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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:
Oct 2015 - Nov 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- State of aggregation: solid/colorless

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Strain: Crl:WI (Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
- Age at study initiation: 36 +/- 1 day (age when supplied); 42 +/- 1 days (age at the bginning of the administration period)
- Weight at study initiation: compareable weight (+/- 20%)
- Housing: 5 animals per (polysulfonate cages supplied by TECNIPLAST)
- Diet (e.g. ad libitum): ad libitum (ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water (e.g. ad libitum):drinking water (ad libitum)
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: polyethylene glycol (PEG) 400
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was applied as a solution. To prepare this solution, the test substance was heated up at 38°C. Then the appropriate amount of test substance was weighed out depending on the desired concentration. Then, PEG 400 was filled up to the desired volume, subsequently mixed with a magnetic stirrer. The test-substance preparations were produced at least weekly and stored at room temperature. The administration volume was 5 mL/kg body weight.

VEHICLE
- Justification for use and choice of vehicle (if other than water): In the context of toxicological studies the stability of the test substance in the vehicle polyethylene glycol 400 has to be verified.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
15 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.
- All animals were checked daily for any abnormal clinically signs before the administration as well as within 2 hours and within 5 hours after the administration. Abnormalities and changes were documented for each animal.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations (DCO) were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 × 37.5 cm with sides of 25 cm high).
- DOC: Abnormal behavior when handled, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmos, feces (appearance/ consistency), urine, pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: before the start of the administration period in order to randomize the animals. During the administration period the body weight was determined on day 0 (start of the administration period) and thereafter at weekly intervals. The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as body weight change.

FOOD CONSUMPTION:
-Food consumption was determined weekly and calculated as mean food consumption in grams per animal and day.

WATER CONSUMPTION: Drinking water consumption was observed by daily visual inspection of the water bottles for any overt changes in volume.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Prior to the start of the administration period on day -3 the eyes of all animals and on study day 91 the eyes of the control and high-dose animals were examined for any changes using an ophthalmoscope

HAEMATOLOGY: Yes
- Time schedule for collection of blood: days 92 and 93 (start of administration period: day 0)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters examined: leukocyte count (WBC), erythrocyte count (RBC), hemoglobin (HCT), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), differential blood count, reticulocytes (RET), prothrombin time (Hepato Quick´s test) (HQT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: days 92 and 93 (start of administrtion period: day 0)
- Animals fasted: Yes
- How many animals: all
- Parameters examined: leukocyte count (WBC), erythrocyte count (RBC), hemoglobin (HCT), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), differtial blood count, reticulocytes (RET), prothrombin time (Hepato Quick´s test) (HQT)

URINALYSIS: Yes
- Time schedule for collection of urine: day 81
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examine: pH, protein (PRO), glucose (GLU), ketones (KET, urobilinogen (UBG), bilirubin (BIL), blood, specific gravity, sediment, color, turbidity (COL, TURB), volume (VOL).

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the administration period
- Dose groups that were examined: all
- Battery of functions tested: functional observation battery (FOB, including home cage observation, open field observation and sensory motor test reflexes) and motor activity assessment.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- The animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology. Female animal No. 80 of test group 3 (150 mg/kg bw/d), which died intercurrently and was
found severely cannibalized on study day 53, was therefore excluded from further investigation as those were no longer possible.
- The following weights were determined in all animals sacrificed on schedule: anesthetized animals, adrenal glands, brain, cauda epididymis, epididymides, heart, kidneys, liver, ovaries, pituitary gland, prostate, seminal vesicles incl. coagulating glands, spleen, testes, thymus, thyroid glands, uterus with cervix.

HISTOPATHOLOGY: Yes
- The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution or in modified Davidson’s solution:
all gross lesions, adrenal glands, aorta, bone marrow (femur), brain, cecum, cervix, coagulating gland, colon, duodenum, epididymis, left (modified Davidson’s solution), esophagus, extraorbital lacrimal glands, eyes with optic nerve (modified Davidson’s solution), femur with knee joint, harderian glands, heart, ileum, jejunum (with Peyer’s patches), kidneys, larynx, liver, lungs, lymph nodes (mesenteric and axillary lymph nodes), mammary gland (male and female), nose (nasal cavity), ovaries, oviducts, pancreas, parathyroid glands, pharynx, pituitary gland, prostate, rectum, salivary, glands (mandibular and sublingual glands), sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord (cervical, thoracic and lumbar cord), spleen, sternum with marrow, stomach (forestomach and glandular stomach), testis, left (modified Davidson’s solution), thymus, thyroid glands, trachea, urinary bladder, uterus, vagina.
The left testis and left epididymis of all male animals sacrificed at scheduled dates were fixed in modified Davidson’s solution, whereas the right testis and epididymis were used for sperm parameters.
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings.
Other examinations:
Estrous cycle determination: Estrous cycle length and normality were evaluated daily for all female animals for a minimum of 3 weeks prior to necropsy.
Sperm parameters: After the organ weight determination, the following parameters were determined in the right testis or right epididymis of all male F0 parental animals and cohort 1A males sacrificed on schedule: Sperm motility examinations were carried out in a randomized sequence.
Statistics:
Clinical examinations: body weight and body weight change were analyzed by a comparison of each group with the control group was performed using DUNNETT's test (two-sided) for the hypothesis of equal means. Rearing, grip strength forelimbs, grip strength hindlimbs, footsplay test, motor activity, estrous cycle were ananalyzed by a non-parametric one-way analysis using KRUSKALWALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise
comparison of each dose group with the control group was performed using WILCOXON test (two-sided) for the equal medians.

Clinical pathology: blood parameters, urine pH, volume and specific gravity were analyzed by non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians. Urinalysis parameters (apart from pH, urine volume, specific gravity, color and turbidity were analyzed pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians. In case of exactly the same numbers of the dose group and the control, no statistical test is performed. Sperm analysis parameters were analyzed pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) with Bonferroni-Holm adjustment for the hypothesis of equal medians; If only control and one dose group are
measured, WILCOXON-test (one-sided) without adjustment were used. For the percentage of abnormal sperms (ABNORMAL6_C) values <6% were set to 6% (cut off 6%).

Pathology: weight parameters were analyzed by non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians.

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS
Treatment-related adverse clinical findings were only observed in female animals of test group 3 (150 mg/kg bw/d) on study day 53. Shortly after test-substance administration, semiclosed eyelids were seen in 3 female animals, slight ataxia was observed in 7 female animals and abdominal position posture was observed in 2 female animals of test group 3 (150 mg/kg bw/d). Taken together, these findings, which only occurred in high-dose female animals, were assessed to be related to test substance administration.
No clinical findings of concern were observed for male animals of test group 3 as well as for male and female animals of test groups 1 and 2 (15 and 50 mg/kg bw/d). Slight salivation after test-substance administration was observed in 2 male and 4 female animals of test group 3 (150 mg/kg bw/d) on single study days. Moderate salivation was observed in one female animal of test group 3 (150 mg/kg bw/d) on study day 53 after testsubstance administration. From the temporary, short appearance immediately after dosing (or shortly before) it was concluded that slight to moderate salivation were induced by a bad taste of the test substance or local affection of the upper digestive tract.
Male animal No. 9 of test group 0 (control) showed a redly encrusted right eye from study days 76 to 83. Thereafter the eyeball was lost. As the animal belonged to the control group the finding was assessed to be incidental.
The left eyelid was swollen in male animal No. 33 of test group 3 (150 mg/kg bw/d) from study day 85 up to the end of the study. The finding was assessed to be incidental and not related to treatment.
MORTALITY
Female animal No. 80 of test group 3 (150 mg/kg bw/d) was found dead and cannibalized on study day 53 before test substance administration. The reason for the spontaneous death could not be evaluated because the animal was found severely cannibalized and a proper pathological examination was not possible anymore.
BODY WEIGHT AND WEIGHT CHANGES
No test substance-related changes of mean body weights and mean body weight change values were observed.
FOOD CONSUMPTION
No test substance-related findings were observed.
WATER CONSUMPTION
No test substance-related, adverse changes with regard to water consumption were observed.
HAEMATOLOGICAL FINDINGS
No treatment-related changes among hematological parameters were observed.
At the end of the administration period in females of test group 2 (50 mg/kg bw/d) mean corpuscular volume (MCV) was lower compared to controls. However, this change was not dose-dependent and, therefore, it was regarded as incidental and not treatment-related.
CLINICAL BIOCHEMISTRY FINDINGS
No treatment-related changes among clinical chemistry parameters were observed.
URINALYSIS FINDINGS
No treatment-related changes among urinalysis parameters were observed.
In males of test group 3 (150 mg/kg bw/d) higher incidences of granulated and epithelial casts were found in the urine sediment, which was most likely due to α2u-globulinuria, a rat-specific effect not relevant for humans (Hard et al., 1993). In females of test group 2 (50 mg/kg bw/d) urine volume was lower compared to controls. This change was not dose-dependent and, therefore, it was regarded as incidental and not treatment-related.
BEHAVIOUR (FUNCTIONAL FINDINGS)
Functional abservational battery:
Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test-substance treated groups and controls, were without a doseresponse relationship or occurred in single rats only, these observations were considered to have been incidental.
The following examinations were performed during FOB and have to be assessed individually:
Home cage observations: No test substance-related effects were observed.
Open field observations: The loss of the right eyeball in male animal No. 9 of test group 0 (control). The left eyelid of male animal No. 33 of test group 3 (150 mg/kg bw/d) was swollen. Both findings were assessed to be incidental and not related to treatment.
Sensorimotor tests/reflexes: No test substance-related effects were observed.
Quantitative parameters: No test substance-related effects were observed.
Regarding the overall motor activity as well as single intervals, no test substance-related deviations to the control animals were noted.
ORGAN WEIGHT FINDINGS
Absolute organ weights: When compared with control group 0 (set to 100%), the mean absolute weight of thyroid glands of female test group 1 animals was significantly decreased. As there was no observable dose-response relationship and the absolute weights of test group 3 female animals were not significantly changed, the decreased thyroid weights of test group 1 females were regarded as incidental. No other mean absolute weight parameters in females and none in males showed significant differences when compared to the control group 0.
Relative organ weights: When compared with control group 0 (set to 100%), the mean relative liver weights of male animals were significantly increased in test groups 1 and 3. Relative weights of the liver of male animals of test group 1 (2.305%) and of test group 3 (2.343%) were slightly above historical controls (2.110-2.299%). Historical control values for terminal body weights of male animals range from 352.84 g to 386.52 g. Terminal body weights of test group 3 animals in this study were in the low range of historical controls (test group 1: 357.75 g) or slightly below this value (test group 3: 349.06 g). This circumstances were assumed to be the reason for the slightly increased relative liver weights in test group 3 animals. The mean absolute liver weights of male animals of test groups 1 and 3 of this study were within historical controls (historical controls: 7.611-8.493 g, test group 3: 8.182 g, test group 1: 8.253 g). Furthermore, there was no clear dose-response relationship observed and no histopathological correlate occurred in test group 3. All other mean relative weight parameters did not show significant differences when compared to the control group 0.
GROSS PATHOLOGICAL FINDINGS
All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
HISTOPATHOLOGICAL FINDINGS
In the kidneys of male animals, a treatment- and dose-related increase in severity of eosinophilic droplets visualized by CAB staining was observed (severity grades up to moderate in some treated animals compared to severity grades up to slight in controls). This was accompanied by a slightly increased incidence of tubular casts in the inner zone of the outer medulla in test group 2 and 3 animals. The incidence and severity of basophilic tubules was similar in all test groups. In animals 37 and 40, for which immunohistochemistry was performed, the staining pattern was similar to that described by Cesta et al. (2013). Therefore, the presence of alpha2µ-globulin (nephropathy) was likely. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
OTHER EFFECTS
No test substance-related effects on estrous cycle length and the number of cycles were obtained.
Concerning motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as sperm head counts in the testis and in the cauda epididymidis no treatment-related effects were observed.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No adverse effects observed.
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical signs

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The administration of 1-Ethinylcyclohexanol by gavage to male and female Wistar rats for 3 months caused test substance-related, adverse signs of systemic toxicity at a dose level of 150 mg/kg bw/d taking clinical findings in female animals into account. Findings related to tubular damage in kidneys of male animals do not represent a risk for humans and were not considered relevant for the determination of the no observed adverse effect level (NOAEL). Therefore, under the conditions of the present study the NOAEL was 150 mg/kg bw/d for male and 50 mg/kg bw/d for female Wistar rats.