/1



 



- umbilical hernia, P/L



 



 



 



1/1



Litter(s) affected, Nb. (%)



5 (23)



5 (25)



10 (50)



19 (95)



Pup(s) affected, Nb. (%)



21 (7)



6 (2)



46 (16)



111 (45)



Nb.: Number; P/L: Nb. of pups / Nb. of litters; In bold: clinical signs considered relevant.


BODY WEIGHTS F1 GENERATION


Table 16 F1 Pup Body Weight and Body Weight Change (g)































































































































Sex



Male



Female



Dose level
(mg/kg bw/day)



0



100



300



800



0



100



300



800



Body weight (g)



Postnatal Day (PND) 1



7.1



7.3


(+3)



7.4


(+4)



6.8


(-4)



6.7



6.9


(+3)



7.0


(+4)



6.5


(-3)



PND 4(a)



10.5



10.4


(-1)



10.4


(-1)



8.6#


(-18)



10.0



9.9


(-1)



9.7


(-3)



8.3**


(-17)



PND 7 



17.7



17.5


(-1)



17.2


(-3)



13.0#


(-27)



16.8



16.8


(0)



16.2


(-4)



12.4#


(-26)



PND 14



37.0



36.6


(-1)



35.5


(-4)



26.9#


(-27)



35.5



35.6


(0)



33.6


(-5)



25.8#


(-27)



PND 21 



59.6



59.0


(-1)



58.0


(-3)



45.3#


(-24)



57.2



57.4


(0)



55.4


(-3)



43.8#


(-23)



Body weight change (g)



PND 1 to 4 



+3.4



+3.1



+2.9



+1.9#



+3.3



+3.0



+2.7



+1.8#



PND 4 to 7 



+7.2



+7.1



+6.8



+4.4#



+6.7



+6.8



+6.5



+4.1#



PND 7 to 14 



+19.4



+19.1



+18.3



+13.9#



+18.7



+18.8



+17.4



+13.5#



PND 14 to 21 



+22.6



+22.5



+22.5



+18.4#



+21.7



+21.9



+21.8



+18.0#



(a): pre-culling. ( ): percentage (%) difference vs. controls. Statistical significance: **: p<0.01; #: p<0.001.


Table 17 Mean Body Weight (g) and Mean Body Weight Change (g) in Cohort 1A Animals
























































































































































































































































Sex



Male



Female



Dose level
(mg/kg bw/day)



0



100



300



800



0



100



300



800



Mean body weight (g)



. Day 1



63



62


(-2)



60


(-5)



51**


(-19)



61



59


(-3)



59


(-3)



49**


(-20)



. Day 8



106



108


(+2)



101


(-5)



85**


(-20)



99



94


(-5)



97


(-2)



78**


(-21)



. Day 15



174



173


(-1)



163*


(-6)



138**


(-21)



145



140


(-3)



142


(-2)



121**


(-17)



. Day 22



247



241


(-2)



227**


(-8)



197**


(-20)



181



174


(-4)



177


(-2)



161**


(-11)



. Day 29



317



306


(-3)



288**


(-9)



255**


(-20)



205



198


(-3)



203


(-1)



185**


(-10)



. Day 36



373



362


(-3)



342**


(-8)



308**


(-17)



224



218


(-3)



224


(0)



208


(-7)



. Day 43



419



404


(-4)



380**


(-9)



341**


(-19)



239



236


(-1)



242


(+1)



224


(-6)



. Day 50



455



436


(-4)



412**


(-9)



367**


(-19)



252



249


(-1)



255


(+1)



238


(-6)



. Day 57



485



467


(-4)



439**


(-9)



389**


(-20)



264



259


(-2)



266


(+1)



249


(-6)



. Day 64



511



490


(-4)



460**


(-10)



409**


(-20)



270



269


(0)



273


(+1)



257


(-5)



Mean body weight change (g)



. Days 1 to 8



+43



+46



+41



+33**



+38



+36



+38



+28**



. Days 8 to 15



+69



+66



+60**



+54**



+47



+46



+46



+44



. Days 15 to 22



+73



+67*



+64**



+58**



+36



+34



+35



+39



. Days 22 to 29



+70



+65*



+62**



+58**



+24



+24



+26



+24



. Days 29 to 36



+56



+56



+54



+54



+19



+21



+21



+23



. Days 36 to 43



+46



+42



+38*



+33**



+15



+18



+19



+16



. Days 43 to 50



+35



+32



+32



+26**



+13



+12



+13



+14



. Days 50 to 57



+31



+31



+27



+23**



+12



+10



+11



+12



. Days 57 to 64



+26



+23



+21



+20*



+6



+10



+7



+7



. Days 1 to 64



+448



+428



+400**



+358**



+209



+210



+214



+207



( ): percentage (%) difference vs. controls. Statistical significance: *: p<0.05; **: p<0.01.


 


Table 18 Mean Body Weight (g) and Mean Body Weight Change (g) in Cohort 1B Animals


















































































































































Sex



Male



Female



Dose level
(mg/kg bw/day)



0



100



300



800



0



100



300



800



Mean body weight (g)



. Day 1



62



64


(+3)



60


(-3)



49#


(-21)



60



60


(0)



57


(-5)



48#


(-20)



. Day 8



108



110


(+2)



104


(-4)



84#


(-22)



97



100


(+3)



94


(-3)



77#


(-21)



. Day 15



175



179


(+2)



167


(-5)



138#


(-21)



144



148


(+3)



139


(-3)



121#


(-16)



. Day 22



245



251


(+2)



231*


(-6)



197#


(-20)



180



187


(+4)



176


(-2)



159#


(-12)



. Day 29



313



316


(+1)



296*


(-5)



255#


(-19)



203



209


(+3)



201


(-1)



186*


(-8)



. Day 36



373



373


(0)



349*


(-6)



303#


(-19)



224



233


(+4)



221


(-1)



209


(-7)



. Day 43



417



414


(-1)



387**


(-7)



338#


(-19)



239



252


(+5)



239


(0)



226


(-5)



. Day 50



453



445


(-2)



419**


(-8)



364#


(-20)



249



263


(+6)



251


(+1)



241


(-3)



. Day 57



482



475


(-1)



445**


(-8)



386#


(-20)



261



273


(-5)



261


(0)



250


(-4)



. Day 64



509



498


(-2)



470**


(-8)



405#


(-20)



270



279


(+3)



270


(0)



261


(-3)



. Day 71



532



518


(-3)



488**


(-8)



421#


(-21)



276



289


(+5)



276


(0)



264


(-4)



















































































































































Sex



Male



Female



Dose level
(mg/kg bw/day)



0



100



300



800



0



100



300



800



Mean body weight change (g)



. Days 1 to 8



+45



+47



+44



+35#



+37



+39



+37



+29#



. Days 8 to 15



+67



+69



+63



+55#



+47



+48



+45



+44



. Days 15 to 22



+70



+72



+65



+59#



+36



+39



+37



+37



. Days 22 to 29



+68



+65



+64



+58#



+24



+22



+26



+27



. Days 29 to 36



+60



+57



+53



+49**



+21



+24



+20



+23



. Days 36 to 43



+45



+42



+38*



+35**



+15



+19



+18



+16



. Days 43 to 50



+35



+31



+32



+26**



+10



+11



+12



+15



. Days 50 to 57



+29



+28



+26



+22**



+12



+10



+9



+9



. Days 57 to 64



+27



+25



+25



+20*



+8



+6



+9



+11



. Days 64 to 71



+23



+20



+19



+15#



+6



+10



+6



+3



. Days 1 to 71



+469



+455



+429*



+372#



+216



+229



+220



+216



( ): percentage (%) difference vs. controls. Statistical significance: *: p<0.05; **: p<0.01; #: p<0.001.


 


Table 19 Mean body weight (g) and Mean body weight change (g) in Cohort 2A animals












































































































































































































Sex



Male



Female



Dose level
(mg/kg bw/day)



0



100



300



800



0



100



300



800



Mean body weight (g)



. Day 1



64



62


(-3)



60


(-6)



49**


(-23)



61



58


(-5)



60


(-2)



47**


(-23)



. Day 8



110



106


(-4)



101


(-8)



82**


(-25)



99



95


(-4)



91


(-8)



76**


(-23)



. Day 15



179



172


(-4)



163


(-9)



137**


(-23)



147



145


(-1)



138


(-6)



121**


(-18)



. Day 22



249



236


(-5)



229


(-8)



199**


(-20)



180



184


(+2)



176


(-2)



160*


(-11)



. Day 29



319



301


(-6)



295


(-8)



260**


(-18)



200



209


(+5)



202


(+1)



185


(-8)



. Day 36



380



356


(-6)



348*


(-8)



313**


(-18)



220



232


(+5)



223


(+1)



207


(-6)



. Day 43



420



390


(-7)



389*


(-7)



348**


(-17)



232



245


(+6)



241


(+4)



222


(-4)



. Day 50



461



426


(-8)



423*


(-8)



374**


(-19)



243



262


(+8)



256


(+5)



239


(-2)



Mean body weight change (g)



. Days 1 to 8



+46



+45



+42



+32**



+38



+37



+32



+28**



. Days 8 to 15



+69



+65



+62



+55**



+48



+50



+47



+46



. Days 15 to 22



+70



+65



+66



+62



+33



+39



+39



+39



. Days 22 to 29



+71



+65



+66



+61**



+20



+25



+26*



+25



. Days 29 to 36



+61



+55



+53



+53



+21



+23



+21



+22



. Days 36 to 43



+41



+34



+40



+34



+12



+13



+19



+15



. Days 43 to 50



+41



+37



+34



+27**



+12



+17



+14



+17



. Days 1 to 50



+397



+365



+363



+326**



+182



+204



+197



+192



(): percentage (%) difference vs. controls. Statistical significance: *: p<0.05; **: p<0.01.


 


FOOD CONSUMPTION F1


Table 20 Mean Food Consumption in Cohort 1A animals (g/animal/day)

























































































































Sex



Male



Female



Dose level
(mg/kg bw/day)



0



100



300



800



0



100



300



800



. Days 1 to 8



9.3



9.9


(+6)



8.8


(-5)



7.1**


(-24)



8.6



8.4


(-2)



8.6


(0)



6.8**


(-21)



. Days 8 to 15



18.0



18.3


(+2)



16.2


(-10)



14.3**


(-21)



14.5



14.1


(-3)



14.5


(0)



12.8


(-12)



. Days 15 to 22



25.8



25.0


(-3)



23.8*


(-8)



21.2**


(-18)



17.6



17.4


(-1)



18.2


(+3)



17.5


(-1)



. Days 22 to 29



28.3



27.3


(-4)



26.8


(-5)



24.9**


(-12)



17.1



16.7


(-2)



18.7


(+9)



18.2


(+6)



. Days 29 to 36



29.7



29.4


(-1)



29.0


(-2)



27.4*


(-8)



16.8



17.1


(+2)



18.5


(+10)



19.4**


(+15)



. Days 36 to 43



28.9



28.4


(-2)



27.7


(-4)



27.0


(-7)



16.9



16.9


(0)



19.0


(+12)



21.4*


(+27)



. Days 43 to 50



28.0



27.8


(-1)



27.8


(-1)



25.5*


(-9)



16.7



17.3


(+4)



19.2


(+15)



20.9*


(+25)



. Days 50 to 57



27.2



27.6


(+1)



26.7


(-2)



25.4


(-7)



16.8



17.0


(+1)



19.2


(+14)



20.8


(+24)



. Days 57 to 64



26.6



26.9


(+1)



26.0


(-2)



25.0


(-6)



16.6



17.1


(+3)



19.2*


(+16)



20.1**


(+21)



( ): percentage (%) difference vs. controls. Statistical significance: *: p<0.05; **: p<0.01.


 


Table 21 Mean Food Consumption in Cohort 1B Animals (g/animal/day)




































































































































Sex



Male



Female



Dose level
(mg/kg bw/day)



0



100



300



800



0



100



300



800



. Days 1 to 8



9.5



10.1


(+6)



9.0


(-5)



7.4**


(-22)



8.4



8.9


(+6)



8.0


(-5)



7.1**


(-15)



. Days 8 to 15



18.1



18.7


(+3)



17.4


(-4)



14.8**


(-18)



14.3



15.3


(+7)



14.2


(-1)



16.5


(+15)



. Days 15 to 22



24.9



26.1


(+5)



24.4


(-2)



22.2**


(-11)



17.2



18.1


(+5)



16.6


(-3)



17.1


(-1)



. Days 22 to 29



28.0



28.4


(+1)



27.5


(-2)



25.8**


(-8)



17.3



19.6


(+13)



18.1


(+5)



18.0


(+4)



. Days 29 to 36



29.3



29.4


(0)



29.4


(0)



27.6


(-6)



17.0



20.0


(+18)



17.6


(+4)



19.0*


(+12)



. Days 36 to 43



29.2



29.2


(0)



29.1


(0)



27.4


(-6)



17.2



22.6


(+31)



18.6


(+8)



18.9


(+10)



. Days 43 to 50



28.2



27.8


(-1)



28.4


(+1)



25.8*


(-9)



16.7



23.0


(+38)



18.4


(+10)



18.7


(+12)



. Days 50 to 57



28.1



28.6


(+2)



27.7


(-1)



25.3


(-10)



17.7



18.4


(+4)



18.0


(+2)



19.3


(+9)



. Days 57 to 64



25.9



26.0


(0)



26.1


(+1)



24.0*


(-7)



15.7



21.4


(+36)



17.2


(+10)



18.6*


(+18)



. Days 64 to 71



26.7



26.8


(0)



26.7


(0)



24.4*


(-9)



17.0



22.9


(+35)



17.8


(+5)



18.4*


(+8)



( ): percentage (%) difference vs. controls. Statistical significance: *: p<0.05; **: p<0.01.


 


THYROID HORMONES F1 GENERATION (COHORT 1A)


Table 22 Thyroid Hormones in Cohort 1A Males






























Dose level
(mg/kg bw/day)



0



100



300



800



HCD


[min. - max.]



T4 (ng/mL ± SD)



48.0 ± 7.8



43.3 ± 5.4


(-10)



44.4 ± 7.3


(-8)



37.2* ± 11.6


(-22)



[31.5 - 50.3]



TSH 
(pg/mL ± SD)



1611 ± 1085



2775 ± 1994


(+72)



1676 ± 821


(+4)



3517* ± 1833


(+118)



[1248 - 2470]



HCD: Historical Control Data (OECD443 - Cohort 1A males, 2017-2020, n = 6 studies). ( ): percentage (%) difference vs. controls. Statistical significance vs. controls: *: p<0.05.


Table 23 Thyroid Hormones in Cohort 1A Females






























Dose level


(mg/kg bw/day)



0



100



300



800



HCD


[min. - max.]



T4 (ng/mL ± SD)



35.1 ± 7.4



31.2 ± 4.7


(-11)



29.6 ± 8.5


(-16)



28.1 ± 6.9


(-20)



[27.5 - 38.8]



TSH 
(pg/mL ± SD)



821 ± 220



1000 ± 339


(+22)



1368* ± 441


(+67)



1716** ± 646


(+109)



[544 - 741]



HCD: Historical Control Data (OECD443 – Cohort 1A females, 2017-2020, n = 6 studies). ( ): percentage (%) difference vs. controls. Statistical significance vs. controls: *: p<0.05; **: p<0.01.


SEXUAL MATURATION F1


Table 24 Mean Age at Balanopreputial Separation and Mean Body Weight on the Day of Occurrence in F1 Males






















































Dose level
(mg/kg/day)



0



100



300



800



HCD


[Min-Max]



Number of males



50



50



49



49



277



Number of positive responses



49



48



49



49



/



Number of negative responses



1



2



0



0



/



Mean age (days ± SD)



48.2 ± 2.9



48.5 ± 3.8



49.3 ± 4.4



51.0** ± 4.2



[43.7-54.1]



Mean body weight (g ± SD)



294.1 ± 27.9



291.4 ± 33.7



284.1 ± 36.4



263.5** ± 27.9



[252.8-324.4]



Statistical significance: **: p<0.01. HCD: Historical Control Data (OECD443 - 2017-2020, n = 4-6 studies). /: not available in HCD.


Table 25 Mean Age at Vaginal Opening and Mean Body Weight on the Day of Occurrence in F1 Females






















































Dose level
(mg/kg/day)



0



100



300



800



HCD


[Min-Max]



Number of females



50



50/49a



50



49



278



Number of positive responses



50



49



50



49



/



Number of negative responses



0



1



0



0



/



Mean age (days ± SD)



33.4 ± 2.1



32.8 ± 4.4



33.1 ± 2.6



36.4** ± 3.3



[31.9-37.2]



Mean body weight (g ± SD)



126.3 ± 14.2



120.8 ± 22.7



120.9 ± 17.1



121.6 ± 22.1



[117.3-175.1]



Statistical significance: **: p<0.01. a: test was performed on 50 females, but body weight was recorded in 49 females. HCD: Historical Control Data (OECD443 2017- 2020, n= 4-6 studies). /: not available in HCD.


MACROSCOPIC EXAMINATIONS F1 GENERATION


Table 26 Test Item-Related Macroscopic Findings in Cohort 1A













































































Sex



Male



Female



Group



1



2



3



4



1



2



3



4



Dose-level (mg/kg bw/day)



0



100



300



800



0



100



300



800



Number of animals



20



20



19



20



20



20



20



19



KIDNEYS; tan discoloration



-



-



1



2



-



-



-



-



LIVER; enlargement



-



-



1



-



-



-



-



-



LIVER; accentuated lobular pattern



-



1



4



2



-



1



1



2



-: not observed in the group.


ORGAN WEIGHTS F1


Table 27 Relevant Changes in Mean Final Body Weights and Organ Weights in Treated Groups of Cohort 1A (% Changes from Controls)
































































































































Sex



Male



Female



Group



2



3



4



2



3



4



Dose-level (mg/kg bw/day)



100



300



800



100



300



800



Examined animals / Number of animals



20/20



19/20



20/20



20/20



20/20



19/20



- Final body weight



-4



-10**



-22**



0



0



-6



- Kidneys


      

.absolute



+4



+9**



+4



+4



+14**



+9



.relative



+8*



+21**



+33**



+5



+13**



+17**



- Liver


      

.absolute



+5



+14**



+14**



+13*



+48**



+68**



.relative



+10



+27**



+46**



+13



+47**



+81**



- Thyroid glands


      

.absolute



-1



+7



+10



0



+16*



+15



.relative



+4



+18*



+40**



+1



+15*



+23**



Statistically significant from controls: *: p<0.05, **: p<0.01. The significance concerned the organ weights values and not the percentages.


Table 28 Relevant Changes in Mean Final Body Weights and Organ Weights in Treated Groups of Cohort 2A (% Changes from Controls)
































































































































Sex



Male



Female



Group



2



3



4



2



3



4



Dose-level (mg/kg bw/day)



100



300



800



100



300



800



Examined animals / Number of animals



10/10



10/10



9/10



10/10



10/10



10/10



- Final body weight



-7



-8



-20**



+8



+7



-1



- Kidneys


      

.absolute



+1



+9



+5



+9



+17



+17



.relative



+9



+18*



+31**



+1



+9



+19



- Liver


      

.absolute



-3



+16



+14



+23*



+51**



+76**



.relative



+5



+25**



+42**



+14



+41**



+79**



- Thyroid glands


      

.absolute



0



+4



+26*



-30**



-16



+1



.relative



+8



+12



+56**



-34**



-21*



+3



Statistically significant from controls: *: p<0.05, **: p<0.01. The significance concerned the organ weights values and not the percentages.


 


Table 29 Relevant Changes in Mean Final Body Weights and Organ Weights in Treated Groups in Cohort 2B (% Changes from Controls)










































































Sex



Male



Female



Group



2



3



4



2



3



4



Dose-level (mg/kg bw/day)



100



300



800



100



300



800



Examined animals / Number of animals



10/10



10/10



10/10



10/10



10/10



10/10



- Final body weight



-7



-8



-24**



-3



-8



-27**



- Thyroid glands


      

.absolute



-1



+14



-25



+17



+28



+19



.relative



+8



+23



-3



+18



+39*



+62**



Statistically significant from controls: *: p<0.05, **: p<0.01. The significance concerned the organ weights values and not the percentages.


HISTOPATHOLOGY F1 GENERATION


Table 30 Main Microscopic Lesions (Incidence and Severity) - Cohort 1A


































































































































Sex



Male



Female



Group



1



2



3



4



1



2



3



4



Dose-level (mg/kg bw/day)



0



100



300



800



0



100



300



800



Number of animals per group



20



20



19



20



20



20



20



19



KIDNEYS; hyaline droplet accumulation



grade 1



-



3



11



9



1



na



na



-



grade 2



1



-



7



6



-



na



na



-



grade 3



-



-



-



3



-



na



na



-



LIVER; hypertrophy; hepatocellular



grade 1



-



8



8



13



-



9



16



12



grade 2



-



-



1



5



-



-



-



2



THYROID GLAND; hypertrophy, follicular cell



grade 1



-



5



10



8



-



2



10



14



grade 2



-



-



-



2



-



-



-



-



-: not observed; na: not applicable.


NEUROBEHAVIOURAL DEVELOPMENT


Table 31 Auditory Startle Reflex on Postnatal Day 23 at 115 dB (mean ± SD) -  Cohort 2A



























































































































































































































Sex



Males



Females



Dose level (mg/kg bw/day)



0



100



300



800



HCD



0



100



300



800



HCD



Trials 1 to 10:



 



 



 



 



 



 



 



 



 



 



. Lat. (ms)



24 ± 0



25* ± 1



25 ± 2



25 ± 1



25 ± 1



24 ± 1



25 ± 1



24 ± 1



24 ± 1



25 ± 1



. Amp. (N)



1.36 ±
0.33



1.39 ± 0.29



1.08 ± 0.25



0.91** ± 0.29



1.64 ± 0.31



1.17 ± 0.30



0.91 ± 0.28



0.96 ± 0.30



0.70** ± 0.16



1.53 ± 0.32



Trials 11 to 20:



 



 



 



 



 



 



 



 



 



 



. Lat. (ms)



24 ± 1



25 ± 0



24 ± 1



24 ± 1



25 ± 1



24 ± 1



25 ± 1



24 ± 1



24 ± 1



24 ± 2



. Amp. (N)



1.16 ±
0.39



1.11 ± 0.28



0.86 ± 0.24



0.75* ± 0.27



1.48 ± 0.34



1.06 ± 0.30



0.79* ± 0.24



0.81 ± 0.25



0.63** ± 0.17



1.25 ± 0.45



Trials 21 to 30:



 



 



 



 



 



 



 



 



 



 



. Lat. (ms)



24 ± 1



25 ± 0



25 ± 2



24 ± 1



25 ± 1



24 ± 1



24 ± 1



24 ± 1



24 ± 1



24 ± 1



. Amp. (N)



1.08 ±
0.33



1.12 ± 0.21



0.79 ± 0.19



0.71** ± 0.23



1.43 ± 0.34



0.91 ± 0.24



0.70 ± 0.22



0.79 ± 0.23



0.59** ± 0.10



1.16 ± 0.36



Trials 31 to 40:



 



 



 



 



 



 



 



 



 



 



. Lat. (ms)



24 ± 1



25 ± 1



24 ± 1



24 ± 1



25 ± 1



24 ± 1



24 ± 1



24 ± 1



24 ± 1



24 ± 1



. Amp. (N)



1.10 ±
0.34



1.06 ± 0.27



0.73* ± 0.25



0.71** ± 0.23



1.41 ± 0.34



0.95 ± 0.25



0.68* ± 0.21



0.79 ± 0.24



0.53** ± 0.20



1.16 ± 0.35



Trials 41 to 50:



 



 



 



 



 



 



 



 



 



 



. Lat. (ms)



25 ± 1



25 ± 1



24 ± 1



24 ± 1



25 ± 1



24 ± 1



25 ± 1



24 ± 1



24 ± 1



24 ± 1



. Amp. (N)



1.05 ± 0.35



1.05 ± 0.28



0.72* ± 0.29



0.64** ± 0.22



1.34 ± 0.33



0.84 ± 0.20



0.68 ± 0.24



0.70 ± 0.19



0.53** ± 0.17



1.18 ± 0.32



Statistically significant difference from controls: *: p<0.05; **: p<0.01. ms: millisecond; N: Newton.


Lat.: Latency. Amp.: Magnitude of response. HCD: Historical Control Data (OECD443 - 2015-2021, n = 3 studies).


NEUROHISTOPATHOLOGY


Table 32 The Main Test Item-Related Measurements in 2A Cohort Animals (And Variations in Percentage Versus Controls)












































































































Group



1M



2M



3M



4M



1F



2F



3F



4F



Dose-level (mg/kg bw/day)



0



100



300



800



0



100



300



800



Total number of submitted animals per group



10



10



10



10



10



10



10



10



L3 L2+6 Cortex thickness (sensory)



Thickness (µm)



3967



4204



4291



3746



3757



 



 



3749



Standard deviation (µm)



184



176


+6%*



143


+8%**



187


-6%*



214



na



na



219


0%



L4-1


Dentate gyrus thickness



Thickness (µm)



695



635



666



606



616



na



na



586



Standard deviation (µm)



69



40


-9%



51


-4%



33


-13%**



29



na



na



60


-5%



L4-3


Entire hippocampus thickness



Thickness (µm)



1638



1639



1650



1488



1520



1628



1500



1426



Standard deviation (µm)



123



39


+9%



115


+1%



99


-9%*



61



77


+7%



67


-1%



74


-6**



*p<0.05; **p<0.01; na: not applicable.

Applicant's summary and conclusion

Conclusions:
The test item, BIS PEROXIDE, was administered daily by oral gavage, at dose levels of 0, 100, 300 or 800 mg/kg bw/day, to sexually-mature male and female rats [parental (P0) generation] starting 10 weeks before mating then continuously through mating, gestation and weaning of their pups (F1 generation). At weaning, the F1 generation was also exposed to graduated doses of the test item and was assigned to Cohorts of animals for reproductive/developmental toxicity.
Systemic toxicity evaluation:
The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 100 mg/kg bw/day based on decreased mean body weight and low mean body weight gain in males from 300 mg/kg bw/day and a series of adverse clinical signs in females at 800 mg/kg bw/day.
Reproductive/developmental toxicity evaluation:
The No Observed Adverse Effect Level (NOAEL) for reproductive/developmental toxicity was considered to be 300 mg/kg bw/day based on low pup body weights at 800 mg/kg bw/day.
Developmental neurotoxicity testing:
The No Observed Effect Level (NOEL) for developmental neurotoxicity was considered to be 100 mg/kg/day and the No Observed Adverse Effect Level (NOAEL) 800 mg/kg/day based on the dose-related decreases in the magnitude of auditory startle test responses from 300 mg/kg/day.
Executive summary:

The objective of this study was to provide general information concerning reproductive and developmental effects that may occur as a result of pre- and post-natal exposure to the test item, BIS PEROXIDE [which was administered daily by the oral route (gavage)], and to evaluate any systemic toxicity in pregnant and lactating females and young and adult offspring.


The dose formulations were administered by oral gavage, to sexually-mature male and female rats [Parental (P) generation] at test item dose levels of 0, 100, 300 and 800 mg/kg/day, daily, starting at least 10 weeks before mating and continuously through mating, gestation and weaning of their pups (F1 generation). At weaning, pups were selected and assigned to cohorts of animals for:



  • reproductive/developmental toxicity testing (cohorts 1A and 1B, without extension to mate the Cohort 1B animals to produce an F2 generation) and,

  • developmental neurotoxicity testing (cohorts 2A and 2B).


The F1 offspring received further treatment with the test item from weaning to adulthood or euthanasia. Clinical observations and pathology examinations were performed on all animals to detect any signs of toxicity, with special emphasis on the integrity and performance of the male and female reproductive systems and the health, growth, development and functioning of the offspring.


Methods


Treatment


P generation and F1 lactating offspring:


Three groups of 24 male and 24 female Sprague-Dawley rats (P generation) received the test item, BIS PEROXIDE (batch No. 2004500112), at 100, 300 or 800 mg/kg/day, daily for at least10 weeks prior to pairing, during pairing, and through gestation and lactation until weaning of the F1 pups [Day 22 post-partum (p.p.)]. The test item was administered orally (gavage,
5 mL/kg). A reference control group of 24 males and 24 females received the vehicle alone
(corn oil), under the same experimental conditions.


F1 generation:


The test item was administered orally, by gavage (5 mL/kg) to groups of 20 rats/sex (Cohorts 1A and 1B) or 10 rats/sex (Cohorts 2A and 2B) at 0 (vehicle, corn oil, only), 100, 300 or 800 mg/kg/day. The treatment schedules were as follows:



  • Cohort 1A: daily from weaning (Day 22 p.p.) until euthanasia (on Days 90 to 93 p.p.),

  • Cohort 1B: daily from weaning (Day 22 p.p.) for at least 10 weeks before euthanasia (after necropsy of Cohort 1A pending no alteration of estrous cycle or sperm parameters, and no macroscopic findings after examination of the reproductive organs),

  • Cohort 2A: daily from weaning (Day 22 p.) until euthanasia (after completion of behavioral testing: on Days 77 to 80 p.p.),

  • Cohort 2B: there was no direct dosing in cohort 2B animals (euthanized on Day 22 p.p.).


Examination of Parental, F1 generation


Clinical signs and mortality were checked daily. Food consumption and body weight were recorded at designated intervals.


P generation males and females were paired until mated or until 14 days had elapsed.


P generation females were allowed to deliver normally and rear their progeny. Pregnancy and litter parameters were recorded.


During lactation, the F1 pups were observed daily for survival and clinical signs. Body weight was measured at designated intervals and the sex-ratio was recorded. In F1, the size of each litter was adjusted on Day 4 p.p. to obtain ten pups per litter. Pup physical development was assessed at designated time-points.


Examination of Cohorts


Cohort 1A: animals were selected for assessment of general toxicity and effects on their reproductive system. Estrous cycle stages were determined daily for all females after the onset of vaginal patency, until the first cornified smear was recorded (estrus), and for 14 days before the end of the treatment period.


Cohort 1B: animals were selected to potentially obtain additional histopathology data.


Cohort 2A: on Day 22 p.p., 20 pups/group (10 males and 10 females/group; 1 male or 1 female/litter; all litters represented by at least 1 randomly selected pup) were selected for neurobehavioral testing followed by neurohistopathology assessment as adults.


Terminal examinations


A macroscopic post-mortem examination was performed on all P and F1 animals (including


F1 pups culled on Day 4 p.p. and F1 pups not selected on Day 22 p.p.). This included 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 ratio of organ weight to body weight (recorded immediately before euthanasia) was calculated and organs were weighed wet as soon as possible after dissection. A microscopic examination was performed on all macroscopic lesions and tissues (complete list) from animals in all groups or the control and high-dose groups.


P generation and Cohort 1A: the first 10 surviving animals/sex/group were fasted (food only) for an overnight period of at least 14 hours prior to blood sampling (for hematology, coagulation and blood biochemistry) and urine collection (for urinalysis) at termination. A series of sperm cell evaluations was performed on surviving males from all groups: motility, morphology, sperm cell head count in testicular/epididymal tissues.


Cohort 1A: splenic lymphocyte subpopulation analysis was performed on 10 surviving animals/sex/group.


Cohorts 2A and 2B: neurohistopathology was performed for all high-dose and control animals following the completion of neurobehavioral testing in Cohort 2A animals or on Day 22 p.p. in Cohort 2B animals.


Thyroid hormone levels (P generation, F1 culled pups and Cohort 1A animals): prior to blood sampling the animals were not fasted (except for animals from which samples were collected for hematology, blood biochemistry and urinalysis). Blood samples were taken on Day 4 p.p.
(F1 pups) for measurement of thyroid hormone (T4) levels. Blood samples were also taken on Day 22 p.p. (F1 pups not selected for Cohorts) and at termination [the first ten surviving males/group and the first ten lactating females/group (P generation animals)] for measurements of thyroid hormone (T4) and Thyroid Stimulating Hormone (TSH) levels.


Results


The concentrations of the dose formulations prepared for the P Generation and Cohorts treatments were all found to be within an acceptable range of variations (measured concentrations at ± 15% of the theoretical concentrations). No test item was found in the control dose formulation.


P generation


Mortality: There were no test-item related deaths.


Clinical signs:



  • in males, there were no adverse clinical signs,

  • in females, at 800 mg/kg/day, close or at the end of the premating period, there was a series of clinical signs (loud breathing, exophthalmos, hunched posture and/or piloerection), which were considered to be adverse based on their severity and/or duration. At 300 and 100 mg/kg/day, there were no adverse clinical signs.


Mean body weight and mean body weight change:



  • in males, from 300 mg/kg/day, there was a lower mean body weight (down to -26% on Days 127 and 134 with p<0.001 at 800 mg/kg/day) and a low mean body weight gain (down to +307 g vs. +475 g in controls on Days 1 to 140 with p<0.001 at 800 mg/kg/day). These findings were considered to be test item treatment-related and adverse (no recovery in terms of body weight or body weight gain). At 100 mg/kg/day, there were no adverse effects,

  • in females, there were no adverse effects during the premating, pregnancy or lactation period. A lower mean body weight was noted at 800 mg/kg/day during the pregnancy period followed by a return towards normal values during the lactation period.


Mean food consumption:



  • in males, at 800 mg/kg/day, mean food consumption was lower on Days 1 to 8 (-13% vs. controls, p<0.01), with a tendency towards a return to control values thereafter and thus, this finding was considered to be non-adverse. At 300 and 100 mg/kg /day, there were no adverse effects,

  • in females, there were no adverse effects during the premating, pregnancy or lactation period.


Estrous cycle, mating and fertility:



  • at 800 mg/kg/day, there was a lower mean number of cycles (1.6 vs. 2.4 in controls, p<0.01) as a consequence of an increased mean duration of cycles (7.3 vs. 5.1 days in controls, not statistically significant), which resulted in a lower percentage of females cycling normally (33.3% vs. 65.2% in controls, not statistically significant). A test
    item-relationship cannot be ruled out, but this finding was considered to be non-adverse based on the absence of an impact on mating and fertility data, and absence of correlated microscopic findings in the female reproductive tract,

  • a 300 and 100 mg/kg /day, when compared to controls, there were no effects on mean estrous cycle parameters,

  • at 800, 300 and 100 mg/kg /day, when compared to controls, there were no effects on mating or fertility data in both sexes.


Delivery data: there were no findings.


Pre-weaning F1 pups:


Mortality: There were no effects on the repartition of deaths in F1 lactating pups and no test item-related findings at macroscopic post-mortem examination of found dead pups.


F1 pup survival during lactation: There were no test item-related effects on the F1 pup viability and lactation indexes.


Clinical observations during lactation: There were no adverse findings.


Body weight and body weight change:



  • at 800 mg/kg/day there were lower mean body weights in males and females from Day 4 p.p. (down to -27% and -26% vs. controls on Day 7 p.p. in males and females, respectively, with p<0.001) and lower mean body weight gains throughout the lactation period. These findings were considered to be adverse,

  • at 300 and 100 mg/kg/day, there were no effects.


Pup development during lactation:



  • there were no effects on Day 1 p.p. mean anogenital distance (AGD) or normalized AGD, in males or females,

  • there were no nipples or areolae in male pups examined on Day 12 p.p.


Cohort 1A


Mortality: There were no test item-related deaths.


Clinical signs: there were no adverse clinical signs.


Body weight, body weight change:



  • at 800 mg/kg/day, there was a lower mean body weight in males (down to -20%, p<0.01) and lower mean body weight gain throughout the treatment period. These findings were considered to be test item treatment-related and adverse (no recovery in terms of body weight or body weight gain). At 800 mg/kg/day, in females, there was a lower mean body weight during the first 3 weeks of the treatment period (down to -21% on Day 8, p<0.01) and a lower mean body weight gain during the first week. Thereafter, mean body weight and mean body weight gain returned towards controls values. Therefore, these findings were considered to be test item-related but not adverse based on their reversibility.

  • at 300 and 100 mg/kg/day, there were no effects.


Food consumption: there were no adverse effects.


Time to first estrous and estrous cycles: there were no effects.


Cohort 1B


Mortality: there were no unscheduled deaths.


Clinical signs: there were no adverse clinical signs.


Body weight, body weight change:



  • at 800 mg/kg/day, there was a lower mean body weight in males (-19% to -22%, p<0.001) and lower mean body weight gain throughout the treatment period. These findings were considered to be test item treatment-related and adverse (no recovery in terms of body weight or body weight gain). At 800 mg/kg/day, in females, there was a lower mean body weight during the first 3 weeks of the treatment period (down to -21% on Day 8, p<0.01) and a lower mean body weight gain during the first week. Thereafter, mean body weight and mean body weight gain returned towards controls values. Therefore, these findings were considered to be test item-related but not adverse based on their reversibility.

  • at 300 and 100 mg/kg/day, there were no effects.


Food consumption: there were no adverse effects.


Cohort 2A


Mortality: there were no test item-related deaths.


Clinical signs: there were no adverse clinical signs.


Body weight and body weight change:



  • in males, at 800 mg/kg/day, there was a lower mean body weight throughout the treatment period (-17% to -25%, p<0.01) and a low mean body weight gain (+326 g vs. +397 g in controls over the Day 1 to 50 period, p<0.01). These findings were considered to be test item treatment-related and adverse (no recovery in terms of body weight or body weight gain).

  • In females, at 800 mg/kg/day, there was a lower mean body weight during the first 4 weeks of the treatment period (down to -23% on Days 1 and 8, p<0.01) and a lower mean body weight gain during the first week. Thereafter, mean body weight and mean body weight gain returned towards controls values. Therefore, these findings were considered to be test item-related but not adverse based on their reversibility.

  • at 300 and 100 mg/kg/day, there were no effects.


Food consumption: there were no effects


Neurobehavioral testing:


Auditory startle test:



  • there were no test item-related effects on the mean latency time,

  • from 300 mg/kg/day and when compared to controls, there were dose-related decreases in the magnitude of the responses (down to -39% vs. controls in males with p<0.01 and
    -44% vs. controls in females with p<0.01 at 800 mg/kg/day). Taking into account the dose level relationship, the amplitudes of the differences and the persistence of this finding over the trials, these changes were considered to be test item-related but non adverse in the absence of findings at auditory reflex evaluation.


Functional Observation Battery: There was no abnormal reactivity to manipulation or to different stimuli.


Motor activity: there were no effects.


Sexual development:


Males:



  • at 800 mg/kg/day, there was an increase in the mean age at balanopreputial separation (51.0 vs. 48.2 days, p<0.01). At this age, the mean body weight of the pups was lower (-10% vs. controls, p<0.01). While a test-item relationship cannot be excluded, the mean values remained within the range of the Historical Control Data. Therefore, in the absence of adverse findings at sperm analysis and in the absence of macro-/microscopic observations in male reproductive organs, these delays were considered to be non-adverse,

  • at 300 and 100 mg/kg/day, there were no effects.


Females:



  • at 800 mg/kg/day, there was an increase in the mean age at vaginal opening (36.4 vs. 33.4 days, p<0.01). While a test-item relationship cannot be excluded, the mean value remained within the range of the Historical Control Data. Therefore, in the absence of adverse findings on estrous cycle and in the absence of macro-/microscopic observations in female reproductive organs, this delay was considered to be non-adverse.,

  • at 300 and 100 mg/kg/day, there were no effects.


Laboratory investigations:


Hematology, coagulation, blood chemistry and urinalysis in P-generation and/or Cohort 1A animals: there were no adverse findings.


Thyroid hormones: there was a decrease in mean T4 level (down to -20 to -22% vs. controls, p<0.05) associated with an increase in mean TSH level (up to +109 to +118% vs. controls, p<0.05). These findings were considered as adaptive changes.


Sperm analysis: there were no effects on sperm parameters (motility, morphology, sperm/testicular numerations and daily sperm production rate) in P-generation or Cohort 1A males.


Lymphocyte subtyping: there were no adverse findings in Cohort 1A animals.


Pathology:


P generation


There were test item-related organ weight increases in the kidneys of males treated at ≥100 mg/kg/day that correlated with enlargement at necropsy and hyaline droplet accumulation at microscopic examination, in the liver at ≥100 mg/kg/day that correlated with enlargement and accentuated lobular pattern at necropsy and hepatocellular hypertrophy at microscopic examination, and in the thyroid glands at ≥100 mg/kg/day that correlated with follicular hypertrophy at microscopic examination. Decreased lymphoid cellularity was seen in males and females and correlated with macroscopic small thymus in males.


Adverse tubular degeneration/necrosis was noted in the kidneys of males treated at
800 mg/kg/day together with pigment and dilatation of tubules. The other findings were considered to be non-adverse.


At quantitative evaluation of primordial follicles or corpora lutea, there were no differences between the high-dose and control groups.


Cohort 1A


There were test item-related organ weight increases in the kidneys at ≥100 mg/kg/day that correlated with tan discoloration at necropsy and hyaline droplet accumulation at microscopic examination, in the liver at ≥100 mg/kg/day that correlated with hepatocellular hypertrophy at microscopic examination and in the thyroid glands at ≥300 mg/kg/day that correlated with follicular hypertrophy at microscopic examination.


At quantitative evaluation of primordial follicles or corpora lutea, there were no differences between the high-dose and control groups.


Cohort 1B


No test item-related organ weight differences, no gross or microscopic findings were noted.


Cohort 2A


There were test item-related organ weight increases in the kidneys of males at ≥300 mg/kg/day, in the liver at ≥100 mg/kg/day that correlated with enlargement at necropsy, and in the thyroid glands in males treated at 800 mg/kg/day.


There were no gross test item-related findings except the enlarged liver in 1/10 females treated at 800 mg/kg/day.


There were no microscopic test item-related lesions.


At neurohistopathology, there were ambiguous test item-related changes in males at
≥ 100 mg/kg/day (cortex and hippocampus) and in females at 800 mg/kg/day (hippocampus).


Cohort 2B


Increased relative-to-body thyroid gland weights were noted in females at ≥ 300 mg/kg/day that correlated with microscopic follicular cell hypertrophy.


There were no gross test item-related findings.


At neurohistopathology, there were no test item-related changes.


Pups


There were no test item-related organ weight differences in non-selected pups or gross findings in any pups.


Conclusion


The test item, BIS PEROXIDE, was administered daily by oral gavage, at dose levels of 0, 100, 300 or 800 mg/kg/day, to sexually-mature male and female rats [parental (P) generation] starting 10 weeks before mating then continuously through mating, gestation and weaning of their pups (F1 generation). At weaning, the F1 generation was also exposed to graduated doses of the test item and was assigned to cohorts of animals for reproductive/developmental toxicity.


Systemic toxicity:


The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 100 mg/kg/day based on adverse decreased mean body weight and low mean body weight gain in males from 300 mg/kg/day and a series of adverse clinical signs in females at 800 mg/kg/day.


Reproductive/developmental toxicity:


The No Observed Adverse Effect Level (NOAEL) for reproductive/developmental toxicity was considered to be 300 mg/kg/day based on low pup body weights at 800 mg/kg/day.


Developmental neurotoxicity:


The No Observed Effect Level (NOEL) for developmental neurotoxicity was considered to be 100 mg/kg/day and the No Observed Adverse Effect Level (NOAEL) 800 mg/kg/day based on the dose-related decreases in the magnitude of auditory startle test responses from 300 mg/kg/day.