Propionamide

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-12-29 to 2021-06-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-12-29 to 2021-06-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: EPA Health Effects Test Guideline OPPTS 870.3650: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

July 2000

Deviations:

yes

Remarks:

Please refer to Principles of method.

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

July 2016

Deviations:

yes

Remarks:

Please refer to Principles of method.

Principles of method if other than guideline:

Deviation from study design:
Body weights and food consumption was inadvertently not recorded for Recovery Groups 1 (control) and 4 (750 mg/kg/day) between Days 7-14 of the Recovery Period. As (partial) recovery of body weight (gain) was noted at Day 7 of the Recovery Period, sufficient data was available for the toxicological evaluation. As food consumption was considered unaffected in all groups throughout the Treatment Period and Week 1 of the Recovery Period, sufficient data was available for the toxicological evaluation.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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 laboratory has general and reproduction/developmental 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 toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: not specified but either Charles River Deutschland, Sulzfeld, Germany or
Charles River Laboratories France, L'Arbresle Cedex, France.
- Females nulliparous and non-pregnant: yes
- Age at initiation of dosing: Males: 10-11 weeks; Females: 13-14 weeks
- Weight at study initiation: Males: 266 to 320 g; Females: 203 to 246 g.
- Fasting period before study: overnight before necropsy
- Housing: On arrival and following the pretest (females only) and pre-mating period + recovery males and females: group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon, MIV type, height 18 cm).
During the mating phase: Main males and females will be cohabitated on a 1:1 basis in Makrolon plastic cages (MIII type, height 18 cm).
During the post-mating phase: males in Makrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Main Females: individually housed in Makrolon plastic cages (MIII type, height 18 cm).
During the lactation phase: Main females will be housed in Makrolon plastic cages (MIII type, height 18 cm). Pups will be housed with the dam, except during locomotor activity monitoring of the dams, when the pups will be kept warm in their home cage .
During locomotor activity monitoring: F0-animals: individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cageenrichment, bedding material, food and water.
The cages will contain appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne
GmbH + CO. KG, Rosenberg, Germany).
- Diet: ad libitum except during designated procedures, pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water: ad libitum except during designated procedures, tap water
- Acclimation period: 9 days prior to start of the pretest period (females) or 8 days before the commencement of dosing (males)

DETAILS OF FOOD AND WATER QUALITY: The feed is analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis are provided by the supplier and are on file at the Test Facility. Periodic analysis of the water is performed, and results of these analyses are on file at the Test Facility.
It is considered that there are no known contaminants in the feed and water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 21
- Humidity (%): 43 to 47
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2020-12-29 To: 2021-03-31

Route of administration:

oral: gavage

Details on route of administration:

The oral route of administration was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.

Vehicle:

water

Details on oral 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 daily and/or weekly (filled out in daily portions) as a solution. The test item was mortared and transferred to a suitable container wherein the vehicle was added. The dosing formulations was stirred for at least 30 minutes and dosed within 24 hours after adding the vehicle to the test item or stored in the refrigerator. The dosing formulations that were removed from the refrigerator were stirred at room temperature for at least 30 minutes before dosing and dosed within 24 hours after removal from the refrigerator. Test item dosing formulations will be kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. No adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.

VEHICLE
- Concentration in vehicle: 24, 60, 150 mg/mL
- Amount of vehicle: 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). No test item was detected in the Group 1 formulation.

Duration of treatment / exposure:

Males: 29 days
For main males this included at least 2 weeks of treatment prior to mating and during the mating period.

Females: at least 56 days (based on females first scheduled for necropsy that delivered pups)
Females were not dosed during littering.

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1

Dose / conc.:

750 mg/kg bw/day (nominal)

Remarks:

Group 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Group 3

Dose / conc.:

120 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

10 males and females per dose (main groups)
5 males and females treates with 0 and 750 mg/kg bw/day (recovery group)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a 14-day dose range finder (DRF) with oral gavage administration of the test item in rats and in an attempt to produce graded responses to the test item.
During the 14-day DRF (dose levels: 500 and 1000 mg/kg /day with female rats only), no mortality was observed up to 1000 mg/kg/day. Test item-related clinical signs included hunched posture and piloerection (both starting at 500 mg/kg/day). At 500 mg/kg/day, body weight was decreased in 2/3 females between Days 1-14 (-2 % and -3 % at Day 14 compared to Day 1) and decreased in 1/3 females between Days 1-4 followed by an increase between Days 4-14 (+3 % at Day 14 compared to Day 1). At 1000 mg/kg/day, body weight was decreased in 1/3 females between Days 1-4 and remained unchanged on the days thereafter (-4 % on Day 14 compared to Day 1). Body weight was decreased in 1/3 females at 1000 mg/kg/day between Days 1-11 which recovered between Days 11-14 to the same level as Day 1. At 1000 mg/kg/day, body weight was decreased in 1/3 females between Days 1-8, which slightly increased between Days 8-14 (-8 % at Day 14 compared to Day 1). At 500 and 1000 mg/kg/day, food consumption was considered normal between Days 1-14, except between Days 8-11 where food consumption was increased. No macroscopic abnormalities were noted in 3/3 females at 500 and 1000 mg/kg/day. Liver weights were considered normal at 500 and 1000 mg/kg/day, while kidney weights were slightly increased at 500 and 1000 mg/kg/day after 14 days of treatment.
Based on the results of the 14-day DRF a dose level of 750 mg/kg/day was considered to be a suitable high-dose level for the Main study, as a moderate decrease in body weight was observed at 1000 mg/kg/day which did not sufficiently increase during the treatment period despite the normal to slightly increased food consumption. The dose levels of 300 and 120 mg/kg/day are included in order to provide information on possible dose-response relationships. The high-dose level should produce some toxic effects, but not death nor obvious suffering. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.
- Fasting period before blood sampling for clinical biochemistry: yes, overnight
- Rationale for selecting satellite groups: The recovery animals were used to study the potential reversibility of possible toxic effects.
- Post-exposure recovery period in satellite groups: 14 days

Positive control:

No positive control was used.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily throughout the study.
- Cage side observations: Animals were observed for general health/mortality and moribundity. Animals were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: During treatment, animals were observed at least once daily, up to the day prior to necropsy. These clinical observations were at least be conducted 3 hours (± 30 minutes) after dosing. Once before the first administration of the test item and at weekly intervals during the treatment and recovery period animals were observed for specific clinical signs in a standard arena.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of treatment (prior to dosing), and weekly thereafter for every study phase. Mated Main females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
Food consumption was quantitatively measured weekly, except for Main males and Main females which are housed together for mating and for Main females without evidence of mating. Food consumption of mated Main females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Water consumption was monitored by visual inspection of the water bottles. If inter group differences were noted, consumption was assessed by weight.

OPHTHALMOSCOPIC EXAMINATION: Yes as part of neurobehavioural examination

HAEMATOLOGY: Yes
- Time schedule for collection of blood: main animals: at the end of the treatment period on the day of scheduled necropsy, recovery animals: at the end of the treatment period and on the day of scheduled necropsy at the end of the recovery period
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes, overnight (max. 24 h), water was available. Main females were not fasted.
- How many animals: 5 per sex per group
- Parameters checked: White Blood Cell Count (WBC), Reticulocytes (absolute), Neutrophils (absolute), Red Blood Cell Distribution Width Gated (RDWG), Lymphocytes (absolute), Hemoglobin, Monocytes (absolute), Hematocrit, Eosinophils (absolute), Mean corpuscular volume (MCV), Basophils (absolute), Mean corpuscular hemoglobin (MCH), Large unstained cells (LUC) (absolute), Red Blood Cell Count (RBC), Mean corpuscular hemoglobin concentration (MCHC), Platelets, Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: main animals: at the end of the treatment period on the day of scheduled necropsy, recovery animals: at the end of the treatment period and on the day of scheduled necropsy at the end of the recovery period
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes, overnight (max. 24 h), water was available. Main females were not fasted.
- How many animals: 5 per sex per group
- Parameters checked: Alanine aminotransferase (ALT), Creatinine, Aspartate aminotransferase (AST), Glucose, Alkaline Phosphatase (ALP), Cholesterol, Total protein, Sodium, Albumin, Potassium, Total Bilirubin, Chloride, Bile Acids, Calcium, Urea, Inorganic Phosphate (Inorg. Phos)

PLASMA/SERUM HORMONES/LIPIDS: Yes, Thyroxine (T4) and Thyroid-Stimulating Hormone (TSH)
- Time schedule for collection of blood: main animals: at the end of the treatment period on the day of scheduled necropsy, recovery animals: at the end of the treatment period and on the day of scheduled necropsy at the end of the recovery period
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes, overnight (max. 24 h), water was available. Main females were not fasted.
- How many animals: 5 per sex per group

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: all males: once during Week 4 of treatment and recovery males grip strength measurement at the end of recovery phase; females: main: once during the last week of lactation (i.e. PND 6-13), recovery: on the first day a Main female is tested and locomotor activity at the end of recovery phase. Tests were performed after clinical observations (including arena observation).
- Dose groups that were examined: 5 main males and 5 main females from each dose group and control, all recovery animals
- Battery of functions tested: sensory activity (hearing ability, pupillary reflex and static righting reflex), grip strength (fore- and hind-limb), motor activity (total movements and ambulations)

IMMUNOLOGY: No

OTHER: Further examinations on reproductive parameters were conducted and are presented in the respective IUCLID section.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, please refer to "Any other information on materials and methods".

HISTOPATHOLOGY: Yes, please refer to "Any other information on materials and methods".

Statistics:

Please refer to "Any other information on materials and methods".

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant clinical signs were noted up to 750 mg/kg/day.
Hunched posture was noted in 1/10, 2/10 and 1/15 females at 120, 300 and 750 mg/kg/day, respectively, on one day in Week 4 of treatment, up to three days in Week 1 and two days in Week 1, respectively, as well as in 1/10 males at 300 mg/kg/day on one day in Week 5 of treatment. This sign was not observed during the Recovery Period. Given the low incidence and in absence of a dose-related trend, this sign was considered of no toxicological relevance.
Lean appearance was recorded once in 1/10 females at 300 mg/kg/day on in Week 1 of treatment. Given the low incidence and in absence of a dose-related trend, this sign was considered of no toxicological relevance.
Incidental findings that were noted included chromodacryorrhea, alopecia, piloerection, scales, scabs and red discoloration of both forelegs. These findings occurred only during the Recovery Period and/or 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 incidence observed, these were considered signs of no toxicological relevance.
No findings were noted during the weekly arena observations in this study.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights and body weight gain were considered to be affected by treatment with the test item in males starting at 300 mg/kg/day and in females at 120 mg/kg/day.
In males at 300 and 750 mg/kg/day, mean body weight and mean body weight gain were decreased throughout the Treatment Period, followed by a partial recovery at 750 mg/kg/day during the 14 days treatment-free Recovery Period. Mean body weights were 8 and 9 % lower compared to the concurrent control group at 300 and 750 mg/kg/day, respectively, at the end of the Treatment Period and 4 % lower compared to the concurrent control group at 750 mg/kg/day at the end of the Recovery Period.
In Main females starting at 120 mg/kg/day, mean body weight was decreased (not always reaching statistical significance) throughout the Treatment Period, except at 120 mg/kg/day from Lactation Day 4 onwards which was similar to the concurrent control group. At the end of the Lactation Period, mean body weights were 8 and 9 % lower compared to the concurrent control group at 300 and 750 mg/kg/day, respectively.
Mean body weight gain at 750 mg/kg/day was decreased up to and including the Post-coitum Period.
In Recovery females at 750 mg/kg/day, mean body weight and mean body weight gain were decreased throughout the Treatment Period (not always reaching statistical significance), followed by full recovery from Day 7 of the Recovery Period onwards. At the end of the Treatment Period, mean body weight was 5 % lower compared to the concurrent control group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption before or after correction for body weight was considered affected by treatment with the test item in Main females starting at 300 mg/kg/day.
In Main females at 300 and 750 mg/kg/day, mean absolute and relative food consumption were decreased throughout the Lactation Period (not always reaching statistical significance). During the last interval of the Lactation Period at 300 and 750 mg/kg/day, mean absolute food consumption was 15 and 20 % lower compared to the concurrent control group, respectively, and mean relative food consumption was 5 and 13% lower compared to the concurrent control group, respectively.
In males and Recovery females, food consumption was considered to be unaffected by treatment with the test item.

Food efficiency:

not examined

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

not specified

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Pupillary reflex reflex was normal in all examined animals up to 750 mg/kg/day.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematology parameters were affected by treatment with the test item in males and females starting at 300 mg/kg/day.
The following changes distinguished treated from control animals. The differences were statistically significant unless indicated otherwise. Relative changes in mean values as compared to the concurrent control group are indicated between parentheses.
In males (end of Treatment and Recovery Periods):
- Lower (0.87 and 0.86x; not statistically significant) mean reticulocyte (RETIC) concentration at 300 and 750 mg/kg/day at the end of the Treatment Period. A partial recovery (0.90x; not statistically significant) was noted at the end of the Recovery Period.
- Higher (1.07x) mean hemoglobin (HGB) concentration at 750 mg/kg/day at the end of the Treatment Period, which recovered to levels similar to the control at the end of the Recovery Period.
- Higher (1.06x) mean hematocrit (HCT) at 750 mg/kg/day at the end of the Treatment Period, which partially recovered (1.03x; not statistically significant) at the end of the Recovery Period.
In Main females (end of Treatment Period):
- Higher (2.67 and 3.00x) mean large unstained cell (LUC) concentration at 300 and 750 mg/kg/day, respectively.
In Recovery females at 750 mg/kg/day (end of Treatment and Recovery Periods):
- Higher (1.79x; not statistically significant) mean neutrophil (NEUT) concentration at the end of the Treatment Period. Partial recovery (1.35x; not statistically significant) was noted at the end of the Recovery Period.
- Higher (1.96x) mean monocyte (MONO) concentration at the end of the Treatment Period. Partial recovery (1.45x; not statistically significant) was noted at the end of the Recovery Period.
- Higher (4.00x; not statistically significant) mean basophil (BASO) at the end of the Treatment Period, which recovered to levels similar to the control at the end of the Recovery Period.
- Higher (1.06x) mean red blood cell (RBC) concentration at the end of the Treatment Period, which recovered to levels similar to the control at the end of the Recovery Period.
- Higher (1.08x each) mean red blood cell distribution width gated (RDWG) at the end of the Treatment and Recovery Period.
- Higher (1.06x) mean HGB concentration at the end of the Treatment Period. Partial recovery (1.03x) was noted at the end of the Recovery Period.
- Higher (1.05 and 1.04x) mean HCT at the end of the Treatment and Recovery Period, respectively.
The higher non-statistically significant LUC concentration in Recovery Females at 750 mg/kg/day at the end of the Treatment Period could be attributed to one outlier and was therefore considered not toxicologically relevant.
Any other (statistically significant) changes in hematology parameters were considered to be unrelated to treatment with the test item due to the absence of a dose-related trend or the occurrence at the end of the Recovery Period alone.

Coagulation parameters of treated rats were considered not to have been affected by treatment with the test item.
In the absence of a dose-related trend, the lower non-statistically significant mean activated partial thromboplastin time (APTT) at 120 and 300 mg/kg/day was considered not to be related to treatment with the test item.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Clinical biochemistry parameters were affected by treatment with the test item in males starting at 300 mg/kg/day and in females starting at 120 mg/kg/day.
The following changes distinguished treated from control animals. The differences were statistically significant unless indicated otherwise. Relative changes in mean values as compared to the concurrent control group are indicated between parentheses.
In males (end of Treatment and Recovery Periods):
- Higher mean alanine aminotransferase (ALT; 2.75 and 1.79x), aspartate aminotransferase (AST; 2.30 and 1.73x) and alkaline phosphatase (ALP; 1.63 and 1.45x) activities at 300 and 750 mg/kg/day, respectively, at the end of the Treatment Period.
At 750 mg/kg/day, partial recovery in ALT (1.08x), AST (1.14x) and ALP (1.11x) was noted at the end of the Recovery Period (all not statistically significant).
- Higher (1.61 and 1.49x) mean total bilirubin (TBIL) concentration at 300 and 750 mg/kg/day, respectively, at the end of the Treatment Period. At the end of the Recovery Period, mean values at 750 mg/kg/day were considered similar to control levels.
- Higher (3.79 and 2.52x) mean bile acid (BILEAC) concentration at 300 and 750 mg/kg/day, respectively (not statistically significant at 750 mg/kg/day), at the end of the Treatment Period, which recovered (0.95x; not statistically significant) at the end of the Recovery Period.
- Lower (0.99 and 0.98x) mean chloride concentration at 300 and 750 mg/kg/day at the end of the Treatment Period. At the end of the Recovery Period, mean values at 750 mg/kg/day were considered similar to control levels.
In Main females (end of Treatment Period):
- Higher mean AST activity (1.88x) at 750 mg/kg/day and mean ALP activity (1.72, 2.01 and 1.83x) at 120, 300 and 750 mg/kg/day, respectively.
- Higher (1.33x) mean TBIL concentration at 750 mg/kg/day.
- Higher (2.21x each; not statistically significant) mean BILEAC concentration at 300 and 750 mg/kg/day.
- Lower (0.83 and 0.82x) mean creatine (CREAT) concentration at 300 and 750 mg/kg/day.
- Higher (1.61 and 1.77x) mean cholesterol (CHOL) concentration at 300 mg/kg/day (not statistically significant) and 750 mg/kg/day.
In Recovery females at 750 mg/kg/day (end of Treatment and Recovery Periods):
- Higher mean ALT (2.00x), AST (2.08x) and ALP (1.34x; not statistically significant) activities at the end of the Treatment Period. Partial recovery for mean ALT (1.58x) and AST (1.32x) activities and no recovery was noted for mean ALP (1.41x) activity at the end of the Recovery Period.
- Higher (2.33x) mean TBIL concentration at the end of the Treatment Period, which partially recovered (1.20x; not statistically significant) at the end of the Recovery Period.
- Higher (4.30x) mean BILEAC concentration at the end of the Treatment Period, which partially recovered (1.84x; not statistically significant) at the end of the Recovery Period.
- Higher (1.60x) mean CHOL concentration at the end of the Treatment Period, which partially recovered (1.28x; not statistically significant) at the end of the Recovery Period.
The changes in ALT, AST and TBIL (males), CREAT (Main females), and ALP and BILEAC (males and Main females) at 300 and 750 mg/kg/day were not dose-related.
The higher non-statistically significant AST activity in Main Females at 300 mg/kg/day at the end of the Treatment Period could be attributed to one outlier and was therefore considered not toxicologically relevant.
Any other (statistically significant) changes in hematology parameters were considered to be unrelated to treatment with the test item due the occurrence at the end of the Recovery Period alone or absence of a dose-related trend.

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

In males:
- Lower (0.77, 0.74 and 0.61x) mean total serum T4 concentration at 120, 300 and 750 mg/kg/day, respectively. Partial recovery (0.72x) at 750 mg/kg/day was noted at the end of the Recovery Period.
- Lower (0.57, 0.36 and 0.35x; not statistically significant) mean total serum TSH concentration at 120, 300 and 750 mg/kg/day, respectively, followed by recovery at the end of the Recovery Period.
In Main females at 750 mg/kg/day:
- Lower (0.89x; not statistically significant) mean total serum T4 concentration at the end of the Treatment Period.
In Recovery females at 750 mg/kg/day:
- Lower (0.61 and 0.25x) mean total serum TSH concentration at the end of the Treatment and Recovery Period, respectively.
The higher non-statistically significant mean total serum TSH concentration in males at 750 mg/kg/day at the end of the Recovery Period could be attributed to one outlier and was therefore considered not toxicologically relevant.
Mean total serum T4 and TSH concentrations in Recovery and Main females, respectively, were considered unaffected by treatment with the test item.
Any other changes in thyroid hormone levels were considered to be unrelated to treatment with the test item due to the absence of a dose-related trend or occurrence at the end of the Recovery Period alone.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

In males at 750 mg/kg/day, mean fore limb grip strength was decreased (0.88x of control) at the end of the Treatment Period. At the end of the Recovery Period, values were comparable to control.
Hind limb grip strength and motor activity (total movements and ambulations) were considered not affected by treatment with the test item.
In Main females at 750 mg/kg/day, mean total movements and ambulations were decreased (0.80 and 0.75x of control, respectively; not statistically significant) at the end of the Treatment Period.
The non-statistically significant increase in mean total movements and ambulations at 100 mg/kg/day were considered to be of no toxicological relevance in absence of a dose response relationship.
Fore and hind limb grip strength at the end of the Treatment Period were considered not affected by treatment with the test item.
In Recovery females at 750 mg/kg/day, mean total movements and ambulations were decreased (0.57 and 0.52x of control, respectively) at the end of the Treatment Period and partially recovered (0.79 and 0.80x of control, respectively) to control levels at the end of the Recovery Period.
Fore and hind limb grip strength at the end of the Treatment Period were considered not affected by treatment with the test item.
All groups showed a similar motor activity habituation profile with in general a decreasing trend in activity over the duration of the test period.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals up to 750 mg/kg/day.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant lower liver weights were recorded at the end of the Treatment Period for males at 120 mg/kg/day (relative to body weight only) and at 300 and 750 mg/kg/day (absolute and relative to body weight). At the end of the 14-day treatment-free Recovery Period, the weight difference from controls was still present, but at a lower magnitude and did not reach statistical significance, suggesting partial recovery.
Statistically significant lower weights were recorded for the adrenal gland in Main females starting at 300 mg/kg/day (absolute and relative to body weights) at the end of the Treatment Period and in Recovery females at 750 mg/kg/day (lower absolute and relative to body weights) at the end of the Recovery Period.
In males at 300 and 750 mg/kg/day, there was a non-significant, lower adrenal gland weight at the end of the Treatment Period. At the end of the 14-day treatment-free Recovery Period, the magnitude of the adrenal gland weight difference from controls in males at 750 mg/kg/day increased and reached statistical significance (absolute and relative to body weight). There was no microscopic correlate for the lower adrenal gland weights in Recovery males.
There were no other test item-related organ weight changes. Remaining statistically significant changes recorded at the end of the Treatment Period in males included lower absolute weight of thyroid glands (at 300 and 750 mg/kg/day), lower absolute weight of prostate gland and seminal vesicles (at 750 mg/kg/day) and higher brain and epididymides relative to body weight (at 300 mg/kg/day). These weight changes were regarded to be related to the lower final body weight of males at the end of the treatment period at 300 and 750 mg/kg/day. At the end of the Recovery Period, statistically significant changes included higher absolute brain weight (males at 750 mg/kg/day) and higher kidney relative to body weight (males at 750 mg/kg/day) and higher thymus relative to body weight (females at 750 mg/kg/day). These occurred only at the end of the recovery period and were regarded to be unrelated to the treatment with the test item.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related gross observations.
All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Atrophy of the adrenal cortex was observed in Main females at 750 mg/kg/day (slight degree).
Pigment (yellow-brown) of the adrenal cortex was observed at a minimal degree in Main females at 300 mg/kg/day and up to a slight degree in Main females at 750 mg/kg/day.
After a 14-day treatment-free Recovery Period, atrophy and pigment were present in Recovery females at 750 mg/kg/day at a minimal degree.

The following test item-related findings were noted in the liver of Main animals:
- Single cell necrosis/apoptosis, starting at 300 mg/kg/day in males (up to slight degree) and females (up to moderate degree).
- Hepatocellular inclusions, starting at 120 mg/kg/day in both sexes (up to slight degree).
- Increased mitosis, defined by more than one or two mitoses per 10 high power field, starting at 300 mg/kg/day in females (minimal degree) and in males (up to slight degree).
- Karyomegaly (i.e. hepatocytes with relative enlarged nuclei), starting in females at 120 mg/kg/day (up to slight degree) and in males starting at 300 mg/kg/day (minimal degree).
- Pigment (yellow-brown, hepatocellular or in Kupfer cells), in females starting at 120 mg/kg/day (minimal degree) and in males starting at 300 mg/kg/day (up to slight degree).
- Vacuolation hepatocellular, in females starting at 300 mg/kg/day (minimal).
The following test item-related liver findings were present in 750 mg/kg/day group animals after a 14-day treatment-free Recovery Period:
- Hepatocellular inclusions, in all males (up to slight) suggesting partial recovery and 2/5 females (minimal).
- Karyomegaly, in 4/5 females (up to slight).
- Pigment, in 2/5 males (minimal) suggesting partial recovery and 4/5 females (up to slight).
- Vacuolation hepatocellular, in 2/5 females (up to slight).

There were no other test item-related histologic changes. 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.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

food consumption and compound intake

histopathology: neoplastic

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Mean Percent Liver and Adrenal Gland Weight Differences from Control Groups

	Main			Recovery
Dose level (mg/kg/day):	120	300	750	750
LIVER (MALES)
Absolute	-8	-21**	-20**	-10
Relative to body weight	-6*	-12**	-13**	-7
ADRENAL GLANDS (FEMALES^a)
Absolute	-14	-28**	-36**	-28**
Relative to body weight	-11	-26**	-30**	-28**
ADRENAL GLANDS (MALES)
Absolute	3	-15	-18	-23*
Relative to body weight	5	-5	-11	-22*
*: P≤0.05, **: P≤0.01 ^a  =  Recovery females were not part of the reproduction phase of the study.

 

Summary Test Item-Related Microscopic Findings of the Adrenal gland.

	Main				Recovery
Dose level (mg/kg/day):	0	120	300	750	0	750
ADRENAL GLANDS (FEMALES)^a,b	5	5	5	5	5	5
Atrophy, cortex
Minimal	-	-	-	-	-	2
Slight	-	-	-	2	-	-
Pigment, cortex
Minimal	-	-	3	1	-	5
Slight	-	-	-	3	-	-

^a  =  Number of tissues examined from each group.
^b  =  Recovery females were not part of the reproduction phase of the study.

 

Summary Test Item-Related Liver Microscopic Findings

	Main				Recovery
Dose level (mg/kg/day):	0	120	300	750	0	750
LIVER (MALES)^a	5	5	5	5	5	5
Single cell necrosis/apoptosis
Minimal	-	-	3	4	-	-
Slight	-	-	1	1	-	-
Hepatocellular inclusions
Minimal	-	4	2	3	-	4
Slight	-	1	2	2	-	1
Increased mitosis
Minimal	-	-	2	-	-	-
Slight	-	-	1	2	-	-
Karyomegaly
Minimal	-	-	2	5	-	-
Pigment
Minimal	-	-	2	3	-	2
Slight	-	-	-	1	-	-
LIVER (FEMALES)^a,b	6	5	5	5	5	5
Single cell necrosis/apoptosis
Minimal	-	-	1	1	-	-
Slight	-	-	1	2	-	-
Moderate	-	-	-	1	-	-
Hepatocellular inclusions
Minimal	-	5	4	4	-	2
Slight	-	-	1	1	-	-
Increased mitosis
Minimal	-	-	2	1	-	-
Karyomegaly
Minimal	-	5	3	3	-	3
Slight	-	-	1	-	-	1
Pigment
Minimal	-	2	3	5	-	3
Slight	-	-	-	-	-	1
Vacuolation hepatocellular
Minimal	-	-	1	2	-	1
Slight	-	-	-	-	-	1

^a  =  Number of tissues examined from each group.
^b  =  Recovery females were not part of the reproduction phase of the study.

 

 

Conclusions:

Based on the results of the combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the parental NOAEL for the test item was established to be 120 mg/kg bw/day.

Executive summary:

The objectives of the study conducted under GLP and with a study design similar to OECD TG 422 were to determine the potential toxic effects of the test item when given orally by gavage for a minimum of 28 days to Wistar Han rats, followed by a 14-day recovery period and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition and early postnatal development.

The recovery animals (used to study the potential reversibility of possible toxic effects) were not mated.

The dose levels were selected based on the results of a 14‑day Dose Range Finder study with oral gavage administration of the test substance in rats.

The study design was as follows:

Group No.		Dose Level (mg/kg/day)	Dose Volume (mL/kg)	Dose Concentration (mg/mL)	Number of Animals
					Males	Females
1	Main Recovery	0 (Vehicle)	5	0	10 5	10 5
2	Main	120	5	24	10	10
3	Main	300	5	60	10	10
4	Main Recovery	750	5	150	10 5	10 5

 

Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity.

The following parameters and end points were evaluated in this study:
mortality/moribundity, clinical signs, functional observations, body weight and food consumption, estrous cycle, clinical pathology, measurement of thyroid hormones T4 and TSH (F₀‑males and -females), gross necropsy findings, organ weights and histopathologic examinations.

Formulation analyses confirmed that formulations of test item in water (Elix) were prepared accurately and homogenously.

One female (No. 62) was euthanized in extremis due to its poor health prior to dosing and was replaced by another female.

Parental toxicity was observed starting at 120 mg/kg/day.

In males, body weight (gain) was decreased starting at 300 mg/kg/day throughout the Treatment Period followed by a partial recovery during the treatment-free Recovery Period.
In Main females, body weight and food consumption was decreased starting at 120 and 300 mg/kg/day, respectively, throughout the Treatment and Lactation Period, respectively.
In Recovery females at 750 mg/kg/day, body weight (gain) was decreased throughout the Treatment Period followed by a full recovery during the treatment-free Recovery Period.
Due to the persistent nature and at the magnitude of these effects, these changes in body weight (in males and Main females starting at 300 mg/kg/day, and in Recovery females at 750 mg/kg/day) and food consumption (in Main females starting at 300 mg/kg/day) were considered adverse.
The decrease in body weight in Main females at 120 mg/kg/day was considered not to be adverse as changes were only minimal.

At 750 mg/kg/day, the lower fore limb grip strength in males and lower motor activity (total movements and ambulation) in Main and Recovery females was considered not to represent an adverse effect on neurobehavior. These results were not supported by other functional observation tests and had no supportive morphological correlates in examined neuronal tissues.

Hematology findings in males comprised higher hemoglobin and hematocrit concentration (both at 750 mg/kg/day), and lower reticulocyte concentration (starting at 300 mg/kg/day), in Main females of higher large unstained cell concentration (starting at 300 mg/kg/day) and in Recovery females of higher neutrophil, monocyte, basophil, red blood cell, hemoglobin and hematocrit concentration, and red blood cell distribution width gated. Except for the changes in red blood cell distribution width gated and hematocrit concentration in Recovery females, (partial) recovery was noted for all other changes at the end of the Recovery Period. These findings were considered non‑adverse since these changes were not associated with any adverse pathological alterations.

Serum level of total T4 concentration was lower in males (starting at 120 mg/kg/day) and Main females (at 750 mg/kg/day) at the end of the Treatment Period. Partial recovery was noted for males at 750 mg/kg/day at the end of the Recovery Period.
Serum levels of total TSH concentration was lower in males (starting at 120 mg/kg/day) and Recovery females at 750 mg/kg/day at the end of the Treatment Period, and fully recovered in males at 750 mg/kg/day, but not in Recovery females, at the end of the Recovery Period alone.

Test item-related findings were noted in the liver of males and Main females starting at 120 mg/kg/day and of Recovery females at 750 mg/kg/day.
At the end of the Treatment Period, a loss of hepatocytes (single cell necrosis/apoptosis) and the presence of yellow-brown pigment (intracytoplasmic in hepatocytes and Kupfer cells) were noted in males and Main females at 300 and 750 mg/kg/day. In addition, there was an increase in the number of cells (increased mitosis) which may indicate a compensatory response to cell loss. This was accompanied by enlarged nuclei (karyomegaly) with occasionally binucleated hepatocytes and hepatocellular inclusions (resembling phagocytosed condensed nuclei) and with lower organ weights in males as well. These findings are suggestive for abnormal cell divisions. After the 14-day treatment-free period in males at 750 mg/kg/day, hepatocellular inclusions and pigment were still present at a subtle lower incidence (for the pigment) and/or mean severity (for the inclusions) suggesting partial recovery.
These microscopic findings reflected changes in clinical biochemistry parameters, consisting of elevated liver enzyme activities (i.e. alanine aminotransferase in males starting at 300 mg/kg/day, aspartate aminotransferase in males starting at 300 mg/kg/day and in Main females at 750 mg/kg/day, and alkaline phosphatase in males and Main females starting at 300 mg/kg/day) and other biomarkers reflecting poor liver function, such as increased total bilirubin concentration (in males starting at 300 mg/kg/day and Main females at 750 mg/kg/day), bile acids (starting at 300 mg/kg/day in males and Main females) and cholesterol concentrations (starting at 300 mg/kg/day in Main females), and decreased creatine concentrations (starting at 300 mg/kg/day in Main females). At the end of the Recovery Period, (partial) recovery was noted for all changes.
Based on the nature of findings (increased cell death and abnormal cell division) and relationship with liver biomarkers associated with liver damage and/or poor liver function, this combination of findings was considered adverse starting at 300 mg/kg/day.
In addition, Main females at 300 and 750 mg/kg/day showed hepatocellular vacuolation at minimal degree, which was regarded non-adverse (Ref. 1 and Ref. 2).

At 120 mg/kg/day at the end of the Treatment Period, hepatocellular inclusions (males and Main females), karyomegaly and pigment (both Main females) were noted. Based on the low severity and in absence of increased mitosis or single cell necrosis, these findings were regarded non-adverse (Ref. 1 and Ref. 2).

In Recovery females at 750 mg/kg/day after the 14-day treatment-free period, microscopic findings noted were hepatocellular inclusions (minimal), pigment (up to slight), karyomegaly (up to slight) and vacuolation (up to slight). These microscopic findings reflected changes in clinical biochemistry parameters, consisting of elevated liver enzyme activities (i.e. alanine aminotransferase and aspartate aminotransferase) and other biomarkers reflecting poor liver function (i.e. total bilirubin, bile acids and cholesterol) at the end of the Treatment Period. Except for changes in alkaline phosphatase activity, partial recovery was noted for all other clinical biochemistry changes at the end of the Recovery Period.
Based on the low severity and in absence of increased hepatocyte mitosis or single cell necrosis, these findings were regarded non‑adverse (Ref. 1 and Ref. 2).

Test item-related findings were noted in the adrenal gland of Main females starting at 120 mg/kg/day and of Recovery females at 750 mg/kg/day. In Main females at the end of the Treatment Period, pigment (yellow-brown, cortical) was noted starting at 300 mg/kg/day and cortical atrophy related to lower organ weights at 750 mg/kg/day. In Recovery females after the 14‑day treatment-free period, both pigment and atrophy were noted at minimal degree. Recorded severities were minimal or slight and there was no direct evidence of cell necrosis or degeneration. Therefore, these finding were considered non-adverse.

Other clinical biochemistry findings comprised of a lower chloride concentration in males starting at 300 mg/kg/day at the end of the Treatment Period followed by full recovery during the 14-day treatment-free Recovery Period and higher alkaline phosphatase concentration in Main females at 120 mg/kg/day. These findings were considered non‑adverse since these changes were not associated with any adverse pathological alterations.

No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. mortality, functional observations (hearing ability, pupillary reflex and static righting reflex), macroscopic examination.

In this study, a reduction of total T4 serum levels were observed in males (starting at 120 mg/kg/day) and Main females (at 750 mg/kg/day) as well as a reduction of total TSH serum levels in males (starting at 120 mg/kg/day) and Recovery females (at 750 mg/kg/day). However, under the conditions of this screening study, no adverse effect was observed that could be linked to the reduction of total T4 and/or TSH, and therefore this reduction was not taken into account when determining the parental NOAEL.

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the parental No Observed Adverse Effect Level (NOAEL) for the test item established to be 120 mg/kg/day (based on the lower body weight (males and Main females) and food consumption (Main females), a subset of disrupted clinical biochemistry parameters (males and Main females), lower liver weights (males) and microscopic findings in the liver at 300 mg/kg/day (males and Main females)).

Note: In this study, a reduction of total T4 serum levels were observed in males (starting at 120 mg/kg/day) and Main females (at 750 mg/kg/day) as well as a reduction of total TSH serum levels in males (starting at 120 mg/kg/day) and Recovery females (at 750 mg/kg/day). However, under the conditions of this screening study, no adverse effect was observed that could be linked to the reduction of total T4 and/or TSH, and therefore this reduction was not taken into account when determining the parental NOAEL.

 

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. Toxicol. Pathol. 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. Toxicol. Pathol.  44(6), 810-24.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

July 2016

Deviations:

yes

Remarks:

Please refer to Principles of method.

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA Health Effects Test Guideline OPPTS 870.3650: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

July 2000

Deviations:

yes

Remarks:

Please refer to Principles of method.

Principles of method if other than guideline:

Deviation from study design:
Body weights and food consumption was inadvertently not recorded for Recovery Groups 1 (control) and 4 (750 mg/kg/day) between Days 7-14 of the Recovery Period. As (partial) recovery of body weight (gain) was noted at Day 7 of the Recovery Period, sufficient data was available for the toxicological evaluation. As food consumption was considered unaffected in all groups throughout the Treatment Period and Week 1 of the Recovery Period, sufficient data was available for the toxicological evaluation.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Propionamide

EC Number:

201-172-1

EC Name:

Propionamide

Cas Number:

79-05-0

Molecular formula:

C3H7NO

IUPAC Name:

propanamide

Test material form:

solid

Test animals

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 laboratory has general and reproduction/developmental 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 toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: not specified but either Charles River Deutschland, Sulzfeld, Germany or
Charles River Laboratories France, L'Arbresle Cedex, France.
- Females nulliparous and non-pregnant: yes
- Age at initiation of dosing: Males: 10-11 weeks; Females: 13-14 weeks
- Weight at study initiation: Males: 266 to 320 g; Females: 203 to 246 g.
- Fasting period before study: overnight before necropsy
- Housing: On arrival and following the pretest (females only) and pre-mating period + recovery males and females: group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon, MIV type, height 18 cm).
During the mating phase: Main males and females will be cohabitated on a 1:1 basis in Makrolon plastic cages (MIII type, height 18 cm).
During the post-mating phase: males in Makrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Main Females: individually housed in Makrolon plastic cages (MIII type, height 18 cm).
During the lactation phase: Main females will be housed in Makrolon plastic cages (MIII type, height 18 cm). Pups will be housed with the dam, except during locomotor activity monitoring of the dams, when the pups will be kept warm in their home cage .
During locomotor activity monitoring: F0-animals: individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cageenrichment, bedding material, food and water.
The cages will contain appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne
GmbH + CO. KG, Rosenberg, Germany).
- Diet: ad libitum except during designated procedures, pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water: ad libitum except during designated procedures, tap water
- Acclimation period: 9 days prior to start of the pretest period (females) or 8 days before the commencement of dosing (males)

DETAILS OF FOOD AND WATER QUALITY: The feed is analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis are provided by the supplier and are on file at the Test Facility. Periodic analysis of the water is performed, and results of these analyses are on file at the Test Facility.
It is considered that there are no known contaminants in the feed and water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 21
- Humidity (%): 43 to 47
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2020-12-29 To: 2021-03-31

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

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 daily and/or weekly (filled out in daily portions) as a solution. The test item was mortared and transferred to a suitable container wherein the vehicle was added. The dosing formulations was stirred for at least 30 minutes and dosed within 24 hours after adding the vehicle to the test item or stored in the refrigerator. The dosing formulations that were removed from the refrigerator were stirred at room temperature for at least 30 minutes before dosing and dosed within 24 hours after removal from the refrigerator. Test item dosing formulations will be kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. No adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.

VEHICLE
- Concentration in vehicle: 24, 60, 150 mg/mL
- Amount of vehicle: 5 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: maximum of 14 consecutive days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 post-coitum
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility if less than 9 females per group showed evidence of mating. The remating period was max. 7 days.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). No test item was detected in the Group 1 formulation.

Duration of treatment / exposure:

Males: 29 days
For main males this included at least 2 weeks of treatment prior to mating and during the mating period.

Females: at least 56 days (based on females first scheduled for necropsy that delivered pups)
Females were not dosed during littering.

Frequency of treatment:

daily

Details on study schedule:

- F1 generation was not mated based on study design of guideline.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

750 mg/kg bw/day (nominal)

Remarks:

Group 1

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Group 2

Doses / concentrations 3

Dose / conc.:

120 mg/kg bw/day (nominal)

Remarks:

Group 3

Doses / concentrations 4

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

10 males and females per dose (main groups)
5 males and females treates with 0 and 750 mg/kg bw/day (recovery group)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a 14-day dose range finder (DRF) with oral gavage administration of the test item in rats and in an attempt to produce graded responses to the test item.
During the 14-day DRF (dose levels: 500 and 1000 mg/kg /day with female rats only), no mortality was observed up to 1000 mg/kg/day. Test item-related clinical signs included hunched posture and piloerection (both starting at 500 mg/kg/day). At 500 mg/kg/day, body weight was decreased in 2/3 females between Days 1-14 (-2 % and -3 % at Day 14 compared to Day 1) and decreased in 1/3 females between Days 1-4 followed by an increase between Days 4-14 (+3 % at Day 14 compared to Day 1). At 1000 mg/kg/day, body weight was decreased in 1/3 females between Days 1-4 and remained unchanged on the days thereafter (-4 % on Day 14 compared to Day 1). Body weight was decreased in 1/3 females at 1000 mg/kg/day between Days 1-11 which recovered between Days 11-14 to the same level as Day 1. At 1000 mg/kg/day, body weight was decreased in 1/3 females between Days 1-8, which slightly increased between Days 8-14 (-8 % at Day 14 compared to Day 1). At 500 and 1000 mg/kg/day, food consumption was considered normal between Days 1-14, except between Days 8-11 where food consumption was increased. No macroscopic abnormalities were noted in 3/3 females at 500 and 1000 mg/kg/day. Liver weights were considered normal at 500 and 1000 mg/kg/day, while kidney weights were slightly increased at 500 and 1000 mg/kg/day after 14 days of treatment.
Based on the results of the 14-day DRF a dose level of 750 mg/kg/day was considered to be a suitable high-dose level for the Main study, as a moderate decrease in body weight was observed at 1000 mg/kg/day which did not sufficiently increase during the treatment period despite the normal to slightly increased food consumption. The dose levels of 300 and 120 mg/kg/day are included in order to provide information on possible dose-response relationships. The high-dose level should produce some toxic effects, but not death nor obvious suffering. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.
- Fasting period before blood sampling for clinical biochemistry: yes, overnight
- Rationale for selecting satellite groups: The recovery animals were used to study the potential reversibility of possible toxic effects.
- Post-exposure recovery period in satellite groups: 14 days

Positive control:

No positive control was used.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily throughout the study.
- Cage side observations: Animals were observed for general health/mortality and moribundity. Animals were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: During treatment, animals were observed at least once daily, up to the day prior to necropsy. These clinical observations were at least be conducted 3 hours (± 30 minutes) after dosing. Once before the first administration of the test item and at weekly intervals during the treatment and recovery period animals were observed for specific clinical signs in a standard arena.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of treatment (prior to dosing), and weekly thereafter for every study phase. Mated Main females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
Food consumption was quantitatively measured weekly, except for Main males and Main females which are housed together for mating and for Main females without evidence of mating. Food consumption of mated Main females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Water consumption was monitored by visual inspection of the water bottles. If inter group differences were noted, consumption was assessed by weight.

- General Reproduction Data
Frequency: Daily from the mating period onwards.
Procedure: Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Palpation was used to aid in confirmation of pregnancy if needed.
The females were allowed to litter. Postnatal day (PND) 1 was defined as the day when a litter was found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). The day prior to PND 1was considered to be the day when the female started to deliver and was defined as PND 0 and used for recording of delivery. Females that were littering were left undisturbed.
Cage debris of pregnant females was examined for evidence of premature delivery. Signs of difficult or prolonged parturition were recorded, if applicable.
Deficiencies in maternal care, such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding, were recorded, if applicable.

OTHER: Further examinations on parameters after repeated exposure were conducted and are presented in the respective IUCLID section.

Oestrous cyclicity (parental animals):

Frequency: Daily vaginal lavage were performed for all females (Main and Recovery) during 14 days prior to treatment (pretest period) and the first 14 days of treatment. For Main females, daily vaginal lavage continued during mating until evidence of copulation is observed.
On the day of necropsy, a vaginal lavage was taken from Main females to determine the stage of estrous. This was done for all Main females, except for females that have to be euthanized in extremis or die spontaneously.
Procedure: Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by serial vaginal lavage procedures.

Sperm parameters (parental animals):

Parameters examined in male parental generation:
testis weight, epididymis weight,
For the testes of all selected Main males of Groups 1 and 4, and all Main males that fail to sire or died before mating detailed qualitative examination will be made, taking into account the tubular stages of the spermatogenic cycle. The examination will be conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings will be noted.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD) on PND 1, pup weight on PND 1, 4, 7, and 13, presence of nipples/areolae in male pups (PND 13). Particular attention was paid to the external reproductive genitals which were examined for signs of altered development; gross evaluation of external genitalia.
Thyroid Hormone
Blood samples were collected from two of the surplus pups (if possible from one male and one female pup) on PND 4. If the target volume of 0.5 mL could not be reached by pooling from two pups, blood from a third surplus pup of the same litter was added.
On PND 14-16, separate blood samples were collected from two pups per litter (from one male and one female, if possible). If the target volume of 1.0 mL/pup could not be reached, a separate blood sample was collected from another pup of the same litter and sex (if possible). Serum for measurement of total T4 was performed for pups from PND 14-16.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death

Postmortem examinations (parental animals):

SACRIFICE
Please refer to "Any other information on materials and methods".

GROSS PATHOLOGY: Yes, please refer to "Any other information on materials and methods".

HISTOPATHOLOGY: Yes, please refer to "Any other information on materials and methods".

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring was sacrificed at PND 14-16.
- These animals were subjected to postmortem examinations (macroscopic)

GROSS NECROPSY
- Gross necropsy consisted of external and internal (only for sex) examinations. External abnormalities were collected and fixed in 10 % buffered formalin.

HISTOPATHOLOGY / ORGAN WEIGTHS
The thyroid was collected from two pups per litter (if possible, from one male and one female pup and preferably from the same pups as selected for (complete) blood collection), and was preserved in 10 % buffered formalin.

Statistics:

Please refer to "Any other information on materials and methods".

Reproductive indices:

Mating index (%): (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 (%): (Number of pregnant females/Number of females mated) x 100
Gestation index (%): (Number of females with living pups on Day 1/Number of pregnant females) 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
Post-implantation survival index was expressed as 100 % when the number of offspring exceeds the number of implantation sites recorded.

Offspring viability indices:

Live birth index (%): (Number of live offspring on Day 1 after littering/Total 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
Viability index (%): (Number of live offspring on Day 4 before culling/Number live offspring on Day 1 after littering) x 100
Lactation index (%): (Number of live offspring on Day 13 after littering/Number live offspring on Day 4 (after culling)) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant clinical signs were noted up to 750 mg/kg/day.
Hunched posture was noted in 1/10, 2/10 and 1/15 females at 120, 300 and 750 mg/kg/day, respectively, on one day in Week 4 of treatment, up to three days in Week 1 and two days in Week 1, respectively, as well as in 1/10 males at 300 mg/kg/day on one day in Week 5 of treatment. This sign was not observed during the Recovery Period. Given the low incidence and in absence of a dose-related trend, this sign was considered of no toxicological relevance.
Lean appearance was recorded once in 1/10 females at 300 mg/kg/day on in Week 1 of treatment. Given the low incidence and in absence of a dose-related trend, this sign was considered of no toxicological relevance.
Incidental findings that were noted included chromodacryorrhea, alopecia, piloerection, scales, scabs and red discoloration of both forelegs. These findings occurred only during the Recovery Period and/or 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 incidence observed, these were considered signs of no toxicological relevance.
No findings were noted during the weekly arena observations in this study.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights and body weight gain were considered to be affected by treatment with the test item in males starting at 300 mg/kg/day and in females at 120 mg/kg/day.
In males at 300 and 750 mg/kg/day, mean body weight and mean body weight gain were decreased throughout the Treatment Period, followed by a partial recovery at 750 mg/kg/day during the 14 days treatment-free Recovery Period. Mean body weights were 8 and 9 % lower compared to the concurrent control group at 300 and 750 mg/kg/day, respectively, at the end of the Treatment Period and 4 % lower compared to the concurrent control group at 750 mg/kg/day at the end of the Recovery Period.
In Main females starting at 120 mg/kg/day, mean body weight was decreased (not always reaching statistical significance) throughout the Treatment Period, except at 120 mg/kg/day from Lactation Day 4 onwards which was similar to the concurrent control group. At the end of the Lactation Period, mean body weights were 8 and 9 % lower compared to the concurrent control group at 300 and 750 mg/kg/day, respectively.
Mean body weight gain at 750 mg/kg/day was decreased up to and including the Post-coitum Period.
In Recovery females at 750 mg/kg/day, mean body weight and mean body weight gain were decreased throughout the Treatment Period (not always reaching statistical significance), followed by full recovery from Day 7 of the Recovery Period onwards. At the end of the Treatment Period, mean body weight was 5 % lower compared to the concurrent control group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption before or after correction for body weight was considered affected by treatment with the test item in Main females starting at 300 mg/kg/day.
In Main females at 300 and 750 mg/kg/day, mean absolute and relative food consumption were decreased throughout the Lactation Period (not always reaching statistical significance). During the last interval of the Lactation Period at 300 and 750 mg/kg/day, mean absolute food consumption was 15 and 20 % lower compared to the concurrent control group, respectively, and mean relative food consumption was 5 and 13% lower compared to the concurrent control group, respectively.
In males and Recovery females, food consumption was considered to be unaffected by treatment with the test item.

Food efficiency:

not examined

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

not specified

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Pupillary reflex reflex was normal in all examined animals up to 750 mg/kg/day.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematology parameters were affected by treatment with the test item in males and females starting at 300 mg/kg/day.
The following changes distinguished treated from control animals. The differences were statistically significant unless indicated otherwise. Relative changes in mean values as compared to the concurrent control group are indicated between parentheses.
In males (end of Treatment and Recovery Periods):
- Lower (0.87 and 0.86x; not statistically significant) mean reticulocyte (RETIC) concentration at 300 and 750 mg/kg/day at the end of the Treatment Period. A partial recovery (0.90x; not statistically significant) was noted at the end of the Recovery Period.
- Higher (1.07x) mean hemoglobin (HGB) concentration at 750 mg/kg/day at the end of the Treatment Period, which recovered to levels similar to the control at the end of the Recovery Period.
- Higher (1.06x) mean hematocrit (HCT) at 750 mg/kg/day at the end of the Treatment Period, which partially recovered (1.03x; not statistically significant) at the end of the Recovery Period.
In Main females (end of Treatment Period):
- Higher (2.67 and 3.00x) mean large unstained cell (LUC) concentration at 300 and 750 mg/kg/day, respectively.
In Recovery females at 750 mg/kg/day (end of Treatment and Recovery Periods):
- Higher (1.79x; not statistically significant) mean neutrophil (NEUT) concentration at the end of the Treatment Period. Partial recovery (1.35x; not statistically significant) was noted at the end of the Recovery Period.
- Higher (1.96x) mean monocyte (MONO) concentration at the end of the Treatment Period. Partial recovery (1.45x; not statistically significant) was noted at the end of the Recovery Period.
- Higher (4.00x; not statistically significant) mean basophil (BASO) at the end of the Treatment Period, which recovered to levels similar to the control at the end of the Recovery Period.
- Higher (1.06x) mean red blood cell (RBC) concentration at the end of the Treatment Period, which recovered to levels similar to the control at the end of the Recovery Period.
- Higher (1.08x each) mean red blood cell distribution width gated (RDWG) at the end of the Treatment and Recovery Period.
- Higher (1.06x) mean HGB concentration at the end of the Treatment Period. Partial recovery (1.03x) was noted at the end of the Recovery Period.
- Higher (1.05 and 1.04x) mean HCT at the end of the Treatment and Recovery Period, respectively.
The higher non-statistically significant LUC concentration in Recovery Females at 750 mg/kg/day at the end of the Treatment Period could be attributed to one outlier and was therefore considered not toxicologically relevant.
Any other (statistically significant) changes in hematology parameters were considered to be unrelated to treatment with the test item due to the absence of a dose-related trend or the occurrence at the end of the Recovery Period alone.

Coagulation parameters of treated rats were considered not to have been affected by treatment with the test item.
In the absence of a dose-related trend, the lower non-statistically significant mean activated partial thromboplastin time (APTT) at 120 and 300 mg/kg/day was considered not to be related to treatment with the test item.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Clinical biochemistry parameters were affected by treatment with the test item in males starting at 300 mg/kg/day and in females starting at 120 mg/kg/day.
The following changes distinguished treated from control animals. The differences were statistically significant unless indicated otherwise. Relative changes in mean values as compared to the concurrent control group are indicated between parentheses.
In males (end of Treatment and Recovery Periods):
- Higher mean alanine aminotransferase (ALT; 2.75 and 1.79x), aspartate aminotransferase (AST; 2.30 and 1.73x) and alkaline phosphatase (ALP; 1.63 and 1.45x) activities at 300 and 750 mg/kg/day, respectively, at the end of the Treatment Period.
At 750 mg/kg/day, partial recovery in ALT (1.08x), AST (1.14x) and ALP (1.11x) was noted at the end of the Recovery Period (all not statistically significant).
- Higher (1.61 and 1.49x) mean total bilirubin (TBIL) concentration at 300 and 750 mg/kg/day, respectively, at the end of the Treatment Period. At the end of the Recovery Period, mean values at 750 mg/kg/day were considered similar to control levels.
- Higher (3.79 and 2.52x) mean bile acid (BILEAC) concentration at 300 and 750 mg/kg/day, respectively (not statistically significant at 750 mg/kg/day), at the end of the Treatment Period, which recovered (0.95x; not statistically significant) at the end of the Recovery Period.
- Lower (0.99 and 0.98x) mean chloride concentration at 300 and 750 mg/kg/day at the end of the Treatment Period. At the end of the Recovery Period, mean values at 750 mg/kg/day were considered similar to control levels.
In Main females (end of Treatment Period):
- Higher mean AST activity (1.88x) at 750 mg/kg/day and mean ALP activity (1.72, 2.01 and 1.83x) at 120, 300 and 750 mg/kg/day, respectively.
- Higher (1.33x) mean TBIL concentration at 750 mg/kg/day.
- Higher (2.21x each; not statistically significant) mean BILEAC concentration at 300 and 750 mg/kg/day.
- Lower (0.83 and 0.82x) mean creatine (CREAT) concentration at 300 and 750 mg/kg/day.
- Higher (1.61 and 1.77x) mean cholesterol (CHOL) concentration at 300 mg/kg/day (not statistically significant) and 750 mg/kg/day.
In Recovery females at 750 mg/kg/day (end of Treatment and Recovery Periods):
- Higher mean ALT (2.00x), AST (2.08x) and ALP (1.34x; not statistically significant) activities at the end of the Treatment Period. Partial recovery for mean ALT (1.58x) and AST (1.32x) activities and no recovery was noted for mean ALP (1.41x) activity at the end of the Recovery Period.
- Higher (2.33x) mean TBIL concentration at the end of the Treatment Period, which partially recovered (1.20x; not statistically significant) at the end of the Recovery Period.
- Higher (4.30x) mean BILEAC concentration at the end of the Treatment Period, which partially recovered (1.84x; not statistically significant) at the end of the Recovery Period.
- Higher (1.60x) mean CHOL concentration at the end of the Treatment Period, which partially recovered (1.28x; not statistically significant) at the end of the Recovery Period.
The changes in ALT, AST and TBIL (males), CREAT (Main females), and ALP and BILEAC (males and Main females) at 300 and 750 mg/kg/day were not dose-related.
The higher non-statistically significant AST activity in Main Females at 300 mg/kg/day at the end of the Treatment Period could be attributed to one outlier and was therefore considered not toxicologically relevant.
Any other (statistically significant) changes in hematology parameters were considered to be unrelated to treatment with the test item due the occurrence at the end of the Recovery Period alone or absence of a dose-related trend.

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

In males:
- Lower (0.77, 0.74 and 0.61x) mean total serum T4 concentration at 120, 300 and 750 mg/kg/day, respectively. Partial recovery (0.72x) at 750 mg/kg/day was noted at the end of the Recovery Period.
- Lower (0.57, 0.36 and 0.35x; not statistically significant) mean total serum TSH concentration at 120, 300 and 750 mg/kg/day, respectively, followed by recovery at the end of the Recovery Period.
In Main females at 750 mg/kg/day:
- Lower (0.89x; not statistically significant) mean total serum T4 concentration at the end of the Treatment Period.
In Recovery females at 750 mg/kg/day:
- Lower (0.61 and 0.25x) mean total serum TSH concentration at the end of the Treatment and Recovery Period, respectively.
The higher non-statistically significant mean total serum TSH concentration in males at 750 mg/kg/day at the end of the Recovery Period could be attributed to one outlier and was therefore considered not toxicologically relevant.
Mean total serum T4 and TSH concentrations in Recovery and Main females, respectively, were considered unaffected by treatment with the test item.
Any other changes in thyroid hormone levels were considered to be unrelated to treatment with the test item due to the absence of a dose-related trend or occurrence at the end of the Recovery Period alone.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

In males at 750 mg/kg/day, mean fore limb grip strength was decreased (0.88x of control) at the end of the Treatment Period. At the end of the Recovery Period, values were comparable to control.
Hind limb grip strength and motor activity (total movements and ambulations) were considered not affected by treatment with the test item.
In Main females at 750 mg/kg/day, mean total movements and ambulations were decreased (0.80 and 0.75x of control, respectively; not statistically significant) at the end of the Treatment Period.
The non-statistically significant increase in mean total movements and ambulations at 100 mg/kg/day were considered to be of no toxicological relevance in absence of a dose response relationship.
Fore and hind limb grip strength at the end of the Treatment Period were considered not affected by treatment with the test item.
In Recovery females at 750 mg/kg/day, mean total movements and ambulations were decreased (0.57 and 0.52x of control, respectively) at the end of the Treatment Period and partially recovered (0.79 and 0.80x of control, respectively) to control levels at the end of the Recovery Period.
Fore and hind limb grip strength at the end of the Treatment Period were considered not affected by treatment with the test item.
All groups showed a similar motor activity habituation profile with in general a decreasing trend in activity over the duration of the test period.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals up to 750 mg/kg/day.

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):

Atrophy of the adrenal cortex was observed in Main females at 750 mg/kg/day (slight degree).
Pigment (yellow-brown) of the adrenal cortex was observed at a minimal degree in Main females at 300 mg/kg/day and up to a slight degree in Main females at 750 mg/kg/day.
After a 14-day treatment-free Recovery Period, atrophy and pigment were present in Recovery females at 750 mg/kg/day at a minimal degree.

The following test item-related findings were noted in the liver of Main animals:
- Single cell necrosis/apoptosis, starting at 300 mg/kg/day in males (up to slight degree) and females (up to moderate degree).
- Hepatocellular inclusions, starting at 120 mg/kg/day in both sexes (up to slight degree).
- Increased mitosis, defined by more than one or two mitoses per 10 high power field, starting at 300 mg/kg/day in females (minimal degree) and in males (up to slight degree).
- Karyomegaly (i.e. hepatocytes with relative enlarged nuclei), starting in females at 120 mg/kg/day (up to slight degree) and in males starting at 300 mg/kg/day (minimal degree).
- Pigment (yellow-brown, hepatocellular or in Kupfer cells), in females starting at 120 mg/kg/day (minimal degree) and in males starting at 300 mg/kg/day (up to slight degree).
- Vacuolation hepatocellular, in females starting at 300 mg/kg/day (minimal).
The following test item-related liver findings were present in 750 mg/kg/day group animals after a 14-day treatment-free Recovery Period:
- Hepatocellular inclusions, in all males (up to slight) suggesting partial recovery and 2/5 females (minimal).
- Karyomegaly, in 4/5 females (up to slight).
- Pigment, in 2/5 males (minimal) suggesting partial recovery and 4/5 females (up to slight).
- Vacuolation hepatocellular, in 2/5 females (up to slight).

There were no other test item-related histologic changes. 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.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

Length and regularity of the estrous cycle were considered affected by treatment with the test item starting at 300 mg/kg/day. Most females had regular cycles of 4 to 5 days.
Disrupted cycles were noted during the Premating Period at 300 mg/kg/day in Main Female Nos. 77, 79 (irregular), Nos. 81, 83 and 84 (inconclusive cycle) and No. 76 (acyclic), and at 750 mg/kg/day in Main Female Nos. 91, 92 and 95 (inconclusive cycle) and Recovery Female No. 97 (irregular). Except for Female No. 79 which had a shortened estrous cycle (3 days), the length and regularity of the estrous cycle of all other aforementioned females were disrupted as these had an extended di-estrous period from Days 4, 5 or 6 for 6-11 days during the Treatment Period. All aforementioned Main females had littered normally.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no morphological findings in the reproductive organs of males which could be attributed to the test item and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

There were 1/10 couples (Male No. 03 and Female No. 53) of the control group that did not mate and 1/10 couples (Male No. 22 and Female No. 72) of the 120 mg/kg/day treated group that did not have offspring and 1/10 females (No. 69) of the 120 mg/kg/day group with implantation sites only.
There were no abnormalities seen in the reproductive organs, which could account for their lack of offspring.
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.


Mating Index
Mating index was not affected by treatment with the test item.
The mating indices were 90, 100, 100 and 100% for the control, 120, 300 and 750 mg/kg/day groups, respectively.
Female No. 53 (control) was not successfully mated. As this occurred in the control group alone, the unsuccessful mating of this females was considered not related to treatment with the test item.


Precoital Time
Precoital time was considered to be affected by treatment with the test item starting at 300 mg/kg/day.
All paired females showed evidence of mating within 4 days, except for Female Nos. 78 (300 mg/kg/day), 93 and 95 (750 mg/kg/day) for which mating took 12, 14 and 14 days, respectively. The longer precoital times were considered a consequence of the extended di estrous period during the mating period.


Number of Implantation Sites
Number of implantation sites was considered not to be affected by treatment with the test item.
The mean number of implantations sites were 14.2, 11.2, 11.7 and 11.8 for the control, 120, 300 and 750 mg/kg/day groups, respectively.
The slightly lower mean number of implantation sites in the test item-treated groups was considered to have arisen as a result of a slightly high mean control value as well as due to a low number of implantation sites of one animal in each group which were within normal limits and without a dose-response relationship. Therefore, the slightly lower mean number of implantation sites in the test item-treated groups was considered not to be related to treatment with the test item.


Fertility Index
Fertility index was considered not affected by treatment with the test item.
The fertility indices were 100, 90, 100 and 100% for the control, 120, 300 and 750 mg/kg/day groups, respectively.
One female (No. 72) at 120 mg/kg/day was not pregnant. In the absence of a dose-related incidence of non-pregnancy, this was considered not to be related to treatment with the test item.


Gestation Index and Duration
Gestation index and duration of gestation were considered not to be affected by treatment with the test item.
The gestation indices were 100, 89, 100 and 100 % for the control, 120, 300 and 750 mg/kg/day groups, respectively.
The failed pregnancy of Female No. 69 (120 mg/kg/day; which had only one implantation site), without related histopathology changes in reproductive organs, was judged to be unrelated to treatment with the test item due to the incidental occurrence and lack of a dose related trend.

Parturition/Maternal Care
No signs of difficult or prolonged parturition were noted among the pregnant 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 Index
The total number of offspring born compared to the total number of uterine implantations was considered to be affected by treatment with the test item at 750 mg/kg/day.
Post-implantation survival index was 97, 95, 97 and 81 % for the control, 120, 300 and 750 mg/kg/day groups, respectively.


Litter Size
Litter size was considered affected by treatment with the test item at 750 mg/kg/day.
Live litter sizes were 13.4, 12.0, 11.2 and 9.5 living pups/litter for the control, 120, 300 and 750 mg/kg/day groups, respectively.
The slightly smaller litter size at 300 mg/kg/day was considered to have arisen as a result of a slightly high mean control value and was therefore considered not to be related to treatment with the test item.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

2

Key result

Dose descriptor:

NOAEL

Remarks:

parental

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

clinical biochemistry

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

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

System:

other: disrupted estrous cycle and possible longer precoital time

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment with the test item up to 750 mg/kg/day.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Viability Index
The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was considered not affected by treatment with the test item.
Viability indices were 100, 100, 96 and 96 % for the control, 120, 300 and 750 mg/kg/day groups, respectively.
Four pups (one each in Litter Nos. 76, 77, 80 and 83) at 300 mg/kg/day and four pups (one each in Litter Nos. 86 and 93, and two in Litter No. 92) at 750 mg/kg/day were found dead or missing on PND 2, 3 or 4. Missing pups were most likely cannibalized. On days prior to its death, no or little milk was noted in the stomach of the pup from Litter No. 83. At necropsy, the left testis was missing of the pup from Litter No. 76 and head scabs were seen on the pup from Litter No. 77. No toxicological relevance was attributed to these dead or missing pups since the mortality incidence remained within the range considered normal for pups of this age.


Lactation Index
The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was considered not to be affected by treatment with the test item.
The lactation indices were 100, 100, 100 and 92 % for the control, 120, 300 and 750 mg/kg/day groups, respectively.
The lower lactation index at 750 mg/kg/day compared to control could be attributed to the high loss of pups in 1/10 litters. Six pups from Litter No. 86 (750 mg/kg/day) went missing on PND 5, 6, 8 or 9 which were most likely cannibalized. One pup displayed hypotonia, lethargy and loss of righting reflex the day prior to the day it went missing. Given the low incidence (pups from only 1/10 litters) at 750 mg/kg/day were affected, no toxicological relevance was attributed to these missing pups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights of pups were considered to be affected by treatment with the test item starting at 300 mg/kg/day.
Mean pup body weights were decreased at 300 and 750 mg/kg/day throughout the Lactation Period (not always statistically significant). At the end of the Lactation Period, mean pup body weights in males, females or combined sexes at 300 mg/kg/day were each 14 % lower than control, and at 750 mg/kg/day, 22, 20 and 21 % lower than control, respectively.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Serum total T4 levels in male and female PND 14-16 pups were considered not to be affected by treatment with the test item.

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

Sex ratio was considered not to be affected by treatment with the test item.

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

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.
The slightly larger (normalized) anogenital distance of male pups is most likely a consequence of the lower pup body weight at PND 1. In addition, a larger anogenital distance in male pups was not considered toxicologically relevant.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Treatment with the test item up to 750 mg/kg/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:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to treatment with the test item.
The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered not to be related to treatment with the test item.

Histopathological findings:

not examined

Description (incidence and severity):

Live Birth Index
The number of live offspring on Day 1 after littering compared to the total number of offspring born was not affected by treatment with the test item.
The live birth indices were 98, 100, 99 and 100 % for the control, 120, 300 and 750 mg/kg/day groups, respectively.
Three pups (Litter No. 58) in the control group and one pup (Litter No. 85) at 300 mg/kg/day were found dead at the first litter check. At necropsy, no milk in the stomach was noted for two pups from Litter No. 58. In addition, only the skeleton was available of the pup from Litter No. 85 due to cannibalism.
No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to other toxic effects:

not specified

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

Mean Percent Liver and Adrenal Gland Weight Differences from Control Groups

	Main			Recovery
Dose level (mg/kg/day):	120	300	750	750
LIVER (MALES)
Absolute	-8	-21**	-20**	-10
Relative to body weight	-6*	-12**	-13**	-7
ADRENAL GLANDS (FEMALES^a)
Absolute	-14	-28**	-36**	-28**
Relative to body weight	-11	-26**	-30**	-28**
ADRENAL GLANDS (MALES)
Absolute	3	-15	-18	-23*
Relative to body weight	5	-5	-11	-22*
*: P≤0.05, **: P≤0.01 ^a  =  Recovery females were not part of the reproduction phase of the study.

 

Summary Test Item-Related Microscopic Findings of the Adrenal gland.

	Main				Recovery
Dose level (mg/kg/day):	0	120	300	750	0	750
ADRENAL GLANDS (FEMALES)^a,b	5	5	5	5	5	5
Atrophy, cortex
Minimal	-	-	-	-	-	2
Slight	-	-	-	2	-	-
Pigment, cortex
Minimal	-	-	3	1	-	5
Slight	-	-	-	3	-	-

^a  =  Number of tissues examined from each group.
^b  =  Recovery females were not part of the reproduction phase of the study.

 

Summary Test Item-Related Liver Microscopic Findings

	Main				Recovery
Dose level (mg/kg/day):	0	120	300	750	0	750
LIVER (MALES)^a	5	5	5	5	5	5
Single cell necrosis/apoptosis
Minimal	-	-	3	4	-	-
Slight	-	-	1	1	-	-
Hepatocellular inclusions
Minimal	-	4	2	3	-	4
Slight	-	1	2	2	-	1
Increased mitosis
Minimal	-	-	2	-	-	-
Slight	-	-	1	2	-	-
Karyomegaly
Minimal	-	-	2	5	-	-
Pigment
Minimal	-	-	2	3	-	2
Slight	-	-	-	1	-	-
LIVER (FEMALES)^a,b	6	5	5	5	5	5
Single cell necrosis/apoptosis
Minimal	-	-	1	1	-	-
Slight	-	-	1	2	-	-
Moderate	-	-	-	1	-	-
Hepatocellular inclusions
Minimal	-	5	4	4	-	2
Slight	-	-	1	1	-	-
Increased mitosis
Minimal	-	-	2	1	-	-
Karyomegaly
Minimal	-	5	3	3	-	3
Slight	-	-	1	-	-	1
Pigment
Minimal	-	2	3	5	-	3
Slight	-	-	-	-	-	1
Vacuolation hepatocellular
Minimal	-	-	1	2	-	1
Slight	-	-	-	-	-	1

^a  =  Number of tissues examined from each group.
^b  =  Recovery females were not part of the reproduction phase of the study.

 

 

Applicant's summary and conclusion

Conclusions:

Based on the results of the combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the parental NOAEL for the test item was established to be 120 mg/kg bw/day. The reproduction NOAEL was determined to be 120 mg/kg bw/day and the developmental NOAEL was 120 mg/kg bw/day.

Executive summary:

The objectives of the study conducted under GLP and with a study design similar to OECD TG 422 were to determine the potential toxic effects of the test item when given orally by gavage for a minimum of 28 days to Wistar Han rats, followed by a 14-day recovery period and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition and early postnatal development.

The recovery animals (used to study the potential reversibility of possible toxic effects) were not mated and consequently were not used for the assessment of reproduction/developmental toxicity.

The dose levels were selected based on the results of a 14‑day Dose Range Finder study with oral gavage administration of the test substance in rats.

The study design was as follows:

Group No.		Dose Level (mg/kg/day)	Dose Volume (mL/kg)	Dose Concentration (mg/mL)	Number of Animals
					Males	Females
1	Main Recovery	0 (Vehicle)	5	0	10 5	10 5
2	Main	120	5	24	10	10
3	Main	300	5	60	10	10
4	Main Recovery	750	5	150	10 5	10 5

 

Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity.

The following parameters and end points were evaluated in this study:
mortality/moribundity, clinical signs, functional observations, body weight and food consumption, estrous cycle, clinical pathology, measurement of thyroid hormones T4 and TSH (F0‑males and -females), gross necropsy findings, organ weights and histopathologic examinations.

In addition, the following reproduction/developmental parameters were determined:
mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4 (PND 4 14-16 pups)).

Formulation analyses confirmed that formulations of test item in water (Elix) were prepared accurately and homogenously.

One female (No. 62) was euthanized in extremis due to its poor health prior to dosing and was replaced by another female.

Parental toxicity was observed starting at 120 mg/kg/day.

In males, body weight (gain) was decreased starting at 300 mg/kg/day throughout the Treatment Period followed by a partial recovery during the treatment-free Recovery Period.
In Main females, body weight and food consumption was decreased starting at 120 and 300 mg/kg/day, respectively, throughout the Treatment and Lactation Period, respectively.
In Recovery females at 750 mg/kg/day, body weight (gain) was decreased throughout the Treatment Period followed by a full recovery during the treatment-free Recovery Period.
Due to the persistent nature and at the magnitude of these effects, these changes in body weight (in males and Main females starting at 300 mg/kg/day, and in Recovery females at 750 mg/kg/day) and food consumption (in Main females starting at 300 mg/kg/day) were considered adverse.
The decrease in body weight in Main females at 120 mg/kg/day was considered not to be adverse as changes were only minimal.

At 750 mg/kg/day, the lower fore limb grip strength in males and lower motor activity (total movements and ambulation) in Main and Recovery females was considered not to represent an adverse effect on neurobehavior. These results were not supported by other functional observation tests and had no supportive morphological correlates in examined neuronal tissues.

Hematology findings in males comprised higher hemoglobin and hematocrit concentration (both at 750 mg/kg/day), and lower reticulocyte concentration (starting at 300 mg/kg/day), in Main females of higher large unstained cell concentration (starting at 300 mg/kg/day) and in Recovery females of higher neutrophil, monocyte, basophil, red blood cell, hemoglobin and hematocrit concentration, and red blood cell distribution width gated. Except for the changes in red blood cell distribution width gated and hematocrit concentration in Recovery females, (partial) recovery was noted for all other changes at the end of the Recovery Period. These findings were considered non‑adverse since these changes were not associated with any adverse pathological alterations.

Serum level of total T4 concentration was lower in males (starting at 120 mg/kg/day) and Main females (at 750 mg/kg/day) at the end of the Treatment Period. Partial recovery was noted for males at 750 mg/kg/day at the end of the Recovery Period.
Serum levels of total TSH concentration was lower in males (starting at 120 mg/kg/day) and Recovery females at 750 mg/kg/day at the end of the Treatment Period, and fully recovered in males at 750 mg/kg/day, but not in Recovery females, at the end of the Recovery Period alone.

Test item-related findings were noted in the liver of males and Main females starting at 120 mg/kg/day and of Recovery females at 750 mg/kg/day.
At the end of the Treatment Period, a loss of hepatocytes (single cell necrosis/apoptosis) and the presence of yellow-brown pigment (intracytoplasmic in hepatocytes and Kupfer cells) were noted in males and Main females at 300 and 750 mg/kg/day. In addition, there was an increase in the number of cells (increased mitosis) which may indicate a compensatory response to cell loss. This was accompanied by enlarged nuclei (karyomegaly) with occasionally binucleated hepatocytes and hepatocellular inclusions (resembling phagocytosed condensed nuclei) and with lower organ weights in males as well. These findings are suggestive for abnormal cell divisions. After the 14-day treatment-free period in males at 750 mg/kg/day, hepatocellular inclusions and pigment were still present at a subtle lower incidence (for the pigment) and/or mean severity (for the inclusions) suggesting partial recovery.
These microscopic findings reflected changes in clinical biochemistry parameters, consisting of elevated liver enzyme activities (i.e. alanine aminotransferase in males starting at 300 mg/kg/day, aspartate aminotransferase in males starting at 300 mg/kg/day and in Main females at 750 mg/kg/day, and alkaline phosphatase in males and Main females starting at 300 mg/kg/day) and other biomarkers reflecting poor liver function, such as increased total bilirubin concentration (in males starting at 300 mg/kg/day and Main females at 750 mg/kg/day), bile acids (starting at 300 mg/kg/day in males and Main females) and cholesterol concentrations (starting at 300 mg/kg/day in Main females), and decreased creatine concentrations (starting at 300 mg/kg/day in Main females). At the end of the Recovery Period, (partial) recovery was noted for all changes.
Based on the nature of findings (increased cell death and abnormal cell division) and relationship with liver biomarkers associated with liver damage and/or poor liver function, this combination of findings was considered adverse starting at 300 mg/kg/day.
In addition, Main females at 300 and 750 mg/kg/day showed hepatocellular vacuolation at minimal degree, which was regarded non-adverse (Ref. 1 and Ref. 2).

At 120 mg/kg/day at the end of the Treatment Period, hepatocellular inclusions (males and Main females), karyomegaly and pigment (both Main females) were noted. Based on the low severity and in absence of increased mitosis or single cell necrosis, these findings were regarded non-adverse (Ref. 1 and Ref. 2).

In Recovery females at 750 mg/kg/day after the 14-day treatment-free period, microscopic findings noted were hepatocellular inclusions (minimal), pigment (up to slight), karyomegaly (up to slight) and vacuolation (up to slight). These microscopic findings reflected changes in clinical biochemistry parameters, consisting of elevated liver enzyme activities (i.e. alanine aminotransferase and aspartate aminotransferase) and other biomarkers reflecting poor liver function (i.e. total bilirubin, bile acids and cholesterol) at the end of the Treatment Period. Except for changes in alkaline phosphatase activity, partial recovery was noted for all other clinical biochemistry changes at the end of the Recovery Period.
Based on the low severity and in absence of increased hepatocyte mitosis or single cell necrosis, these findings were regarded non‑adverse (Ref. 1 and Ref. 2).

Test item-related findings were noted in the adrenal gland of Main females starting at 120 mg/kg/day and of Recovery females at 750 mg/kg/day. In Main females at the end of the Treatment Period, pigment (yellow-brown, cortical) was noted starting at 300 mg/kg/day and cortical atrophy related to lower organ weights at 750 mg/kg/day. In Recovery females after the 14‑day treatment-free period, both pigment and atrophy were noted at minimal degree. Recorded severities were minimal or slight and there was no direct evidence of cell necrosis or degeneration. Therefore, these finding were considered non-adverse.

Other clinical biochemistry findings comprised of a lower chloride concentration in males starting at 300 mg/kg/day at the end of the Treatment Period followed by full recovery during the 14-day treatment-free Recovery Period and higher alkaline phosphatase concentration in Main females at 120 mg/kg/day. These findings were considered non‑adverse since these changes were not associated with any adverse pathological alterations.

No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. mortality, functional observations (hearing ability, pupillary reflex and static righting reflex), macroscopic examination.

Reproductive toxicity was observed starting at 300 mg/kg/day.

Estrous cycle was disrupted (mostly an extended di-estrous period) in 5/10 females and 4/15 females at 300 and 750 mg/kg/day, respectively. In addition, precoital time was increased in 1/10 females and 2/10 females at 300 and 750 mg/kg/day, respectively. Due to the high incidence and possible effect on precoital time, the disrupted estrous cycle starting at 300 mg/kg/day was considered as adverse.

Developmental toxicity was observed starting at 300 mg/kg/day.

Post-implantation index and smaller litter size were decreased at 750 mg/kg/day. In addition, the body weight of pups at 300 and 750 mg/kg/day were decreased throughout the Lactation Period. Taken together, these findings were considered adverse.

In this study, a reduction of total T4 serum levels were observed in males (starting at 120 mg/kg/day) and Main females (at 750 mg/kg/day) as well as a reduction of total TSH serum levels in males (starting at 120 mg/kg/day) and Recovery females (at 750 mg/kg/day). However, under the conditions of this screening study, no adverse effect was observed that could be linked to the reduction of total T4 and/or TSH, and therefore this reduction was not taken into account when determining the parental NOAEL.

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the parental No Observed Adverse Effect Level (NOAEL) for the test item established to be 120 mg/kg/day (based on the lower body weight (males and Main females) and food consumption (Main females), a subset of disrupted clinical biochemistry parameters (males and Main females), lower liver weights (males) and microscopic findings in the liver at 300 mg/kg/day (males and Main females)). The reproduction NOAEL was determined to be 120 mg/kg/day (based on the disrupted estrous cycle and possible longer precoital time starting at 300 mg/kg/day) and the developmental NOAEL was 120 mg/kg/day (based on the lower body weight of pups at 300 mg/kg/day).

 

Note: In this study, a reduction of total T4 serum levels were observed in males (starting at 120 mg/kg/day) and Main females (at 750 mg/kg/day) as well as a reduction of total TSH serum levels in males (starting at 120 mg/kg/day) and Recovery females (at 750 mg/kg/day). However, under the conditions of this screening study, no adverse effect was observed that could be linked to the reduction of total T4 and/or TSH, and therefore this reduction was not taken into account when determining the parental NOAEL.

 

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. Toxicol. Pathol. 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. Toxicol. Pathol.  44(6), 810-24.