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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:
2012-12-11 - 2013-10-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (GLP)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
adopted 21 Sep 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report):Hydrocypivalic acid neopentylglycolester
- Physical state: solid, flakes/whit
- Analytical purity: 99.0 %
- Lot/batch No.: 95078568E0
- Stability under test conditions: the stability of the test substance under storage conditions over the test period was guaranteed.
- Storage condition of test material: room temperature; protect against moisture; avoid temperatures >40°C
- Other: the test substance was homogeneous (visually)

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Crl:WI(Han) from Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
- Age at study initiation: 33 ± 1 days when supplied, 42 ± 1 days at the start of the administration period
- Housing: 5 animals per cage in polysulfonate cages supplied by TECNIPLAST, Hohenpeißenberg, Germany (floor area about 2065cm2).
Motor activity measurements were conducted in polycarbonate cages (floor area about 800 cm2) supplied by TECNIPLAST, Hohenpeißenberg,
Germany
- Diet: ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland; ad libitum
- Water: drinking water from water bottles; ad libitum


ENVIRONMENTAL CONDITIONS (fully air-conditioned rooms in which central air conditioning)
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
drinking water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Hydroxypivalic acid neopentylglycolester was applied as a solution. To prepare this solution, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, drinking water was filled up to the desired volume, subsequently released manually. The test-substance preparations were produced at least weekly.

VEHICLE
Concentration in vehicle: 100, 300 and 1000 mg/kg bw dose groups.
The concentration control analyses revealed values in the expected range of the target concentrations. The means of the nominal concentrations of the samples taken at the beginning of the study were in a range of 98.8-104.5% of the nominal concentrations. The means of the nominal concentrations of the samples from test substance preparations prepared at the end of the study were also in a range of 99.5-105.0% of the nominal
concentrations.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance in drinking water at room temperature for a period of 7 days was demonstrated before the start of the study
(BASF project No.: 01Y0084/03Y007; PART III, Supplement).
The concentration control analyses revealed values in the expected range of the target concentrations. The means of the nominal concentrations of the samples taken at the beginning of the study were in a range of 98.8-104.5% of the nominal concentrations. The means of the nominal concentrations of the samples from test substance preparations prepared at the end of the study were also in a range of 99.5-105.0% of the nominal
concentrations.
Duration of treatment / exposure:
92 (male rats) and 95 days (female rats)
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the following dose levels were selected for the present study: 1000 mg/kg bw/day as highest dose, 300 mg/kg bw/day as mid dose, and 100 mg/kg bw/day as low dose.
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
All animals were checked daily for any clinically abnormal 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.
Time schedule: a check for moribund and dead rats was made twice daily on working days and once daily on Saturdays, Sundays and public#
holidays.

DETAILED CLINICAL OBSERVATIONS: Yes
Cage side observations included: abnormal behavior in handling, fur, skin, posture, salivation, respiration, activity/ arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, assessment of the feces discharged during the examination (appearance/consistency), assessment of the urine discharged during the examination, pupil size.

BODY WEIGHT: Yes
Time schedule for examinations: before the start of the administration period, on study 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 study day 0 was calculated as body weight change.

FOOD CONSUMPTION
Food consumption was determined weekly over a period of 1 day and calculated as mean food consumption in grams per rat and day.

WATER CONSUMPTION
Drinking water consumption was determined weekly as representative value over a period of
3 days and calculated as mean water consumption in grams per animal and day.

OPHTHALMOSCOPIC EXAMINATION: Yes
Time schedule for examinations: prior to the start and at the end of the administration period .
At the end of the administration period, i.e. study day 85, the eyes of animals in test groups 0 (control) and 3 (1000 mg/kg bw/d) were examined for any changes using an ophthalmoscope.

HAEMATOLOGY: Yes
Parameters examined: leukocyte count (WBC), erythrocyte count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), differential blood count, reticulocytes,
prothrombin time (Hepato Quick’s test; HQT).

CLINICAL CHEMISTRY: Yes
Parameters examined: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT), sodium (NA), potassium (K), Chloride (Cl), Inorganic phosphate (INP), calcium (Ca), urea (UREA), creatinine (CREA), glucose (GLUC), total bilirubin (TBIL), total protein (TPROT), albumin (ALB), globulins (GLOB), triglycerides (TRIG), cholesterol (CHOL).

URINALYSIS: Yes
Time schedule for collection of urine: day 85
Parameters examined: pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity, sediment, color (turbidity), volume.


SPERM PARAMETERS:
Motolity; morphology, head count (cauda epididymis), head count (testis)

NEUROBEHAVIOURAL EXAMINATION: Yes
A functional observational battery (FOB) was performed in all animals at the end of the administration period starting at about 10:00 h. At least one hour before the start of the FOB the rats were transferred singly to polycarbonate cages. Drinking water was provided ad libitum whereas no food was offered during the measurements. The FOB started with passive observations without disturbing the rats, followed by removal from the home cage, open field observations in a standard arena and sensory motor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.

Home cage observations:
The rats were observed in their closed home cages; any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the rats. Attention was paid to:
1. Posture
2. Tremors
3. Convulsions
4. Abnormal movements
5. Gait abnormalities

Open field observations:
The rats were transferred to a standard arena (50 × 50 cm with sides of 25 cm height) and observed for at least 2 minutes. Following parameters were examined:
1. Behavior on removal from the cage
2. Fur
3. Skin
4. Salivation
5. Nasal discharge
6. Lacrimation
7. Eyes/ pupil size
8. Posture
9. Palpebral closure
10. Respiration
11. Tremors
12. Convulsions
13. Abnormal movements/ stereotypes
14. Gait abnormalities
15. Activity/ arousal level
16. Feces excreted within 2 minutes (number/ appearance/ consistency)
17. Urine excreted within 2 minutes (amount/ color)
18. Rearing within 2 minutes

Sensory motor tests/reflexes
The rats were removed from the open field and subjected to following sensory motor or reflex tests:
1. Reaction to an object being moved towards the face (Approach response)
2. Touch sensitivity (Touch response)
3. Vision (Visual placing response)
4. Pupillary reflex
5. Pinna reflex
6. Audition (Auditory startle response)
7. Coordination of movements (Righting response)
8. Behavior during handling
9. Vocalization
10. Pain perception (Tail pinch)
11. Grip strength of forelimbs
12. Grip strength of hindlimbs
13. Landing foot-splay test
14. Other findings

Measurement of motor activity
The motor activity assessment (MA) was carried out in all animals per sex and group at the end of the administration period. Motor activity was measured on the same day as FOB was performed. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For this purpose, the animals were placed in new clean polycarbonate cages for the time of measurement. Eighteen beams were allocated per cage. The numbers of beam interrupts were counted over 12 intervals of 5 minutes. The sequence at which the animals were placed in the polycarbonate cages was selected at random. Motor activity measurements were carried out starting at 14:00 h. On account of the measuring variant "staggered", the starting time varied according to the time needed to place the animals in the cages. For each animal, measurement started individually when the 1st beam was interrupted and was finished exactly 1 hour later. The animals did not receive any food or water during the measurements. After the transfer of the last animal the room of measurement was darkened. The program required a file name for the measured data to be
stored. This name consisted of the reference number and a serial number.

OTHER:
Estrous cycle determination
Vaginal smears for estrous cycle determination were be prepared in the morning and evaluated according to the timetable for at least 3 weeks. Differentiation was conducted for the evaluation of cycle length and normality. Moreover, at the scheduled necropsy a vaginal smear was microscopically examined to determine the stage of the estrous cycle for each female animal. The samples were disposed after examination.
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. 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 vesicle with coagulating glands, spleen, testes, thymus, thyroid glands, uterus with cervix

HISTOPATHOLOGY: Yes; the following organs or tissues were fixed in 4% buffered formaldehyde solution or in modified Davidson’s solution: all gross lesions, adrenal glands, aorta, bone marrow (femur), brain, cecum, cervix, coagulation glands, colon, duodenum, epididymides left(modified Davidsons´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 Davidsons´s solution), thymus, thyroid glands, trachea, urinary bladder, uterus, vagina.
The left testis and left epididymis of all animals sacrificed at scheduled dates were fixed in modified Davidson’s solution, whereas the right testis and epididymis was used for sperm parameters
Statistics:
- Clinical observations: body weight and body weight change were analyzed by a comparison of each group with the control group using DUNNETT's test (two-sided) for the hypothesis of equal means. Feces, rearing, grip strength forelimbs, grip strength hindlimbs, footsplay test and motor activity 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.
- Clinical pathology: Blood parameters: parameters with bidirectional changes gravity 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. Parameters with unidirectional changes were analysed pairwise with the control group using the WILCOXON-test (one-sided) with Bonferroni-Holm adjustment for the hypothesis of equal medians. Urinalysis parameters (apart from pH, urine volume, specific gravity, color and turbidity pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.Urine pH ,volume, specific gravity ,color and turbidity were analyzed by non-parametric one-way analysis using KRUSKAL-WALLIS test . Spermanalysis parameters were analysed by pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) with Bonferroni-Holm adjustment.
- 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 AND MORTALITY
No rat died prematurely in the present study.
During clinical examinations treatment-related, adverse effects were observed directly after test substance administration at a dose level of 1000 mg/kg bw/d. Head shaking was observed in all male and female animals. Further signs of systemic toxicity, e.g. unsteady gait (slight impairment of coordination), semi-closed or completely closed eyelids, were observed in several male and female animals. These findings were reversible and only observed between 2 to 5 hours after application. The effect was of neuropharmacological nature as it could not be observed later and no neuropathological lesions were observed. No changes of toxicological relevance were observed in animals at dose levels of 100 and 300 mg/kg bw/d.

BODY WEIGHT AND WEIGHT GAIN
No test substance-related changes of body weight and body weight change were observed in any test group.

FOOD CONSUMPTION
No test substance-related effects on food consumption were obtained.

WATER CONSUMPTION
No test substance-related effects on water consumption were obtained. Over the entire application period the observed deviations in water-consumption values for male and female animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d) were assessed as being incidental and not related to
treatment.

OPHTHALMOSCOPIC EXAMINATION
There were no treatment-related findings. All apparent findings were assessed as being incidental in nature since they occured in individual animals only and did not show a dose-response relationship.

HAEMATOLOGY
No treatment-related changes among hematological parameters were observed. At the end of the study, in females of test group 3 (1000 mg/kg bw/d) absolute eosinophil counts were higher compared to controls, but the values were within the historical control range (absolute eosinophil counts 0.06-0.12 Giga/L). Therefore, this alteration was regarded as incidental and not treatment-related.

CLINICAL CHEMISTRY
No treatment-related changes among clinical chemistry parameters were observed. At the end of the study, in males of test groups 1 to 3 (100, 300 and 1000 mg/kg bw/d) glucose levels were lower compared to controls, but the means were within the historical control range (5.11-7.16 mmol/L
In females of test group 1 (100 mg/kg bw/d) inorganic phosphate levels were lower compared to controls, but the values were not dose-dependently changed. Therefore, the alterations of both mentioned parameters were regarded as incidental and not treatment-related.

URINALYSIS
No treatment-related changes among urinalysis parameters were observed. In males of test group 3 (1000 mg/kg bw/d) transitional epithelial cells and granulated and epithelial cell cast were found in the urine sediment. The casts were already observed in males of test group 2 (300 mg/kg bw/d). These effects were typical for male rats of this age. They were not found in the corresponding females. These findings were often observed in
α2u-globulinuria which was regarded as a rat specific finding without human relevance.
In rats of both sexes of test group 3 (1000 mg/kg bw/d), urine pH value was lower compared to controls. This effect per se was not regarded as an adverse effect. In females of this test group calcium oxalate, phosphate, tyrosin-like crystals and crystals of unknown content were
found in the urine sediment. No histopathologic finding could be correlated with the occurrence of these crystals and therefore, these changes were regarded as treatmentrelated, but not adverse. In males of test groups 1 and 2 (100 and 300 mg/kg bw/d) urine volume was increased and
specific gravity of the urine was decreased. These parameters were not dose-dependently changed and therefore the alterations were regarded as incidental and not treatment-related.

SPERM PARAMETERS
Concerning the motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as the sperm head counts in the testis and in the cauda epididymidis treatment-related effects were not observed.

CLINICAL PATHOLOGY
No treatment related, adverse effects were observed up to a dose level of 1000 mg/kg bw/d.

NEUROBEHAVIOUR
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 dose-response relationship or occurred in single rats only, these observations were considered to have been
incidental
Home cage observation: No test substance-related effects were observed.
Open field observations:
OneFemale animal of test group 3 (1000 mg/kg bw/d) showed piloerection and another female animal of the same test group showed respiratory
sounds. Taking the findings obtained during the clinical examinations into account, a relation to treatment was given.
No test substance-related effects were observed in male animals of test group 3 (1000 mg/kg bw/d) as well as in male and female animals of test groups 1 and 2 (100 and 300 mg/kg bw/d).
Sensorimotor tests/reflexes:
No test substance-related effects were observed.Swelling in the left anogenital region was observed in one female animal of test group 1
(100 mg/kg bw/d). Another female animal of test group 3 (1000 mg/kg bw/d) showed respiratory sounds during this tests. Both findings were assessed as being incidental and not related to treatment.
Quantitative parameters:
No test substance-related effects were observed.
Regarding the overall motor activity as well as single intervals, no test substance-related deviations were noted for male and female animals.
Female animals of test groups 2 (300 mg/kg bw/d) and 3 (1000 mg/kg bw/d) had an increased number of beam interrupts in single interval 8. These changes were assessed as being incidental and not related to treatment.

ORGAN WEIGHTS
Regarding pathology, the relative liver weights in males and females of test group 3 (1000 mg/kg bw/d) and the absolute and relative kidney weights of males of test group 3 (1000 mg/kg bw/d) were significantly increased. A treatment-related effect could not be excluded, but because there was no histopathological correlate, the weight increase was considered to be non-adverse. Thus, no treatment-related adverse findings were observed with regard to organ weights, gross lesions and microscopic findings.

ESTROUS CYCLE
No test substance-related effects on estrous cycle lenght and the number of cycles were obtained.

SPERM PARAMETERS
Concerning the motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as the sperm head counts in the testis and in the cauda epididymidis treatment-related effects were not observed.

Effect levels

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: transient clinical signs at 1000 mg/kg bw/d

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion