Diisopropylbiphenyl and triisopropylbiphenyl

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

OECD 443 in rats

NOAEL parental general toxicity: 50 mg/kg bw/day

NOAEL paretnal reproduction toxicity: 50 mg/kg bw/day

NOAEL developmental toxicity (F1) (pre-weaning): 50 mg/kg bw/day

NOAEL developmental toxicity (F1) (post-weaning): 50 mg/kg bw/day

NOAEL developmental neurotoxicity (F1): 150 mg/kg bw/day

NOAEL developmental immunotoxicity (F1): 150 mg/kg bw/day

Link to relevant study records

Reference

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:

16 Jul 2019 - 18 Oct 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Health and Youth Inspectorate, Ministry of Health, Welfare and Sport, Utrecht, The Netherlands

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:

The study design was based on a final ECHA decision (CCH-D-2114373832-44-01/F)

- Premating exposure duration for parental (P0) animals : Ten weeks premating exposure duration was required because there was no substance specific information in the dossier supporting shorter premating exposure duration as advised in the ECHA Guidance on information requirements and chemical safety assessment ECHA Chapter R.7a, Section R.7.6 (version 6.0, July 2017).

- Basis for dose level selection : The highest dose level was chosen to induce some toxicity to allow comparison of effect levels and effects of reproductive toxicity with those of systemic toxicity. The dose level selection should be based upon the fertility effects with the other cohorts being tested at the same dose levels. As there was no relevant data to be used for dose level setting available, a range-finding study was performed.

- Inclusion/exclusion of extension of Cohort 1B: ECHA concluded that Cohort 1B shall not be extended to include mating of the animals and production of the F2 (for details see ECHA decision).

- Termination time for F2 : A F2 generation was not requested by ECHA and accordingly not included in the study.

- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B : Based on the available data from a 28-day oral toxicity study, during which adverse effects on the nervous system were noted, ECHA considered that the criteria to include Cohorts 2A and 2B was met.

- Inclusion/exclusion of developmental immunotoxicity Cohort 3 : Based on the available data, ECHA considered that the criteria to include Cohort 3 were not met and concluded that the developmental immunotoxicity Cohort 3 need not to be included.

- Route of administration : oral (gavage)

- Other considerations, e.g. on choice of species, strain, vehicle and number of animals: The study was performed in the rat, as this is the default species according to the OECD guideline 443.

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. The testing laboratory has general and reproduction/developmental/neurological historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive and neurological toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: P animals 6 wks; F1 animals 3 wks
- Weight at study initiation: (P) Males: 154 - 200 g; Females: 125 - 161 g; (F1) Males: 40 - 64 g; Females: 37 - 66 g
- Fasting period before study: no
- Housing: On arrival, prior to mating and during the post-weaning period, animals were group housed (up to 5 animals of the same sex and same dosing group and cohort together) in polycarbonate cages (Macrolon type IV; height 18 cm).
During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (type III; height 18 cm).
During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, type IV; height 18 cm). Females were individually housed in Macrolon plastic cages (type III, height 18 cm).
During the lactation phase, females were housed in Macrolon plastic cages (type III, height 18 cm). Pups were housed with the dam until termination or weaning (on PND 21).
During locomotor activity monitoring, F1-animals in Cohort 2A were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water for a maximum of 2 hours.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany, ad libitum)
- Water: Municipal tap water via water bottles, ad libitum)
- Acclimation period: 13 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 24
- Humidity (%): 45 - 66
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 23 Jun 2019 To: 07 Feb 2020

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared at least weekly as a solution, formulated in daily portions maximally 8 days in advance and stored in the refrigerator protected from light. On each day of dosing, the dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing. Also when formulations were prepared on the day of dosing, formulations were stirred at room temperature for at least 30 minutes before dosing.

VEHICLE
- Justification for use and choice of vehicle: Trial preparations were performed to select the suitable vehicle and to establish a suitable formulation procedure.
- Concentration in vehicle: 2.5, 12.5 and 37.5 mg/mL, respectively
- Amount of vehicle (if gavage): 4 mL/kg

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: max. 14 days
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged individually in Macrolon plastic cages (type III, height 18 cm)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were performed using a validated analytical procedure (HPLC-UV). Dose formulation samples were collected on three occasions during the study (weeks 1, 11 and 22 of treatment). On all three occasions achieved concentrations were determined for all groups and homogeneity was determined for the low and high dose group. Stability analysis was previously performed and demonstrated that the test item is stable in the vehicle when stored at room temperature under normal laboratory light conditions for at least 24 hours, in a refrigerator (2-8°C) for at least 8 days, and in a freezer (≤ -15°C) for at least 21 days (3 weeks).

Results of accury and homogeneity analysis:
No test item was detected in the Group 1 formulations.
The concentrations analyzed in the formulations of Groups 2, 3 and 4 prepared for use in study Week 1 and Week 11 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). The concentrations analyzed in the formulations of Groups 2 and 3 prepared for use in study Week 22 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).
For the formulation of Group 4 prepared for use in study Week 22, the mean accuracy was below the target concentration (i.e. 87% of target). The difference from target was minor and previous analyses performed in study Weeks 1 and 11 demonstrated that accuracies of the Group 4 formulations met the predefined specifications. Therefore, it was concluded that overall Group 4 formulations were prepared sufficiently accurate for the purpose of this study.
The formulations of Groups 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Duration of treatment / exposure:

P Males: At least 10 weeks prior to mating and then up to necropsy (11-12 weeks)
P Females: At least 10 weeks prior to mating, then through mating, gestation and at least until LD 21 (16-18 weeks)
Cohort 1A and 1B: PND 21 up to at least PND 91 or PND 98, respectively.
Cohort 2A: PND 21 up to at least PND 77

A few F0-female (one in Group 1, one in Group 2, four in Group 3 and two in Group 4) were not dosed on one occasion day as these females were littering at the moment of dosing. The omission of one day of dosing over a period of several weeks was considered not to affect the toxicological evaluation.
Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk, or from exposure to maternal urine/feces.
From weaning onwards (PND 21), F1-animals of Cohorts 1A, 1B, 1C and 2A were dosed up to and including the day before scheduled necropsy. The F1-animals of Cohort 2B, Cohort Surplus and Spares (not assigned to one of the cohorts) were not dosed.

Frequency of treatment:

daily, 7 days/week

Details on study schedule:

- F1 parental animals were not mated in this study.
- Age at mating of the mated animals in the study: 16 weeks

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

P animals: 25 males and females per dose
F1A cohort: 20 males and females per dose
F1B cohort: 20 males and females per dose
F2A cohort: 10 males and females per dose
F2B cohort: 10 males and females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a dose range finder study. In this study, the test item was administered daily by oral gavage to 8 Wistar Han rats of both sexes at dose levels of 10, 30, 100 and 300 mg/kg bw/day for 13-18 weeks (including a 10 week period prior to mating). Moreover, from PND 21-28, F1-animals received the test item via daily oral gavage. There were three test item related mortalities in F0-females (one at 30 mg/kg bw/day and two at 300 mg/kg bw/day). These females died during or shortly after delivery of their litter. Main macroscopic findings in these females were many grey-white foci on the liver and enlarged liver and the main cause of moribundity was slight to marked coagulative necrosis in the liver. Main findings included: An increased food consumption at 300 mg/kg bw/day in males from the third week of treatment onwards and in females from the third week of treatment until the lactation phase, during which food consumption was somewhat decreased. Reduced body weight and body weight gain in males at 300 mg/kg bw/day from the third week of treatment onwards (a similar trend was observed at 100 mg/kg bw/day). Test item-related increased liver weights were present in males starting at 30 mg/kg bw/day and in females starting at 100 mg/kg bw/day and increased thyroid gland weights were present in males starting at 30 mg/kg bw/day and in females at 300 mg/kg bw/day. Test item-related macroscopic findings were enlarged liver and black-brown discolouration of the liver (present in males and females treated at 100 and 300 mg/kg bw/day). Moreover, enlarged thyroid gland was present in males and females at 300 mg/kg bw/day. Duration of gestation was increased at 300 mg/kg bw/day and mean litter size was decreased at 300 mg/kg bw/day to approximately 4 living pups/litter (a similar but less pronounced trend was observed at 100 mg/kg bw/day). Moreover, mean number of implantation sites at 300 mg/kg bw/day was significantly reduced. Body weight, sex ratio and clinical signs of pups were considered unaffected by treatment. In F1-animals, absolute and relative liver weights were increased at 30 (females only), 100 and 300 mg/kg bw/day (males and females). Moreover, in F1-females absolute and relative thyroids weights were increased at 300 mg/kg bw/day (a similar trend was observed in F1-males).

In addition to the above mentioned dose range finder study, a 28-day study and a prenatal developmental toxicity study with oral exposure of the test item in rats were considered for dose level selection of the current study.

In the 28-day toxicity study (Simon, 2014), the test item was administered daily by oral gavage to 10 Wistar Han rats of both sexes at dose levels of 35, 150 and 600 mg/kg bw/day. No test item-related mortality occurred during the study. Clinical signs noted in males and females were salivation at 150 and 600 mg/kg bw/day towards the end of the treatment period. Main findings included: An initial decrease in food consumption at 600 mg/kg bw/day, followed by an increased food consumption until the end of treatment. Reduced body weight gain in males at 600 mg/kg bw/day during the whole treatment phase. Slightly increased body weight gain in females at 600 mg/kg bw/day, although absolute body weights were unaffected. Prostate gland and seminal vesicles as well as epididymides were decreased in males at 150 and 600 mg/kg bw/day, showing statistical significance at 600 mg/kg bw/day. Weights of ovaries were decreased at 600 mg/kg bw/day and weights of uterus were decreased at 150 and 600 mg/kg bw/day. In addition to changes in the organ weights, follicular degeneration and disturbance of estrous cycle in the uterus were noted microscopically at 600 mg/kg bw/day. A slight increase in kidney weights was observed at 600 mg/kg bw/day. Absolute and relative liver weights and thyroid weights were markedly increased at all dose levels associated with changes in biochemical parameters, pituitary and thyroid hormone levels (TSH ↑, T3 and T4 ↓), macroscopic findings (liver) and microscopic findings. Macroscopic evaluation revealed enlarged livers in all animals at 600 mg/kg bw/day. Microscopic findings in the liver included diffuse or centrilobular hypertrophy (at all dose levels) with granular eosinophilic cytoplasm and focal (in 2/10 females at 600 mg/kg bw/day) or single cell necrosis (at all dose levels in males). In this study, a no observed adverse effect level (NOAEL) could not be established after 4 weeks of oral treatment at dose levels of 35, 150 and 600 mg/kg/day.

In the prenatal developmental toxicity study (de Groot, 2020), the test item was administered daily by oral gavage to 22 time-mated female Wistar Han rats from Day 6 to 20 post-coitum at dose levels of 0, 50, 150, 450 mg/kg bw/day. Piloerection was observed in 8/22 females at 450 mg/kg bw/day, mainly at the end of the treatment period. Maternal toxicity was observed at 450 mg/kg bw/day, consisting of body weight loss observed in approximately half of the females between post-coitum Day 6-9, resulting in a mean body weight gain of zero. This was followed by statistically significant reduced body weight gain throughout the treatment period. The observed body weight effects coincided with statistically significantly reduced food consumption at 450 mg/kg bw/day between post-coitum Day 6-12, after which it returned to normal values and remained within the normal range of biological variation over the remainder of the treatment period. Body weight gain corrected for gravid uterus was also lower at 450 mg/kg bw/day. Enlarged livers were noted in females treated at 450 mg/kg bw/day. Moreover, a test item-related increase in liver weights was observed at all dose levels. TSH serum levels were increased at 450 mg/kg bw/day and a similar, not statistically significant trend was observed at 150 mg/kg bw/day. Additionally, a decrease in total T4 and total T3 was observed at 150 and 450 mg/kg bw/day. A test-item related increase in the mean % of early resorptions, and consequently % total resorptions and % post-implantation loss per litter was observed at 450 mg/kg bw/day. Mean male, female and combined fetal body weights were significantly reduced at 450 mg/kg bw/day. Based on the results in this prenatal developmental toxicity study the maternal No Observed Adverse Effect Level (NOAEL) was established at 50 mg/kg bw/day, whereas the developmental NOAEL was set at 150 mg/kg bw/day.

Based on the above mentioned results and considering the long treatment period of 13-16 weeks in the EOGRTS in light of the quite severe maternal toxicity observed at dose levels down to 300 mg/kg bw/day, also in combination with the pronounced decreased litter size at 300 mg/kg bw/day (resulting in insufficient offspring to fill the required Cohorts), dose levels of 10, 50 and 150 mg/kg bw/day were selected.

- Rationale for animal assignment: random

- Fasting period before blood sampling for clinical biochemistry: The selected F0-animals and Cohort 1A animals were fasted overnight with a maximum of 24 hours before blood sampling, but water was available.

Positive control:

not applicable

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, in the morning and at the end of the working day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first administration of the test item, and at weekly intervals during the treatment period (after dosing)

BODY WEIGHT: Yes
- Time schedule for examinations: P generation: on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 14 and 21.
Live pups were weighed individually on PND 1, 4, 7, 13 and 21. After weaning F1 animals were weighed weekly. In addition, for each female and male the body weight was recorded on the day of acquisition of vaginal patency and balanopreputial separation, respectively.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: on a regular basis throughout the study by visual inspection of the water bottles

HAEMATOLOGY AND COAGULATION
- Blood was collected from the retro-orbital sinus under isoflurane anaesthesia from P and F1A animals (10 selected rats/sex/group) in the morning of scheduled sacrifice between 7.00 and 10.30 a.m.. Animals were fasted prior to blood sampling. The following parameters were analysed: red blood cells, haemoglobin, haematocrit, mean corpuscular volume, red blood cell distribution width, mean corpuscular haemoglobin concentration, mean corpuscular haemoglobin, reticulocyte count (absolute), platelets, white blood cells, neutrophils (absolute), lymphocytes (absolute), monocytes (absolute), eosinophils (absolute), basophils (absolute), large unstained cells (absolute).

A blood smear was prepared from each haematology sample. Blood smears were labeled, stained, and stored. Blood smears were evaluated when required to confirm analyser results.

The following coagulation parameters were determined: activated partial thromboplastin timeand prothrombin time

CLINICAL CHEMISTRY
- Blood was collected from the retro-orbital sinus under isoflurane anaesthesia from P and F1A animals (10 selected rats/sex/group) in the morning of scheduled sacrifice between 7.00 and 10.30 a.m.. Animals were fasted prior to blood sampling. The following parameters were analysed: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total protein, albumin, total bilirubin, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic.

THYROID HORMONES
- Blood was collected from the retro-orbital sinus under isoflurane anaesthesia from P and F1A animals (10 selected rats/sex/group) in the morning of scheduled sacrifice between 7.00 and 10.30 a.m.. Furthermore, pooled blood was collected from PND 4 pups and Cohort 2B pups terminated on PND 21/22. Blood samples were processed to serum. Serum of F0-animals, Cohort 1A animals and Cohort 2B animals (i.e. PND 21-22 pups) was used for measurement of both T4 and TSH. Due to insufficient sample volume, serum from Cohort 2B was diluted. Pooled serum of culled PND 4 pups was used for measurement of T4 only.

URINALYSIS
- Urine was sampled overnight for 15-20 hours with absence of food from P and F1A animals (10 selcted rats/sex/group) during the last week of dosing. The following parameters were analysed: volume, specific gravity, clarity, colour, pH, blood, white blood cells, bilirubin, urobilinogen, protein, ketones, nitrite; from the sediment: white blood cells (WBC-sed.), red blood cells (RBC-sed.), casts, epithelial cells, crystals, bacteria, other

Oestrous cyclicity (parental animals):

Daily vaginal lavage was performed for all F0-females beginning 14 days prior to mating and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.
A vaginal lavage was also taken from all females surviving until scheduled necropsy, except for females euthanized in extremis.

During the first period, daily vaginal lavage was performed for all Cohort 1A females starting on the day of onset of vaginal patency and was minimally continued until the first estrous was determined, in order to determine the time interval between these two events. During the second period, daily vaginal lavage was performed from PND 75 to 88.
The estrous cycle data of the first period is not reported.

Sperm parameters (parental animals):

Parameters examined in all P and F1A males:
testis weight, epididymis weight, sperm motility and progressive motility, sperm morphology, sperm count in epididymides, enumeration of cauda epididymal sperm reserve

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); from 2 excess pups/litter blood samples for thyroid hormones were taken before they were killed and macroscopically examined (externally and internally).

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups, sexual maturation (age and body weight at vaginal opening, age and body weight at balano-preputial separation). Particular attention was paid to the external reproductive genitals which examined for signs of altered development.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: Cohort F2A and F2B were examined for possible signs of developmental neurotoxicity. The following methods were applied: acoustic startle response between PND 23-25 (F2A and a selection of 1A and 1C animals), functional obersation battery (FOB) between PND 63-75 (F2A animals); neurohistopathology and morphometric analysis for control and high dose F2A and F2B animals on PND 76-90 and 21-22, repectively.

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: Spleen samples (approximately half of the spleen) were taken from 10 selected rats per sex per group from the F1 Cohort 1A adults at necropsy and were analysed using a validated analytical method for the relative percentage of T-cells, T-helper cells, T-cytotoxic cells, B-cells and Natural Killer (NK)-cells. The ratio of T-helper cells/ T-cytotoxic cells (Th/Tc) was calculated. The % lymphoid cells of peripheral blood mononuclear cells (PBMC) were determined using the Forward Scatter and Side Scatter.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after successful mating and a minimum of 10 weeks of treatment
- Maternal animals: LD 23-25

GROSS NECROPSY
- Gross necropsy consisted of a full post mortem examination, with special attention being paid to the reproductive organs.
The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.

HISTOPATHOLOGY / ORGAN WEIGHTS
The organs identified in Attachment "Tissue Collection and Preservation for the P Generation" under Attached Background Material were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals euthanized in extremis. Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of the aberrant organ was taken and recorded in the raw data. Organ to body weight ratios (using the terminal body weight) were calculated.
Representative samples of the tissues were collected from all animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution), unless otherwise indicated. Identified tissues were embedded in paraffin, sectioned at a thickness of 2-4 micrometers, mounted on glass slides, and stained with hematoxylin and eosin (HE). They were then were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology. A peer review on the histopathology data was performed by a second pathologist.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as F1 cohort animals were sacrificed at PND 4 (culling) or between PND 22 and 24.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Animals not selected to one of the F1 cohorts were were externally examined, with particular attention to the external reproductive genitals to examine signs of altered development, and sex was determined (both externally and internally).
- Scheduled necropsy of Cohort 1A was conducted on PND 89-95. All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.
- Scheduled necropsy of Cohort 1B was conducted on ≥ PND 97. Cohort 1B animals were not deprived of food overnight before necropsy. These animals were weighed and deeply anaesthetized using isoflurane and subsequently exsanguinated. All animals were subjected to a limited examination, with special attention being paid to the reproductive organs.
- Scheduled necropsy of Cohort 1C was conducted after positive determination of vaginal patency or balanopreputial separation. Cohort 1C animals were not deprived of food overnight before necropsy and no terminal body weight was recorded. The animals were deeply anaesthetized using isoflurane and subsequently exsanguinated. All animals were subjected to a limited examination, with special attention being paid to the reproductive organs.
- Cohort 2A and 2B
Scheduled necropsy of Cohort 2A was conducted on PND 76-90. Scheduled necropsy of Cohort 2B was conducted on PND 21-22. The animals were not deprived of food overnight before necropsy and no terminal body weight was recorded. The animals were first anaesthetized using isoflurane, followed by blood sampling for measurement of thyroid hormones, and subsequently sacrificed by whole body (in situ) perfusion using heparinized saline (0.9% NaCl) followed by a 4% paraformaldehyde solution (adjusted to pH 7.4; HCl, KCl, NaH2PO4 x H2O, Na2HPO4 x 2H2O, paraformaldehyde and NaOH, aqua dest.).
All animals were subjected to a limited examination, with special attention being paid to the reproductive organs. After perfusion, the cranium was removed, exposing the brain. The skull including the brain was placed in 10% buffered formalin and allowed to fix for at least 7 days prior to removal from the skull. The fixed brains were removed and weighed, and the length and maximum width of the brain was measured for all animals selected for neuropathology . Subsequently, the brain was fixed in 10% buffered formalin together with selected PNS tissues.
- Cohort Surplus
Scheduled necropsy of Cohort Surplus was conducted on PND 22. Cohort Surplus animals were not deprived of food overnight before necropsy and a terminal body weight was recorded. All animals were subjected to a limited examination, with special attention being paid to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGTHS
The organs identified in Attachment "Tissue Collection and Presevration for the F1 Generation" under Attached Background Material were prepared for microscopic examination and weighed, respectively.

Cohort 1A: In addition to the histopathology procedures described above, HE stained step sections of ovaries and corpora lutea at a thickness of 5 micrometers (5 step sections in total, including the routine section) were prepared for the Cohort 1A animals of Group 1 and 4 for quantitative evaluation of follicles (primordial and small growing follicles counted together), as well as corpora lutea. As a treatment-related effect was suspected, this was extended to animals in the intermediate dose groups (Groups 2 and 3).

Cohort 2A and 2B: In addition to the procedures described above, the entire brain from all groups was processed to the block stage up front at the same time to avoid effects of fixation duration on morphometry. Sections of the brains of all Cohort 2A and 2B animals (all groups) were also stained for myelin and cell bodies using Luxol Fast Blue and Cresyl Violet. For morphometric analysis, 3 consecutive sections were taken from neocortical, hippocampal and cerebellar areas to ensure homologous sections are obtained.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed according to sex and occasion. Descriptive statistics number, mean and standard deviation were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Parametric
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). For the motor activity data set (at least 3 groups) parametric (ANOVA) tests on group means were applied with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six different covariance structures, were used in order to select the best fitting statistical model.

Non-Parametric
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).

Incidence
An overall Fisher’s exact test was used to compare all groups. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Reproductive indices:

Mating Index Males (%): (Number of males mated / Number of males paired) x 100

Mating Index Females (%): (Number of females mated / Number of females paired) x 100

Precoital time: Number of days between initiation of cohabitation and confirmation of mating

Fertility Index Males (%): (Number pregnant females / Number of males mated) x 100

Fertility Index Females (%): (Number pregnant females / Number of females mated) x 100

Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Post-implantation survival index (%): (Total number of offspring born / Total number of uterine implantation sites) x 100

Live Birth Index (%) = (Number of live offspring on Day 1 after littering / Number of offspring born) x 100

Percentage live males at First Litter Check (%): (Number of live male pups at First Litter Check / Number of live pups at First Litter Check) x 100

Percentage live females at First Litter Check (%): (Number of live female pups at First Litter Check / Number of live pups at First Litter Check) x 100

Offspring viability indices:

Viability Index (%): (Number of live offspring on Day 4 Postpartum / Number of live offspring on Day after littering) x 100

Weaning Index (%): (Number of live offspring on Day 21 after littering / Number of live offspring on Day 4 (after culling) ) x 100

Percentage live males at weaning (%): (Number of live male pups on Day 21 after littering / Number of live pups on Day 21 after littering) x 100

Percentage live females at weaning (%): (Number of live female pups on Day 21 after littering / Number of live pups on Day 21 after littering) x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

After approximately 11 weeks of treatment, the majority of animals at 150 mg/kg bw/day started to show an increased activity of the forelegs a few minutes after dosing. It consisted of repeated short periods of ‘digging’ in the sawdust and lasted for a couple of minutes only. This behaviour was seen first in males, and started a few days later also in females.
Salivation was observed after dosing among animals of the 10 mg/kg bw/day group (males only), 50 and 150 mg/kg bw/day groups (both sexes) from the first weeks of treatment onwards. Considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing) it was considered to be a physiological response rather than a sign of systemic toxicity.

Other clinical signs noted during the treatment period included alopecia, scabs, wounds, a crusted mass, red discoloration and thickening of the ear(s), exophthalmos, chromodacryorrhoea, diarrhoea, piloerection, hunched posture, and a broken tail apex. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the low incidence observed and in absence of a dose-relation, these were considered signs of no toxicological relevance.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Two high dose females treated at 150 mg/kg bw/day were euthanized for humane reasons due to a poor condition:
One female was sacrificed in extremis on Day 23 post-coitum due to delivery difficulties. Prior to its death it presented with piloerection. At necropsy, six dead fetuses and nine early resorptions were found in its uterus (no live fetuses). This presence of dead fetuses in the uterus was regarded as the main cause of moribundity. Other gross findings included an accentuated lobular pattern of the liver and enlarged spleen, which correlated histopathologically with mainly centrilobular hepatocellular hypertrophy and extramedullary hematopoiesis, respectively.
A second female was euthanized in extremis as it was noted with complications during delivery on Day 23 post-coitum. This female which showed piloerection and a pale appearance, had delivered nine dead pups and two live pups, but its litter was not found completed yet (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). At the time the dam was sacrificed, all its eleven pups were dead. No macroscopic abnormalities were noted for this female at necropsy. Histopathological examination revealed pyometra as main cause of moribundity, probably caused by the presence of dead fetuses.

Note: One control female was euthanized, as she had a total litter loss on Day 2 of lactation.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In males treated at 150 mg/kg bw/day, slightly lower body weights and body weight gains compared to concurrent controls were noted from Week 7 of the premating period onwards, lasting until the end of the mating period. Changes compared to concurrent controls were slight only (not always reaching statistical significance). A similar trend was observed for males treated at 50 mg/kg bw/day, but changes compared to the concurrent control group did not reach statistical significance. Overall these changes in body weight were relatively slight only. Mean terminal body weight in males at 50 and 150 mg/kg bw/day was 0.95x and 0.93x of control, respectively (reaching statistical significance at the high dose only).
No treatment-related changes in body weight and body weight gain were noted in females up to 150 mg/kg bw/day. The slightly lower mean body weight and body weight gain observed towards the end of gestation (Days 17 and 20 post-coitum) was considered to be secondary to the smaller litter size observed at this high dose level. .
Any remaining statistically significant changes in body weights or body weight gains (incl. the higher mean body weight gain in high dose females at Day 22 of premating) were considered to be unrelated to treatment with the test item as no trend was apparent regarding dose and/or changes were not consistent in time.

For details see Attachment "Summary of P animal body weights and body weight gains" under Attached Background Material.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No toxicologically relevant changes in food consumption before or after allowance for body weight were noted in males and females treated up to 150 mg/kg bw/day.
In males treated at 50 and 150 mg/kg bw/day, a slightly lower food consumption (absolute and relative to body weight) was observed in the first week of premating. No toxicological relevance was attached to this finding, as changes compared with the concurrent control group were relatively slight (0.95-0.97x of control; reaching no statistical significance for relative food consumption in males at 50 mg/kg bw/day), occurred in the absence of a clear dose-related trend and were transient only. From Week 2 of premating onwards, absolute food consumption was within the concurrent control range, while food consumption after correction for body weight was slightly higher in males at 150 mg/kg bw/day compared to the concurrent controls (1.03-1.07x and 1.14x of control during premating and in the first week of the mating period, respectively; not always statistically significant).
Also in females at 150 mg/kg bw/day, a trend towards slightly higher food intake (absolute and relative to body weight) was noted during pre-mating and post-coitum. Changes compared with the concurrent control group were relatively slight only (relative food consumption: 1.04-1.11x and 1.06-1.14x of control during pre-mating and post-coitum, respectively) and did not always reach statistical significance.
The lower absolute and relative food intake in females at 150 mg/kg bw/day during lactation (relative: 0.91-0.89x of control; not always reaching statistical significance) could be attributed to the many smaller litters in this high dose group and hence a lower caloric demand.
The slightly higher absolute food consumption in females at 50 mg/kg bw/day during post-coitum and lactation (not always statistically significant) could directly be related to the relatively high body weight in this mid dose group. After correction for body weight, mean values of relative food intake were comparable to the concurrent control group. The higher mean observed from Days 7-11 post-coitum was considered to be the result of a slightly low control mean.
Any other statistically significant changes in food consumption before or after correction for body weight were considered to be unrelated to treatment since no trend was apparent regarding dose and duration of treatment.

For details see Attachment "Summary of P animal food consumption" under Attached Background Material).

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

No intergroup differences were noted by visual inspection of water bottles.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no differences noted in hematological parameters between control and treated rats that were considered to be related to treatment with the test item.
The mean reticulocyte count recorded in females at 150 mg/kg bw/day was slightly low at 101.7 109/L (0.71x of control, not statistically significant) and slightly below the available historical range*. However, no toxicological relevance was attached to this finding as all correlating parameters were within normal ranges.
Any statistically significant changes were considered to be of no toxicological relevance as they occurred in the absence of a treatment-related distribution and/or remained within the range considered normal for rats of this age and strain.

Coagulation parameters of treated rats were considered not affected by treatment.

For details see Attachment "Summary of P animal haematology and coagulation data" under Attached Background Material.

------------------------
*Historical control data for Wistar Han rats, F0-females (period 2017-2019):
Reticulocyte count (109/L): mean: 204.4; P5 - P95: 108.40 – 288.90 (n=87).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The following statistically significant changes distinguished treated from control animals. Relative changes in mean values as compared to the concurrent control group are indicated between parentheses. Relevant historical control data are provided by footnote
- Mean alkaline phosphatase (ALP) activity was increased in males at 150 mg/kg bw/day (2.06x; above historical control range).
- Mean total protein concentrations were increased in females at 50 and 150 mg/kg bw/day (1.06x and 1.08x, respectively; above historical control range at 150 mg/kg bw/day).
- Mean albumin concentrations were increased in males and females at 50 and 150 mg/kg bw/day (1.04x and 1.10x (males), respectively, and 1.06x and 1.10x (females), respectively; all above historical control range, except for males at 50 mg/kg bw/day).
- Mean total bilirubin concentration was decreased in females at 150 mg/kg bw/day (0.78x; within historical control range).
- Mean glucose concentration was increased in males at 50 and 150 mg/kg bw/day (1.21x and 1.25x, respectively; both levels above historical control range).
- Mean chloride concentration was increased in females at 50 and 150 mg/kg bw/day (1.02x and 1.03x, respectively; both levels within historical control range).
- Mean calcium concentration was increased in males and females at 150 mg/kg bw/day (1.02x and 1.03x, respectively; both levels within historical control range).

Remaining statistically significant changes were seen in the low dose and/or mid dose group only, or occurred with no apparent dose-related response. These were therefore considered to be unrelated to treatment with the test item.

For details see Attachment "Summary of P animal clinical biochemistry data" under Attached Background Material.

THYROID HORMONE ANALYSES:
Treatment-related changes were observed in the high dose group only:
- Mean TSH levels were increased in males and females at 150 mg/kg bw/day (5.07x and 10.77x of control, respectively; both levels above historical control range).
- Mean total T4 level was decreased in males at 150 mg/kg bw/day (0.64x of control; below historical control range).

For details see Attachment "Summary of P animal thyroid hormones" under Attached Background Material.

-------------------------
Historical control data for Wistar Han rats, F0-generation (period 2017-2019):
ALP (U/L) - males: mean: 95; P5-P95: 62.0-132.0 (n=90).
Total protein (g/L) - females: mean: 64.4; P5-P95: 59.10-70.30 (n=91).
Albumin (g/L) - males: mean: 32.5; P5-P95: 30.90-34.60 (n=90).
- females: mean: 34.4; P5-P95: 32.10-36.70 (n=91).
Total bilirubin (µmol/L) - females: mean: 2.4; P5-P95: 1.50-3.40 (n=91).
Glucose (mmol/L) - males: mean: 8.45; P5-P95: 6.730-10.410 (n=90).
Chloride (mmol/L) - females: mean: 100; P5-P95: 95.0-104.0 (n=91).
Calcium (mmol/L) - males: mean: 2.59; P5-P95: 2.470-2.730 (n=90).
- females: mean: 2.63; P5-P95: 2.460-2.850 (n=91).
TSH (µIU/mL) - males: mean: 0.181; P5-P95: 0.0310-05200 (n=90).
- females: mean: 0.215; P5-P95: 0.0350-0.6420 (n=87).
Total T4 (µg/dL) - males: mean: 5.93; P5-P95: 4.100-7.820 (n=90).

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urinalysis did not reveal any treatment-related changes

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in the liver and thyroid glands of males and females, the kidneys of males and the urinary bladder of females, and are summarized in Table 2.

LIVER
Hepatocellular hypertrophy, mainly centrilobular, was present starting at 50 mg/kg bw/day up to moderate in males and up to slight degree in females. This finding correlated with the higher liver weight and with the macroscopic enlarged and thickened liver.

THYROID GLAND
An increased incidence and severity of follicular cell hypertrophy was present starting at 50 mg/kg bw/day up to moderate degree in males and up to slight degree in females. This finding correlated with the higher thyroid gland weight.
An increased incidence and severity of colloid alteration was present starting at 50 mg/kg bw/day up to slight degree in males and females.

KIDNEYS
An increased incidence and severity of hyaline droplet accumulation was present in males starting at 50 mg/kg bw/day up to moderate degree.

URINARY BLADDER
Hyperplasia of the urothelium was present in a single female at 150 mg/kg bw/day at minimal degree, and hypertrophy of the urothelium was present in females at 150 mg/kg bw/day at minimal degree.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

REPRODUCTIVE ORGANS
Histopathological examination of reproductive tissues revealed no evidence of a treatment-related effect on reproduction.
Couples that did not succeed in producing healthy offspring are summarized in Table 3.
No abnormalities were seen in the reproductive organs or mammary gland (examined for one control female with total litter loss only), that could explain the absence of pregnancy, or account for their lack of healthy offspring, except for one high dose female which was observed with pyometra.

SPERMATOGENESIS STAGING
Stage dependent qualitative evaluation of spermatogenesis in the testis was performed. The testes revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.

Histopathological findings: neoplastic:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Length and regularity of the estrous cycle were unaffected by treatment with the test item.
Most females had regular cycles of 4 to 5 days. An irregular cycle of 2, 3 and 8 days was noted for three low dose females, and an irregular cycle of 2 days was noted for one female each in the mid and high dose group, respectively. All these females with irregular cycle had normal litters. Given their incidental nature, no dose response and absence of an apparent correlation to pregnancy status, these findings did not indicate a relation with treatment.
For details see Attachment "Summary of P females estrous cycle patterns" under Attached Background Material.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Sperm motility, morphology and concentration were considered unaffected by treatment with the test item.
Mean number of sperm cells with abnormal head was statistically significantly higher at 150 mg/kg bw/day compared with the control mean. However, it should be noted that the difference in means was very slight only (mean of three cells in the high dose group vs. one cell in the control group). Furthermore, mean number of sperm cells with abnormal heads in the high dose group remained within historical control range , and mean numbers of sperm cells with normal morphology were similar in all groups. Therefore, this change was considered not to have been caused by treatment with the test item.

For details see Attachment "Summary of P male sperm measures" under Attached Background Material.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Mating index for both males and females was not affected by treatment with the test item. Mating was confirmed for all females, except for three mid dose females. In the absence of a dose-related trend, it this was considered unrelated to treatment with the test item.

Precoital time was unaffected by treatment. Mean precoital time was 2.0, 1.6, 3.4 and 2.7 days for the control, 10, 50 and 150 mg/kg bw/day groups, respectively. The majority of females showed evidence of mating within 5 days, except for three females in the 50 mg/kg bw/day group for which it took 7, 9 and 14 days, respectively, before mating could be confirmed, and one female in the 150 mg/kg bw/day group for which it took 14 days. Except for one mid dose female which was observed with an irregular cycle of 2 days all of these females had regular estrous cycles. In the absence of a dose-related trend, the incidentally longer precoital times were considered unrelated to treatment.

The number of implantation sites was unaffected by treatment up to 50 mg/kg bw/day. At 150 mg/kg bw/day, a statistically significantly lower mean number of implantation sites was recorded (10.0 vs 11.8 in the control group). At the individual level it was noted that 4/23 pregnant females at 150 mg/kg bw/day had less than seven implantation sites, i.e. the lowest number of implantations in the concurrent controls (1/24). For comparison, all females in the 10 and 50 mg/kg bw/day groups had more than seven implantation sites.

The fertility index was considered to be unaffected by treatment with the test item. The fertility indices were 96%, 96%, 95% and 92% for the control, 10, 50 and 150 mg/kg bw/day groups, respectively, for both males and females. The number of non-pregnant females versus mated females was 1/25, 1/25, 1/22 and 2/25 in the control, 10, 50 and 150 mg/kg bw/day groups, respectively. As these cases of non-pregnancy remained within the normal biological variation, these were considered not to be related to treatment with the test item.

DEVELOPMENTAL DATA
At 150 mg/kg bw/day, three pregnant females did not have live offspring, which resulted in a gestation index of 87% vs. 100% in the controls. One of the two females euthanized in extremis had six dead fetuses in its uterus in addition to nine early resorptions (no living pups), and all eleven pups of the other female had died before necropsy. The third female had four implantation sites only.
All pregnant females in the control, 10 and 50 mg/kg bw/day groups had live offspring, resulting in a gestation index of 100% for these groups.

Duration of gestation was considered to be unaffected by treatment up to 150 mg/kg bw/day. Duration of gestation was 21.0, 21.5, 21.8 and 21.7 days in the control, 10, 50 and 150 mg/kg bw/day groups, respectively. The slightly, but statistically significantly longer mean duration of gestation observed at 50 and 150 mg/kg bw/day was regarded unrelated to treatment with the test item as no trend was apparent regarding dose. All values remained within the normal range of biological variation*.

PARTURITION / MATERNAL CARE
Signs of difficult and prolonged parturition were noted for two high dose females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

POST-IMPLANTATION SURVIVAL
At 150 mg/kg bw/day, post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was lower, i.e. 70% vs. 92% for the control group.
The total number of offspring born compared to the total number of uterine implantations was unaffected by treatment at 10 and 50 mg/kg bw/day. Post-implantation survival index was 92% for each the control, 10 and 50 mg/kg bw/day groups.

LITTER SIZE
At 150 mg/kg bw/day, mean litter size was statistically significantly lower than the control mean (7.7 vs 10.8 in the control group). At the individual level it was noted that 10/20 females with live offspring on lactation Day 1 had less than eight pups, i.e. the lowest number of living pups per concurrent control litter (1/24 control female) when excluding the control female with one pup only (No. 119) as this was considered an outlier. For comparison, only 2/24 and 1/21 females in the 10 and 50 mg/kg bw/day groups, respectively, had less than eight live pups.
Up to 50 mg/kg bw/day, litter size remained unaffected by treatment with the test item. Mean litter sizes were 10.8, 10.4 and 11.6 living pups/litter for the control, 10 and 50 mg/kg bw/day groups, respectively.
At the individual level, there was one control female which had delivered only two pups of which one was dead at first litter check. Such small litters are occasionally seen in this type of study. As this was a control female, a possible relation to treatment with the test item could be excluded.

PUP SEX RATIO
Sex ratio was considered not to be affected by treatment.
The statistically significantly lower proportion of male pups in the 10 and 50 mg/kg bw/day group was not regarded as treatment-related. It was rather caused by the relatively high proportion of males in the control group. All values remained within the normal range of biological variation.

LIVE BIRTH INDEX
A lower live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was observed in the 150 mg/kg bw/day group. The live birth indices were 100, 99, 100 and 96% for the control, 10, 50 and 150 mg/kg bw/day groups, respectively.
In total 7 pups in the 150 mg/kg bw/day group were found dead at first litter check: i.e. 5/11 pups in Litter No. 181, and 2/9 pups in Litter No. 185. For comparison, the number of dead pups at first litter check was 1, 3 (all from the same litter) and 0 in the control, 10 and 50 mg/kg bw/day groups, respectively.

VIABILITY INDEX
The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was unaffected by treatment up to 150 mg/kg bw/day. Viability indices (number of live offspring on PND 1 as a percentage of total number of offspring born) were 99, 100, 100 and 98% for the control, 10, 50 and 150 mg/kg bw/day groups, respectively.
Two pups of the control group (one in Litter Nos. 119 and 124 each), one pup in the 10 mg/kg bw/day group (Litter No. 146), and three pups in the 150 mg/kg bw/day group (two in Litter No. 185, and one in Litter No. 200), were found dead or missing between PND 2 and PND 4. Pups found missing were most likely cannibalized. In case of control Litter No. 119, this was the last pup that was lost (total litter loss; first pup was found dead at first litter check). No toxicological relevance was attributed to these dead/missing pups since the mortality incidence remained within the range considered normal for pups of this age.

WEANING INDEX
The number of live offspring at weaning (PND 21) compared to the number of live offspring on PND 4 (after culling) was unaffected by treatment up to 150 mg/kg bw/day. The weaning indices were 99, 99, 100 and 100% for the control, 10, 50 and 150 mg/kg bw/day groups, respectively.
One pup in the control group went missing on PND 7, and one pup in the 10 mg/kg bw/day group died spontaneously on PND 16. The missing pup was most likely cannibalized. No toxicological relevance was attributed to these dead pups since the mortality incidence remained within the range considered normal for pups of this age, and no trend was apparent regarding dose.

For details see Attachment "Summary of P female mating performance_fertility_duration of gestation_litter performance" under Attached Background Material.

-----------------------------
* Historical control data for Wistar Han rats; F0-animals (period 2017-2019):
Duration of gestation (days): mean = 21.4; P5 – P95 = 21.00 – 22.00 (n=212 animals)

Discussion on increased TSH levesl:
There was a marked increase in TSH in males and females (5.07x and 10.77x of control, respectively), and decrease in total T4 in males (0.64x of control) at 150 mg/kg bw/day. It should be noted that these changes in thyroid hormone concentrations occurred in conjunction with markedly increased liver weights and correlating hepatocellular hypertrophy. This finding may be indicative of hepatic microsomal enzyme induction leading to increased metabolism of thyroid hormones and chronic thyroid stimulating hormone (TSH) stimulation of the thyroid gland. Possible adversity of the effects on thyroid hormones could not be assessed within this type of study.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

clinical biochemistry

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive performance

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

urinary

Organ:

bladder

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

during lactation:
No clinical signs occurred among pups that were considered to be related to treatment up to 150 mg/kg bw/day.
One pup in the 50 mg/kg bw/day group was observed with its right eye closed and secretion of red fluid from its eye on PND 19 and 20. This finding was regarded to be caused by an accident (e.g. inflicted during a fight with its littermates). Another pup was noted with diarrhoea on PND 13. A relation to treatment could be excluded as this latter pup was a control pup treated with the vehicle only. The nature and incidence of other clinical signs observed remained within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant.

from weaning onwards:
Salivation was observed after dosing among animals of the 10 mg/kg bw/day group (males only), and the 50 and 150 mg/kg bw/day groups (both sexes). Onset of salivation was seen earlier (starting in Week 2 vs. Weeks 5-9) and at a higher incidence at 150 mg/kg bw/day (both sexes), compared to the lower dose levels. Considering the nature and minor severity of the effect (predominantly at a slight degree) and its time of occurrence (i.e. after dosing) it was considered to be a physiological response rather than a sign of systemic toxicity.
Other clinical signs noted during the treatment period included slight lethargy, rales, alopecia, scabs, wounds, a broken or missing tail (apex), chromodacryorrhoea, and ptosis. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the low incidence observed, these were considered signs of no toxicological relevance.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

No mortality occurred that was considered to be related to treatment with the test item.

Cohort 1B one female in the control group was sacrificed on Day 6 of treatment due to its poor condition. In the period before its death it presented with slight lethargy and piloerection (Days 3 and 5), quick breathing and slightly pale appearance (Day 3), and hunched posture (Day 5). On treatment Day 6 (PND 25) it had lost 14 gram of her initial weight of 47 gram (taken from study daybook). At necropsy, watery-cloudy fluid was found in the thoracic cavity, which is indicative for that complications during the gavage procedure were the underlying cause for the animal’s poor condition.
Cohort 1B one male of the low dose group (at 10 mg/kg bw/day) was found missing after the pre-dose observation on Day 2 of treatment. No explanation could be obtained for this from earlier
observations performed for this animal.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

during lacation:
Body weights of pups were considered not to be affected by treatment.
In general higher mean values for body weights (male and female pups separately or combined) were recorded in all treated groups compared to the concurrent control group. This difference was relatively slight (maximum of 7% in males and 6% in females on PND 21), reaching statistical significance in the 50 and 150 mg/kg bw/day groups incidentally only. It was considered to be caused by the relative low values in the control group, rather than to be related to treatment with the test item.

Historical control data for Wistar Han rats; F1-pups PND 21 (period 2017-2019):
Body weight PND 21 (g) – males: mean = 49.8; P5 – P95 = 42.00-58.00 (n=378).
– females: mean = 48.3; P5 – P95 = 42.00-55.00 (n=403)

from weaning onwards:
No changes in body weight or body weight gain were noted that were considered to be related to treatment.
The slightly, not always statistically significantly higher body weight gain observed in females at 50 and 150 mg/kg bw/day when compared to the concurrent controls from Day 29 of treatment onwards was considered not related to treatment as a dose-related trend was absent and means for absolute body weights were comparable to those of the concurrent controls.

For details see Attachment "Summary of F1 animal body weight data" under Attached Background Material.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

from weaning onwards:
No toxicologically relevant changes in food consumption (before and/or after correction for body weight) were noted across the dose groups.
The trend towards slightly higher food consumption (before and/or after correction for body weight) observed in females at 50 and 150 mg/kg bw/day was considered not toxicologically relevant as changes compared to the concurrent control group were very slight only (not always reaching statistical significance) and/or occurred in the absence of a dose-related trend.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

from weaning onwards:
Cohort 1A:
No changes in hematology parameters were noted in males or females that were considered to be toxicologically relevant.
When compared to the concurrent control group, a decreased number of red blood cells was observed in males at 150 mg/kg bw/day (0.96x of control). Values of 3/10 high dose males were
below the lowest control value (ranging between 0.90-0.95x of mean control value). As changes were only minimal, occurred in the absence of effects on correlating parameters, and mean value remained within the range of historical control data, no toxicological significance was attached to this finding.

No changes in coagulation parameters were noted in males or females that were considered to be toxicologically relevant.
The shorter prothrombin time observed at 50 and 150 mg/kg bw/day in males (0.96 and 0.97x of control) and females (0.94 and 0.95x of control; not statistically significant at 150 mg/kg bw/day) were considered not to represent a change of toxicological relevance based on the direction of change, its minimal magnitude and the absence of a dose-related response.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

before weaning:
T4 and TSH levels on PND4 and 21/22:
Serum levels of total T4 in male and female pups combined, culled at PND 4, were statistically significantly lower at 150 mg/kg bw/day when compared to controls (0.48x; below historical control range).
Historical control data for Wistar Han rats; F1-pups PND 4 (period 2017-2019):
Total T4 (µg/dL) – males and females combined: mean = 1.54; P5 – P95 = 1.050-2.170 (n=155).

In PND 21/22 females (Cohort 2B), a dose-related increase in concentrations of total T4 was noted from 10 mg/kg bw/day onwards. This change was considered to be unrelated to treatment, as differences when compared to the concurrent control group were minimal and mean values remained within the available historical control range. Means for total T4 in treated males and TSH in treated males and females were comparable to the control means.

Historical control data for Wistar Han rats; F1-female pups PND 21/22 (period 2017-2019):
Total T4 (µg/dL) – Cohort 2B (PND 21-22): mean = 4.83; P5 – P95 = 3.180-6.940 (n=20).
– Cohort Surplus (PND 22): mean = 4.61; P5 – P95 = 3.095-6.235 (n=80).

from weaning onwards:
For details see Attachment "Summary of F1 animal clinical biochemistry" under Attached Background Material.
Cohort 1A:
The following statistically significant changes were noted in males and females at 50 and 150 mg/kg bw/day. Relative differences in mean values as compared to the control group are indicated between parentheses. Relevant historical control data are provided below.
- Increased alanine aminotransferase (ALAT) activity in males at 150 mg/kg bw/day (1.28x; slightly above historical control range).
- Increased alkaline phosphatase (ALP) activity in males at 150 mg/kg bw/day (1.80x; above historical control range).
- Increased total protein concentration in females at 150 mg/kg bw/day (1.11x; slightly above historical control range).
- Increased albumin concentrations in males at 150 mg/kg bw/day (1.10x; above historical control range) and females at 50 and 150 mg/kg bw/day (1.05 and 1.15x, respectively; above historical control range for 150 mg/kg bw/day).
- Decreased total bilirubin concentration in females at 150 mg/kg bw/day (0.79x; within historical range).
- Increased glucose concentration in females at 150 mg/kg bw/day (1.23x; slightly above historical control range).
- Increased calcium concentration in females at 150 mg/kg bw/day (1.04x; within historical control range).
Any other statistically significant changes in clinical chemistry parameters were not considered to be related to treatment as they occurred in the absence of a dose-related trend (increased levels of glucose and potassium in males at 50 mg/kg bw/day; decreased levels of bile acids in females at 10 and 50 mg/kg bw/day)

Historical control data for Wistar Han rats; F1-generation (period 2017-2019):
ALAT (U/L) – F1-males: mean = 32.1; P5 – P95 = 25.60-40.70 (n=90).
ALP (U/L) – F1-males: mean = 114; P5 – P95 = 77.0-158.0 (n=90).
Total protein (g/L) – F1-females: mean = 66.5; P5 – P95 = 61.40-71.40 (n=90).
Albumin (g/L) – F1-males: mean = 33.2; P5 – P95 = 31.20-34.90 (n=90).
– F1-females: mean = 35.7; P5 – P95 = 32.80-39.10 (n=90).
Total bilirubin (µmol/L) – F1-females: mean = 2.1; P5 – P95 = 1.30-3.10 (n=90).
Glucose (mmol/L) – F1-females: mean = 7.42; P5 – P95 = 5.910-9.410 (n=90).
Calcium (mmol/L) – F1-females: mean = 2.60; P5 – P95 = 2.480-2.700 (n=90).

Thyroid hormone analysis:
Treatment-related changes are given below. Relative differences in mean values as compared to the control group are indicated between parentheses. Relevant historical control data are provided below.
- Mean TSH activities were increased in males and females at 150 mg/kg bw/day (5.24x and 8.45x of control, respectively; both above historical control range).
Also in males at 10 and 50 mg/kg bw/day, higher mean concentrations of TSH were noted (3.68x and 3.80x of control). These changes did not reach statistical significance, occurred in the absence of clear dose-proportionality, and means remained within the historical control data range. However, in light of the magnitude of this change a possible relation to treatment with the test item could not be excluded.
- Mean total T4 concentration was decreased in males at 150 mg/kg bw/day (0.61x of control; below historical control range).

Historical control data for Wistar Han rats; F1-generation Cohort 1A (period 2017-2019):
TSH (µIU/mL) – F1-males: mean = 0.192; P5 – P95 = 0.0360-0.5360 (n=90).
TSH (µIU/mL) – F1-females: mean = 0.076; P5 – P95 = 0.0170-0.1950 (n=90).
Total T4 (µg/dL) – F1-males: mean = 6.26; P5 – P95 = 4.200-8.190 (n=90).

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

from weaning onwards:
Cohort 1A:
The number of females with the maximum score 3 for crystals was increased from 2/10 in the control group to 7/10 in the 150 mg/kg bw/day group. This increase was regarded as test item-related, but not adverse, as all values remained within the historical control range.
Urinary parameters of treated males were considered to be unaffected by treatment with the test item.
A trend towards higher mean volume of urine was noted for males at 150 mg/kg bw/day compared to concurrent controls (not statistically significant). This increase was mainly attributed to one male, of which the value was 3.75x of the mean control value. Latter change occurred in the absence of any correlating abnormalities at the organ level. As all other volumes remained within the historical control range15, it was considered not related to treatment with the test item.

For details see Attachment "Summary of F1 animal urinalysis" under Attached Background Material.

Historical control data for Wistar Han rats; F1-generation Cohort 1A (period 2017-2019):
Volume of urine (mL) – F1-males: mean = 10; P5 – P95 = 4.0-18.0 (n=90).
Crystals (score 0/3) – F1-females: mean = 2; P5 – P95 = 0.0-3.0 (n=90).

Sexual maturation:

no effects observed

Description (incidence and severity):

from weaning onwards:
Balanopreputial separation (prepuce opening) in males and vaginal patency (vaginal opening), occurrence of first estrus, and time between vaginal opening and first estrus in females were considered not to be affected by treatment with the test item.
At 50 and 150 mg/kg bw/day, mean time to balanopreputial separation (BPS) was statistically significantly reduced compared to the control group (41.4 and 41.3 days, respectively, vs. 42.6 days in controls). It should be noted that means did not show a dose-related trend and were similar to the available historical control mean. Moreover, the body weight at attainment of BPS was similar for all groups indicating no developmental advancement.
Therefore, this apparent earlier onset of balanopreputial separation was regarded unrelated to treatment with the test item.
In all treated groups, mean time to vaginal opening (VO) was statistically significantly shorter than in controls (31.4, 28.4 and 29.6 days, respectively, at 10, 50 and 150 mg/kg bw/day, vs. 32.3 days in controls). Mean body weight on the day of vaginal opening was also statistically significantly lower at 50 and 150 mg/kg bw/day (79 and 82 gram, respectively, vs. 94 gram in controls). Finally, the time point of first estrous was statistically significantly earlier at 50 and
150 mg/kg bw/day (PND 32.5 and 32.8, respectively, vs. PND 37.2 in controls). This was regarded to be related to the slightly earlier onset of vaginal patency. Time until first estrus was on average 4 days for all groups. These variations in onset of vaginal opening, body weight at time point of vaginal opening, and day of acquiring first estrous were considered not to be caused by treatment with the test item, as changes did not show a dose-related trend and values remained within the available control range.

Historical control data for Wistar Han rats; F1-generation (period 2017-2019):
Balanopreputial separation (PND): mean = 41.4; P5 – P95 = 39-44 (n=519).
Body weight on day of balanopreputial separation (gram): mean = 174; P5 – P95 = 151.0-197.0 (n=519).
Vaginal patency (PND): mean = 31.4; P5 – P95 = 27-35 (n=526).
Body weight on day of vaginal patency (gram): mean = 96; P5 – P95 = 74.0-115.8 (n=524).
First estrus (PND): mean = 35.2; P5 – P95 = 30.05-39.00 (n=180).
Interval between vaginal patency and first estrus (days): mean = 3.7; P5 – P95 = 1-7 (n=176).

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

before weaning:
Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment with the test item.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

before weaning:
Treatment up to 150 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

before weaning:
A treatment-related increase in liver weight was observed in Cohort Surplus pups (PND 22; both sexes) at 150 mg/kg bw/day. Changes compared to controls are shown in table 4. All changes reached statistical significance, except for absolute liver weight in female pups of Cohort Surplus. It should be noted that for Cohort Surplus pups liver organ weight is not required by the applicable guideline, and no internal control data are available; it was added to this study as the liver is a known target organ of the test item.
Fresh weights of brain, thyroids, thymus and spleen in Cohort Surplus pups (PND 22; both sexes) were unaffected by treatment up to 150 mg/kg bw/day. Any (statistical significant) changes in absolute and/or relative organ weights between treated and control animals were considered to be unrelated to treatment with the test item as changes were relatively slight (reaching no statistical significance), occurred in the absence of a dose-related trend and/or remained in the normal range of biological variation.

from weaning onwards:
Cohort 1A (see table 5):
There was a higher liver and thyroid gland weight in males and females (absolute and relative to body weight) at 50 and 150 mg/kg bw/day. In addition, there was a higher kidney weight at 150 mg/kg bw/day (absolute and relative to body weight) in males and females, and in females there was a higher heart weight and adrenal gland at 150 mg/kg bw/day (absolute and relative to body weight).
Any other significant organ weight changes were relative to body weight only or were considered to be secondary to the small decrease in total body weight at 150 mg/kg bw/day (pituitary gland, heart and testes in males, and ovaries in females).
At the individual level, it was noted that in both the control and high dose group there was one Cohort 1A female with significantly lower ovary weights compared to the remaining females in each group. Both females presented with extended estrus. However, ovary weights of the remaining two high dose females with extended estrus were within the same range as for females with normal estrous cycle. As such, there was no clear relation between extended estrus and low ovary weights.

Cohort 1B see table 6:
There was a higher liver weight in males and females (absolute and relative to body weight) at 50 and 150 mg/kg bw/day. In addition, a higher thyroid gland weight (absolute and relative to body weight) was noted at 50 mg/kg bw/day (males only) and 150 mg/kg bw/day (both sexes). A higher adrenal weight was observed in females at 50 and 150 mg/kg bw/day (reaching no statistical significance for relative organ weight at 50 mg/kg bw/day).
No toxicological relevance was attached to the slightly, but statistically significantly higher mean liver weight (relative to body weight only) observed in males at 10 mg/kg bw/day as the difference with concurrent controls was minimal (< 10%).
The statistically significantly higher mean ovary weights (absolute and relative to body weight) at 150 mg/kg bw/day remained within the historical control data range or were only slightly higher (relative organ weight). Moreover, no test item-related change in ovary weight was observed in Cohort 1A animals that were necropsied on approximately the same time point. Therefore, this change was considered not to be related to treatment with the test item.
Other organ weights and organ to body weight ratios among the dose groups were similar to control levels.

Internal control data for Wistar Han rats; F1-animals (period 2018-2019):
Ovary weight - absolute (gram): mean = 0.142; P5 – P95 = 0.1070-0.1710 (females, Cohort 1B; n=184).
Ovary weight - relative (% from terminal body weight): mean = 0.058; P5 – P95 = 0.0404-0.0716 (females, Cohort 1B; n=182).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

before weaning:
No macroscopic findings were noted among pups sacrificed at the end of the lactation period that were considered to be related to treatment.

from weaning onwards:
Cohort 1A
Test item-related macroscopic alterations at the end of the treatment period in animals of Cohort 1A were observed in the liver of animals treated at 50 mg/kg bw/day (females) and 150 mg/kg bw/day (both males and females). These alterations consisted of:
- An enlarged liver in 8/20 males treated at 150 mg/kg bw/day. This correlated with microscopic hepatocellular hypertrophy and the higher liver weight.
- Red-brown or black-brown discoloration of the liver in 1/20 females at 50 mg/kg bw/day, and 5/20 males and 3/20 females treated at 150 mg/kg bw/day.

Cohort 1B
Test item-related macroscopic alterations at the end of the treatment period in animals of Cohort 1B were observed in the liver of animals treated at 50 mg/kg bw/day (females) and 150 mg/kg bw/day (both males and females). These alterations consisted of:
- An enlarged liver in 16/20 males and 2/20 females treated at 150 mg/kg bw/day.
- Red-brown or black-brown discoloration of the liver in 2/20 males and 4/20 females treated at 150 mg/kg bw/day.

Cohort 1C
There were no gross observations in animals of Cohort 1C treated at 10 and 50 mg/kg bw/day.
Note: Not sufficient numbers of pups were available in the 150 mg/kg bw/day group to start a Cohort 1C.

Cohort 2A
Test item-related macroscopic alterations at the end of the treatment period in animals of Cohort 2A were observed in the liver of males treated at 150 mg/kg bw/day. These alterations consisted of:
- An enlarged liver in 9/10 males treated at 150 mg/kg bw/day.

Cohort 2B
There were no test item-related gross observations in animals of Cohort 2B.

Cohort Surplus
There were no test item-related gross observations in animals of Cohort Surplus.

All of the (remaining) recorded macroscopic findings in either cohort were within the range of background gross observations encountered in rats of this age and strain.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

from weaning onwards:
Cohort 1A:
Test item-related microscopic findings were noted in the liver and thyroid glands of males and females as shown in table 7.

Liver
Hepatocellular hypertrophy, mainly centrilobular, was present starting at 50 mg/kg bw/day up to moderate degree in males and females. This finding correlated with the higher liver weight and with the macroscopic enlarged liver.

Thyroid gland
An increased incidence and severity of follicular cell hypertrophy was present starting at 50 mg/kg bw/day up to moderate degree in males and females. This finding correlated with the higher thyroid gland weight. An increased incidence and severity of colloid alteration was present starting at 50 mg/kg bw/day in males and at 150 mg/kg bw/day in females up to slight degree.

The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations. This included the ovaries of females observed with extended estrus as well.

Ovarian Follicle Counts
There were no test item-related effects on the ovarian follicle counts or corpora lutea counts in the 150 mg/kg bw/day group females (Cohort 1A) when compared to control group females. This included the females observed with extended estrus as well.
Any variation between group mean counts represented biological variability and were not statistically significant.

Spermatogenesis Staging
Stage-dependent qualitative evaluation of spermatogenesis in the testis was performed in Cohort 1A males of the control and 150 mg/kg bw/day group. The testes revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Estrous Cyle - Cohort 1A:
An increased incidence of females with extended estrus was observed in the high dose group: three females at 150 mg/kg bw/day had at least five consecutive days of estrus, compared to one control female and no females in the 10 and 50 mg/kg bw/day groups. This higher incidence of extended estrus in the 150 mg/kg bw/day group was considered to be test item-related. Of special note is that one of the three females remained in estrus during the entire 14-days observation period.
In addition, 2/20 females at 10 mg/kg bw/day were observed with an irregular cycle (i.e. one cycle of 3 or 6 days, next to a normal cycle of 4-5 days). Given the incidental nature and the absence of a dose-related incidence, the occurrence of an irregular estrous cycle in two low dose females was considered unrelated to treatment with the test item.

For details see Attachment "Summary of F1 animal estrous cycle patterns" under Attached Background Material.

Sperm analysis:
Sperm motility, concentration and morphology were considered not affected by treatment with the test item. All mean values remained within the normal range of biological variation.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional tests in Cohort 2A:
Acoustic startle response was considered to be unaffected by treatment with the test item.
Detailed clinical observations revealed no symptoms that were considered to be related to
treatment.
The clinical symptoms that were observed were considered to be within the normal range of behavioural findings for this type of study, and were generally also observed in control animals. These findings were therefore considered not to be related to treatment with the test item.
Mean rectal temperature of animals treated at 10, 50 and 150 mg/kg bw/day (both sexes) were in the same range as for concurrent controls.
Motor activity of animals treated up to 150 mg/kg bw/day (both sexes) was considered not affected by treatment. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
Functional observation parameters (hearing ability, pupillary reflex, foot splay and grip strength) were not affected by treatment with the test item.

For details see Attachment "Summary of F1 animal developmental neurotoxicity data" under Attached Background Material.

Developmental immunotoxicity:

no effects observed

Description (incidence and severity):

Splenic lymphocyte subpopulation:
There were no test item-related effects on splenic lymphocyte subpopulations observed.

Neuropathology and Morphometry - Cohort 2A and 2B:
Brain weights:
Fixed brain weights at PND 21-22 (Cohort 2B) and PND 76-86 (Cohort 2A) were unaffected by treatment up to 150 mg/kg bw/day.

Brain Dimension:
Brain dimensions (length and width of brain) at PND 21-22 (Cohort 2B) and PND 76-86 (Cohort 2A) were unaffected by treatment up to 150 mg/kg bw/day.

Brain Histopathology:
In F1-animals of Cohorts 2B (PND 21-22) and 2A (PND 76-86), there were no test item-related effects on the H&E or Luxol Fast Blue/Cresyl Violet stained sections of brain or peripheral nerves in the control or high-dose group males or females.

Brain Morphometry:
There was no evidence of delevelopmental neurotoxicity in the brain of test item-treated animals at either PND 21-22 or PND 76-82. Morphometric analysis of the brain at PND 21-22 revealed no statistically significant differences in animals potentially exposed to the test item at a dose of 150 mg/kg bw/day compared to control animals.
At PND 76-82, a statistically significantly lower thickness in the parietal cortex was observed in high dose females treated at 150 mg/kg bw/day, compared to concurrent controls. There was a (non-significant) opposite effect in the same region in high dose females at PND 21-22, and there were no differences in any of the other brain levels. Moreover, no test item-related changes were observed in the brain of males treated at 150 mg/kg bw/day. Therefore, the single significant difference in the parietal cortex thickness in high dose females was considered spurious and not related to treatment with the test item. There were no other statistically significant differences in animals treated at 150 mg/kg bw/day when compared to control animals.

Key result

Dose descriptor:

NOAEL

Remarks:

developmmental (pre-weaning)

Generation:

F1

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOAEL

Remarks:

developmental (post-weaning)

Generation:

F1

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Remarks:

developmental neurotoxicity

Generation:

F1

Effect level:

>= 150 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed up to the highest dose tested

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 150 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed up to the highest dose tested

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Table 1. Mean Percent Organ Weight Differences from Control Groups – P-Generation Males and Females

	Males			Females
Dose level (mg/kg):	10	50	150	10	50	150
Body Weight	0	-5	-7^**	-1	3	-2
Liver
Absolute	7	23^**	71^**	-1	14^*	37^**
Relative to body weight	6^*	29^**	85^**	0	10^*	39^**
Thyroid gland
Absolute	4	21^**	33^**	7	16^*	29^**
Relative to body weight	4	28^**	45^**	7	12	30^**
Kidneys
Absolute	3	4	14^*	0	7^*	5
Relative to body weight	2	8^**	22^**	0	3	6^*
Adrenal glands
Absolute	2	0	12**	-2	3	5
Relative to body weight	0	0	23**	-3	0	3

*: P<0.05, **: P<0.01 (Dunnett-test based on pooled variance); test item-related changes shaded in grey

Test item-related findings are shaded grey.

 

Table 2. Summary Test Item-Related Microscopic Findings - P-Generation Males and Females

	Males				Females
Dose level (mg/kg bw/day):	0	10	50	150	0	10	50	150
Liver^a	25	25	25	25	25	25	25	25
Hypertrophy hepatocellular
Minimal	-	-	13	7	-	-	12	11
Slight	-	-	7	15	-	-	-	13
Moderate	-	-	-	3	-	-	-	-
Thyroid gland^a	25	25	25	25	25	25	25	25
Hypertrophy follicular cell
Minimal	5	7	7	3	4	2	11	12
Slight	-	-	13	13	-	-	4	13
Moderate	-	-	2	9	-	-	-	-
Alteration colloid
Minimal	1	2	13	9	-	-	5	19
Slight	-	-	3	15	-	-	-	2
Kidney^a	25	25	25	25	25	25	25	25
Hyaline droplet accumulation
Minimal	14	12	11	3	-	-	-	-
Slight	1	3	14	12	-	-	-	-
Moderate	-	-	-	7	-	-	-	-
Urinary bladder^a	25	-	-	25	25	25	25	25
Hyperplasia urothelium
Minimal	-	-	-	-	-	-	-	1
Hypertrophy urothelium
Minimal	-	-	-	-	-	-	-	6

^a = Number of tissues examined from each group. Test item-related findings are shaded grey.

Table 3. Correlation of Histopathology Findings with In-Life Reason for Males that Failed to Sire and Females that Failed to Deliver Healthy Pups - P-Generation

Group	Dose level mg/kg	Female/Male Nos.	In-Life Reason	Histopathology
1	0	111/11	Not pregnant	-/-
		119/19	Total litter loss – PND 2	-/-
2	10	143/43	Not pregnant	-/-
3	50	152/52	Not mated	-/-
		160/60	Not pregnant	-/-
		165/65	Not mated	-/-
		174/74	Not mated	-/-
4	150	176/76	No live offspring	Pyometra
		183/83	Not pregnant	-/-
		188/88	Implantation sites only	-/-
		194/94	No live offspring	Dead fetuses in uterus (macroscopic finding)
		198/98	Not pregnant	-/-

 -  = No histopathologic correlate.

Table 4: Mean percent liver organ weight difference from control groups – F1 Generation cohort Surplus, males and females
	Males			Females
Dose level (mg/kg bw/day)	10	50	150	10	50	150
Body weight	13	8	6	0	0	-6
Liver
- absolute	16	14	23**	5	3	7
- relative to body weight	2	7	16**	5	3	13**
**: p<0.01 (Dunnett-test based on pooled variance)

Table 5: Mean percent organ weight difference from control groups – F1 Generation cohort 1A, males and females
	Males			Females
Dose level (mg/kg bw/day)	10	50	150	10	50	150
Body weight	0	-2	-5	-3	-3	-4
Liver
- absolute	8	26**	67**	1	17**	59**
- relative to body weight	8	29**	75**	4	21**	66**
Thyroid gland
- absolute	1	5	18**	3	15	40**
- relative to body weight	0	8	22**	6	19	46**
Kidneys
- absolute	1	5	10**	0	3	7**
- relative to body weight	2	8*	17**	3	5*	12**
Adrenal glands
- absolute	-2	2	3	5	7	31**
- relative to body weight	0	6	6	10	10	38**
Heart
- absolute	0	-2	1	-2	4	7*
- relative to body weight	0	0	5*	0	7*	11**
*: p<0.05; **: p<0.01 (Dunnett-test based on pooled variance); test item related changes in are shown in bold

Table 6: Mean percent organ weight difference from control groups – F1 Generation cohort 1B, males and females
	Males			Females
Dose level (mg/kg bw/day)	10	50	150	10	50	150
Body weight	3	0	-5	0	0	0
Liver
- absolute	9	23**	57**	4	21**	57**
- relative to body weight	6*	23**	66**	3	21**	57**
Thyroid gland
- absolute	-2	19*	22**	3	11	27**
- relative to body weight	-4	20**	28**	2	11	28**
Adrenal glands
- absolute	0	-2	6	-2	12*	27**
- relative to body weight	0	0	14	0	15	30**
Ovaries
- absolute	n.a.	n.a.	n.a.	5	10	16*
- relative to body weight	n.a.	n.a.	n.a.	5	10	16*
*: p<0.05; **: p<0.01 (Dunnett-test based on pooled variance); test item related changes in are shown in bold; n.a.: not applicable

Table 7: Summary Test Item-Related Microscopic Findings – F₁Generation Cohort 1A Males and Females

	Males				Females
Dose level (mg/kg bw/day):	0	10	50	150	0	10	50	150
Liver^a	20	20	20	20	20	20	20	20
Hypertrophy hepatocellular
Minimal	-	-	9	1	-	-	12	1
Slight	-	-	5	9	-	-	4	9
Moderate	-	-	-	10	-	-	-	10
Thyroid gland^a	20	20	20	20	20	20	20	20
Hypertrophy follicular cell
Minimal	5	6	3	3	1	2	9	4
Slight	-	1	9	8	-	-	3	9
Moderate	-	-	7	8	-	-	-	5
Alteration colloid
Minimal	-	1	5	8	-	-	-	8
Slight	-	-	3	2	-	-	-	2

^a = Number of tissues examined from each group. Test item-related findings are shaded grey.

Conclusions:

The objective of this study was to provide an evaluation of the pre- and postnatal effects of test substance on development as well as a thorough evaluation of systemic toxicity in pregnant and lactating females and young and adult offspring of Wistar Han rats. Detailed examination of key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, and physical and functional development until adulthood, was expected to identify specific target organs in the offspring. In addition, the study provided and/or confirmed information about the effects of the test item on the integrity and performance of the adult male and female reproductive systems. Specifically, but not exclusively, the following parameters were considered: gonadal function, the estrous cycle, epididymal sperm maturation, mating behavior, conception, pregnancy, parturition, and lactation. Furthermore, the information obtained from the developmental neurotoxicity assessments characterized potential neurodevelopmental effects of the test item. The dose levels in this study were selected to be 0, 10, 50 and 150 mg/kg bw/day.
P0-Generation - Parental Results
No toxicologically relevant changes were observed at 10 mg/kg bw/day.
At 150 mg/kg bw/day, two females were euthanized for humane reasons on Day 23 post-coitum due to poor condition. The main cause of moribundity in these females was considered to be the presence of dead fetuses in the uterus, which in one female had resulted in pyometra.
Increased activity of the forelegs was noted in the majority of animals following approximately 11 weeks of treatment at 150 mg/kg bw/day. It consisted of repeated short periods of ‘digging’ in the sawdust which started several minutes after dosing and lasted for a few minutes only. The cause of this repetitive behavior in the parental P0-animals could not be elucidated. No other indications for any neurological disorder could be found in the P0-generation, based on the outcome of observations for clinical signs and histopathological examination of the brain and central nerve system. Neither was this behavior seen in the siblings of the P0-animals which were potentially exposed to the test item in utero and via maternal milk during lactation, and were dosed with the test item from weaning onwards
(PND 21) for a maximum of 11-12 weeks (Cohorts 1A and 1B). Nor were there any treatment-related findings in F1-animals of Cohorts 2A and 2B, designated to extensive neurotoxicity testing. Therefore, this short and transient period of ‘digging’ was considered test item-related, but not adverse.
Lower body weights and body weight gains were noted in males treated at 150 mg/kg bw/day from Week 7 of the premating period onwards, lasting until the end of the mating period. Changes compared to concurrent controls were slight only, resulting in a mean terminal body weight of 0.93x of control. The slightly lower body weight gain in high dose males occurred despite a normal food intake during premating and mating. In females of the high dose group, food intake was even slightly higher compared to the concurrent controls during pre-mating and post-coitum, while mean body weight was similar for both groups. This is indicative for a lower food efficiency in males and females at 150 mg/kg bw/day, i.e. more food has to be ingested to reach the same growth in the animal’s mass. It was considered to be test itemrelated, but not adverse given the relatively slight magnitude of the effect.
Body weight gain in males at 50 mg/kg bw/day when compared to the concurrent control group was only marginally lower (mean terminal body weight: 0.95x of control) and occurred in the absence of a higher relative food intake. As such no toxicological relevance was attached to this finding.
Test item-related findings were noted in the liver, thyroid glands, urinary bladder, kidneys, and adrenal glands of males and/or females at 10, 50 and/or 150 mg/kg bw/day. Higher liver weights started in males at 10 mg/kg bw/day and in females at 50 mg/kg bw/day and correlated (starting at 50 mg/kg bw/day) with microscopic hepatocellular hypertrophy, mainly centrilobular that was observed in males and females starting at 50 mg/kg bw/day. Correlating necropsy findings were at the highest dose (150 mg/kg bw/day) only and consisted of enlarged and/or thickened liver observed in males and females. In males at 150 mg/kg bw/day, the considerable increase in liver weight (71% (absolute) and 85% (relative to body weight) above control means) in combination with up to moderate hepatocellular hypertrophy was considered adverse. In addition, in males there was a small, but significant, increase in alkaline phosphatase activity. For females at 150 mg/kg bw/day there was a slightly lower increase in liver weight (less than 40%) and the hepatocellular hypertrophy was up to slight and therefore this combination was considered of concern, but at present non-adverse. No microscopic correlate was found for the necropsy finding of dark-red/black-brown discolouration of the liver, incidentally observed in males and females at 150 mg/kg bw/day. Higher thyroid gland weights started in males and females at 50 mg/kg bw/day. In addition, there was an increased incidence and severity of follicular cell hypertrophy and colloid alteration present in males and females starting at 50 mg/kg bw/day, and the thyroid gland was macroscopically enlarged at 50 mg/kg bw/day in males and at 150 mg/kg bw/day in males and females. In males and females at 150 mg/kg bw/day, the combination of the considerable increase in thyroid gland weight (males 33% (absolute) and 45% (relative to body weight) and females 29% (absolute) and 30% (relative to body weight) above control means) with the high incidences and severities of follicular cell hypertrophy and colloid alteration was considered adverse. In addition, there was a marked increase in TSH in males and females (5.07x and 10.77x of control, respectively) and decrease in total T4 in males (0.64x of control) at 150 mg/kg bw/day. It should be noted that these changes in thyroid hormone concentrations occurred in conjunction with markedly increased liver weights and correlating hepatocellular hypertrophy. This finding may be indicative of hepatic microsomal enzyme induction leading to increased metabolism of thyroid hormones and chronic thyroid stimulating hormone (TSH) stimulation of the thyroid gland. Possible adversity of the effects on thyroid hormones could not be assessed within this type of study.
The urothelial hyperplasia and hypertrophy in the urinary bladder of females treated at 150 mg/kg bw/day was considered adverse based on the hyperplastic nature. These changes occurred in absence of any macroscopically visible changes at necropsy. The increase in kidney weight in males at 150 mg/kg bw/day (relative to body weight and absolute) could not be readily attributed to the histopathology and was considered non-adverse in the absence of correlating macroscopic or microscopic kidney findings. In addition, the increased hyaline droplet accumulation in the kidney of males starting at 50 mg/kg bw/day was considered non-adverse in the absence of any degenerative findings. For females any differences in kidney weight including those that reached statistical significance were considered not to be test item-related due to a lack of dose-related pattern and variability in group values.
Finally, the increase in adrenal gland weight in males at 150 mg/kg bw/day could not be readily attributed to the histopathology and was considered non-adverse in the absence of macroscopic or microscopic adrenal gland findings.
Observed changes in clinical biochemistry parameters in males and/or females that were considered to be related to treatment included slightly increased concentrations of albumin, calcium, glucose, total protein and chloride, and slightly decreased total bilirubin at 50 and/or 150 mg/kg bw/day. No test item-related changes were noted in any of the remaining parameters investigated in this study (i.e. hematology, coagulation and urinalysis parameters) up to the highest dose level tested (150 mg/kg bw/day).
P0-Generation - Reproductive Results
No reproductive toxicity was observed up to 50 mg/kg bw/day.
A test item-related lower number of implantation sites was recorded for females treated at 150 mg/kg bw/day, which was considered to be adverse. No treatment-related changes were noted in any of the remaining reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, estrous cycle, sperm parameters, and histopathological examination of reproductive organs, incl. spermatogenic profiling) up to the highest dose level tested (150 mg/kg bw/day).
P0-Generation / F1-Generation (Pre-Weaning) - Developmental Results
No developmental toxicity was observed up to 50 mg/kg bw/day.
At 150 mg/kg bw/day, three pregnant females did not have live offspring. As a consequence, a lower gestation index was observed in the high dose group (87% vs. 100% in the controls). Two females had signs of difficult and prolonged parturition and were euthanized on Day 23 post-coitum for humane reasons. One of these two females had six dead fetuses in its uterus in addition to nine early resorptions (no living pups), and all eleven pups of the other female
had died before necropsy. The third female had four implantation sites only. Post-implantation loss was significantly higher in the 150 mg/kg bw/day group resulting in a lower post-implantation survival index (70% vs. 92% in the controls). This change could only in part be attributed to the three pregnant females that failed to deliver live offspring (see above). For high dose females that had live offspring on lactation Day 1, mean litter size was significantly lower than the control mean (7.7 vs. 10.8 in the controls). Also a lower live birth index was noted in the 150 mg/kg bw/day group. This lower value, caused by two litters with
a few dead pups, was still within the normal range of biological variation. However, in combination with the other developmental effects seen at this high dose level, it was considered treatment-related. Moreover, based on the type of changes observed, these findings were regarded as adverse. Lower serum levels of total T4 were noted in PND 4 pups (male and female pup combined) in the 150 mg/kg bw/day group (0.48x of control). This change was considered treatment-related, as the mean was below the historical control range.
A treatment-related increase in liver weight was observed in Cohort Surplus pups (PND 22; both sexes) at 150 mg/kg bw/day. The difference compared to the control group was relatively slight, but without histopathological examination of the livers no conclusion on possible adversity could be drawn.
No test item-related changes were noted in any of the remaining developmental parameters investigated in this study for P0-animals and F1-pups until weaning (i.e. duration of gestation, viability and weaning indices, sex ratio, maternal care, and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, thyroid hormone levels (total T4 and TSH in PND 21/22 pups), macroscopic examination, and organ weights of brain, thyroid glands, thymus and spleen) up to the highest dose level tested (150 mg/kg bw/day).
F1-Generation (Post-Weaning) - Developmental Results
(Developmental) toxicity was observed from 50 mg/kg bw/day onwards during the postweaning phase. A test item-related higher incidence of females with extended estrous was observed in the 150 mg/kg bw/day group (three high dose females vs. one female in the control group, and none in the intermediate groups). No abnormalities were found in the reproductive organs of these females, neither macroscopically nor microscopically, and also ovarian follicle counts and corpora lutea counts for these females were within normal ranges. However, based on the type of change, this higher incidence of extended estrous in females at 150 mg/kg bw/day was considered to be adverse.
Exposure of the F1-animals to test substance in utero, through nursing during lactation, and via oral gavage following weaning was associated in Cohort 1A with alterations in liver, thyroid gland, kidney, adrenal gland and heart. Higher liver weights started in males and females at 50 mg/kg bw/day and correlated with microscopic hepatocellular hypertrophy, mainly centrilobular that was observed in males and females starting at 50 mg/kg bw/day. In males and females at 150 mg/kg bw/day, the considerable increase in liver weight (males 67% (absolute) and 75% (relative to body weight) and females 59% (absolute) and 66% (relative to body weight) above control means) in combination with up to moderate hepatocellular hypertrophy was considered adverse. In addition, in males there was a small, but significant, increase in alanine aminotransferase and alkaline phosphatase. No microscopic correlate was found for the necropsy finding of redbrown/black-brown discolouration of the liver, observed mainly in males and females at 150 mg/kg bw/day. Higher thyroid gland weight was noted in males and females at 150 mg/kg bw/day. In addition, there was an increased incidence and severity of follicular cell hypertrophy present in males and females starting at 50 mg/kg bw/day, and colloid alteration in males starting at 50 mg/kg bw/day and in females at 150 mg/kg bw/day. In males and females at 150 mg/kg bw/day, the combination of the considerable increase in thyroid gland weight (males 18% (absolute) and 22% (relative to body weight) and females 40% (absolute) and 46% (relative to body weight) above control means) with the high incidences and severities of follicular cell hypertrophy and colloid alteration was considered adverse. In addition, there was a marked increase in TSH in males and females (5.24x and 8.45x of control, respectively) and decrease in total T4 in males (0.61x of control) at 150 mg/kg bw/day. Also in males at 10 and 50 mg/kg bw/day, higher mean concentrations of TSH were noted (3.68x and 3.80x of control). These changes did not reach statistical significance, occurred in the absence of clear dose-proportionality, and means remained within the historical control data range. However, in light of the magnitude of this change a possible relation to treatment with the test item could not be excluded. It should be noted that these changes in thyroid hormone concentrations occurred in
conjunction with markedly increased liver weights and correlating hepatocellular hypertrophy. This finding may be indicative of hepatic microsomal enzyme induction leading to increased metabolism of thyroid hormones and chronic thyroid stimulating hormone (TSH) stimulation of the thyroid gland. Possible adversity of the effects on thyroid hormones could not be assessed within this type of study. The increased organ weights at 150 mg/kg bw/day for kidney in males and females, and adrenal gland and heart in females could not be readily attributed to the histopathology and was considered non-adverse in the absence of correlating macroscopic or microscopic findings in these organs.
Similar to animals in Cohort 1A, also in Cohort 1B treatment-related changes in liver, thyroid and adrenal gland weight were observed. Higher liver weights started in males and females at 50 mg/kg bw/day and correlated with the gross finding of enlarged livers at necropsy. In addition, higher thyroid gland weights were observed at 50 mg/kg bw/day (males only) and 150 mg/kg bw/day (both sexes), and higher adrenal gland weights were noted at 50 and 150 mg/kg bw/day (females only). These changes in thyroid and adrenal gland weight
occurred in absence of any gross observations for either organ. It should be noted that for both Cohorts 1A and 1B the duration of treatment was almost the same (i.e. necropsy from PND 89-95 and PND 94-106, respectively). No histopathology was performed for Cohort 1B (not required by the OECD443 testing guideline). Animals of Cohort 2A were treated for a shorter duration (i.e. necropsy from PND 76-86). Enlarged livers were observed in males at 150 mg/kg bw/day. No data on liver weight or liver histopathology is available for Cohort 2A (not required by the OECD443 testing guideline). There were no test item-related gross observations in animals of Cohort 1C treated at 10 and 50 mg/kg bw/day, and animals of Cohorts 2B and Surplus. Note: Due to the limited number of pups available in the 150 mg/kg bw/day group, no Cohort 1C at this high dose was added to the study and priority was given to allocate high dose pups to the other mandatory cohorts.
Remaining changes in clinical biochemistry parameters in males and/or females of Cohort 1A that were considered to be related to treatment at 50 and/or 150 mg/kg bw/day included increased concentrations of total protein, albumin and glucose. As these changes occurred in the absence of correlating findings at the organ level, they were considered non-adverse. Urinalysis revealed an increase in Cohort 1A females with a relatively high number of crystals in their urine (i.e. with maximum score 3) in the 150 mg/kg bw/day group. This
change occurred in the absence of altered pH-value of the urine. It was regarded as test item-related, but not adverse, as all values remained within the historical control range and no abnormalities at the organ level were evident. No other test item-related effects were recorded for developmental parameters in F1-animals, including viability/mortality, clinical signs, body weight/body weight gain, food consumption, functional tests, balanopreputial separation (prepuce opening), vaginal patency (vaginal opening), occurrence of first eostrus, time between vaginal opening and first estrous, length and regularity of the estrous cycle, and sperm motility, concentration and morphology, hematology, coagulation or urinary parameters. Histopathologically, no test-item related effects were noted at stage-dependent qualitative evaluation of spermatogenesis of males of Cohort 1A and morphology of reproductive organs in both sexes.
F1-Generation (Post-Weaning) - Developmental Neurotoxicity
No treatment-related changes in in-life or post-mortem developmental neurotoxicity endpoints were noted in Cohort 2 animals.
F1-Generation (Post-Weaning) - Developmental Immunotoxicity
No treatment-related changes in developmental immunotoxicity endpoints (i.e. splenic lymphocyte subpopulations, lymphoid histopathology and organ weights) were observed.

In conclusion, based on the results of this extended one generation reproductive toxicity study (including Cohorts 1 and 2), the following No Observed Adverse Effect Level (NOAEL) of the test substance were established:
General toxicity (P0): 50 mg/kg bw/day (based on lesions in the liver (males), thyroid gland (both sexes) and urinary bladder (females) at 150 mg/kg bw/day).
Reproduction (P0): 50 mg/kg bw/day (based on decreased numbers of implantation sites in F0-females and a higher incidence of extended estrus in F1-females at 150 mg/kg bw/day).
Developmental toxicity (F1) (until weaning on PND 21): 50 mg/kg bw/day (based on premature maternal deaths, increased post-implantation loss, decreased gestation and live birth indices, decreased litter size, and increased pup liver weight at 150 mg/kg bw/day).
Developmental toxicity (F1) (post-weaning): 50 mg/kg bw/day (based on lesions in the liver and thyroid gland in males and females at 150 mg/kg bw/day).
Developmental neurotoxicity (F1): at least 150 mg/kg bw/day.
Developmental immunotoxicity (F1): at least 150 mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable study (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex IX, 8.7, of Regulation (EC) No 1907/2006.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The reproductive toxicity potential of the registration substance was investigated in a GLP compliant study in rats (Han Wistar) according to OECD 443 (Peter, 2020). This Extended One Generation Reproductive Toxicity Study included Cohorts 1A and 1B (without extension to a second generation), Cohorts 1C, 2A and 2B (developmental neurotoxicity) and Cohort Surplus. Wistar Han rats were treated with Diisopropylbiphenyl and triisopropylbiphenyl by daily oral gavage at dose levels of 10, 50 and 150 mg/kg bw/day. The animals of the control group received the vehicle, corn oil, alone. P0-males were treated for 12 weeks, including 10 weeks prior to mating and during the mating and post-mating period, up to and including the day before scheduled necropsy. P0-females were treated for a minimum of 16 weeks, including 10 weeks prior to mating, the variable time to conception, the duration of pregnancy and at least 21 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 14-15 weeks. From weaning onwards (PND 21), F1-animals of Cohorts 1A, 1B, 1C and 2A were dosed up to and including the day before scheduled necropsy (i.e. for a minimum of 3 weeks). The F1-animals of Cohort Surplus, Cohort 2B and Spares (not assigned to one of the cohorts) were not dosed.

Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels.

P0-Generation - Parental Results

No toxicologically relevant changes were observed at 10 mg/kg bw/day.

At 150 mg/kg bw/day, two females were euthanized for humane reasons on Day 23 post-coitum due to poor condition. The main cause of moribundity in these females was considered to be the presence of dead fetuses in the uterus, which in one female had resulted in pyometra.

Increased activity of the forelegs was noted in the majority of animals following approximately 11 weeks of treatment at 150 mg/kg bw/day. It consisted of repeated short periods of ‘digging’ in the sawdust which started several minutes after dosing and lasted for a few minutes only. The cause of this repetitive behavior in the parental P0-animals could not be elucidated. No other indications for any neurological disorder could be found in the P0-generation, based on the outcome of observations for clinical signs and histopathological examination of the brain and central nerve system. Neither was this behavior seen in the siblings of the P0-animals which were potentially exposed to the test item in utero and via maternal milk during lactation, and were dosed with the test item from weaning onwards (PND 21) for a maximum of 11-12 weeks (Cohorts 1A and 1B). Nor were there any treatment-related findings in F1-animals of Cohorts 2A and 2B, designated to extensive neurotoxicity testing. Therefore, this short and transient period of ‘digging’ was considered test item-related, but not adverse.

Lower body weights and body weight gains were noted in males treated at 150 mg/kg bw/day from Week 7 of the premating period onwards, lasting until the end of the mating period. Changes compared to concurrent controls were slight only, resulting in a mean terminal body weight of 0.93x of control. The slightly lower body weight gain in high dose males occurred despite a normal food intake during premating and mating. In females of the high dose group, food intake was even slightly higher compared to the concurrent controls during pre-mating and post-coitum, while mean body weight was similar for both groups. This is indicative for a lower food efficiency in males and females at 150 mg/kg bw/day, i.e. more food has to be ingested to reach the same growth in the animal’s mass. It was considered to be test item-related, but not adverse given the relatively slight magnitude of the effect. Body weight gain in males at 50 mg/kg bw/day when compared to the concurrent control group was only marginally lower (mean terminal body weight: 0.95x of control) and occurred in the absence of a higher relative food intake. As such no toxicological relevance was attached to this finding.

Test item-related findings were noted in the liver, thyroid glands, urinary bladder, kidneys, and adrenal glands of males and/or females at 10, 50 and/or 150 mg/kg bw/day. Higher liver weights started in males at 10 mg/kg bw/day and in females at 50 mg/kg bw/day and correlated (starting at 50 mg/kg bw/day) with microscopic hepatocellular hypertrophy, mainly centrilobular that was observed in males and females starting at 50 mg/kg bw/day. Correlating necropsy findings were at the highest dose (150 mg/kg bw/day) only and consisted of enlarged and/or thickened liver observed in males and females. In males at 150 mg/kg bw/day, the considerable increase in liver weight (71% (absolute) and 85% (relative to body weight) above control means) in combination with up to moderate hepatocellular hypertrophy was considered adverse. In addition, in males there was a small, but significant, increase in alkaline phosphatase activity. For females at 150 mg/kg bw/day there was a slightly lower increase in liver weight (less than 40%) and the hepatocellular hypertrophy was up to slight and therefore this combination was considered of concern, but at present non-adverse (Ref. 1; Ref. 2). No microscopic correlate was found for the necropsy finding of dark-red/black-brown discolouration of the liver, incidentally observed in males and females at 150 mg/kg bw/day. Higher thyroid gland weights started in males and females at 50 mg/kg bw/day. In addition, there was an increased incidence and severity of follicular cell hypertrophy and colloid alteration present in males and females starting at 50 mg/kg bw/day, and the thyroid gland was macroscopically enlarged at 50 mg/kg bw/day in males and at 150 mg/kg bw/day in males and females. In males and females at 150 mg/kg bw/day, the combination of the considerable increase in thyroid gland weight (males 33% (absolute) and 45% (relative to body weight) and females 29% (absolute) and 30% (relative to body weight) above control means) with the high incidences and severities of follicular cell hypertrophy and colloid alteration was considered adverse (Ref. 1; Ref. 2). In addition, there was a marked increase in TSH in males and females (5.07x and 10.77x of control, respectively), and decrease in total T4 in males (0.64x of control) at 150 mg/kg bw/day. It should be noted that these changes in thyroid hormone concentrations occurred in conjunction with markedly increased liver weights and correlating hepatocellular hypertrophy. This finding may be indicative of hepatic microsomal enzyme induction leading to increased metabolism of thyroid hormones and chronic thyroid stimulating hormone (TSH) stimulation of the thyroid gland. Possible adversity of the effects on thyroid hormones could not be assessed within this type of study.

The urothelial hyperplasia and hypertrophy in the urinary bladder of females treated at 150 mg/kg bw/day was considered adverse based on the hyperplastic nature. These changes occurred in absence of any macroscopically visible changes at necropsy. The increase in kidney weight in males at 150 mg/kg bw/day (relative to body weight and absolute) could not be readily attributed to the histopathology and was considered non-adverse in the absence of correlating macroscopic or microscopic kidney findings. In addition, the increased hyaline droplet accumulation in the kidney of males starting at 50 mg/kg bw/day was considered non-adverse in the absence of any degenerative findings (Ref. 1). For females, any differences in kidney weight including those that reached statistical significance were considered not to be test item-related due to a lack of dose-related pattern and variability in group values. Finally,the increase in adrenal gland weight in males at 150 mg/kg bw/day could not be readily attributed to the histopathology and was considered non-adverse in the absence of macroscopic or microscopic adrenal gland findings.

Observed changes in clinical biochemistry parameters in males and/or females that were also considered to be related to treatment included slightly increased concentrations of albumin, calcium, glucose, total protein and chloride, and slightly decreased total bilirubin at 50 and/or 150 mg/kg bw/day. No test item-related changes were noted in any of the remaining parameters investigated in this study (i.e. hematology, coagulation and urinalysis parameters) up to the highest dose level tested (150 mg/kg bw/day).

P0-Generation - Reproductive Results

No reproductive toxicity was observed up to 50 mg/kg bw/day.

A test item-related lower number of implantation sites was recorded for females treated at 150 mg/kg bw/day, which was considered to be adverse. No treatment-related changes were noted in any of the remaining reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, oestrous cycle, sperm parameters, and histopathological examination of reproductive organs, incl. spermatogenic profiling) up to the highest dose level tested (150 mg/kg bw/day).

P0-Generation / F1-Generation (Pre-Weaning) - Developmental Results

No developmental toxicity was observed up to 50 mg/kg bw/day.

At 150 mg/kg bw/day, three pregnant females did not have live offspring. As a consequence, a lower gestation index was observed in the high dose group (87% vs. 100% in the controls). Two females had signs of difficult and prolonged parturition and were euthanized on Day 23 post-coitum for humane reasons. One of these two females had six dead fetuses in its uterus in addition to nine early resorptions (no living pups), and all eleven pups of the other female had died before necropsy. The third female had four implantation sites only. Post-implantation loss was significantly higher in the 150 mg/kg bw/day group resulting in a lower post-implantation survival index (70% vs. 92% in the controls). This change could only in part be attributed to the three pregnant females that failed to deliver live offspring (see above). For high dose females that had live offspring on lactation Day 1, mean litter size was significantly lower than the control mean (7.7 vs. 10.8 in the controls). Also a lower live birth index was noted in the 150 mg/kg bw/day group. This lower value, caused by two litters with a few dead pups, was still within the normal range of biological variation. However, in combination with the other developmental effects seen at this high dose level, it was considered treatment-related. Moreover, based on the type of changes observed, these findings were regarded as adverse.

Lower serum levels of total T4 were noted in PND 4 pups (male and female pup combined) in the 150 mg/kg bw/day group (0.48x of control). This change was considered treatment-related, as the mean was below the historical control range.

A treatment-related increase in liver weight was observed in Cohort Surplus pups (PND 22; both sexes) at 150 mg/kg bw/day. The difference compared to the control group was relatively slight, but without histopathological examination of the livers no conclusion on possible adversity could be drawn. No test item-related changes were noted in any of the remaining developmental parameters investigated in this study for P0-animals and F1-pups until weaning (i.e. duration of gestation, viability and weaning indices, sex ratio, maternal care, and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, thyroid hormone levels (total T4 and TSH in PND 21/22 pups), macroscopic examination, and organ weights of brain, thyroid glands, thymus and spleen) up to the highest dose level tested (150 mg/kg bw/day).

F1-Generation (Post-Weaning) - Developmental Results

(Developmental) toxicity was observed from 50 mg/kg bw/day onwards during the post-weaning phase.

A test item-related higher incidence of females with extended oestrous was observed in the 150 mg/kg bw/day group (three high dose females vs. one female in the control group, and none in the intermediate groups). No abnormalities were found in the reproductive organs of these females, neither macroscopically nor microscopically, and also ovarian follicle counts and corpora lutea counts for these females were within normal ranges. However, based on the type of change, this higher incidence of extended oestrous in females at 150 mg/kg bw/day was considered to be adverse.

Exposure of the F1-animals to test substance in utero, through nursing during lactation, and via oral gavage following weaning was associated in Cohort 1A with alterations in liver, thyroid gland, kidney, adrenal gland and heart. Higher liver weights started in males and females at 50 mg/kg bw/day and correlated with microscopic hepatocellular hypertrophy, mainly centrilobular that was observed in males and females starting at 50 mg/kg bw/day. In males and females at 150 mg/kg bw/day, the considerable increase in liver weight (males 67% (absolute) and 75% (relative to body weight) and females 59% (absolute) and 66% (relative to body weight) above control means) in combination with up to moderate hepatocellular hypertrophy was considered adverse (Ref. 1; Ref. 2). In addition, in males there was a small, but significant, increase in alanine aminotransferase and alkaline phosphatase. No microscopic correlate was found for the necropsy finding of red-brown/black-brown discolouration of the liver, observed mainly in males and females at 150 mg/kg bw/day. Higher thyroid gland weight was noted in males and females at 150 mg/kg bw/day. In addition, there was an increased incidence and severity of follicular cell hypertrophy present in males and females starting at 50 mg/kg bw/day, and colloid alteration in males starting at 50 mg/kg bw/day and in females at 150 mg/kg bw/day. In males and females at 150 mg/kg bw/day, the combination of the considerable increase in thyroid gland weight (males 18% (absolute) and 22% (relative to body weight) and females 40% (absolute) and 46% (relative to body weight) above control means) with the high incidences and severities of follicular cell hypertrophy and colloid alteration was considered adverse (Ref. 1; Ref. 2). In addition, there was a marked increase in TSH in males and females (5.24x and 8.45x of control, respectively), and decrease in total T4 in males (0.61x of control) at 150 mg/kg bw/day. Also in males at 10 and 50 mg/kg bw/day, higher mean concentrations of TSH were noted (3.68x and 3.80x of control). These changes did not reach statistical significance, occurred in the absence of clear dose-proportionality, and means remained within the historical control data range. However, in light of the magnitude of this change a possible relation to treatment with the test item could not be excluded. It should be noted that these changes in thyroid hormone concentrations occurred in conjunction with markedly increased liver weights and correlating hepatocellular hypertrophy. This finding may be indicative of hepatic microsomal enzyme induction leading to increased metabolism of thyroid hormones and chronic thyroid stimulating hormone (TSH) stimulation of the thyroid gland. Possible adversity of the effects on thyroid hormones could not be assessed within this type of study.

The increased organ weights at 150 mg/kg bw/day for kidney in males and females, and adrenal gland and heart in females could not be readily attributed to the histopathology and was considered non-adverse in the absence of correlating macroscopic or microscopic findings in these organs.

Similar to animals in Cohort 1A, also in Cohort 1B treatment-related changes in liver, thyroid and adrenal gland weight were observed. Higher liver weights started in males and females at 50 mg/kg bw/day and correlated with the gross finding of enlarged livers at necropsy. In addition, higher thyroid gland weights were observed at 50 mg/kg bw/day (males only) and 150 mg/kg bw/day (both sexes), and higher adrenal gland weights were noted at 50 and 150 mg/kg bw/day (females only). These changes in thyroid and adrenal gland weight occurred in absence of any gross observations for either organ. It should be noted that for both Cohorts 1A and 1B the duration of treatment was almost the same (i.e. necropsy from PND 89-95 and PND 94-106, respectively). No histopathology was performed for Cohort 1B (not required by the OECD443 testing guideline). Animals of Cohort 2A were treated for a shorter duration (i.e. necropsy from PND 76-86). Enlarged livers were observed in males at 150 mg/kg bw/day. No data on liver weight or liver histopathology is available for Cohort 2A (not required by the OECD443 testing guideline). There were no test item-related gross observations in animals of Cohort 1C treated at 10 and 50 mg/kg bw/day, and animals of Cohorts 2B and Surplus. Note: Due to the limited number of pups available in the 150 mg/kg bw/day group, no Cohort 1C at this high dose was added to the study and priority was given to allocate high dose pups to the other mandatory cohorts. Remaining changes in clinical biochemistry parameters in males and/or females of Cohort 1A that were considered to be related to treatment at 50 and/or 150 mg/kg bw/day included increased concentrations of total protein, albumin and glucose. As these changes occurred in the absence of correlating findings at the organ level, they were considered non-adverse.

Urinalysis revealed an increase in Cohort 1A females with a relatively high number of crystals in their urine (i.e. with maximum score 3) in the 150 mg/kg bw/day group. This change occurred in the absence of altered pH-value of the urine. It was regarded as test item-related, but not adverse, as all values remained within the historical control range and no abnormalities at the organ level were evident.

No other test item-related effects were recorded for developmental parameters in F1-animals, including viability/mortality, clinical signs, body weight/body weight gain, food consumption, functional tests, balanopreputial separation (prepuce opening), vaginal patency (vaginal opening), occurrence of first oestrous, time between vaginal opening and first oestrous, length and regularity of the oestrous cycle, and sperm motility, concentration and morphology, hematology, coagulation or urinary parameters. Histopathologically, no test-item related effects were noted at stage-dependent qualitative evaluation of spermatogenesis of males of Cohort 1A and morphology of reproductive organs in both sexes.

F1-Generation (Post-Weaning) - Developmental Neurotoxicity

No treatment-related changes in in-life or post-mortem developmental neurotoxicity endpoints were noted in Cohort 2 animals.

F1-Generation (Post-Weaning) - Developmental Immunotoxicity

No treatment-related changes in developmental immunotoxicity endpoints (i.e. splenic lymphocyte subpopulations, lymphoid histopathology and organ weights) were observed.

CONCLUSION

In conclusion, based on the results of this extended one generation reproductive toxicity study (including Cohorts 1 and 2), the following No Observed Adverse Effect Level (NOAEL) of

Diisopropybiphenyl and trisisopropylbiphenyl were established:

General toxicity (P0): 50 mg/kg/day (based on changes in the liver (males), thyroid gland (both sexes) and urinary bladder (females) at 150 mg/kg/day).

Reproduction (P0): 50 mg/kg bw/day (based on decreased numbers of implantation sites in P0-females and a higher incidence of extended oestrous in F1-females at 150 mg/kg bw/day).

Developmental toxicity (F1) (until weaning on PND 21): 50 mg/kg bw/day (based on premature maternal deaths, increased post-implantation loss, decreased gestation and live birth indices, decreased litter size, , and increased pup liver weight at 150 mg/kg bw/day).

Developmental toxicity (F1) (post-weaning): 50 mg/kg bw/day (based on lesions in the liver and thyroid gland in males and females at 150 mg/kg bw/day).

Developmental neurotoxicity (F1): at least 150 mg/kg/day.

Developmental immunotoxicity (F1): at least 150 mg/kg/day.

Note: Possible adversity of the observed increase in TSH (males and females) and decrease in total T4 (males) in both P0- and F1-animals and decreased total T4 in PND 4 pups could not be assessed within this type of study and was therefore not taken into account when determining the NOAEL for general and developmental toxicity. It should be noted that these treatment-related changes in thyroid hormone concentrations occurred in the presence of liver hypertrophy in P0- and F1-animals.

 

References:

Ref. 1: Kerlin, R., Bolon, B., Burkhardt, J., Francke, S., Greaves, P., Meador, V., Popp, P. (2016). Scientific and Regulatory Policy Committee: Recommended (“Best”) Practice for Determining, Communicating, and Using Adverse Effect Data from Nonclinical Studies. Toxicologic Pathology. 44(2), 147-162.

Ref. 2: Palazzi X, Burkhardt JE, Caplain H, Dellarco V, Fant P, Foster JR, Francke S, Germann P, Gröters S, Harada T, Harleman J, Inui K, Kaufmann W, Lenz B, Nagai H, Pohlmeyer-Esch G, Schulte A, Skydsgaard M, Tomlinson L, Wood CE, Yoshida M (2016). Characterizing "Adversity" of Pathology Findings in Nonclinical Toxicity Studies: Results from the 4th ESTP International Expert Workshop. Toxicologic Pathology. 44(6), 810-24.

Effects on developmental toxicity

Description of key information

OECD 414 in rats

NOAEL maternal general toxicity: 50 mg/kg bw/day

NOAEL maternal developmental toxicity: 150 mg/kg bw/day

NOAEL embryo-fetal toxicity: 150 mg/kg bw/day

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 Oct 2018 - 18 Feb 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted in 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 2008

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 1998

GLP compliance:

yes (incl. QA statement)

Remarks:

Health and Youth Inspectorate, Ministry of Health, Welfare and Sport, Utrecht, The Netherlands

Limit test:

no

Species:

rat

Strain:

other: Han Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Deutschland, Sulzfeld, Germany
- Age at study initiation: 11-15 weeks
- Weight at study initiation: 175 - 261 g
- Fasting period before study: no
- Housing: individually in Macrolon plastic cages (MIII type, height 18 cm) containing appropriate bedding (Lignocel S 8-15)
- Diet: pelleted rodent diet SM R/M-Z (SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: municipal tap water in bottles, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 22
- Humidity (%): 41 - 55
- Air changes (per hr): at least 10; fresh ait (no re-circulation)
- Photoperiod: 12 hrs dark / 12 hrs light):

IN-LIFE DATES: From: 02 Dec 2018 To: 20 Dec 2018

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared as daily portions maximally 8 days in advance and stored in the refrigerator protected from light. On each day of dosing, the dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing. Also when formulations were prepared on the day of dosing, formulations were stirred at room temperature for at least 30 minutes before dosing.
Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle and the test item. No correction was made for the purity/composition of the test item. Any residual volumes were discarded.

VEHICLE
- Justification for use and choice of vehicle: Trial preparations were performed at the Test Facility to select the suitable vehicle. Corn oil was identified as a suitable vehicle.
- Concentration in vehicle: 12.5, 37.5 and 112.5 mg/mL
- Amount of vehicle: 4 mL/kg bw
- Supplier and lot/batch no.: Sigma-Aldrich, batch nos. MKC F8882 and MKCG 3257

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability analyses performed before study start in conjunction with the method development and validation study demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
During the study, dose formulation samples were collected for analysis during week 1 and week 2 of dosing. At both occasions samples for determination of achieved concentration were taken from all dose groups and samples for determination of homogeneity were take from the low and high dose group. Analyses were performed using a validated analytical procedure.
Results: The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). No test item was detected in the vehicle control (Group 1) formulations. The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Details on mating procedure:

Time-mated animals were received from the breeder.

Duration of treatment / exposure:

Day 6 -20 of gestation

Frequency of treatment:

daily, 7 days/week

Duration of test:

15 days

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

22 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of the dose range finder study.
In this dose range finding study in the pregnant rat, and in an attempt to produce graded responses to the test item. In this dose range finder groups of 6 presumed pregnant female rats were treated at 35, 150 and 600 mg/kg bw/day once daily from day 6 to 20 post coitum inclusive. Animals in the control group (n = 6) received the vehicle, corn oil, only. In this study no mortality occurred. Clinical signs noted were piloerection at 600 mg/kg bw/day. No body weight gain was observed at 600 mg/kg bw/day from post-coitum Day 6 to 9, followed by a growth rate that was comparable to the concurrent control group (similar trend at 150 mg/kgbw/day). Food consumption was decreased at 600 mg/kgbw/day between post-coitum Days 6-9 and 9-12, which returned to control levels afterwards. Enlarged livers were noted in 3/6 females at 600 mg/kg bw/day with concurrent moderately to severely increased liver weights at 150 and 600 mg/kg bw/day. Although not statistically significant, thyroid weights were slightly increased at 150 and 600 mg/kg bw/day (not dose-dependent). Number of corpora lutea, implantation sites, viable and dead fetuses, late resorptions, pre- and post-implantation loss and sex ratio were unaffected up to 600 mg/kg bw/day. At 600 mg/kg bw/day, 1/6 females had early resorptions only. Lower fetal body weights were noted at 600 mg/kg bw/day (same trend at 150 mg/kg bw/day). External examination of the fetuses did not reveal any abnormalities up to 600 mg/kg bw/day.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, in the morning and at the end of the working day
- Cage side observations were included.

CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily, beginning on Day 2 post-coitum and lasting until the day prior to necropsy

BODY WEIGHT: Yes
- Time schedule for examinations: on Days 2, 6, 9, 12, 15, 18 and 21 post-coitum

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Time schedule: measured for Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation Day 21
All animals (including animals found dead or sacrificed before planned necropsy and females with early delivery) were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). The thyroid gland, uterus and liver were weighed at necropsy for all scheduled euthanasia animals.

THYROID HORMONES:
Blood of F0-animals (except for animals which were found dead and females that delivered their offspring early) were collected on the day of scheduled necropsy bewteen 07:00 and 09:00 a.m. from the jugular vein. Animals were not fasted overnight. Blood samples at a target volume of 1 mL were collected into tubes without anticoagulant. Blood samples were processed for serum, and serum was analyzed for triiodothyronine (T3), thyroxine (T4) and thyroid-stimulating hormone (TSH).

HISTOPATHOLOGY:
Thyroid gland of all animals of Groups 1 and 4 were embedded in paraffin, sectioned at a thickness of 2-4 µm, mounted on glass slides, and stained with hematoxylin and eosin. As treatment-related changes in the thyroid gland were expected after the evaluation of Group 1 and 4 animals, histological examination of the thyroid gland was extended to Group 2 and 3 animals.

ORGAN WEIGHTS:
At scheduled necropsy the following organ weights were recorded : thyroid gland (paired organ weight), liver, uterus

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number and distribution of live and dead fetuses: Yes
- Sex of each fetus based on anogenital distance: Yes

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [half per litter]

Statistics:

please refer to "Any other information and methods"

Indices:

Body Weight Gains: Calculated against the body weight on Day 6 post-coitum
Corrected Body Weight Gains: Body weight on Day 21 post-coitum minus the body weight on Day 6 post-coitum and the weight of gravid uterus.
Relative Food Consumption: Calculated against the body weight for scheduled intervals.
Organ Weight Relative to Body Weight: Calculated against the body weight on Day 21 post-coitum.

Pre-implantaion loss (%) = ((no. of corpora lutea - no. of implantation sites)/no. of corpora lutea) x 100
Post-implantaion loss (%) = ((no. of implantation sites - no. of live fetuses)/no. of implantation sites) x 100

The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison, calculating the number of affected fetuses as a mean litter proportion on a total group basis, where:
Viable fetuses affected/litter (%) = ((no. of viable fetuses affected/litter)/number of viable fetuses/litter)) x 100

Historical control data:

Historical control data from studies performed during 2014 to 2017 (37 studies) are appended to the report.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Piloerection was observed in 8/22 high dose females on two or more consecutive days, mainly at the end of the treatment period, and in a single mid dose female on the last day of treatment.
Salivation (slight) seen after dosing among individual mid and high dose females, mainly in the second part of the treatment period, was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign is considered to be a physiological response related to the taste of the test item rather than a sign of systemic toxicity.
Other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One high dose female was found dead in her cage prior to dosing on Day 9 of treatment (Day 14 post-coitum). Blood was noted in her cage and on her vagina (externally). No clinical signs of toxicity were noted for this animal on the days preceding her death. For this female, 4% body weight loss was recorded between post-coitum Day 6-9, which recovered between Day 9-12 (13% body weight gain in this period). Food consumption for this animal was also decreased between Day 6-9, but was considered within normal range between Day 9-12. No macroscopic abnormalities were noted at necropsy. This female was pregnant and had 15 implantations in uterus (5 early resorptions and 10 normal developing implantations). Given the low incidence and in the absence of signs of ill health at the time of her death, this mortality was considered not test item-related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In approximately half of the high dose females body weight loss (up to 6%) was observed between post-coitum Day 6-9, resulting in a mean body weight gain of zero. This was followed by statistically significantly reduced body weight gain throughout the treatment period, with 5% lower body weights at the end of the treatment period compared to concurrent controls (not statistically significant). A trend towards a reduced body weight and body weight gain was also observed in the mid dose between post-coitum Day 6-15, reaching statistical significance for body weight gain only (post-coitum Day 9 and 15). For details see Attachment "Summary of female body weights and body weight gains" under Attached Background Material.
Body weight gain corrected for gravid uterus was also lower at 450 mg/kg bw/day. Mean corrected weight gain in this group was 20.3 g (9.4%) versus 28.8 g (13.1%) in the control group. A similar trend was observed at 150 mg/kg bw/day (20.7 g; 9.5%), although not reaching statistical significance. (for details see Attachment "Summary of corrected body weight gains" under Attached Background Material).
No toxicologically relevant changes in body weight were noted in the low dose group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A dose-related reduction in food consumption before and after allowance for body weight was observed at 150 and 450 mg/kg bw/day between post-coitum Day 6-12. Absolute and relative food consumption were decreased up to 21 and 15%, respectively at 150 mg/kg bw/day, and up to 37 and 33%, respectively at 450 mg/kg bw/day. Thereafter, food consumption returned to normal values and remained within the normal range of biological variation over the rest of the treatment period.
No toxicologically relevant changes in food consumption before or after allowance for body weight were noted in the low dose group. For details see Table 1.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The following statistically significant changes distinguished treated from control animals (relative changes in mean values as compared to the concurrent control group are indicated between parentheses):
- increased alanine transaminase (ALAT) values at 150 and 450 mg/kg bw/day (1.32 and 1.79x of control, respectively)
- increased aspartate aminotransferase (ASAT) values at 450 mg/kg bw/day (1.20x of control)
- decreased total protein values at 450 mg/kg bw/day (0.82x of control)
- decreased total bilirubin values at 150 and 450 mg/kg bw/day (0.74 and 0.74x of control, respectively)
- increased urea at 50, 150 and 450 mg/kg bw/day (1.20, 1.27 and 1.37x of control, respectively)
- decreased creatinine at 450 mg/kg bw/day (0.91x of control)
- increased chloride at 450 mg/kg bw/day (1.02x of control)
- decreased calcium at 450 mg/kg bw/day (0.95x of control)

Although statistically significant, these alterations in clinical biochemistry parameters were considered not toxicologically relevant due to the minimal magnitude of the change (calcium, chloride and total protein values), the absence of a dose response (bilirubin), or the absence of biological relevance (decreased creatinine). Urea values achieving a level of statistical significance when compared to controls, were considered to have arisen as a result of slightly low control values and were considered to be of no toxicological significance. Given the limited number of parameters investigated in this type of study (i.e. limited collection of tissues and no histopathological evaluation), further evaluation of these findings was considered not possible.

A statistical significant increase in serum levels of TSH was observed for F0 females at 450 mg/kg bw/day (6.2 x of control). A similar trend was observed at 150 mg/kg bw/day (2.4 x of control), although the value did not reach statistical significance. Total T3 and T4 were reduced at 150 and 450 mg/kg bw/day, reaching statistical significance for total T4 at 150 mg/kg bw/day only (0.73x of control). Although mean T4 values remained within the historical control range*, most individual T4 values were below detection limit (1.00 µg/dL) and therefore actual mean T4 values at 450 mg/kg bw/day are lower than indicated. Mean T3 and mean TSH values were respectively below and above the limits of the historical control range*, although also for T3 all samples (except for one at both 150 and 450 mg/kg bw/day), were below the detection limit (40 ng/dL), which means that the actual mean T3 values at 150 and 450 mg/kg bw/day are lower than the indicated means (see Table 2).
The observed decrease in total T3/T4 together with the increase in TSH might be related to the increased liver weights observed at 150 and 450 mg/kg bw/day and the non-adverse follicular cell hypertrophy in the thyroid glands that was noted at increased incidence and severity at 450 mg/kg bw/day. Possible adversity of the effects on thyroid hormones could not be assessed within this type of screening study and was therefore not taken into account when determining the parental NOAEL.

* Historical control data for thyroid hormone analyses in pregnant Wister Han rats (period 2018-2019):
TSH mean = 0.440, P5–P95 = 0.195–0.819 (n=30)
Total T3 mean = 66.1, P5–P95 = 51.0–84.5 (n=28)
Total T4 mean = 2.08, P5–P95 = 1.47–3.03 (n=29)

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There was a test item-related increase in liver weights at all doses. Absolute liver weights and liver/body weight ratios were increased with statistical significance for females treated at 50, 150 and 450 mg/kg bw/day (1.16, 1.33 and 1.74x of control for absolute liver weights and 1.18, 1.36 and 1.84x of control, for relative liver weights), see Table 3.

No changes in thyroid or uterus weights were noted.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Enlarged livers were observed in 8/22 females treated at 450 mg/kg bw/day versus 0/22 in the concurrent control group. This correlated with the dose dependent increase in liver weights at 450 mg/kg bw/day and increased liver enzymes (ALAT and ASAT) observed at 150 and 450 mg/kg bw/day. Based on the incidence observed, this was considered test item-related.
As no histopathology of the livers was performed, taking into account the magnitude of the effect on the liver weights and the increased liver enzymes at 150 and 450 mg/kg, adversity of these findings could not be excluded. In the absence of increased liver enzymes at 50 mg/kg and as the increase in liver weights was less than 20 percent, the liver findings at 50 mg/kg were considered non-adverse.


Watery fluid in the uterus was found in one pregnant high dose female. At the incidence observed this finding was considered not related to treatment. For one pregnant female each at 50 and at 150 mg/kg bw/day, the uterine content was hemorrhagic/contained clotted blood. There was no indication for early delivery in these females and this finding was therefore considered not toxicologically relevant.
Other findings that were noted among control and/or treated animals were considered to be of no toxicological significance, since they remained within the range of biological variation for rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related thyroid gland findings were noted in females treated at 450 mg/kg bw/day. Increased incidence and severity of follicular cell hypertrophy was present in the thyroid glands at increased incidence in females treated at 450 mg/kg bw /day up to slight degree (see Table 4).
Based on the low severity, which was within background severity for this finding, and the lack of organ weight changes in the thyroid gland this was considered non-adverse.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Description (incidence and severity):

A summary of maternal performance including numbers of pregnant and non-pregnant animals is given in Table 5. No effects of treatment were noted up to and including 450 mg/kg bw/day.

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

At 450 mg/kg bw/day, the mean % of total resorptions and the mean % post-implantation loss per litter were increased (11.2% vs concurrent control value of 3.9% for both). This value was outside the historical control range. The increase in post-implantation loss at 450 mg/kg bw/day was mainly attributed to one female, which had total post-implantation loss (11 early resorptions, 11 corpora lutea). However, post-implantation loss was still somewhat increased after excluding values of this female from calculations (6.3% vs concurrent control value of 3.9%, respectively).

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

At 450 mg/kg bw/day one female had resorptions only. All other pregnant females had litters with viable fetuses.

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

At 450 mg/kg bw/day, the mean % of early resorptions per litter was increased (10.6% vs concurrent control value of 3.9%), although not reaching statistical significance. This value was outside the historical control range. The increase in early resorptions was mainly attributed to one female, which had total post-implantation loss (11 early resorptions, 11 corpora lutea). However, percent of early resorptions was still somewhat increased after excluding values of this female from calculations (5.6% vs concurrent control value of 3.9%, respectively).

Dead fetuses:

no effects observed

Description (incidence and severity):

No dead fetuses were noted in any dose group.

Changes in pregnancy duration:

effects observed, treatment-related

Description (incidence and severity):

One high dose female was suspected of delivering her litter early on Day 20 post-coitum although no pups were found. This suspicion is based on an observation taken from the study day book and the marked body weight loss observed for this female between Day 18 and Day 21 post-coitum (i.e. 52 g). Pups missing were most likely cannibalised. From post-coitum Day 18-20, this female was noted with piloerection and at necropsy, pale discolouration of her whole body was noted. At necropsy, one late resorption and 10 early resorptions were recorded; however, since this female was suspected to have had an early delivery, the early resorptions could have been empty implantation sites.
As an early delivery at Day 20 post-coitum is rare for rats a relation with the test item could not be excluded.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Four females were not pregnant: two females in the control group, one female at 150 mg/kg bw/day and one female at 450 mg/kg bw/day. These cases of non-pregnancy were considered unrelated to the test item as it occurred in the same incidence in the treated as in the vehicle control group.

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

150 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

early or late resorptions

pre and post implantation loss

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean male and female fetal body weights were significantly reduced at 450 mg/kg bw/day (relative difference to concurrent controls: -7 and -6%, respectively), as well as mean combined fetal body weights (relative difference to concurrent controls -6%). Values were below the lower limit of the historical control range*. Even though the difference was only 6%, fetal body weights have a small variation in general and as mean values at 450 mg/kg bw/day were below the lower limit of the historical control range, these findings were considered toxicologically relevant.

Mean female and combined (male and female) fetal weights were slightly higher (relative difference to concurrent controls: 4% for both) at 50 mg/kg bw/day. Taking into consideration the direction (increase) of the effect and since values were within the historical control range of the Test Facility, this finding was considered not toxicologically relevant (for details see Table 6).

* HCD of fetal examinations are presented in Attachment "Historical Control Data of Fetal Examinations" under Attached Background Information.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Mean litter sizes as well number of live offspring were considered unaffected by treatment up to 450 mg/kg bw/day (for details see Table 6).

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male:female ratio was unaffected by treatment up to 450 mg/kg bw/day.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Mean litter sizes were considered unaffected by treatment up to 450 mg/kg bw/day.

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on external morphology following treatment up to 450 mg/kg bw/day.
The only fetus with an external finding was one mid dose fetus. This fetus had exencephaly and open eyes. Skeletal examination substantiated the skull finding and revealed small mandibles and anomalies of the sternum, vertebrae and ribs. Due to the single occurrence in the mid dose group, these malformations were considered to be of spontaneous origin.
External variations were not seen in any group.

For a summary of fetal malformations and variations (absolute numbers and percentages) see Attachment "Summary of fetuses and litters with malformations and variations" under Attached Background Material.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

The number of fetuses with bent ribs was statistically significant higher at 450 mg/kg bw/day. Mean litter incidences of this variation were 10.9%, 8.9%, 15.7% and 29.8% per litter in the control, 50, 150 and 450 mg/kg bw/day groups, respectively and the high dose incidence was above the historical control maximum value of 27.4% per litter. Bent ribs represent a common finding in prenatal toxicity studies with a large variety of compounds (but are rarely observed in untreated controls) and the underlying cause is most likely an interference with the chondrification and ossification process of the ribs. Various biologic activities may interfere with these processes and maternal toxicity seems to be an important causative factor for bent ribs. Regardless of the cause, as it has been demonstrated previously that bent ribs are completely reversible, this finding was considered not adverse.

Several ossification parameters indicated reduced ossification at 450 mg/kg bw/day compared to the control group. Increased incidences were observed for unossified metacarpals and metatarsals (18.7% versus 12.0% per litter), reduced ossification of the skull (11.9% versus 5.8 % per litter) and reduced ossification of vertebral centra (4.3% versus 0.0% per litter). None of these increases was statistically significant and only the incidence of unossified metacarpals and metatarsals was above the historical control value (17.6% per litter)*. The delayed skeletal ossification appears to be associated with impaired fetal growth as shown by the body weights of fetuses with unossified metacarpals and metatarsals in both the control and 450 mg/kg bw/day group. Mean weights of both affected control (4.7 g) and 450 mg/kg (4.6 g) fetuses were below their group mean values (5.2 and 4.9 g, respectively).
Noteworthy in the 150 mg/kg bw/day group, is the variation of unossified metacarpals and metatarsals that occurred at a statistically significant lower incidence compared to control Group. Mean litter incidences of this finding were 12.0%, 5.6%, 2.3% and 18.7% per litter in the control, 50, 150 and 450 mg/kg bw/day groups, respectively. Because there was no dose-response for this finding, the low value at the mid-dose level was considered a chance finding.
The other variations that were noted occurred in the absence of a dose-related incidence trend, infrequently and/or at frequencies that were within the range of available historical control data*. Therefore, they were considered not treatment-related.

Three skeletally malformed fetuses were observed in this study. Besides the mid dose fetus with exencephaly, small mandibles and anomalies of the sternum, vertebrae and ribs that was described above, another mid dose fetus and one low dose fetus had a vertebral anomaly with or without associated rib anomaly. Due to the low incidence and absence of a dose-relationship, these malformations were considered not treatment-related.

For a summary of fetal malformations and variations (absolute numbers and percentages) see Attachment "Summary of fetuses and litters with malformations and variations" under Attached Background Material.
* HCD of fetal examinations are presented in Attachment "Historical Control Data of Fetal Examinations" under Attached Background Information.

Visceral malformations:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on visceral morphology following treatment up to 450 mg/kg bw/day.
The only visceral malformation was observed at cephalic examination of one high dose fetus. This fetus had a small right eye and as it occurred singly and was seen previously among historical control fetuses, it was considered not to be treatment-related.
Only two visceral variations (small supernumerary liver lobes and convoluted ureter) were observed in this study and at the low incidence these occurred, they were not considered to be related to treatment.

For a summary of fetal malformations and variations (absolute numbers and percentages) see Attachment "Summary of fetuses and litters with malformations and variations" under Attached Background Material.

Other effects:

no effects observed

Description (incidence and severity):

The anogenital distance before and after correction for body weight was unaffected by treatment up to 450 mg/kg bw/day (see Table 7).

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

450 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Table 1. Maternal food consumption (g/animal/day)

Post coitum		Dose level (mg/kg bw/day)
		0 (control)	50	150	450
Days 2- 6	Mean	20	23	21	21
	SD	2.4	9.8	1.7	2.5
	N	20	22	21	21
Days 6 -9	Mean	19	18	15**	12**
	SD	2.6	2.1	2.6	2.1
	N	20	22	21	21
Days 9-12	Mean	20	19	17**	14**
	SD	2.2	1.8	2.8	3.8
	N	20	22	21	21
Days 12-15	Mean	21	21	20	19
	SD	2.4	2.2	2.4	2.1
	N	20	22	21	20
Days 15 -18	Mean	23	23	22	21
	SD	1.9	2.5	2.5	2.9
	N	20	22	21	20
Days 18-21	Mean	21	21	20	19
	SD	3.1	2.3	3.4	3.9
	N	20	22	21	20
Mean of means		20	21	19	18
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**)

Table 2. Maternal thyroid hormone determination – Pregnant females

		Dose level (mg/kg bw/day)
		0 (control)	50	150	450
TSH (µIU/mL)	Mean	0.384	0.479	0.930	2.369**
	SD	0.186	0.231	0.715	1.696
	N	20	22	21	19
Total T3 (ng/dL)	Mean	61.1	53.6	42.4	53.6
	SD	9.3	10.1	--	--
	N#	17	13	1	1
Total T4 (µg/dL)	Mean	2.12	2.37	1.55*	1.86
	SD	0.65	0.53	0.48	0.96
	N##	19	19	19	5
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) #      Missing values were below the detection limit of 40 ng/dL ##    Missing values were below detection limit of 1.00 µg/dL Historical control data for thyroid hormone analyses in pregnant Wister Han rats (period 2018-2019): TSH mean = 0.440, P5–P95 = 0.195–0.819 (n=30) Total T3 mean = 66.1, P5–P95 = 51.0–84.5 (n=28) Total T4 mean = 2.08, P5–P95 = 1.47–3.03 (n=29)  Table 3. Maternal organ weights (g) – Pregnant females     Dose level (mg/kg bw/day)     0 (control) 50 150 450 Body weight Mean 322 319 317 307   SD 31 24 28 33   N 20 22 21 19             Liver Mean 10.47 12.17** 13.98** 18.22**   SD 1.45 1.29 1.45 1.43   N 20 22 21 19             Thyroids Mean 0.020 0.021 0.021 0.022   SD 0.007 0.005 0.006 0.005   N 20 22 21 19 */** Dunnett-test based on pooled variance significant at 1% (**)  Table 4. Thyroid gland histopathology findings – all females   Dose level (mg/kg bw/day)   0 (control) 50 150 450 No. of organs examined 22 22 22 22 Thyroid glands               Follicular cell hypertrophy                        Minimal 2 3 6 9                Slight 0 0 0 7   Table 5. Summary of maternal performance   Dose level (mg/kg bw/day)   0 (control) 50 150 450 Females on study 22 22 22 22 Females that aborted or delivered early 0 0 0 1 Females that died 0 0 0 1 Females examined at scheduled necropsy 22 22 22 20       Nongravid (non-pregnant) 2 0 1 1       Gravid (pregnant) 20 22 21 19       With resportions only 0 0 0 1       With viable fetuses 20 22 21 18 Total females gravid 20 22 21 21 Table 6. Summary of fetal data Parameter   Dose level (mg/kg bw/day)     0 (control) 50 150 450 No. of gravid females   20 22 21 19             Total number of viable fetuses   215 218 233 187             Viable fetuses/litter Mean 10.8 9.9 11.1 9.8   SD 2.31 2.39 1.73 3.10             Viable fetuses % Mean 96.1 94.6 98.0 88.8   SD 6.00 9.62 3.68 24.50             Corpora lutea Mean 12.2 10.5 11.3 11.1   SD 2.33 2.30 1.77 1.70             Pre-implantation loss/litter Mean 0.9 1.0 0.8 1.1   SD 1.19 1.17 1.03 1.51             Pre-implantation loss (%) Mean 7.6 8.6 6.6 7.6   SD 9.96 11.14 8.73 9.53             Early resorptions/litter Mean 0.5 0.6 0.2 1.2   SD 0.69 1.01 0.44 2.78             Late resorptions/litter Mean 0.0 0.0 0.0 0.1   SD 0.00 0.00 0.00 0.23             Post-implantation loss/litter Mean 0.5 0.6 0.2 1.3   SD 0.69 1.01 0.44 2.77             Post-implantation loss (%) Mean 3.9 5.4 2.0 11.2   SD 6.00 9.62 3.68 24.50             Male fetal weight (g) Mean 5.4 5.5 5.4 5.0**   SD 0.27 0.22 0.31 0.41             Female fetal weight (g) Mean 5.1 5.3* 5.1 4.8**   SD 0.34 0.26 0.30 0.28             Fetal weight (g) Mean 5.2 5.4* 5.2 4.9**   SD 0.24 0.20 0.31 0.32 */** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**)  Table 7. Summary of fetal anogenital distances (AGD) Parameter   Dose level (mg/kg bw/day)     0 (control) 50 150 450 Male AGD (mm) Mean 2.71 2.67 2.61 2.57   SD 0.155 0.200 0.283 0.296             Corrected male AGD by fetal weight (mm/g) Mean 1.55 1.51 1.48 1.51   SD 0.088 0.117 0.167 0.170             Female AGD (mm) Mean 1.35 1.34 1.32 1.38   SD 0.219 0.231 0.163 0.207             Corrected female AGD by fetal weight (mm/g) Mean 0.79 0.77 0.77 0.82   SD 0.128 0.134 0.096 0.131

Conclusions:

Timed-pregnant Wistar rats were treated with the test item, reaction mass of diisopropyl-1,1’-biphenyl and tris-(1-methylethyl)-1,1’biphenyl, by daily oral gavage from gestation Days 6 up to and including Day 20. Groups of 22 females each received the test item formulated in corn oil at a volume of 4 mL. No test item-related mortality was noted during the study. Piloerection was observed in 8/22 females at 450 mg/kg bw/day on two or more consecutive days, mainly at the end of the treatment period and in a single female at 150 mg/kg bw/day on the last day of treatment. Maternal toxicity was observed at 450 mg/kg bw/day, consisting of body weight loss observed in approximately half of the females between post-coitum Day 6-9, followed by statistically significant reduced body weight gain throughout the treatment period. The observed body weight effects coincided with statistically significant reduced food consumption at 450 mg/kg bw/day between post-coitum Day 6-12.
Macroscopic evaluation revealed enlarged livers in females treated at 450 mg/kg bw/day. Moreover, a test item-related increase in liver weights was observed at all dose levels (up to 1.18, 1.36 and 1.84x compared to concurrent controls at 50, 150 and 450 mg/kg bw/day, respectively for relative liver weights). Also, in line with these findings are the increased ALAT and ASAT values at 450 mg/kg bw/day and the increased ALAT value at 150 mg/kg bw/day. Taking into account the magnitude of the effect on the liver weights and the increased liver enzymes at 150 and 450 mg/kg bw/day, adversity of these finding could not be excluded. In the absence of increased liver enzymes at 50 mg/kg bw/day and as the increase in liver weights was less than 20 percent, the liver findings at 50 mg/kg bw/day were considered non-adverse.
A marked increase in serum levels of TSH was observed for high dose females and a similar, not statistically significant trend was observed in mid dose females. Additionally, a decrease in total T4 and total T3 was observed in mid and high dose females. The observed decrease in total T3/T4 together with the increase in TSH is likely related to the increased liver weights observed at 150 and 450 mg/kg bw/day and the non-adverse follicular cell hypertrophy in the thyroid glands that was noted at increased incidence and severity at 450 mg/kg bw/day. Possible adversity of the effects on thyroid hormones could not be assessed within this type of study and was therefore not taken into account when determining the parental NOAEL.
At 450 mg/kg bw/day, one female was suspected of delivering her litter early on Day 20 post-coitum although no pups were found. As an early delivery at Day 20 post-coitum is rare for rats a relation with the test item could not be excluded.
A test item-related increase in the mean % of early resorptions, and consequently % total resorptions and % post-implantation loss per litter was observed at 450 mg/kg bw/day.
Mean male and female fetal body weights were significantly reduced at 450 mg/kg bw/day.
Skeletal examination showed an increase in the number of high dose fetuses with bent ribs. Additionally, several ossification parameters indicated reduced ossification at 450 mg/kg bw/day compared to the control group. These variations were considered not a direct effect of the test item but secondory the the observed lower fetal weights.
No treatment-related toxicologically significant changes were noted in any of the remaining developmental parameters investigated in this study (i.e. litter size, sex ratio, (corrected) anogenital distance, external, visceral malformations and developmental variations).
Based on the above reported findings the no-observed-adverse-effect level (NOAEL) for maternal general toxicity was considered to be 50 mg/kg bw/day, the NOAEL for developmental and embryo-fetal toxicity was considered to be 150 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable study (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex IX, 8.7, of Regulation (EC) No 1907/2006.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Toxicity to reproduction: other studies

Additional information

Developmental toxicity was tested in a GLP compliant study in rats (Han Wistar) according to OECD 414 (de Groot, 2020). In this study 22 timed-mated rats per dose level were treated daily by gavage with 50, 150 and 450 mg test item/kg bw/day suspended in corn oil from gestation day 6 to day 20. The animals of the control group received vehicle only (corn oil).

Examinations of maternal animals included clinical observations, mortality, body weight, food consumption, gross pathological examinations, organ weights of thyroid gland and liver and determination of serum thyroid hormones (TSH, T3 and T4).Additionally selected clinical chemistry parameters were determined. On day 21 of gestation fetuses were delivered by caesarian section. The following parameters were determined at caesarian section: number of corpora lutea, number of implantations, uterus weight, number of resorptions, number of pregnant, number of live/dead fetuses, sex of live fetuses, individual weights and anogenital distance of foetuses, as well as external (all fetuses), visceral (one half of the fetuses) and skeletal one half of the fetuses) malformations and variations.

There were no test item-related mortalities. Piloerection was observed in 8/22 females at 450 mg/kg bw/day on two or more consecutive days, mainly at the end of the treatment period and in a single female of the 150 mg/kg group on the last day of treatment. 

Maternal toxicity was observed at 450 mg/kg bw/day, consisting of body weight loss observed in approximately half of the females between post-coitum Day 6-9. This was followed by statistically significant reduced body weight gain throughout the treatment period. The observed body weight effects coincided with statistically significant reduced food consumption at 450 mg/kg bw/day between post-coitum Day 6-12. Body weight gain corrected for gravid uterus was also lower at 450 mg/kg bw/day.

A similar trend towards a reduced body weight and body weight gain was observed at 150 mg/kg between post-coitum Day 6-15. Body weight gain corrected for gravid uterus was also lower at 150 mg/kg bw/day, although not reaching statistical significance. Moreover, food consumption was statistically significant reduced at 150 mg/kg bw/day between post-coitum Day 6-12. As no body weight loss was observed at 150 mg/kg bw/day and there were no differences in body weights at end of the treatment period, the body weight and body weight gain effects at 150 mg/kg bw/day were considered not to be toxicologically relevant.

Macroscopic evaluation revealed enlarged livers in females treated at 450 mg/kg bw/day. Moreover, a test item-related increase in liver weights was observed at all dose levels (up to 1.18, 1.36 and 1.84x compared to concurrent controls at 50, 150 and 450 mg/kg, respectively for relative liver weights). Also, in line with these findings are the increased ALAT and ASAT values at 450 mg/kg bw/day and the increased ALAT value at 150 mg/kg bw/day. Taking into account the magnitude of the effect on the liver weights and the increased liver enzymes at 150 and 450 mg/kg bw/day, adversity of these finding could not be excluded.

In the absence of increased liver enzymes at 50 mg/kg bw/day and as the increase in liver weights was less than 20 percent, the liver findings at 50 mg/kg bw/day were considered non-adverse.

A marked increase in serum levels of TSH was observed for F0 females at 450 mg/kg bw/day and a similar, not statistically significant trend was observed at 150 mg/kg bw/day. Additionally, a decrease in total T4 and total T3 was observed at 150 and 450 mg/kg bw/day. The observed decrease in total T3/T4 together with the increase in TSH is likely related to the increased liver weights observed at 150 and 450 mg/kg bw/day and the non-adverse follicular cell hypertrophy in the thyroid glands that was noted at increased incidence and severity at 450 mg/kg bw/day. Possible adversity of the effects on thyroid hormones could not be assessed within this type of study and was therefore not taken into account when determining the parental NOAEL. The mean numbers of corpora lutea, implantation sites and pre-implantation loss in the control and test groups were similar and in the range of normal biological variation.

At 450 mg/kg bw/day one female was suspected of delivering her litter early on Day 20 post-coitum although no pups were found. As an early delivery at Day 20 post-coitum is rare for rats a relation with the test item could not be excluded.

A test item-related increase in the mean % of early resorptions, and consequently % total resorptions and % post-implantation loss per litter was observed at 450 mg/kg bw/day. For both early resorptions and post-implantation loss, values were outside the historical control range. The increase in % of early resorptions and % of post-implantation loss at 450 mg/kg bw/day was mainly attributed to one female, which had early resorptions only (11 corpora lutea). It should be mentioned that this female had a marked body weight loss between Day 6-12 post-coitum and that her food consumption was very low between Day 9-12 post-coitum. After excluding values of this female from calculations, % of early resorptions and % of post-implantation loss at 450 mg/kg was still somewhat increased. 

Mean male and female fetal body weights were significantly reduced at 450 mg/kg bw/day (relative difference to concurrent controls: -7 and -6%, respectively), as well as mean combined fetal body weights (relative difference to concurrent controls -6%). Even though the difference was only 6%, fetal body weights have a small variation in general and as mean values at 450 mg/kg were below the lower limit of the historical control range, these findings were considered toxicologically relevant.

Skeletal examination showed an increase in the number of fetuses with bent ribs at 450 mg/kg bw/day. Additionally, several ossification parameters indicated reduced ossification at 450 mg/kg bw/day compared to the control group. These variations were considered not a direct effect of the test item but secondary to the reduced fetal weights at this dose level.

No treatment-related toxicologically significant changes were noted in any of the remaining developmental parameters investigated in this study (i.e. litter size, sex ratio, (corrected) anogenital distance, external, visceral malformations and developmental variations).

Based on the above reported findings the no-observed-adverse-effect level (NOAEL) for maternal general toxicity was considered to be 50 mg/kg bw/day, the NOAEL for developmental and embryo-fetal toxicity was considered to be 150 mg/kg bw/day.

Justification for classification or non-classification

In an OECD 443 study evidence of reproduction effects were observed in P0 animals as well in F1 animals occuring together with other toxic effecs. However, the adverse effects on reproduction is considered not to be secondary non-specific consequence of the other toxic effects. In conclusion, the test substance meet the classification criteria for reproduction toxicity (Category 2, H361fd) according to Regulation EC No 1272/2008.

In an OECD 414, developmental effects observed in the offspring and/or maternal animals are considered as a secondary, non-specific consequence of maternal toxicity and not as indication of specific developmental toxicity. In conclusion, the test substance does not meet the classification criteria for developmental toxicity according to Regulation EC No 1272/2008.