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Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
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 vehicle analytically confirmed
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals 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:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat

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.

Justification for classification or non-classification

No classification is required for repeated dose toxicity according to Regulation (EC) No 1272/2008, Annex I.

 

Reason: The derived NOAELs (300 mg/kg bw for males; 100 mg/kg bw for females) were set based on effects “of minimal to moderate degree” on the mesenterial lymph nodes attributed to intestinal absorption of the test article and therefore a port of entry effect. There is no indication of adverse effects related to systemic toxicity. Thus, a classification for repeated dose toxicity according to regulation (EC) No 1272/2008 is not warranted for the substance.