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Description of key information

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:
From 25 Nov 2020 to 23 Apr 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
2018
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
- Physical Description: Yellow to brownish liquid
Species:
rat
Strain:
other: Crl: WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 6-7 weeks
- Condition: Outbred, SPF-Quality
-Source: Charles River Deutschland, Sulzfeld, Germany or Charles River Laboratories France, L´ Arbresle Cedex, France
- Weight at study initiation: Males between 155 and 215 g and females between 115 and 146 g
- Housing: Polycarbonate cages (Makrolon type IV, height 18 cm) containing sterilized wooden fibers as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. Up to 5 animals of the same sex and same dosing group together. During locomotor activity monitoring, animals are were individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.
- Diet: Ad libitum, SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany. During motor activity measurements, animals did not have access to food for a maximum of 2 hours.
- Water: Municipal tap water, Freely available to each animal via water bottles.
- Acclimation period: at least 5 days
- For environmental enrichment, animals were provided with paper (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom), except when interrupted by study procedures/activities. For animal welfare reasons, animals were provided with wooden sticks (Swedish aspen wood, Bioservices, Uden, The Netherlands).

DETAILS OF FOOD AND WATER QUALITY: Periodic analysis of the water was performed, and analysis for nutritional components and environmental contaminants in the feed were provided by the supplier. It is considered that there were no known contaminants in the water or feed that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24 (Target), 19 to 21 (Actual mean)
- Humidity (%): 40 to 70 (Target), 39 to 59 (Actual mean)
- Air changes (per hr): Ten or more
- Photoperiod (hrs dark / hrs light): 12 /12 (except during designated procedures)

IN-LIFE DATES: From: 8 Dec 2020 To: 10 Mar 2021
Route of administration:
oral: gavage
Details on route of administration:
The oral route of exposure was selected because ECHA has evaluated this the most
appropriate route of administration for the study.
Vehicle:
paraffin oil
Remarks:
Specific gravity: 0.85 g/cm3
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Dose formulations were divided into aliquots where required to allow to be dispensed on each dosing occasion. Formulations (w/w) were prepared at least weekly, homogenized to visually acceptable levels, and stored at 2-8°C.
Test item dosing formulations were kept at room temperature until dosing. An adjustment was made for specific gravity of the test item and vehicle. No correction was made for the purity and composition of the test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle was chosen based on trial preparations. These trial preparations were caried out prior to the start of the study to assess the suitability of the formulation procedure and used a representative dosing concentration and volume. These trials were not performed as part of this study and were not used for dosing.
- Amount of vehicle (if gavage): 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected in week 1, 6 and 13 of dosing. The accuracy of preparation (concentration) was analysed for all groups, homogeneity was checked for groups 2 and 4 from the top, middle and bottom of the preparations.
Analyses were performed using a validated method. Concentration results were considered acceptable if mean sample concentration results were within or equal to ±10% solutions of theoretical concentration. Homogeneity results were considered acceptable if the relative standard deviation (RSD) of concentrations of ±10% for each group.
Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20254769) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Once daily, 7 days a week
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
(Group 2)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
(Group 3)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
(Group 4)
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 results of a 28-day repeated dose toxicity study with oral exposure of the test substance in rats, performed by the Sponsor, and in an attempt to produce graded responses to the test item. In this 28-d rat study (OECD 407) rats received 0, 100, 300 and 1000 mg/kg bw/day test item by daily oral gavage with paraffin oil as vehicle. No effects on clinical signs, body weight, food consumption, functional observations or clinical pathology were seen. Based on histopathological finding in the mesenterial lymph nodes (foamy macrophage aggregates), the NOAEL was set at 300 mg/kg bw/day for males and 100 mg/kg bw/day for females. For the current study, dose levels were selected with the rational that the high-dose level should produce some toxic effects, but not excessive lethality that would prevent meaningful evaluation. 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
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily a check for cage side observations was performed; from Day 1 at 0 to 1 hours postdose. ; At least twice daily beginning upon arrival through termination a check was done for mortality, except on days of receipt and necropsy where frequency was at least once dialy.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly; from Week 1 and throughout the study, and on the day of necropsy.


BODY WEIGHT: Yes
- Time schedule for examinations: Weekly; from at least Day 1 and throughout the study.

FOOD CONSUMPTION: yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, weekly; from at least Day 1 and throughout the study.

FOOD EFFICIENCY: no
- 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: Yes
- Time schedule for examinations: Monitored on regular basis throughout the study by visual inspection of the water bottles.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pretreatment and week 13
- Dose groups that were examined: Pretreatment Period - All animals once (including spare animals); Dosing Period - All Group 1 and 4 animals during Week 13.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day of necropsy between 7.00 and 10.30 a.m.
- Anaesthetic used for blood collection: Yes (isoflurane )
- Animals fasted: Yes
- How many animals: all (10/sex/dose)
- Parameters examined: White blood cell (WBC), Neutrophils (absolute), Lymphocytes (absolute), Monocytes (absolute), Eosinophils (absolute), Basophils (absolute), Large unstained cells (LUC) (absolute), Red blood cell (RBC), Reticulocytes (absolute), Red blood cell distribution width (RDW), Hemoglobin, Hematocrit, Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelets, Prothrombin time (PT), Activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day of necropsy between 7.00 and 10.30 a.m.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all (10/sex/dose)
- Parameters examined: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST),
Alkaline phosphatase (ALP), Total protein, Albumin, Total bilirubin, Urea, Creatinine, Glucose, Cholesterol, Triglycerides, HDL and LDL Cholesterol,Sodium, Potassium, Chloride, Calcium, Inorganic phosphate (Inorg. Phos)

PLASMA/SERUM HORMONES/LIPIDS: Yes
- Time schedule for collection of blood: day of necropsy between 7.00 and 10.30 a.m.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all (10/sex/dose)
- Parameters examined: Triiodothyronine (T3), Thyroxine (T4), Thyroid stimulating hormone (TSH)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once during the Dosing Period, during Week 12-13. These tests were performed after clinical observations (including arena observation, if applicable).
- Dose groups that were examined: The first 5 animals per sex per group
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

ARENA OBSERVATIONS: yes
- Arena observations were performed on all animals, once before the first administration of the test item and weekly during the Treatment Period. Animals were observed for clinical signs outside the home cage in a standard arena.

ESTROUS STAGE DETERMINATION: yes
- Time schedule for examinations: End of Treatment - on the day of necropsy, a vaginal smear was taken to determine the stage of estrous from all animals. This was done for all females.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Study 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.


ORGAN WEIGHTS: Yes
- Organs weighed at necropsy: brain, epididymis, adrenal gland, pituitary gland, prostate gland,
seminal vesicle gland (including coagulation gland), thyroid gland, heart, kidney, liver, ovary,
spleen, testis, thymus, uterus/cervix.

HISTOPATHOLOGY: Yes
- The following tissues were examined for the control group and the 1000 mg/kg bw/day dose group: aorta artery, bone marrow, sternum, brain, cervix, epididymides, eye, adrenal gland, mammary gland, parathyroid gland, pituitary gland, prostate gland, seminal vesicle gland (including coagulation gland), thyroid gland, gross lesions/masses, gut-associated lymphoid tissue, heart, kidney, cecum, colon, rectum, liver, lung, lymph node (mandibular and mesenteric site), skeletal muscles, optic and sciatic nerve, ovary, pancreas, skin, duodenum, ileum, jejunum, spinal cord, spleen, stomach, testis, thymus, trachea, urinary bladder, uterus, vagina.
- The following tissues were examined for all groups: gross lesions/masses
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. The following pairwise
comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Data Collected in Provantis:
- Parametric/Non-parametric: Levene’s test was used to assess the homogeneity of group variances. The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively.
- Non-Parametric: The groups were compared using an overall Kruskal-Wallis test. If the overall Kruskal-Wallis test was found to be significant, then the above pairwise comparisons were conducted using Dunn’s test.
- ANCOVA: data corresponding to a response variable of interest and to a related covariate were
submitted to an analysis of covariance (ANCOVA), and if found to be significant, then pairwise comparisons were conducted using Dunnett’s test.
- Incidence: A Fisher’s exact test was used to conduct pairwise group comparisons of interest.

Data Collected/Processed in Toxdata
- Parametric/Non-parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-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: Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).
- Incidence: An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The abovepairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
See attachment 1 - clinical signs
Abnormal breathing sounds were noted in one female at 100 mg/kg bw/day on Day 36. As this only occurred in one animal at a single occasion and lacked a dose-related response, it was considered not test item-related.
Any other clinical signs noted during the Dosing Period (e.g. fur loss, skin lesions, scabs and discolored eye lid) 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 and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test item.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
See attachment 2- body weight
Body weights and body weight gain of treated animals remained in the same range as controls over the study period.
Food consumption and compound intake (if feeding study):
no effects observed
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related ophthalmology findings in Week 13. The nature and incidence of ophthalmology findings noted during the Pre-treatment 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:
effects observed, treatment-related
Description (incidence and severity):
See attachment 3- haematology
No test item-related hematology parameter changes were observed in males up to 300 mg/kg
bw/day and in females at 100 mg/kg bw/day. At 1000 mg/kg bw/day an increase in white blood cell, neutrophil, lymphocyte, monocyte, basophil and large unstained cell count was observed in males and females. The neutrophil and monocyte counts in females at 300 mg/kg bw/day were also increased.
No test item-related effects on coagulation parameters were observed.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
See attachment 4- Clinical biochemistry
No test item-related clinical chemistry parameter changes were observed in males and females at 100 mg/kg bw/day. An increase in alanine aminotransferase and aspartate aminotransferase (ALT and AST) activities were noted in males at 300 and 1000 mg/kg bw/day (1.74x and 2.49x of control for ALT, and 2.01x and 3.30x of control for AST, respectively, not statistically significant at 300 mg/kg bw/day). ALT and AST activities were also increased in females at 1000 mg/kg bw/day (each 1.58x of control). Furthermore, an increased alkaline phosphatase (ALP) was noted in males at 1000 mg/kg bw/day (1.12x of control, not statistically significant) and an
increase in calcium concentration was noted in females at 300 and 1000 mg/kg bw/day (1.06x
and 1.07x of control, respectively, not statistically significant) (see Table 1 in "Any other information on results incl. tables").
Other values despite of achieving a level of statistical significance, when compared to controls, occurred in the absence of a dose-related distribution and/or were, given the magnitude of change, considered to be of no toxicological significance.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
See attachment 5 - Behaviour
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was similar between control and high dose animals. 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:
effects observed, treatment-related
Description (incidence and severity):
See attachment 6 - Organ weight findings including organ body weight ratios
In females at 1000 mg/kg bw/day, a slightly higher liver weight was observed and was statistically significant relative to body weight and correlated with the microscopic observation of histiocytic infiltrates.
In males and females at 1000 mg/kg bw/day, mildly higher spleen weight of similar
magnitude was observed and was statistically significant relative to body weight and/or
absolute value. In males this correlated with a slightly higher incidence/severity of
extramedullary hematopoiesis; there was no microscopic correlate in females.
There were no other test item-related organ weight changes (see Table 2 in "Any other information on results incl. tables").
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
See attachment 7 - Gross pathology
There were few macroscopic observations in males at 1000 mg/kg bw/day that were interpreted to be test item-related and included pale foci in the liver (caudate process and medial lobe) of one male and an enlarged spleen in one male.
The remainder of the 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 8 - Histopathology
In the jejunum of males and females at 1000 mg/kg bw/day, minimal histiocytic infiltrates were present multifocally in the lamina propria of the villi.
In the liver of males and females at 300 and 1000 mg/kg bw/day, multifocal histiocytic infiltrates were present scattered throughout the sections. Additionally, at 1000 mg/kg bw/day, minimal or mild necrosis was present.
In the mesenteric lymph node of males and females at 300 and 1000 mg/kg bw/day multifocal
histiocytic infiltrates were present ranging from minimal to moderate. A low incidence and severity of histiocytic infiltrates was present in females at 100 mg/kg bw/day however this was generally comparable to that observed in the control animals and not ascribed to the test item.
In the spleen of males at 300 and 1000 mg/kg bw/day, there was a slightly higher incidence and severity of extramedullary hematopoiesis present when compared to the control males which was considered test item-related. In females the incidence of extramedullary hematopoiesis was more variable and without a dose relationship thus a relationship to the test item was not suspected.
In the testis, a low incidence of mononuclear cell infiltrates consisted of small individual foci in the capsular or subcapsular region were noted in a few males at 300 mg/kg bw/day (1/10) and 1000 mg/kg bw/day (3/10). This was interpreted to be within the range of background findings and not test item-related due to the low incidence and focal nature and often unilateral nature and was primarily composed of lymphocytes (not histiocytic cells as the test item-related infiltrates seen in other organs).
In the brain of one control female axonal dystrophy was noted in the medulla oblongata (specifically, the cuneate and gracile nuclei). This observation was bilaterally symmetrical and characterized by the presence of numerous spheroids in these nuclei. Axonal dystrophy was also present bilaterally in the dorsal funiculus of the spinal cord as a few spheroids present at each examined segment (cervical, thoracic and lumbar). This is a spontaneous lesion that is reported to occur in ageing rats (Kaufman et al., Eisenbrandt et al).
The remaining histologic changes were considered to be incidental findings or 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.
There were no toxicologically significant changes in the estrous stage.
Details on results:
As the hematology changes (white blood cell, neutrophil, lymphocyte, monocyte, basophil and large unstained cell count) occurred in absence of a histopathological correlation, these findings were considered to be not adverse.
Microscopically, histiocytic infiltrates were observed in the jejunum of males and females at 1000 mg/kg bw/day, and in the mesenteric lymph node and liver of males and females starting at 300 mg/kg bw/day. In the liver of both sexes at 1000 mg/kg bw/day these findings were accompanied by increased liver enzymes (aspartate aminotransferase and alkaline phosphatase) and by foci of necrosis. The foci of necrosis were considered to be adverse. Despite the observation of histiocytic infiltrates in the mesenteric lymph node up to a moderate degree in males and females at 300 and 1000 mg/kg bw/day, their presence was not accompanied by necrosis or degeneration, nor by indication of dysfunction (e.g. no indication
of obstruction of lymphatic drainage or morphologic alteration in the number or arrangement
of lymphoid cells), thus alone was considered non-adverse.
In the spleen a slight increase in the incidence and severity of extramedullary hematopoiesis in males at 300 and 1000 mg/kg bw/day correlated at 1000 mg/kg bw/day to higher spleen weight and a single instance of spleen enlargement. Higher spleen weight was also noted in females at 1000 mg/kg bw/day but without a histological correlation. The spleen findings were considered to be non-adverse due to the minimal degree and only the slight variation from background observations, or lack of histologic correlate.
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Table 1 Test Item-Related Hematology Changes






















































































Groups223344
Dose level (mg/kg bw/day)10020030030010001000
SexMFMFMF
White blood cells----1,27x1,51x
Neutrophils---1,68x1,21x1,83x
Lymfocytes----1,28x1,47x
Monocytes---1,75x1,72x1,78x
Basophils----2,33x3,75x
Large unstained cells----1,54x2,71x

M = Males F = Females


A dash (-) indicates absence of change. Numerical values indicate fold change of the treated group mean value relative to the control group mean value. 


 


Table 2 Mean Percent Liver And Spleen Weight Differences from Control Groups













































































 MMMFFF
Dose level (mg/kg bw/dag)10030010001003001000
Liver      
Absolute3580110
Relative to body weight2370310*
Spleen      
Absolute21825*41230**
Relative to body weight01423*41430**

*: P<0.05, **: P<0.01; shaded values were considered test item-related.

Conclusions:
In conclusion, administration of the test substance by once daily oral gavage for
90 days was well tolerated in Wistar Han rats at dose levels up to and including 1000 mg/kg
bw/day. An adverse morphologic alteration was present in the liver of males and females at
1000 mg/kg bw/day and consisted of foci of necrosis. The other alterations observed in
clinical pathology, organ weight changes and histopathology were considered to be not
adverse. Based on these results, the No Observed Adverse Effect Level (NOAEL) was
considered to be 300 mg/kg bw/day.
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
(2008)
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
Stability in vehicle analytically confirmed
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain: Wistar (Hsd Cpb:WU)
- Source: Harlan Nederland
- Age at delivery: 6 weeks
- Weight at study initiation: males 194.5 (186-204) g, females 143.0 (129-155) g
- Housing: Individually in Makrolon® cages Type IIa until tattooing, thereafter 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:
paraffin oil
Details on oral exposure:
Administration volume: 2 mL/kg bw

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

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item gave a solution in the vehicle for which stability was analytically verified.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test substance content (all doses including 0 mg/kg) was checked twice during the study.
For analysis a portion of the dosage form was filled into a sodium chloride cell. A high resolution FTIR spectrum was scanned and evaluated. Quantification is based upon the concentration-dependent absorption bands due to the oscillation of ester function bond which occurs near 1750/cm.
Duration of treatment / exposure:
30 days (males), 31 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: In a pilot study the test substance was administered orally by gavage to three male and three female rats per group in daily doses of 0 and 1000 mg/kg bw for 2 weeks. Survival was not affected by the treatment. At clinical observations no findings were observed. Body weight development was not affected by the treatment. The feed and water intake in treated groups was comparable to that in the respective control groups. Determination of organ weights revealed no relevant differences to the respective control values. Based on these results the following dose scheme was used for the present study: 0-100-300-1000 mg/kg body weight.
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 condition and behavior of all animals were checked and recorded daily (“in cage observation”) some time after administration of the last animal.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Open Field Observation (OFO) weekly
A careful clinical examination including observation outside the home cage in a standard arena was performed once prior to treatment and in a weekly interval up to necropsies in all animals. Any clinical signs and abnormalities including changes in skin, fur, eyes, mucous membranes occurrence of secretions and excretions, autonomic activity, changes in gait, posture, and response to handling, clonic/tonic movements, stereotypes and bizarre behavior were recorded as well as time of onset, location and grading. Findings and abnormalities were recorded either using a coding system or uncoded.

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION:
- Time schedule for examinations: weekly
The following parameters were calculated: mean daily food intake per kg body weight; 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

WATER CONSUMPTION: Yes
- Time schedule for examinations: weekly

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 30
The blood samples were collected in the morning from the retro-orbital venous plexus of non-fasted animals anesthetized with CO2/air.
- How many animals: all dose groups and controls
- Parameters checked: 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 30
The blood samples for determination of glucose concentrations were taken in the morning from one of the caudal veins of non-fasted, non-anesthetized animals. The blood samples used for determining the other parameters in peripheral blood were collected in the morning from the retro-orbital venous plexus on non-fasted animals anesthetized with CO2/air.
- How many animals: all dose groups and controls
- Parameters checked: Alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, bile acids, total bilirubin, albumin, total protein, cholesterol, creatinine, urea, glucose, sodium, potassium.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Functional Observational Battery (FOB) on Day 23/24; Motor Activity (MA) and locomotor activity (LMA) on Day 23/24; The examinations were performed earliest about 30 min. after treatment.
- Dose groups that were examined: all dose groups and controls
- FOB - Battery of functions tested: home cage observation (posture, piloerection, gait abnormalities, involuntary motor movements, vocalizations, others), observations during handling (ease of removing, reaction to being handled, muscle tone, palpebral closure, lacrimation, nasal discharge, salivation, stains, others), open field observations (piloerection, respiratory abnormalities, posture, involuntary motor movements, stereotypy, bizarre behavior, gait abnormalities, vocalizations, arousal, rearing, defecation, urination), reflex/physiological observations (approach response, touch response, auditory response, tail pinch response, pupil size, pupil response, righting reflex, grip strength, landing foot splay, body temperature, body weight)
- MA: MA and locomotor activity (LMA) were examined as activity for the entire 60-minute session (Summary Session MA and LMA) and activity during each 10-minute interval (Summary Interval MA and LMA). MA was measured as the number of beam interruptions that occurred during the test session. LMA will be measured by eliminating consecutive counts for a given beam. Thus, for LMA, only one interruption of a given beam was counted until the animal relocated in the maze and interrupted one of the other beams. Habituation was evaluated as a decrement in activity during the test session.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (all dose groups and controls), on Days 31/32
- Organ Weights: 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), prostate, seminal vesicles, ovaries (both) and uterus.

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 (joint, bone marrow), 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, Vagina, Zymbal's glands, Physical identifier.
Deviating from this livers, mesenteric lymph nodes and thyroid glands 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 clinico-chemical 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
No animal died during the study. Therefore, survival was not affected by the treatment with the test substance.
At clinical observations no test substance-related clinical findings were noted.

BODY WEIGHT AND WEIGHT GAIN
Body weight development was not affected by the treatment.

FOOD CONSUMPTION
Food intake in all treated groups was comparable to that of the respective control group.

WATER CONSUMPTION
Water intake in all treated groups was comparable to that of the respective control group.

HAEMATOLOGY
Hematological investigation gave no evidence for test substance-related effects on red blood or blood coagulation up to 1000 mg/kg. The statistically significantly increase of erythrocyte count and hematocrit value at 1000 mg/kg in females is not regarded to reflect a treatment-related effect as the differences to control were slight, all individual values for erythrocyte count were within the range of historical control values and with respect to hematocrit only two of the individual values exceeded the upper 2-range of historical control data.
The investigation gave no evidence for treatment-related effects on differential blood count or blood coagulation up to 1000 mg/kg in males and females.

CLINICAL CHEMISTRY
Determination of enzyme activities in peripheral blood revealed in males statistically significantly decreased activities of ASAT at 1000 mg/kg and of ALAT in all dose groups. However, the differences to control were slight, none of the individual values exceeded the lower 2-range of historical control data, there was no dose dependence with respect to ALAT and as generally rather an increase than a decrease in these enzyme activities is regarded to be indicative for an adverse effect, this was not regarded to be of toxicological relevance.
Determination of TSH-concentration in peripheral blood showed in female's highest values at 300 and 1000 mg/kg, in males TSH concentrations were inconspicuous.

NEUROBEHAVIOUR
The functional observational battery did not reveal any evidence for a treatment-related effect.
At motor/locomoter activity measurements the determination did not to reveal a significant effect in males up to 1000 mg/kg and in females up to 300 mg/kg. At 1000 mg/kg values for motor activity and locomotor activity were highest compared to the other female groups. The differences with respect to MA were slight and were somewhat more pronounced for LMA. Therefore, and as the values compared to historical data are slightly higher, a treatment-related effect may be assumed. However, as statistical significance was absent in any case and there was no correlation to the other investigations this was not regarded to be an adverse effect.

ORGAN WEIGHTS
Determination of organ weights at necropsy gave no evidence for treatment-related effects in males and females up to 1000 mg/kg.

GROSS PATHOLOGY AND HISTOPATHOLOGY (NON-NEOPLASTIC)
At necropsy distinct lobulation of the liver was found in males beginning at 100 mg/kg (incidence: 0-1-1-2). At histopathology hypertrophy of centrilobular hepatocytes was observed in 3/5 males at 1000 mg/kg up to a slight degree. Correlation to liver lobulation observed at necropsy was only found in one 1000 mg/kg male. Females were not affected.
Fat storage of the liver was slightly increased in both genders of the dosed groups, but without dose-correlation this finding was assessed to be incidental. It was concluded that liver hypertrophy might indicate a non-adverse adaptive effect.
At histopathology thyroidal follicular cell hypertrophy with colloidal decrease was raised in females at 300 mg/kg by incidence (0-1-3-3) and at 1000 mg/kg also by severity. Only a marginal increase by incidence (1-1-2-3) could be found in males at 1000 mg/kg. Determination of TSH-concentration in peripheral blood showed in females highest values at 300 and 1000 mg/kg. From the 6 animals showing histopathological changes of thyroids 4 animals showed high values of TSH concentration. However, in two females TSH values were inconspicuous, and with the exception of the value of animal No. 37 no other value exceeded the upper 2s range of historical control values, the differences to control were slight and statistical significance was absent. Therefore, although there might be a slight correlation to the histopathological findings, the changes observed are not regarded to be of toxicological relevance. It was concluded that thyroid hypertrophy might indicate a non-adverse adaptive effect.
The mesenterial lymph node showed foamy macrophage aggregates of minimal to moderate degree in all males and females at 1000 mg/kg and in one female at 300 mg/kg. A PAS stain detecting carbohydrate macromolecules was minimally to slightly positive in these areas. Macrophage aggregates in the mesenterial lymph node reflect a compound-related effect, which might be attributed to intestinal resorption of the test article. No such findings were detected in distal lymph nodes (popliteal, mandibular).
Determination of organ weights at necropsy gave no evidence for treatment-related effects in males and females up to 1000 mg/kg. Gross and histopathological investigation into other organs and tissues gave no evidence for treatment-related effects.
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: histopathology of mesenterial lymph nodes (foamy macrophage aggregates)
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: histopathology of mesenterial lymph nodes (foamy macrophage aggregates)
Critical effects observed:
no
Executive summary:

A subacute oral toxicity study according to OECD TG 407 was conducted with the test item formulated in paraffin oil and administered by gavage to 5 male and 5 female Wistar rats per dose group. Doses were 0 (vehicle control), 100, 300 and 1000 mg/kg bw per day.

At clinical observations no test substance-related findings were noted. The functional observational battery did not reveal any evidence for a treatment-related effect. At motor/locomotor activity measurements the values were highest for the females of 1000 mg/kg but without statistical significance or correlations to other investigations no adverse effect was concluded. Body weight development as well as food and water intake was not affected by the treatment. Hematological investigation gave no evidence for test substance-related effects.

At necropsy distinct lobulation of the liver was found in males beginning at 100 mg/kg. At histopathology hypertrophy of centrilobular hepatocytes was observed in 3/5 males at 1000 mg/kg up to a slight degree. Correlation to liver lobulation observed at necropsy was only found in one 1000 mg/kg male. It was concluded that liver hypertrophy might indicate a non-adverse adaptive effect.

Thyroidal follicular cell hypertrophy with colloidal decrease was raised in females at 300 mg/kg by incidence and at 1000 mg/kg also by severity. Only a marginal increase could be found in males at 1000 mg/kg. Determination of TSH-concentration in peripheral blood showed in females highest values at 300 and 1000 mg/kg. Although there might be a slight correlation to the histopathological findings the changes observed are not regarded to be of toxicological relevance. It was concluded that thyroid hypertrophy might indicate a non-adverse adaptive effect. It is well known that there are species differences in thyroid system physiology between rodents and humans with the rat being especially sensitive to secondary responses of the thyroid system to liver enzyme induction (thyroid hormone imbalance).

The mesenterial lymph node showed foamy macrophage aggregates of minimal to moderate degree in all males and females at 1000 mg/kg and in one female at 300 mg/kg. A PAS stain detecting carbohydrate macromolecules was minimally to slightly positive in these areas. Macrophage aggregates in the mesenterial lymph node reflect a test substance-related effect, which might be attributed to intestinal resorption of the test article. No such findings were detected in distal lymph nodes (popliteal, mandibular). Gross and histopathological examinations of the intestine were unobtrusive.

Determination of organ weights at necropsy gave no evidence for treatment-related effects in males and females up to 1000 mg/kg. Gross and histopathological investigation into the other organs and tissues gave no evidence for treatment-related findings.

Therefore the no-observed-adverse-effect level (NOAEL) was 300 mg/kg body weight in males and 100 mg/kg body weight in females based on effects in mesenterial lymph nodes.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subchronic
Species:
rat
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

A subacute oral toxicity study according to OECD TG 407 was conducted with the test item formulated in paraffin oil and administered by gavage to 5 male and 5 female Wistar rats per dose group. Doses were 0 (vehicle control), 100, 300 and 1000 mg/kg bw per day.


At clinical observations no test substance-related findings were noted. The functional observational battery did not reveal any evidence for a treatment-related effect. At motor/locomotor activity measurements the values were highest for the females of 1000 mg/kg, but without statistical significance or correlations to other investigations no adverse effect was concluded. Body weight development as well as food and water intake was not affected by the treatment. Hematological investigation gave no evidence for test substance-related effects.


At necropsy distinct lobulation of the liver was found in males beginning at 100 mg/kg. At histopathology hypertrophy of centrilobular hepatocytes was observed in 3/5 males at 1000 mg/kg up to a slight degree. Correlation to liver lobulation observed at necropsy was only found in one 1000 mg/kg male. It was concluded that liver hypertrophy might indicate a non-adverse adaptive effect.


Thyroidal follicular cell hypertrophy with colloidal decrease was raised in females at 300 mg/kg by incidence and at 1000 mg/kg also by severity. Only a marginal increase could be found in males at 1000 mg/kg. Determination of TSH-concentration in peripheral blood showed in females highest values at 300 and 1000 mg/kg. Although there might be a slight correlation to the histopathological findings the changes observed are not regarded to be of toxicological relevance. It was concluded that thyroid hypertrophy might indicate a non-adverse adaptive effect. It is well known that there are species differences in thyroid system physiology between rodents and humans with the rat being especially sensitive to secondary responses of the thyroid system to liver enzyme induction (thyroid hormone imbalance).  


The mesenterial lymph node showed foamy macrophage aggregates of minimal to moderate degree in all males and females at 1000 mg/kg and in one female at 300 mg/kg. A PAS stain detecting carbohydrate macromolecules was minimally to slightly positive in these areas. Macrophage aggregates in the mesenterial lymph node reflect a test substance-related effect, which might be attributed to intestinal absorption of the high average molecular weight test article. No such findings were detected in distal lymph nodes (popliteal, mandibular). Gross and histopathological examinations of the intestine were unobtrusive. 


Determination of organ weights at necropsy gave no evidence for treatment-related effects in males and females up to 1000 mg/kg. Gross and histopathological investigation into the other organs and tissues gave no evidence for treatment-related findings.


Therefore the no-observed-adverse-effect level (NOAEL) was 300 mg/kg body weight in males and 100 mg/kg body weight in females based on effects in mesenterial lymph nodes attributed to intestinal absorption of the high average molecular weight test article and therefore a port of entry effect. There is no indication of adverse effects related to systemic toxicity.


 


A subchronic oral toxicity study according to OECD TG 408 was conducted with the test item formulated in paraffin oil and administered by gavage to 10 male and 10 female Wistar rats per dose group. Doses were 0 (vehicle control), 100, 300 and 1000 mg/kg bw per day.


At 100 mg/kg bw/day, no test item-related findings were observed.


At 300 mg/kg bw/day, test item-related findings comprised increased neutrophil and monocyte counts, histiocytic infiltrates in the mesenteric lymph node and liver of males and females and a slight increase in the incidence and severity of extramedullary hematopoiesis in the spleen of males only. These findings were considered to be non-adverse. 


At 1000 mg/kg bw/day, test item-related findings consisted of increased white blood cell parameters and increased spleen weight in males and females, and a slight increase in the incidence and severity of extramedullary hematopoiesis in the spleen with a single instance of macroscopically enlarged spleen in males. Furthermore, multifocally histiocytic infiltrates were present in the jejunum, mesenteric lymph node and liver of males and females. In the liver this was accompanied by increased liver enzyme activities. These findings were considered to be non-adverse. In addition, foci of hepatocellular necrosis were present in the liver of males and females, which were considered to be adverse. 


In conclusion, administration of the test substance by once daily oral gavage for
90 days was well tolerated in Wistar Han rats at dose levels up to and including 1000 mg/kg
bw/day. An adverse morphologic alteration was present in the liver of males and females at
1000 mg/kg bw/day and consisted of foci of necrosis. The other alterations observed in
clinical pathology, organ weight changes and histopathology were considered to be not
adverse. Based on these results, the No Observed Adverse Effect Level (NOAEL) was
considered to be 300 mg/kg bw/day.

Justification for classification or non-classification

A classification for repeated dose toxicity (STOT-RE) according to regulation (EC) No 1272/2008 is not warranted for the substance.