Registration Dossier

Administrative data

Description of key information

The NOAEL for subchronic repeated oral dose toxicity was determined with 300 mg/kg bw/day for male rats based on slight to moderate histopathological findings in the liver and with 1000 mg/kg bw/day for female rats.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019 - 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
2018
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 1907170617
- Expiration date of the lot/batch: 03 October 2020

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability of the test substance and storage conditions: stability verified; no specific storage conditions needed
- Stability of the test substance in the vehicle: analytically confirmed
Stability for at least 6 hours at room temperature and for at least 8 days at 2-8°C is confirmed at 500 mg/mL, Test Facility Study No. 20215813.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The substance 2-Acetone, condensation product with phenol was filled at 130 °C into plastic covered Duran glass bottles. During the cooling down a vacuum was build which tightens the cap. Therefore, the bottles need to be heated up to 60°C in a cabinet heater before the cap could be loosed. Decanting was not possible at this temperature. Therefore, the bottles need to be heated up to 100-120 °C in a cabinet heating with loosed cap. Then decanting was possible in a fume cupboard with appropriate heat protection gloves.
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Crl:WI(Han) rats
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: males 137-175 g and females 113 to 146 g
- Fasting period before study: no
- Housing: up to 5 animals of the same sex and dosing group in Macrolon type IV cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days before start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): targetet 18-24 °C, actual mean 20-21°C
- Humidity (%): targetet 40-70%, actual mean 47-58%
- Air changes (per hr): at least 10 air changes/hours
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

IN-LIFE DATES: From 2019-12-17 to 2020-06-02
Route of administration:
oral: gavage
Vehicle:
other: polyethylene glycol 400, specific gravity 1.125
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

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. The vehicle was used in available repeated dose studies with the substance.
- Amount of vehicle (if gavage): 5 mL/kg bw

METHOD of FORMULATION:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. For Weeks 1 and 2 the dosing formulations were prepared daily as a solution and dosed within 6 hours after preparation of the formulation. From Week 3 onwards the dosing formulations were prepared weekly as a solution (based on available stability results), filled out in daily portions and stored in the refrigerator protected from light. The dosing formulations were removed from the refrigerator and stirred for at least 30 minutes before dosing.
Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.
Adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability measurements were conducted as part of the method development and validation study (20215812). The stability of the test item in the vehicle was assured prior to the start of the main study.
Accuracy, homogeneity and stability were determined for formulations prepared for use for use in Week 1, Week 6 and Week 13.
Duplicate samples (approximately 500 mg) were taken at middle position (50% height) or at top, middle and bottom position (90%, 50% and 10% height). The samples taken at 90%, 50% and 10% height were also used for the determination of the homogeneity of the formulations.
For determination of stability, additional samples were taken at 50% height and stored at room temperature under normal laboratory light conditions for 6 hours. For the determination of stability in a refrigerator, duplicate samples of approximately 500 mg were accurately weighed into glass vessels and stored in a refrigerator (4-8°C) for 8 days.

RESULTS:
Accuracy
The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).
A small response at the retention time of the test item was observed in the chromatograms of the Group 1 formulation prepared for use in Week 6. It was considered not to derive from the formulation since a similar response was obtained in the analytical blanks. In all other formulations of Group 1, no test item was detected.
Homogeneity
The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).
Stability
Formulations of Group 2 and Group 4 were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours and in a refrigerator (2-8°C) for at least 8 days.
Duration of treatment / exposure:
90 days
Frequency of treatment:
once daily
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected based on the result of previously performed toxicity studies in Wistar Han rats with oral gavage (data on file at Sponsor site). In a 28-day study (OECD 407), rats received 0, 100, 300 and 1000 mg/kg/day test item by daily oral gavage with polyethylene glycol (PEG400) as vehicle. There were no clinical signs or effects on body weight, food consumption, functional observations or clinical pathology up to 1000 mg/kg/day. In males at 1000 mg/kg/day, findings were noted on the liver which were considered an indication of effects on fat metabolism. Females, however, were not affected.
The NOAEL of the 28-day study was determined to be 300 mg/kg/day for the males and 1000 mg/kg/day for the females. In a teratology study (OECD 414), rats received 0, 100, 300 and 1000 mg/kg/day test item by daily oral gavage with polyethylene glycol (PEG400) as vehicle. No maternal or developmental toxicity was observed up to 1000 mg/kg/day.
- Rationale for animal assignment (if not random):
Animals were assigned to groups at random at the discretion of the biotechnician. Body weight variation did not exceed ± 20% of the sex mean.
- Fasting period before blood sampling for clinical biochemistry: yes, overnight (maximal 24 hours)
Positive control:
not adequate
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Mortality: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly; from Week 1 and throughout the study, and on the day of necropsy
- Arena observations: Once before the first administration of the test item and weekly during the Treatment Period.
Once prior to start of treatment and at weekly intervals, this was also performed outside the home cage in a standard arena (collected under Test Facility Study No. 510847 for logistic reasons and reported under Test Facility Study No. 509882). The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly; fasted weight on the day of necropsy.

FOOD CONSUMPTION: Yes
- Food consumption: weekly; quantitatively measured per cage.

WATER CONSUMPTION: Yes
Water consumption was monitored by visual inspection of the water bottles on a regular basis throughout the study.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Dose groups that were examined: all animals at pretest; groups 1 and 4 main study animals during week 13.
Since no treatment-related ophthalmologic findings were noted in Week 13, the eyes of the rats of Groups 2 and 3 were not examined.
Procedure: The eyes were examined using an ophthalmoscope after application of a mydriatic agent (Tropicol 5 mg/mL solution, THEA Pharma, Wetteren, Belgium).

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 12-13
- Dose groups that were examined: first 5 animals/sex/group
- Battery of functions tested:
- hearing ability / pupillary reflex / static righting reflex (score 0 = normal/present, score 1 = abnormal/absent)
- fore- and hind-limb grip strength were recorded as the mean of three measurements
- locomotor activity (recording period: 1 hour under normal laboratory light conditions, using a computerized monitoring system). Total movements and ambulations were reported. Ambulations represents movements characterized by a relocation of the entire body position like walking, whereas total movements represents all movements made by the animals, including ambulations but also smaller or finer movements like grooming, weaving or movements of the head

ESTROUS CYCLE DETERMINATION: Yes
On the day of necropsy, a vaginal smear was taken to determine the stage of estrus from all females.

HAEMATOLOGY: Yes
Blood samples were analyzed for the parameters given in OECD 408. Blood samples were processed for plasma, and plasma was analyzed.

CLINICAL CHEMISTRY: Yes
Blood samples were processed for plasma or serum, and analyzed for the parameters specified in OECD 408.
After measurement of thyroid hormones using the Immulite, part of the remaining volume was used for measurement of T3 using LC-MS. This measurement was performed according to the bioanalytical method validated in Test Facility Study No. 20213516.
General data concerning chemicals, reagents, preparation of stock solutions, calibration standards and quality control samples (QCs) as well as the analytical conditions were stored under Test Facility Study No. 20215813.
After receipt, the samples were stored in an ultra-low freezer (≤ -75°C) until analysis. Any remaining samples were returned to storage and discarded after for the retention period.
The analysis was based on the following guidelines:
• European Medicines Agency (EMA). Guideline on Bioanalytical Method Validation. EMEA/CHMP/EWP/192217/2009, 01 February 2012.
• Guidance for industry: Bioanalytical Method Validation, U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER) and Center for Veterinary Medicine (CVM), May 2018

URINALYSIS: No

IMMUNOLOGY: No
Sacrifice and pathology:
Animals surviving until scheduled euthanasia were weighed, and euthanized using isoflurane, followed by exsanguination. Animals were fasted (overnight with a maximum of 24 hours) before their scheduled necropsy.

GROSS PATHOLOGY: Yes
Animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.
Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available.
The organs listed in OECD 408 were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together. Organ to body weight ratio (using the terminal body weight) were calculated.

Representative samples of the tissues identified in the following table were collected from all animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands), unless otherwise indicated.

HISTOPATHOLOGY: Yes
All tissues as defined in OECD 408 were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology. Target tissues identified by the study pathologist during microscopic evaluation were communicated to the Study Director; tissues were evaluated and reported.
A peer review on the histopathology data was performed by a second pathologist.

Statistics:
Statistics for Data Collected in Provantis
All statistical analyses were performed within the respective study phase, unless otherwise noted.
-Descriptive, interferential and parametric/non-parameteric (ANOVA F-test or Kruskal-Wallis test; Dunnett's or Dunn's test) statistical analysis used.
-ANCOVA (Dunnett's test)
-Incidence (Fisher's exact test

Statistics for Data Collected/processed in Toxdata
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.
- Parametric (Dunnett-test); For the motor activity data set (at least 3 groups) parametric (ANOVA) tests on group means were applied with Bonferroni correction for multiple testing.
- Non-parametric (Steel-test)
- Incidence (overall Fisher's exact test)

Computerized systems
Critical computerized systems used in the study are listed below or presented in the appropriate Phase Report. All computerized systems used in the conduct of this study have been validated; when a particular system has not satisfied all requirements, appropriate administrative and procedural controls were implemented to assure the quality and integrity of data.

Retention and Disposition of records, samples, and specimens
All study-specific raw data, electronic data, documentation, study plan, samples, specimens, and final report were archived at the Test Facility by no later than the date of final report issue. At least two years after issue of the final report, the Sponsor will be contacted.
Electronic data generated by the Test Facility were archived as noted above, except that data collected using Provantis and Dispense, files stored on SDMS (Study Plan (amendments) and reporting files) and study deviations were archived at the Charles River Laboratories facility location in Wilmington, Massachusetts, USA.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
see attachment 1 - clinical signs:
No toxicologically-relevant clinical signs were noted during daily detailed clinical observations and no findings were noted during the arena observations in this study.
Salivation seen after dosing among all groups in a dose-related trend 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 related to dosing rather than a sign of systemic toxicity.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
see attachment 2 - body weights:
Body weights and body weight gain were considered to have been unaffected by treatment.
Any statistically significant changes in body weight gain were considered to be unrelated to treatment since no trend was apparent regarding duration of treatment.
Description (incidence and severity):
see attachment 3 - food consumption:
From start of treatment onwards, food consumption was decreased (up to -13.3% compared to control) in females treated at 1000 mg/kg/day.
Food consumption of males was similar to the control level over the study period.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No ophthalmology findings were noted that were considered to be related to treatment.
The nature and incidence of ophthalmology findings noted during the Pretreatment Period and in Week 13 was similar among the groups, and occurred within the range considered normal for rats of this age and strain. These findings were therefore considered to be unrelated to treatment with the test item.
Haematological findings:
no effects observed
Description (incidence and severity):
see attachment 4 - hematology:
Hematological parameters were considered not to have been affected by treatment.
Any statistically significant changes in hematology parameters were considered to be unrelated to treatment with the test item as these occurred in the absence of a dose-related trend.
Coagulation parameters of treated rats were considered not to have been affected by treatment with the test item.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
see attachment 5 - clinical chem summary and Table 1
For males treated at 1000 mg/kg/day, total bilirubin concentrations were decreased (0.84x of control). In addition, urea concentrations were increased (1.19x of control) and creatinine concentrations were decreased (0.89x of control) for males treated at 1000 mg/kg/day.
For females treated at 1000 mg/kg/day, total bilirubin concentrations were decreased (0.73x of control). (see Table 1)
Other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.

Thyroid hormone analysis (see Table 1)
A decreased concentration of T4 hormone was measured for males treated at 1000 mg/kg/day (0.76x of control).
A decreased concentration of TSH hormone (not statistically significant) was measured for males and females of all treatment groups. In absence of a dose-response relationship, and as mean values remained within the historical control range (see subtext Table 1) these changes were considered not toxicologically relevant.
No test item-related changes were noted for T3 hormone levels up to 1000 mg/kg/day.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
see attachment 6 - functional findings:
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength and motor activity was similar between treated and control groups.
All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
see attachment 7 - organ weights:
There were no test item-related alterations in organ weights.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
see attachment 8 - gross pathological findings:
The liver of one male treated at 1000 mg/kg/day showed prominent lobular architecture which was consistent with the hepatocellular cytoplasmic alteration observed microscopically.
All other recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see attachment 9 - histopathological findings:
Test item-related microscopic findings after treatment with the test item were noted in the liver of the 1000 mg/kg/day group males and are summarized in Table 2.
Centrilobular cytoplasmic alteration was observed in the liver of 5/10 males treated at 1000 mg/kg/day (3 minimal, 2 mild). These animals also showed centrilobular hepatocellular vacuolation at minimal degree and single cell necrosis in the centrilobular area at minimal degree. The cytoplasmic alteration was characterized by increased basophilia and fine granular cytoplasm. Several of the cells in the same area also showed hepatocellular cytoplasmic vacuoles of varying size. The livers of the females at 1000 mg/kg/day did not show test item-related histopathological changes. Based on these findings the livers of the 100 and 300 mg/kg/day treated males were also examined microscopically. Microscopic examination of these tissues revealed no histopathological changes related to treatment with the test item.
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.
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no adverse effects up to and including the limit dose
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes

Table 1: Thyroid hormone levels, total bilirubin, urea and creatinine concentrations (day 92 relative to start day)

 sex (10 animals per group)  males    
   
 females         
 dose in mg/kg bw/day   control   100   300   1000  control  100   300   1000
 T3 (ng/mL) mean  0.309  0.312  0.351  0.343  0.541  0.516  0.487  0.463
 T4 (ng/mL) mean  43.49  40.63  41.42  33.08**  35.52  30.06  31.27  30.62
 TSH (mU/L) mean #  0.2619  0.0833  0.1416  0.0605  0.1506  0.0594  0.0650  0.0746
 total bilirubin (µmol/L)  2.38  3.32  2.09  1.99*  3.1  2.75  2.83  2.20**
 Urea (mmol/L)  7.08  7.90  8.16  8.44*  7.12  7.39  7.26  7.74
 Creatinine (µmol/L)  32.6  30.8  30.1  29.1** 32.4  31.3  31.2  31.4

Anova & Dunnett: * = p <= 0.05

Anova & Dunnett: ** = p <= 0.01

# Historical Control Range TSH hormone Wistar Han rats (2019-2020)

             Males (mU/L):       mean = 0.1840; P5-P95 = 0.0352 – 0.5412 (n=182)

             Females (mU/L):       mean = 0.0887; P5-P95 = 0.0100 – 0.2685 (n=174)

A decreased concentration of T4 hormone was measured for males treated at 1000 mg/kg bw/day (0.76x of control).

A decreased concentration of TSH hormone (not statistically significant) was measured for males and females of all treatment groups.

In absence of a dose-response relationship and as mean values remained within the historical control range, these changes were considered not toxicologically relevant.

No test item-related changes were noted for T3 hormone levels up to 1000 mg/kg/day.

Table 2: Test Item-Related Microscopic Findings in the liver – Scheduled Euthanasia Animals (Day 92)

   males control  males 100 mg/kg bw  males 300 mg/kg bw  males 1000 mg/kg bw  females control  females 1000 mg/kg bw
 no. of tissues examined  10  10  10  10  10  10
 liver cytoplasmic alteration minimal  0  0  0  3  0  0
 liver cytoplasmic alteration mild  0  0  0  2  0  0
 hepatocellular vacuolation, minimal  0  0  0  5  0  0
 single cell necrosis, centrilobular minimal  0  0  0  5  0  0
 infiltration, mononuclear cell, minimal  2  5  1  2  1  2
Conclusions:
NOAEL subchronic oral toxicity study in rats: 300 mg/kg bw/day for males, 1000 mg/kg bw for females
Executive summary:

A subchronic oral gavage toxicity study in rats following OECD TG 408 was performed with the substance. Based on a low toxicity profile observed in available studies the dose levels were selected with 0, 100, 300 and 1000 mg/kg bw/day. Formulation analyses confirmed that the test item formulations in polyethylene glycol 400 were stable, accurate and homogenous.

A lower food consumption (up to -13.3% compared to controls) was noted for females treated at 1000 mg/kg/day during the study period. As there was no test item-related effect on body weight (gain) in these animals during the study period, the effect on food consumption was considered not adverse.

Furthermore, a marked decrease in T4 hormone concentration was noted for males at 1000 mg/kg/day only (minus 24%). There was no histopathological correlate in the thyroid gland. No other effects on thyroid hormones were observed.

At necropsy, prominent lobular architecture of the liver of one male at 1000 mg/kg/day was noted, which was consistent with the hepatocellular cytoplasmic alteration observed microscopically in 5/10 males at this dose level (3 minimal, 2 mild). Other microscopic changes in the liver noted for the 5 affected males were vacuolation at minimal degree and single cell necrosis in the centrilobular area at minimal degree. This combination of alterations is interpreted to represent degenerative findings and is therefore considered to be adverse. Microscopic examination of the livers of the 100 and 300 mg/kg bw/day treated males as well as the 1000 mg/kg bw/day treated females revealed no such histopathological changes.

No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. mortality, clinical appearance, body weight, functional observations, ophthalmology, hematology, clinical chemistry, coagulation and organ weights).

In conclusion, administration of the test item by once daily oral gavage was well tolerated in rats at levels up to 1000 mg/kg/day. Adverse morphologic alterations were present in the liver of males treated at 1000 mg/kg/day and consisted of centrilobular cytoplasmic alteration, hepatocellular vacuolation and single cell necrosis. Based on these results, the no-observed-adverse-effect level (NOAEL) was considered to be 300 mg/kg/day for male rats and 1000 mg/kg bw for female rats.

In this study, a marked reduction of total T4 was observed in males treated at 1000 mg/kg/day which was considered to be test item-related. However, under the conditions of this study no adverse effect was observed that could be linked to the reduction of total T4 and therefore this reduction was not taken into account when determining the parental male NOAEL.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
June - Nov 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
(2008)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
Stability in the solvent was analytically proved.
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain Wistar (Hsd Cpb:WU)
- Source: Harlan Nederland, AD Horst, Netherlands
- Age at delivery of animals: 6 weeks
- Weight at study initiation: males 222 (210-238) g, females 173 (164-183) g
- Housing: from tattooing to necropsy in groups with 2 or 3 animals per cage in Makrolon cages type IV
- Diet and water: ad libitum
- Acclimation period: approximately 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): approximately 55
- Air changes (per hr): ≥ 10
- Photoperiod (hrs dark / hrs light): 12 /12
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on oral exposure:
Administration volume: 5 mL/kg bw

PREPARATION OF DOSING SOLUTIONS:
The formulations were prepared as needed and taking into account the analytically determined stability.

VEHICLE
polyethylene glycol 400
- Justification for use and choice of vehicle (if other than water): The test item gave a solution in vehicle for which stability was analytically verified.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Content checks on formulations (including controls) were determined twice during the study.
The samples were quantified by reversed phase (C18) HPLC with UV-detection (DAD, wavelength 200 nm). Standard solutions of the authentic test item were used for calibration.
Duration of treatment / exposure:
28 days (males), 29 days (females)
Frequency of treatment:
once daily
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: according to results obtained in a previous dose toleration study (female rats, oral administration) performed within a developmental toxicity study (see IUCLID-chapter 7.8.2)
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: for Morbidity and Mortality twice daily (once daily on weekends and public holidays); General Clinical Observations (in-cage) daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Open Field Observation weekly (once before the start of treatment);
Animals were placed into a standard arena (open field) for behavioral observations. Any clinical signs (findings) and abnormalities were recorded. Body surfaces and orifices, posture, general behaviour, breathing and excretory products were assessed. Findings and abnormalities were recorded either using a coding system or uncoded.

BODY WEIGHT: Yes
- Time schedule: just prior to first treatment and daily thereafter until last weighing immediately before necropsy.

FOOD AND WATER INTAKE: Yes
- Time schedule for examinations: weekly
On the basis of this data the following was calculated:
- for each interval: mean daily food intake per animal, mean daily food intake per kg body weight;
- for the total period: measurement of mean food intake per animal and day, mean food intake per kg body weight and day;
- cumulative food intake per animal and cumulative food intake per kg body weight.
Comparable calculations were done for the water intake.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 28
The blood samples were collected in the morning from the retro-orbital venous plexus of non-fasting animals anesthetized with or CO2/air.
- How many animals: all dose groups and controls
- Parameters examined: Differential blood count, erythrocyte morphology, erythrocyte count, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular volume, hemoglobin concentration, hematocrit, leucocyte count, reticulocyte count, thrombocyte count, thromboplastin time (Hepato-Quick).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 28
The blood samples for determination of glucose concentrations were taken from the caudal vein of non-fasting, non-anesthetized animals. The blood samples used for determinating the other parameters were collected in the morning from the retro-orbital venous plexus of non-fasting animals anesthetized with or CO2/air.
- How many animals: all dose groups and controls
- Parameters examined: Alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, albumin, cholesterol, creatinine, total protein, urea, glucose, gall acids, potassium, sodium.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Functional Observational Battery on day 24 (males) and day 25 (females); Motor/Locomotor Activity on day 24 (males) and day 25 (females).
- Dose groups that were examined: all dose groups and controls
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (all dose groups and controls)
Animals were sacrificed at day 29 (males) and day 30 (females) by exsanguination under deep diethyl ether anesthesia, necropsied and their organs and tissues subjected to thorough gross pathological examination. The following organs of the animals killed at the end of the treatment were weighed before fixation: Brain, heart, liver, spleen, kidneys (both), thymus, adrenal glands (both), epididymides (both), testes (both), seminal vesicles with coagulation gland, prostate, ovaries (both) and uterus. The organ weights are specified in both absolute and relative terms.

HISTOPATHOLOGY: Yes
The following organs and tissues were fixed and histopathologically evaluated for the control and highest dose group:
Abnormalities, Adrenal glands, Aorta, Brain (cerebrum, cerebellum, brain stem), Epididymides, Esophagus, Eyes, Eyelids, Exorbital lacrimal glands, Femur (with joint), Harderian glands, Head (with skull cap), Nasal Cavity, Heart, Intestine, Peyer's patches, Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum, Kidneys, Larynx, Liver, Lungs, Lymph nodes, mandibular, Lymph nodes, mesenteric, Lymph nodes, popliteal, Optic nerves, Ovaries, Oviducts, Pancreas, Pharynx, Pituitary gland, Prostate, Salivary glands (parotid, submandibular, sublingual), Sciatic nerve, Seminal vesicles (incl. coagulation glands), Skeletal muscle (thigh), Skin (mammary region), Spinal cord (cervical, thoracic, lumbar), Spleen, Sternum with Bone Marrow , Stomach, Testes, Thymus, Thyroid glands (with parathyroids), Tongue, Trachea, Ureters, Urethra, Urinary bladder, Uterus (with cervix), Vagina, Zymbal's glands, Physical identifier.
Deviating from this the livers from all dose groups were evaluated histopathologically.

Statistics:
Statistical evaluations on body and organ weight data were done using the Dunnett-test in connection with a variance analysis. Evaluating clinical pathology parameters an analyses of variance followed by a Dunnett test, an adjusted Welch test or a Kruskal-Wallis test was performed. For all these tests SAS® routines were used.
All variables that were not dichotomous were described by sex, dose group and time point using appropriate measures of central tendency (mean, median) and general variability (standard deviation, minimum, maximum).
For the statistical evaluation of samples drawn from continuously distributed random variables three types of statistical tests were used. The choice of the test being a function of prior knowledge obtained in former studies. Provided that the variables in question were approximately normally distributed with equal variances across treatments, the Dunnett test was used, if heteroscedasticity appeared more likely, a p value adjusted Welch test was applied. If the evidence based on experience with historical data indicated, that the assumptions for a parametric analysis of variance cannot be maintained, distribution-free tests in lieu of ANOVA were carried out, i.e. the Kruskal-Wallis test followed by adjusted Mann-Whitney-Wilcoxon tests (U tests) where appropriate.
With respect to data collected in the functional observational battery categorical variables were analyzed with a repeated measures analysis of variance followed by a one-way analysis of variance using the SAS procedure PROC CATMOD. As part of the one-way analysis, contrasts were performed to compare the results of each treated group with those of the control group. The logic of the analysis plan for continuous variables was analogous to that of the categorical variables, but using the SAS procedure PROC GLM.
Statistics of MA/LMA will be generated with an evaluation step of the SPADER (=Safety Pharmacology Automated Data Evaluation and Reporting) application.
Details on results:
CLINICAL SIGNS AND MORTALITY
Survival was not affected by the treatment with the test substance up to 1000 mg/kg.
The behaviour and clinical appearance were not toxicologically relevantly changed in both sexes up to 1000 mg/kg.

BODY WEIGHT AND WEIGHT GAIN
Body weight development was not toxicologically relevantly changed at any dose.

FOOD AND WATER INTAKE
Food and water intake were not toxicologically relevantly changed in both sexes up to 1000 mg/kg.

HAEMATOLOGY
Red and white blood parameters as well as blood coagulation were not toxicologically relevantly changed in any dose group.

CLINICAL CHEMISTRY
Enzyme activities, substrates and electrolytes in peripheral blood were not affected by the test substance up to 1000 mg/kg.

NEUROBEHAVIOUR
FOB did not reveal any test substance-induced toxicologically relevant findings in males and females up to 1000 mg/kg. Motor and locomotor activities did not reveal any indications of a neurotoxicological potential of the test substance in males and females up to 1000 mg/kg.

ORGAN WEIGHTS
Liver weights were slightly increased in males at 1000 mg/kg. Females were not affected.

GROSS PATHOLOGY
Necropsy revealed distinct lobulation and surface changes in livers of males at 1000 mg/kg. Females were not affected. Gross pathological investigation did not give any indication of test substance-related functional or morphological alterations in the other organs examined.

HISTOPATHOLOGY: NON-NEOPLASTIC
Histopathological evaluation revealed centrilobular vacuolation together with eosinophilia in males at 1000 mg/kg. The vacuolation was proved as hepatocellular fat deposition and might indicate an (adverse) influence on fat metabolism. Females were not affected.
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: slightly increased liver weights, distinct lobulation and surface changes of liver at necropsy and histopathological correlate (hepatocellular fat deposition)
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: no toxicological relevant changes within all investigations
Critical effects observed:
no
Executive summary:

An oral subacute dose study according to OECD TG 407 with the test item administered by gavage as formulation in polyethylene glycol was conducted using 5 male and 5 female Wistar rats per dose group and doses of 0 (vehicle control), 100, 300 and 1000 mg/kg bw per day.

Survival was not affected by the treatment up to the highest dose. Clinical and functional observations as well as motor activity measurements did not reveal any indications of test substance-induced effects. Body weight development as well as food and water intake was not affected by the test substance at any dose. Hematological and clinico-chemical parameters were not toxicologically relevantly changed. In males liver weights were slightly increased and necropsy revealed distinct lobulation and surface changes at 1000 mg/kg. These morphological observations corresponded to centrilobular vacuolation together with eosinophilia in this group. The vacuolation was proved as hepatocellular fat deposition and might indicate an (adverse) influence on fat metabolism. Females were not affected. Gross and histopathological investigation did not give any indication of test substance-related functional or morphological alterations in the other organs examined.

Therefore the no-observed-adverse-effect level (NOAEL) was 300 mg/kg body weight in males based on liver findings and 1000 mg/kg body weight in females.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Organ:
liver
other: of male rats only

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

A subchronic oral gavage toxicity study in Wistar rats following OECD TG 408 was performed with the substance. Based on the experiences gained in a subacute oral toxicity study the dose levels for the OECD TG 408 were selected with 0, 100, 300 and 1000 mg/kg bw/day, formulated in PEG 400.

A marked decrease in T4 hormone concentration was noted for males at 1000 mg/kg/day only (minus 24%). There was no histopathological correlate in the thyroid gland. No other effects on thyroid hormones were observed.

Microscopic change became obvious in the liver of high dosed males only. Hepatocellular cytoplasmic alteration were observed in 5/10 males at this dose level (3 minimal, 2 mild). Other microscopic changes in the liver noted for the 5 affected males were vacuolation at minimal degree and single cell necrosis in the centrilobular area at minimal degree. This combination of alterations is interpreted to represent degenerative findings and is therefore considered to be adverse. Microscopic examination of the livers of the 100 and 300 mg/kg bw/day treated males as well as the 1000 mg/kg bw/day treated females revealed no histopathological changes.

No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. mortality, clinical appearance, body weight, functional observations, ophthalmology, hematology, clinical chemistry, coagulation and organ weights).

In conclusion, administration of the test item by once daily oral gavage was well tolerated in rats at levels up to 1000 mg/kg/day. Adverse morphologic alterations at minimal to mild degree were present in the liver of males treated at 1000 mg/kg/day and consisted of centrilobular cytoplasmic alteration, hepatocellular vacuolation and single cell necrosis. Based on these results of this subchronic study, the no-observed-adverse-effect level (NOAEL) was considered to be 300 mg/kg/day for male rats and 1000 mg/kg bw for female rats.

In this study, a marked reduction of total T4 was observed in males treated at 1000 mg/kg/day which was considered to be test item-related. However, under the conditions of this study no adverse effect was observed that could be linked to the reduction of total T4 and therefore this reduction was not taken into account when determining the parental male NOAEL.

The findings in the subchronic study are well in line with the findings of a subacute oral toxicity study, in which liver of males rats was also slightly affected at 1000 mg/kg bw/day only. Slight morphological changes corresponded to centrilobular vacuolation together with eosinophilia in this group. The vacuolation was proved as hepatocellular fat deposition and might indicate an (adverse) influence on fat metabolism. Females were not affected. The NOAEL was consistently 300 mg/kg bw for males due to liver findings and 1000 mg/kg bw for females in the subacute oral toxicity study..

Justification for classification or non-classification

No classification required for Repeated Dose Toxicity according to Regulation (EC) No 1272/2008, Annex I.