Propionamide

Endpoint summary

Description of key information

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 (reference 7.5.1-1).

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

120 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study is considered of sufficient quality as it was conducted in accordance with generally accepted scientific standards under GLP.

System:

hepatobiliary

Organ:

liver

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

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  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 bw/day.

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

At 750 mg/kg bw/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 bw/day), and lower reticulocyte concentration (starting at 300 mg/kg bw/day), in Main females of higher large unstained cell concentration (starting at 300 mg/kg bw/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 bw/day) and Main females (at 750 mg/kg bw/day) at the end of the Treatment Period. Partial recovery was noted for males at 750 mg/kg bw/day at the end of the Recovery Period.
Serum levels of total TSH concentration was lower in males (starting at 120 mg/kg bw/day) and Recovery females at 750 mg/kg bw/day at the end of the Treatment Period, and fully recovered in males at 750 mg/kg bw/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 bw/day and of Recovery females at 750 mg/kg bw/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 bw/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 bw/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 bw/day, aspartate aminotransferase in males starting at 300 mg/kg bw/day and in Main females at 750 mg/kg bw/day, and alkaline phosphatase in males and Main females starting at 300 mg/kg bw/day) and other biomarkers reflecting poor liver function, such as increased total bilirubin concentration (in males starting at 300 mg/kg bw/day and Main females at 750 mg/kg bw/day), bile acids (starting at 300 mg/kg bw/day in males and Main females) and cholesterol concentrations (starting at 300 mg/kg bw/day in Main females), and decreased creatine concentrations (starting at 300 mg/kg bw/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 bw/day.
In addition, Main females at 300 and 750 mg/kg bw/day showed hepatocellular vacuolation at minimal degree, which was regarded non-adverse (Ref. 1 and Ref. 2).

At 120 mg/kg bw/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 bw/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 bw/day and of Recovery females at 750 mg/kg bw/day. In Main females at the end of the Treatment Period, pigment (yellow-brown, cortical) was noted starting at 300 mg/kg bw/day and cortical atrophy related to lower organ weights at 750 mg/kg bw/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 bw/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 bw/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 bw/day) and Main females (at 750 mg/kg bw/day) as well as a reduction of total TSH serum levels in males (starting at 120 mg/kg bw/day) and Recovery females (at 750 mg/kg bw/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 bw/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 bw/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 bw/day) and Main females (at 750 mg/kg bw/day) as well as a reduction of total TSH serum levels in males (starting at 120 mg/kg bw/day) and Recovery females (at 750 mg/kg bw/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.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes according to Regulation (EC) No 1272/2008 (CLP). Based on available data on repeated dose toxicity the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighteenth time in Regulation (EU) 2022/692.