Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From December 5, 2011 to August 06, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Version / remarks:
1981
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3200 (Repeated Dose Dermal Toxicity -21/28 Days)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Commission Regulation (EC) No 440/2008
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
[(3S,4R,4aR,6S,6aS,12R,12aS,12bS)-3-(cyclopropanecarbonyloxy)-6,12-dihydroxy-4,6a,12btrimethyl-11-oxo-9-(pyridin-3-yl)-1,2,3,4,4a,5,6,6a,12a,12b-decahydro-11H,12Hbenzo[f]pyrano[4,3-b]chromen-4-yl]methylcyclopropanecarboxylate
Cas Number:
915972-17-7
Molecular formula:
C33H39NO9
IUPAC Name:
[(3S,4R,4aR,6S,6aS,12R,12aS,12bS)-3-(cyclopropanecarbonyloxy)-6,12-dihydroxy-4,6a,12btrimethyl-11-oxo-9-(pyridin-3-yl)-1,2,3,4,4a,5,6,6a,12a,12b-decahydro-11H,12Hbenzo[f]pyrano[4,3-b]chromen-4-yl]methylcyclopropanecarboxylate
Test material form:
solid
Details on test material:
- Analytical purity: >94 %
Specific details on test material used for the study:
Lot number: 080722
Expiry: 31 July 2013
Appearance: solid/yellow

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Details on species / strain selection:
The rat is a frequently used Iabaratory animal, and there is comprehensive experience with this animal species. Moreover, the rat has been proposed as a suitable animal species by the OECD and the EPA.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
Source: Charles River Laboratories, Research Models and Services GmbH, Germany
Age at treatment start: 63 ± 1 days
Weight at dosing: 260.3-264.0 g (males) and 176.7-180.6 g (females)
Housing: Housed individually in Makrolon type M III cages (Becker &Co., Castrop-Rauxel, Germany, floor area of about 800 cm2).
Diet: ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland. ad libitum
Water: tap water in bottles, ad libitum
Acclimation period: about 6 days

ENVIRONMENTAL CONDITIONS
Temperature (°C): 20 - 24 °C
Humidity (%): 30-70 %;
Air changes (per hr): 15 per hour
Photoperiod (hrs dark/hrs light): 12 hours/day (light on at 06:00 and off at 18:00)

Administration / exposure

Type of coverage:
semiocclusive
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
Suspension of 1%
Details on exposure:
TEST SUBSTANCE PREPARATION
To prepare this suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, drinking water containing 1% carboxymethylcellulose was filled up to the desired volume, subsequently released with Ultraturax. During administration of the test substance, preparations were kept homogeneaus by stirring with a magnetic stirrer. The test-substance preparations were produced at least once a week.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance in drinking water containing 1 % carboxymethylcellulose at room temperature over a period up to 5 days was demonstrated before the administration period. At the beginning of the administration period samples were taken from the lowest and highest concentration for homogeneity analysis.Additional concentration control analyses of the test substance preparations were performed in samples of all concentrations at the start of the administration period. The test substance was homogeneously distributed ranging form 90-110 % of the nominal concentration.
Duration of treatment / exposure:
4 weeks (males: 21 applications, females: 22 applications); applied uniformly to the clipped dorsal skin (dorsal and dorsolateral parts of the trunk; at least 10 % of the body surface)
Frequency of treatment:
Every week day
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 rats/sex/group
Control animals:
yes, concurrent no treatment
Details on study design:
RANDOMISATION
Prior to the first detailed clinical observation, the rats were distributed according to weight among the individual test groups, separated by sex. The weight variation of the rats used did not exceed 20 percent of the mean weight of each sex. The list of randomization instructions was compiled with a computer.

The test substance was applied uniformly to the clipped dorsal skin (dorsal and dorsolateral parts of the trunk; at least 10 % of the body surface) using 3 mL syringes. The application volume was 5 mL/kg bw, based upon the latest individual body weight determination. The skin was covered for at least 6 hours after administration using a semiocclusive dressing, consisting of 4 layers of porous gauze dressing and a stretch bandage. After removal of the dressing, the treated skin was washed with lukewarm water. Control animals received the vehicle, only.

The first clipping was carried out at least 24 hours before the randomization. The rats were reclipped at least once a week (depending on hair growth).

All rats were sacrificed after a fasting period (withdrawal of food) of at least 16 hours.
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS
- Mortality and moribundity: twice daily on working days, once daily on Saturdays, Sundays and public holidays
- Clinically abnormal signs: daily

DETAILED CLINICAL OBSERVATIONS
- 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).
- The following parameters were examined: abnormal behavior during “handling”, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmus, feces (appearance/consistency), urine, pupil size

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

DRINKING WATER CONSUMPTION
- daily visual inspection of the water bottles for any overt changes in volume.

BODY WEIGHT
- before the start of the administration period
- during the administration period 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.

FUNCTIONAL OBSERVATIONAL BATTERY(FOB)
- at the end of the administration period.
- passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests and reflex tests.
- The findings were ranked according to the degree of severity, if applicable.

Home cage:
The animals were observed in their closed home cages for: posture, tremor, convulsions, abnormal movements, impairment of gait, other findings

Open field:
The animals were transferred to a standard arena (50 x 50 cm with sides of 25 cm high) and observed for at least 2 minutes for: behavior when removed from cage, fur, skin, salivation, nose discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements, impairment of gait, activity/arousal level, feces (number of fecal pellets/appearance/consistency) within two minutes, urine (appearance/quantity) within two minutes, number of rearings within two minutes

Sensorimotor tests/reflexes
The animals were removed from the open field and subjected to following sensorimotor or reflex tests: approach response, touch response, vision ("visual placing response"), pupillary reflex, pinna reflex, audition ("startle response"), coordination of movements ("righting response"), behavior during "handling", vocalization, pain perception ("tail pinch"), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test, other findings

Motor activity assessment
- at the end of the administration period.
- same day as the FOB was performed.
- The examinations were performed using the TSE Labmaster System. 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. 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.

OPHTHALMOSCOPY
- prior to the start of the administration period.
- At the end of the administration period, i.e. study day 28, the eyes of animals in test groups 0 (control) and 3 (1000 mg/kg bw/d) were examined for any changes using an ophthalmoscope after administration of a mydriatic.

HAEMATOLOGY
Time schedule for collection of blood: Blood was drawn in the morning
Anaesthetic used for blood collection: Yes ; isoflurane
How many animals: 10 animals/sex/group
Parameters in the table below were examined: Leukocyte count (WBC), Erythrocyte count (RBC), Haemoglobin (HGB), Haematocrit (HCT), Mean corpuscular volume (MCV), Prothrombin time (Hepato Quick’s test; HQT), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes
Blood smears were prepared and stained according to Wright without being evaluated due to non-ambiguous results of the differential blood cell counts

CLINICAL CHEMISTRY
Time schedule for collection of blood: Blood was drawn in the morning
How many animals: 10 animals/sex/group
The following parameters were 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)
Sacrifice and pathology:
NECROPSY
The animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.

ORGAN WEIGHTS AND FIXATION
- all animals sacrificed at scheduled dates
- anesthetised animals, adrenal glands, brain, epididymides, heart, kidneys, liver ovaries, spleen, testes, thymus, thyroid glands, uterus with cervix
- fixed in 4 % formaldehyde solution or in modified Davidson’s solution: all gross lesions, adrenal glands, aorta, brain, bone marrow (femur), cecum, cervix, colon, duodenum, epididymides, esophagus, eyes with optic nerve (modified Davidson's solution), heart, ileum, jejunum, kidneys, larynx, liver, lungs, lymph nodes (axillary and mesenteric lymph nodes), mammary gland, nose (nasal cavity), ovaries, pancreas, parathyroid glands, pharynx, pituitary gland, prostate, rectum, salivary glands (mandibular and sublingual glands), sciatic nerve, seminal vesicles, skeletal muscle, skin treated and untreated, spinal cord (cervical, thoracic and lumbar cord), stomach (forestomach and glandular stomach), spleen, target organs, testes (modified Davidson's solution), thymus, thyroid glands, trachea, urinary bladder, uterus

From the livers, one additional slice of the Lobus dexter medialis and the Lobus sinster lateralis was fixed in Carnoy's solution and embedded in paraplast.

HISTOPATHOLOGY
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings.
All organs were stained with hematoxylin and eosin (H&E), lungs as well embedded in paraplast (test group 1 and 2)
Statistics:
CLINICAL EXAMINATIONS
Body weight, body weight change and food consumption: A comparison of each group with the control group was performed using DUNNETT's
test (two-sided) for the hypothesis of equal means
Feces, rearing, grip strength forelimbs, grip strength hindlimbs, foot-splay test, motor activity: Non-parametric one-way analysis using KRUSKAL- WALLIS test (two-sided). lf 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:
For parameters with bidirectional changes: 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
For parameters with unidirectional changes: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians

PATHOLOGY
Weight parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). lf 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 the WILCOXON test for the hypothesis of equal medians

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No treatment related clinical signs of toxicity were observed throughout the study.

.
Dermal irritation:
effects observed, non-treatment-related
Description (incidence and severity):
One female animal of test group 2 (300 mg/kg bw/d) and three female animals of test group 3 (1000 mg/kg bw/d) showed lesions caused by repeated shearing. The finding was assessed as being not related to the test substance.
Mortality:
no mortality observed
Description (incidence):
No mortality was observed in this study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight and body weight changes were not affected by treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No clear treatment-related effects on food consumption were observed.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No test-substance influence on water consumption was observed.
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related effects observed.

All apparent findings were assessed as being incidental in nature since they occurred in individual animals only and did not show a dose-response relationship.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes in the hematological parameters were found.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes in the clinical chemistry parameters were found.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
FOB
There were no treatment related effects observed.

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.

MOTOR ACTIVITY MEASUREMENT
The single interval No. 5 was significantly decreased in male animals of test groups 1, 2 and 3 ( 100, 300 and 1000 mg/kg bw/d). The overall motor activity was significantly increased in female animals of test group 1 (100 mg/kg bw/d).

However, there were no significant deviations in the overall motor activity (summation of all intervals) in male animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d) and no significant deviations in the single intervals in female animals of test group 1 (100 mg/kg bw/d) in comparison to the concurrent control group. Thus, these findings were assessed as being not relevant.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment related changes in absolute or relative organ weight were observed.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All gross lesions observed in test animals occurred singularly and were considered to be spontaneaus in origin and are not related to treatment.

Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
In the treated skin, minimal multifocal hyperkeratosis of parakeratotic type (parakeratosis) was observed through all test groups including also control animals. The following incidence was found:
1000 mg/kg bw/d
Male: 4/10; female 5/10
300 mg/kg bw/d
Male 2/10; female: 4/10
100 mg/kg bw/d
Male: 2/10; female: 2/10
Control
Male: 1/10; female 1/10

The increased incidence observed in females of the 300 mg/kg bw/d dose group and males and females the 1000 mg/kg bw/d dose groups rather regarded as incidental since the severity of the parakeratosis remained minimal in both treated as well as control animals.

All other histopathological findings occurred either individually or were biologically equally distributed over the control group and the treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Histopathological findings: neoplastic:
not examined

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Remarks on result:
other: There were no adverse local and systemic effects at any dose level, including the limit dose.

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion