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Toxicological information

Repeated dose toxicity: dermal

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Administrative data

Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Test material form:
solid - liquid: suspension
Remarks:
dispersion containing micronized ETH50 (nano form)
Details on test material:
Please refer to "Confidential details on test material".

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, l'Arbresle, France
- Age at study initiation: approximately 8 weeks old
- Body weight at acclimatization: 244 g (range: 222 g to 274 g) for the males, 166 g (range: 144 g to 186 g) for the females
- Housing: individually housed in suspended wire-mesh cages (43 x 21.5 x 18 cm)
- Diet: SSNIFF R/M-H pelleted maintenance diet, (SSNIFF Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water (filtered with a 0.22 µm filter), ad libitum
- Acclimation period: 7 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 50 ± 20 %
- Air changes: approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod: 12 hours / 12 hours, light/dark cycle

Administration / exposure

Type of coverage:
open
Vehicle:
other: Mixture of 80% ointment with 20% aqueous solution with 0.5% carboxymethylcellulose
Details on exposure:
TEST SITE
- Area of exposure: corresponding to approximately 10 % of body surface area (i.e. from 45 to 50 cm in males and from 30 to 35 cm in females according to their age/growth)
- % coverage: uncovered
- Time intervals for shavings or clipplings: animals were clipped whenever necessary, at least 4 hours before dosing and at least once a week

REMOVAL OF TEST SUBSTANCE
- Washing: using purified water and dried with a cotton pad
- Time after start of exposure: After a period of at least 6 hours after each application

TEST MATERIAL
- Amount applied: 2.5 mL/kg bw/day
- Concentration: The test item was administered as a suspension in the vehicle.
The test item was mixed with the required quantity of vehicle in order to achieve the concentrations of 60, 200 and 400 mg/mL (expressed as active component) or 126, 420 and 840 g/mL (expressed as test item as received).
The placebo control dosage form was obtained by mixing the required quantity of vehicle and placebo in order to achieve the concentration of 840 mg of placebo/mL. Therefore, concentration of excipients in the placebo control dosage form was equivalent to the concentration of excipients administered in the highest dosage group


USE OF RESTRAINERS FOR PREVENTING INGESTION:
- yes
- animals of high dose group II (1000 mg of active ingredient/kg/day) wore a protective plastic collar for a period of at least 6 hours after each application, in order to prevent ingestion of the test item
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
From each dosage form, duplicate samples were taken at three different levels of the container (top, middle, bottom) just after preparation (day 0) and after 3, 4 and 9 days of storage at room temperature and away from light; each aliquot sampled was analyzed immediately after sampling for concentration of the test tern to evaluate the homogeneity. The mean concentrations (n=6) measured on days 0, 3, 4 and 9 (mean of homogeneity assay) were taken as actual values for the stability test.
Duration of treatment / exposure:
The dosage forms were administered daily for a period of at least 13 weeks (i.e. 91 to 92 days according to the necropsy schedule).
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 150, 500 and 1000 mg/kg bw/day
Basis:
other: corresponding to 0, 315, 1050 and 2100 mg of test item formulation/kg bw/day,
No. of animals per sex per dose:
Group 1 Control (untreated) Principal 15 males + 15 females ( 0 mg/kg bw/day)
Group 2 Control (placebo/vehicle) Principal 15 males + 15 females, satellite 3 males + 3 females ( 0 mg/kg bw/day)
Group 3 low dose Principal 10 males + 10 females, satellite 3 males + 3 females ( 150 mg/kg bw/day)
Group 4 mid dose Principal 10 males + 10 females, satellite 3 males + 3 females ( 500 mg/kg bw/day)
Group 5 high dose I Principal 15 males + 15 females, satellite 3 males + 3 females ( 1000 mg/kg bw/day)
Group 6 high dose II Principal 15 males + 15 females, satellite 3 males + 3 females ( 1000 mg/kg bw/day)
Control animals:
other: untreated and placebo group
Details on study design:
- Dose selection rationale: the dose-levels were selected on the basis of the results of a preliminary 2-week cutaneous toxicity study performed in the same species (CIT/Study No. 32403)
- Rationale for selecting satellite groups: the satellite animals were allocated to toxicokinetics investigations

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily
- Mortality/Viability: Once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a week until the end of the study
- From day 1 to week 4, before each application, the site of application was examined for possible signs of irritation and edema on principal animals. From week 5, the site was examined weekly during the detailed clinical examination.
Cutaneous reactions at the application site were scored before each daily treatment according to the scale specified in OECD Guideline No. 404
- During the treatment period, the presence of chromodacryorrhea and/or chromorhinorrhea in animals of group 6 (due to the wearing of collar) was documented

FUNCTIONAL OBSERVATION BATTERY (FOB) (principal animals)
- All animals of groups 2 and 5 were evaluated once at the end of the treatment period. This included a detailed clinical examination, measurement of reactivity to manipulation or to different stimuli and motor activity. All animals were observed in the cage, in the hand and in the standard arena.
The following parameters were assessed and graded:
"touch escape" or ease of removal from the cage, in the hand: fur appearance, salivation, lachrymation, piloerection, exophthalmos, reactivity to handling, pupil size (presence of myosis or mydriasis), in the standard arena (2-minute recording): grooming, palpebral closure, defecation, urination, tremors, twitches, tonic and clonic convulsions, gait, arousal (hypo- and hyper-activity), posture, stereotypy, behavior, breathing, ataxia and hypotonia.
Then, the following parameter measurements, reflexes and responses were recorded:
touch response, forelimb grip strength, pupillary reflex, visual stimulus response, auditory startle reflex, tail pinch response, righting reflex,
landing foot splay, rectal temperature.
Motor activity of all animals was measured once by automated infra-red sensor equipment over a 60-minute period.


BODY WEIGHT: Yes
- Time schedule for examinations: once before group allocation, on the first day of treatment, and then once a week until the end of the study

FOOD CONSUMPTION:
- Time schedule for examinations: once a week
- Food consumption for each animal determined calculated as g food / animal / d: Yes

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- on all principal animals before the beginning of the treatment period and on all principal animals of the placebo control and high-dose groups 1 (groups 2 and 5) on one occasion at the end of the treatment period.


HAEMATOLOGY: Yes
- Time schedule for collection of blood: For principal animals at the end of the treatment period and for the recovery animals at the end of the treatment-free period (2 weeks).
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- Parameters examined: erythrocytes, hemoglobin, mean cell volume, packed cell volume, mean cell hemoglobin concentration, mean cell hemoglobin, thrombocytes, leucocytes, differential white cell count with cell morphology: neutrophils, eosinophils, basophils, lymphocytes, monocytes, prothrombin time, activated partial thromboplastin time, fibrinogen
- Other: Blood samples were taken from the orbital sinus of the animals (before the daily treatment)
- Bone marrow: Bone marrow smears were prepared from the femoral bone of all animals killed on completion of the treatment and treatment-free periods.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see HAEMATOLOGY
- Animals fasted: Yes
- How many animals: for principal animals at the end of the treatment period and for the recovery animals at the end of the treatment-free period
- Parameters examined: sodium, potassium, chloride, calcium, inorganic phosphorus, glucose, urea, creatinine, total bilirubin, total proteins, albumin, albumin/globulin ratio, total cholesterol, triglycerides, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase

URINALYSIS: Yes
- Time schedule for collection of urine: see HAEMATOLOGY
- Metabolism cages used for collection of urine: Yes (overnight period of at least 14 hours, urine was collected in the presence of thymol crystals)
- Animals fasted: Yes
- Parameters examined:
Quantitative: volume, pH, specific gravity; semi-quantitative: proteins, glucose, ketones, bilirubin, nitrites, blood, urobilinogen, leucocytes, erythrocytes, cylinders, magnesium ammonium phosphate crystals, calcium phosphate crystals, calcium oxalate crystals, cells;
qualitative: appearance, colour


PLASMA LEVELS OF THE TEST ITEM (SATELLITE ANIMALS)
- Blood samples for the determination of plasma levels of the test item were taken from each satellite animal in order to confirm systemic exposure to the test item as follows:
- on day 8, before the daily dose application,
- in week 13, before terminal sacrifice for groups 2 to 4, and on the first day of the treatment-free period for groups 5 and 6.

Sacrifice and pathology:
Sacrifice
On completion of the treatment or treatment-free period, after at least 14 hours fasting, all surviving principal animals were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and sacrificed by exsanguination.

ORGAN WEIGHTS: yes
The body weights were recorded for all principal animals before sacrifice at the end of the treatment and treatment-free periods.

The following organ weights were recorded on the scheduled dates of necropsy:

adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus, thyroid including parathyroid gland.

The organs were weighed wet as soon as possible after dissection. The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.

Macroscopic post-mortem examination
A complete macroscopic post-mortem examination was performed on all principal animals and on one found dead satellite female (group 6). This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues.

Preservation of tissues
For all study animals, excluding found dead, the tissues were preserved in 10 % buffered formalin except for the eyes with optic nerves and Harderian glands, and the testes and epididymides, which were fixed in Davidson's fixative. A bone marrow smear, for the determination of the bone marrow differential cell count, was prepared from the femur of each animal sacrificed on completion of the treatment and treatment-free periods.
Additional skin samples were taken from each principal animal (at the end of the treatment and treatment-free periods) as follows:
dose-site skin were taken in two separate pieces of 1cm x 1 cm approximately, and cut to include some of the underlying subcutaneous musculature, each piece was placed separately and immediately into liquid nitrogen to freeze the tissue completely, each sample was separately placed into a suitable container, labelled with animal number and study group number, samples were held frozen, dose-group separately, at -80 °C. One of these samples was randomly selected for one male and one female each only, from groups 1 (control), 2 (placebo), 4 (mid-dose) and 5 (high-dose). Other samples were kept frozen at -80 °C.

Microscopic examination
A microscopic examination was performed on:
- all the tissues for animals of the placebo control group and high-dose groups (groups 2, 5 and 6) sacrificed at the end of the treatment period and for 2 animals that died.
- all the macroscopic lesions of all the animals of the untreated group (group 1), low- and intermediate-dose groups (groups 3 and 4), sacrificed on completion of the treatment period.

All preserved tissue samples were processed, embedded and cut at an approximate thickness of approximately 4 micrometers, and stained with hematoxylin and eosin (except testes and epididymides which were stained with hematoxylin/PAS).

HISTOPATHOLOGY: Yes
A microscopic examination was performed on:
adrenals, aorta, brain (including medulla / pons cerebellar and cerebral cortex), cecum, colon, duodenum, epididymides, esophagus, eyes with Harderian glands, femoral bone with articulation, heart, ileum (with Peyers patches), jejunum, kidneys, liver, lungs with bronchi, lymph nodes (mandibular and mesenteric), mammary gland area, optic nerves, ovaries (with oviducts), pancreas, pituitary gland, prostate, rectum, salivary glands (sublingual and submandibular), sciatic nerves, seminal vesicles, skeletal muscle, skin, spinal cord (cervical, thoracic and lumbar), spleen, sternum with bone marrow, stomach with forestomach, testes, thymus, thyroids with parathyroids, tongue, trachea, ureters, urinary bladder, uterus (horns and cervix), vagina and macroscopic lesions.




Statistics:
Statistical analysis was performed as follows:
by comparison of untreated group (group 1) and placebo/vehicle control group (group 2); assigned control group: group 1,
by comparison of treated groups (groups 3 to 6) and vehicle control group (group 2); assigned control group: group 2,
by comparison of high-dose groups (group 5 versus group 6); assigned control group: group 5.

- Kolmogorov-Lilliefors test, log transformation if necessary; normality of distribution
- Bartlett test (three or more groups) or Fisher test (two groups); homogeneity of variances
- Bartlett test, if not homogenous: Dunn test
- Bartlett test, if homogenous: Dunnett test
- Fisher test, if not homogenous: Mann-Whitney / Wilcoxon test
- Fisher test, if homogenous: Students t-test

Results and discussion

Results of examinations

Details on results:
Mortality:
No test item-related deaths occurred.
One placebo control principal male was found dead during week 13. No relevant clinical signs were noted prior to death. At macroscopic post-mortem examination, red colored lungs were noted associated with red fluid in thoracic cavity. As it was noted in the control group,
this death was unrelated to the test item. One satellite female from the high-dose group II was found dead during week 10. No clinical signs occurred prior to death. A definitive cause of death was not established for this rat; however, moderate pulmonary edema was diagnosed as a terminal event. Since mortality only occurred in a single animal of this group before the end of the treatment period and since no other deaths occurred in the any other dose-groups, this isolated death was considered to be of no toxicological significance and unrelated to the treatment with the test item.

Clinical signs:
Scabs were noted at the application site in 6/15 males and 5/15 females from 5 given 1000 mg/kg bw/day (with no protective collar). This finding resolved by the end of the dosing period for almost all the animals except in 2/15 group 5 males. Clinical signs related to pain, such as abnormal vocalization and/or hyperactivity were observed, mainly from week 5, within the 30-minute period after treatment and generally for less than 30 minutes, in animals given the test item at the high dose-level of 1000 mg/kg bw/day, but mainly in the high-dose group with no protective collar.

Detailed clinical examination and Functional Observation Battery:
The functional test battery showed no test item treatment-related changes in any neurotoxicological parameter. Motor activity was not affected by the test item treatment.

Body weight:
When compared to untreated animals, a slightly lower mean body weight gain was observed in placebo control males (-15 % when compared to untreated controls) during the treatment period. This effect was attributed to the placebo treatment. In all males given 1000 mg/kg/day (groups 5 and 6) statistically significantly (p<0.05) lower mean body weight gain was recorded, principally from week 2 and during the whole study period. Mean body weight and mean body weight gains from both female high-dose groups (groups 5 and 6) were similar during the full study period. The group 5 and 6 males given 1000 mg/kg bw/day gained 13 and 9 grams more weight respectively than placebo controls during the treatment-free period, thereby showing full reversal of their body weight trends observed during the treatment period.

Food consumption:
Mean food consumption was not affected by the placebo or test item treatment in any period.

Ophthalmology:
There were no ophthalmological findings at the end of the treatment period.

Hematology and blood biochemistry:
There were no differences of toxicological significance between control and test item treated animals in any hematological or blood biochemical parameters. The lower glucose and triglycerides concentrations observed in test item high-dose group males (groups 5 and 6) at the end of the treatment period were considered to be related to the treatment procedure.

Urinalysis:
There were no disturbances in any urinalysis parameters at the end of the treatment period.

Organ weight:
Organ weight changes were recorded in the thymus of group 5 males at 1000 mg/kg bw/day, and the adrenal glands of group 5 females. Although no histopathologic changes were observed in these tissues in rats of this dose group, the changes were considered as non-specific indicators of a stress-related response.

Macroscopic post-mortem examination:
Except for scabs noted in the treated application site of 2 group 5 males at 1000 mg/kg bw/day, no test item related macroscopic findings were observed.

Microscopic examination
Epidermal hyperplasia and associated hyperkeratosis were noted in rats from groups 2 (0 mg/kg bw/day), 5 and 6 (both 1000 mg/kg bw/day), which received similar doses of excipients either directly or mixed with test item. These findings were considered likely to reflect mild non-specific irritant-effects related to the mechanical preparation of the application site and repeated treatments with equal excipient concentrations to the application site.
Minimal increased lymphocytolysis was recorded in a few rats of groups 5 and 6 (i.e. mainly females of group 5). This finding may reflect a minimal non-specific stress-related response related to the treatment procedure.


Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The dermal administration of the test item to rats up to and including 1000 mg/kg bw/d did not cause any biologically significant test item-related adverse effects.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Chemical analysis:

The results of the chemical analysis demonstrated satisfactory homogeneity and concentration of the test item in the vehicle for the respective dose formulation samples tested.

Plasma levels of the test item:

Both on day 8 and in week 13, plasma levels of the placebo group (group 2) were below quantifiable limit of 0.8 ng/mL. All group 3 to 6 animals showed quantifiable amounts of the active ingredient in plasma (up to 12 ng/ml ETH50) that persisted through the reversibility period. However, clear dose- and time- relationships could not be demonstrated because of the wide range of results. Overall, it can be said that during the study period, measurable amounts of the test item active ingredient could be found in plasma samples, suggesting that systemic exposure occurred following repeated dermal dosing.

Applicant's summary and conclusion