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Toxicological information

Repeated dose toxicity: oral

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020

Materials and methods

Test guidelineopen allclose all
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Clear colourless viscous liquid
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.

Test animals

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

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

open allclose all
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

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.