2-ethylhexyl 2-([1,1'-biphenyl]-4-ylcarbonyl)benzoate

Endpoint summary

Description of key information

Study conducted to recognised testing guidelines with GLP certification.

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:

04 March 2021 to 09 July 2021

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Specific details on test material used for the study:

Physical Description: Colourless to pale yellow liquid (determined by Charles River Den Bosch)
Purity/Composition: 96.69%
Storage Conditions: At room temperature
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation: males were 10-11 weeks old, females were 13-14 weeks old
- Weight at study initiation: males weighed between 301 and 333g, females 200 and 244 g.
- Fasting period before study: F0-males were fasted overnight with a maximum of 24 hours before blood sampling
- Housing: polycarbonate cages (Macrolon, MIV type, height 18 cm.)
- Diet (e.g. ad libitum): Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. During motor activity measurements, animals had no access to food for a maximum of 2 hours.
- Water (e.g. ad libitum): Municipal tap water was freely available to each animal via water bottles. During motor activity measurements, animals had no access to water for a maximum of 2 hours.
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY: It is considered that there were no known contaminants at levels in the feed that would interfere with the objectives of the study. It is considered that there were no known contaminants at levels in the water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 21 °C
- Humidity (%): 47 to 73%
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

IN-LIFE DATES: From: 24 August 2020 To: 02 March 2020

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): not reported
- Mixing appropriate amounts with (Type of food): not reported
- Storage temperature of food: not reported

VEHICLE
- Justification for use and choice of vehicle (if other than water): Trial preparations were performed to select the suitable vehicle and to establish a suitable formulation procedure. These trials were not performed as part of this study and these preparations were not used for dosing. Raw data of these trials will be retained by the Test Facility.
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): not reported
- Lot/batch no. (if required):
- Purity: 96.69%

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were performed using a validated analytical procedure (Test Facility Reference No. 20221845).

Duplicate sets of samples (approximately 500 mg) were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% of target concentration.

Duration of treatment / exposure:

The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 28 days.

Frequency of treatment:

Once daily

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a 10-day Dose Range Finder with oral administration of the test item in rats.
- Rationale for animal assignment (if not random):
- Fasting period before blood sampling for clinical biochemistry: F0-males were fasted overnight with a maximum of 24 hours before blood sampling
- Rationale for selecting satellite groups: not reported
- Post-exposure recovery period in satellite groups: not reported
- Section schedule rationale (if not random):
- Other:

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Not specified
- Time schedule: not reported
- Cage side observations checked in table [No.?] were included: not reported

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

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

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

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: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: Not specified
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Not specified
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (identity) / No / Not specified
- Animals fasted: Yes / No / Not specified
- How many animals:
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Animals fasted: Yes
- How many animals: 80
- Parameters checked in table [No.?] were examined: not specified

PLASMA/SERUM HORMONES/LIPIDS: Not specified
- Time of blood sample collection:
- Animals fasted: Yes / No / Not specified
- How many animals:

URINALYSIS: No
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / Not specified
- Animals fasted: Yes / No / Not specified
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

IMMUNOLOGY: No
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.

Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.

The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Salivation seen after dosing among animals of the 100, 300 and 1000 mg/kg/day dose groups was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response rather than a sign of systemic toxicity.
Incidental findings that were noted included alopecia, chromodacryorrhea, piloerection and scabs. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered not to be signs of toxicological relevance.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The statistically significant decrease in body weight gain in males at 100 mg/kg/day in Week 3 of the mating period may be attributed to poor hydration and thus was considered to be unrelated to treatment with the test item.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption before or after correction for body weight was similar to the control level up to 1000 mg/kg/day.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

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.
• Mean alkaline phosphatase concentration was increased (1.31, 1.45 and 1.50x of control, respectively) in males at 100 (not statistically significant), 300 and 1000 mg/kg/day. The (statistically significant) increase in mean alkaline phosphatase concentration in males at all dose levels was considered a result of a relatively low mean control, and was therefore considered to be of no toxicological relevance.
• Mean potassium concentration was decreased (0.91x of control) in females at
1000 mg/kg/day.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

A test item-related enlarged liver was observed in 1/10 females treated at 300 mg/kg/day and in 2/10 females at 1000 mg/kg/day.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

An increased incidence and severity of extramedullary hematopoiesis in the spleen was recorded in 4/5 males of the 300 mg/kg/day group (2 minimal, 2 mild) and in 5/5 males of the 1000 mg/kg/day group (2 minimal, 3 mild), compared to background severities in 3/5 control group males (3 minimal) and 1/5 males at 100 mg/kg/day (minimal).

Centrilobular hepatocellular hypertrophy in the liver was recorded in 1/5 females of the 300 mg/kg/day group (minimal) and in 2/5 females of the 1000 mg/kg/day group (2 minimal).

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw (total dose)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Wistar Han rats were treated with C991 by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg/day. The rats of the control group received the vehicle, polyethylene glycol 400, alone.

Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation (for 50-55 days). Females that failed to deliver pups were treated for 40-41 days.

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

Parental results

No parental toxicity was observed up to the highest dose level tested (1000 mg/kg/day). Non-adverse test item-related morphologic alterations were present in males at 300 and 1000 mg/kg/day in the liver (increased weights), in females at 300 and 1000 mg/kg/day in the liver (macroscopically enlarged livers and centrilobular hepatocellular hypertrophy) and in males at 300 and 1000 mg/kg/day in the spleen (extramedullary hematopoiesis).

The decreased potassium concentration in females at 1000 mg/kg/day 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/moribundity, clinical appearance, functional observations (motor activity, grip strength, hearing ability, pupillary reflex and static righting reflex), body weight, food consumption, hematology, clotting parameters, male T4 thyroid hormone levels).

Reproductive results

No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg/day).

No test item-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

Developmental results

No developmental toxicity was observed up to the highest dose level tested (1000 mg/kg/day).

No test item-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care, litter size and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopic examination).

Conclusions:

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following No Observed Adverse Effect Levels (NOAELs) of C991 were established:
Parental NOAEL: at least 1000 mg/kg/day.
Reproduction NOAEL: at least 1000 mg/kg/day.
Developmental NOAEL: at least 1000 mg/kg/day.

Executive summary:

The objectives of this study were to determine the potential toxic effects of C991 when given orally by gavage for a minimum of 28 days to Wistar Han rats, and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition and early postnatal development.

In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated.

The following parameters and end points were evaluated in this study: mortality/ moribundity, clinical signs, functional observations, body weight and food consumption, estrous cycle determination, clinical pathology, measurement of thyroid hormone T4 (F0-males), 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 14-16 pups)).

Formulation analyses confirmed that formulations of test item in polyethylene glycol 400 were prepared accurately and homogenously.

No parental toxicity was observed up to 1000 mg/kg/day.

Non-adverse test item-related morphologic alterations were present in males at 300 and 1000 mg/kg/day in the liver (increased weights), in females at 300 and 1000 mg/kg/day in the liver (macroscopically enlarged livers and centrilobular hepatocellular hypertrophy) and in males at 300 and 1000 mg/kg/day in the spleen (extramedullary hematopoiesis).

The decreased potassium concentration in females at 1000 mg/kg/day were considered non-adverse since these changes were not associated with any adverse pathological alterations.

No reproductive toxicity was observed up to 1000 mg/kg/day.

No developmental toxicity was observed up to 1000 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Experimental exposure time per week (hours/week):

168

Species:

rat