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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:
15 May 2014 - 22 August 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-compliant guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
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)
Qualifier:
according to
Guideline:
other: Japanese Chemical Substances Control Law 1973, Notification of Mar. 31 2012 by MHLW (0331 No.7), METI (No. 5) and MOE (No. 110331009)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-ethanol
- Physical state: White powder
- Stability under test conditions: stable over at least 6 hours at room temperature and over 8 days in a refrigerator under nitrogen.
- Storage condition of test material: In refrigerator (2-8°C) in the dark under nitrogen

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation:
- Housing: Group housing of 5 animals per sex in Macrolon cages (MIV type, height 18 cm) with sterilized sawdust as bedding material and paper as cage-enrichment. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage without cageenrichment
or bedding material.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). During motor activity measurements, animals had no access to food.
- Water: Free access to tap-water except during motor activity measurements
- Acclimation period: At least 5 days before the start of treatment under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature: 18 to 24°C
- Humidity: 40 to 70%
- Air changes: at least 10 air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Remarks:
specific gravity 1.036
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing. From Day 16 onwards, formulations (w/w) were prepared on a weekly basis. Formulations were homogenized to a visually acceptable level and were heated to a maximum of 48.5°C for a maximum of 63 minutes to obtain visual homogeneity. Formulations were allowed to cool down below 40°C before dosing. No correction was made for the purity of the test substance. Formulations were placed on a magnetic stirrer during dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations and on information from the Sponsor.
- Concentration in vehicle:
- Amount of vehicle (if gavage): 10 ml/kg dose volume
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations, in Weeks 1, 6 and 13). Stability in vehicle over 6 hours and at room temperature under normal laboratory light conditions and over 8 days in the refrigerator under nitrogen was also determined (highest and lowest concentration, in Week 1).
The concentrations analysed 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%). The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%). Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours and under nitrogen in a refrigerator for at least 8 days.
Duration of treatment / exposure:
At least 90 days. Animals were dosed up to the day prior to necropsy
Frequency of treatment:
Once daily, 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Doses / concentrations
Remarks:
Doses / Concentrations:
30, 150, 750 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels are based on a previously conducted 28-day study where dose levels of 100, 300 and 1000 mg/kg were tested. Signs of toxicity were seen only in animals treated at 1000 mg/kg and included signs of irritation in the glandular stomach (1/5 males showing a small ulcer, and 4/5 males showing chronic inflammation) and increased liver weights in females (relative weights were increased with approximately 18% compared to controls; without a microscopic correlate). Relevant signs were primarily noted for males only. A NOAEL of 300 mg/kg was determined for males and a NOAEL of 1000 mg/kg was determined for females.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily. The time of death was recorded as precisely as possible.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards, detailed clinical observations were made in all animals immediately after dosing (based on the absence of a peak effect in occurrence of clinical signs in a 28-day oral toxicity study). Once prior to start of treatment and at weekly intervals, this was also performed outside the home cage in a standard arena. 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

FOOD CONSUMPTION
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at pretest: All animals (including spare animals); at Week 13: Group 1 and 4 animals.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: between 7.00 and 10.30 a.m. on the day of necropsy at the end of the treatment
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: White blood cells, Differential leucocyte count, Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin,
Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets, Prothrombin time, Activated Partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: between 7.00 and 10.30 a.m. on the day of necropsy at the end of the treatment
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Total Protein, Albumin, Total Bilirubin, Urea,
Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During Week 12-13 of treatment
- Dose groups that were examined: Group 1 and 4 animals, locomotor activity was peformed on all animals.
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex. 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).
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Animals were deprived of food overnight (with a maximum of 24 hours) prior to scheduled necropsy. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded. Rats found dead were subjected to a full post mortem examination as soon as possible after death and always within 24 hours.

HISTOPATHOLOGY: Yes
Samples of the following tissues and organs were collected from all animals at necropsy and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands):
Ovaries, Adrenal glands, Pancreas, Aorta, Peyer's patches [jejunum, ileum] if detectable, Brain [cerebellum, mid-brain, cortex], Pituitary gland, Caecum, (Preputial gland), Cervix Prostate gland, (Clitoral gland), Rectum, Colon, Salivary glands - mandibular, sublingual, Duodenum, Sciatic nerve, Epididymides*, Seminal vesicles including coagulating gland, Eyes with optic nerve [if detectable] and Harderian gland*, (Skeletal muscle), Skin, Female mammary gland area, Spinal cord -cervical, midthoracic, lumbar, (Femur including joint), Spleen, Heart, Sternum with bone marrow, Ileum, Stomach, Jejunum, Testes*, Kidneys, Thymus, Larynx, Thyroid including parathyroid [if detectable], (Lacrimal gland, exorbital), (Tongue), Liver, Trachea, Lung, infused with formalin, Urinary bladder, Lymph nodes - mandibular, mesenteric, Uterus, (Nasopharynx), Vagina, Oesophagus, All gross lesions

* Fixed in modified Davidson's solution, prepared at WIL Research Europe using Formaldehyde 37-40%, Ethanol, Acetic acid- glacial and Milli-Ro water. Tissues were transferred to formalin after fixation for at least 24 hours.

Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

All organ and tissue samples were processed, embedded in paraffin wax, cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin. The following slides were examined by a pathologist:
- all tissues collected at the scheduled sacrifice from all Group 1 and 4 animals,
- all tissues from male no. 18 (Group 2) which died spontaneously,
- all gross lesions.
All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings. Histopathology was subjected to a peer review.

ORGAN WEIGHTS
The following organ weights and terminal body weight were recorded from the surviving animals on the scheduled day of necropsy:
Adrenal glands, Spleen, Brain, Testes, Epididymides, Thymus, Heart, Uterus (including cervix), Kidneys, Prostate**, Liver, Seminal vesicles including coagulating glands**, Ovaries, Thyroid including parathyroid
** weighed when fixed for at least 24 hours.
Statistics:
The following statistical methods were used to analyze the data:

- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Ref. 1; manyto-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (Ref. 2; many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Ref. 3) was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test (Ref. 4) was applied to motor activity data to determine intergroup differences.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY
No mortality occurred that was considered to be of toxicological relevance. One male at 30 mg/kg (no.18) was found dead on Day 29. Necropsy of this animal revealed a perforation of the oesophagus and watery-clear contents in the thoracic cavity, which strongly suggests that this death was due to a gavage incident. Also, since no further mortality occurred in this dose group or higher dose groups, this death was considered to be unrelated to treatment with the test substance.
Animals at 750 mg/kg showed an increased incidence of rales. No abnormalities were noted during weekly arena observations. Regurgitation, laboured respiration and/or rales were incidentally noted for one or two males and/or females at 30, 150 and 750 mg/kg during the first few weeks of treatment. At 750 mg/kg, rales occurred in a higher number of animals than at 30 and 150 mg/kg, but remained of minimal severity and generally occurred on a few days only for each of these individual animals. Also, laboured respiration and/or rales at 30 and 150 mg/kg were noted at a similar incidence in control animals. Therefore, these findings were considered to be partly related to the dose volume administered, and considered to be of no toxicological relevance. Salivation seen after dosing among all animals at 30, 150 and 750 mg/kg (and incidentally also for some control animals) during the larger part of the treatment period was considered to be a physiological response rather than a sign of systemic toxicity, considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). For the test substance treated groups, this sign may be related to taste of the test substance. Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological significance.

BODY WEIGHT AND WEIGHT GAIN
Body weights and body weight gain was considered to have been unaffected by treatment.
The statistically significantly lower body weight gain of males between Weeks 5 and 7 was minor in nature and did not become more pronounced as treatment progressed. Absolute body weight remained similar to control levels throughout treatment. Therefore, these changes were considered to be unrelated to treatment.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption before or after correction for body weight remained similar to the control level over the study period.

OPHTHALMOSCOPIC EXAMINATION
No ophthalmology findings were noted that were considered to be related to treatment.
The nature and incidence of ophthalmology findings noted during pretest 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 substance.

HAEMATOLOGY
Males at 750 mg/kg showed a lower activated partial thromboplastin time (APTT). Other haematological parameters of treated rats were considered not to have been affected by treatment. Any statistically significant changes in haematology parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend and/or remained within the range considered normal for rats of this age and strain.

CLINICAL CHEMISTRY
The following statistically significant changes in clinical biochemistry parameters were considered to be related to treatment:
- Higher alanine aminotransferase activity in females at 750 mg/kg,
- Lower total protein level in males at 750 mg/kg,
- Lower albumin level in males at 750 mg/kg.
Other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment since no clear dose-related trend was noted, means remained essentially within the range considered normal for rats of this age and strain and/or control levels were considered
slightly low (sodium). In case of the lower total bilirubin level recorded for males at 750 mg/kg, an increase would be expected in case of target organ toxicity.

NEUROBEHAVIOUR
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 WEIGHTS
Organ weights and organ to body weight ratios of treated animals were similar to those of control animals.

GROSS PATHOLOGY
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.
The incidence of necropsy findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, did not show a dose-related incidence trend and/or had no treatment-related histopathological correlates. These necropsy findings were therefore considered to be of no toxicological relevance.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no microscopic findings recorded which could be attributed to treatment with the test substance.
All microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.

Effect levels

Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw (total dose)
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
From the results presented in this report a definitive No Observed Adverse Effect Level (NOAEL) for the test article of 750 mg/kg was established.
Executive summary:

In a 90d repeated dose study following OECD guideline 408 and under GLP quality control, the test substance, formulated in propylene glycol, was administered daily for at least 90 days by oral gavage to SPF-bred Wistar rats at dose levels of 0, 30, 150 and 750 mg/kg body weight. One control group and three treated groups were tested, each consisting of 10 males and 10 females.

Evaluated parameters Chemical analyses of formulations were conducted in Weeks 1, 6 and 13 of the study to assess accuracy and homogeneity. Stability was determined over 6 hours at room temperature and over 8 days in the refrigerator (under nitrogen).

The following parameters were evaluated: clinical signs daily; functional observation tests in Week 12-13; body weight and food consumption weekly; ophthalmoscopy at pretest and in Week 13; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.

Formulation analyses confirmed that formulations of test substance in propylene glycol were prepared accurately and homogenously, and were stable over at least 6 hours at room temperature and over 8 days in a refrigerator under nitrogen. The increased incidence of rales among animals at 750 mg/kg was considered to be of no toxicological relevance since this clinical sign remained of minimal severity and generally occurred on a few days only for each of the individual animals showing this symptom. No signs of ill-health were

recorded among any animal and body weight and food consumption values were considered to have been unaffected by treatment.

Clinical pathology changes recorded at 750 mg/kg consisted of a higher alanine aminotransferase activity in females, and lower total protein and albumin level in males. Since these changes occurred in the absence of supportive histopathological (liver) lesions, these were considered not to be adverse in nature. The lower activated partial thromboplastin time of males at 750 mg/kg was considered to be of no toxicological relevance since this change was slight in nature (mean remained within the range considered normal for rats of this age and strain), and an increase would be expected in case of target organ toxicity. No treatment-related changes were noted in any of the other parameters investigated in this study (i.e. functional observations, ophthalmoscopy, macroscopic examination, organ weights and microscopic examination). From the results presented in this report a definitive No Observed Adverse Effect Level (NOAEL) of 750 mg/kg was established.