Registration Dossier

Administrative data

Description of key information

In a 90-day repeated dose toxicity test (comparable to OECD TG 408, acc. to GLP) by oral

gavage in male and female Sprague Dawley rats no toxicity occurred up to the limit dose of 1000 mg/kg bw/day ETH50. The NOAEL was determined to be 1000 mg/kg bw/day.
In a 90-day dermal repeated dose toxicity test (according to OECD TG 411) in male and female Wistar rats no adverse test substance related effects were noted up to the limit dose of 1000 mg/kg bw/day ETH50. The NOAEL was determined to be 1000 mg/kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004-09-28 - 2005-09-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Reliable guideline study performed under GLP.
Qualifier:
according to guideline
Guideline:
other: Note for guidance on repeated dose toxicity. Committee for proprietary medicinal products (CPMP/SWP/1042/99). European Agency for Evaluation of Medicinal Products, 27 July 2000.
Deviations:
no
Principles of method if other than guideline:
Test design comparable with OECD TG 408;
extended to the assessment of the following parameters: T3, T4, TSH, testosterone (males), estradiol and progesterone (females).
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
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 6 weeks old
- Body weight at acclimatization: 214 g (range: 202 g to 232 g) for the males, 155 g (range: 131 g to 171 g) for the females
- Housing: individually housed in suspended wire-mesh cages (43 x 21.5 x 18 cm)
- Diet: A04 C pelleted maintenance diet (SAFE, Villemoisson, Epinay-sur-Orge, France), ad libitum
- Water: tap water (filtered with a 0.22 µm filter), ad libitum
- Acclimation period: 8 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

IN-LIFE DATES:
- From: 2004-10-06 To: 2005-01-07
Route of administration:
oral: gavage
Vehicle:
other: 0.5 % (w/v) carboxymethylcellulose in purified water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a suspension in the vehicle. The test item was ground to a fine powder using a mortar and pestle, and then mixed with the required quantity of vehicle in order to achieve the concentrations of 10, 50 and 200 mg/mL, and then homogenized using a magnetic stirrer. The test item dosage forms were prepared on a weekly basis depending on their stability (i.e. 9 days) and were stored at +4° C prior to use and protected from light, according to the storage conditions of the test item.

VEHICLE
- Justification for use and choice of vehicle: solubility of the test item in 0.5 % CMC
- Concentration in vehicle: 10, 50 or 200 mg/mL
- Amount of vehicle: constant dose volume of 5 mL/kg bw/d
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of dose formulations of the test were analysed to check homogeneity, concentration and stability. Actual concentrations of the test item in the dosage forms were determined on weeks 1, 4, 8 and 13.
Duration of treatment / exposure:
90 days
Frequency of treatment:
once daily, 7 days/week
Remarks:
Doses / Concentrations:
0, 50, 250, 1000 mg/kg bw/d (groups 1,2,3,4, respectively)
Basis:
actual ingested
No. of animals per sex per dose:
Main study: 10 animals (all groups)
Recovery: 6 animals (control and high dose group only)
Toxicokinetics: 6 animals (low, mid and high dose group)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Results of a 2-week toxicity range finding oral study (CIT/Study No. 28340 TSR):

The administration of the test item by oral route (gavage) to Sprague-Dawley rats for 14 days was well tolerated when given at 300, 600 or 1000 mg/kg bw/day. The only variation was a decrease in the white blood cell and lymphocyte counts in females treated at 1000 mg/kg bw/day. No findings were noted at histopathology.
- Post-exposure recovery period in satellite groups: 4 weeks
Positive control:
not applicable
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

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
- Time and dose group schedule for examinations: all principal animals before the beginning of the treatment period and on all principal animals on one occasion at the end of the treatment (week 12)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During week 13: All principal animals from the groups 2 and 3 (low and mid dose group) and the last 10 animals from the groups 1 and 4; at the end of the treatment free period (week 18): The first 6 animals from groups 1 and 4 (control and high dose group)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all surviving main group animals
- 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)
- As no anaemia was observed, the reticulocyte count was not determined. As no relevant abnormalities were observed during hematological investigations, the bone marrow differential cell count was not determined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see HAEMATOLOGY
- Animals fasted: Yes
- How many animals: determined for all principal animals from the groups 2 and 3 and for the last 10 animals from the groups 1 and 4 during week 13 and for the first 6 animals from groups 1 and 4 at the end of the treatment free period (week 18)
- 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)
- 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

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: week 12
- Dose groups that were examined: all surviving animals
- Functional Observation Battery (FOB), detailed clinical examination; e.g. sensory activity / grip strength / motor activity

OTHER:
HORMONAL ASSAYS:
- Time schedule for collection of blood: week 6, 13 for all principal animals from the groups 2 and 3 and for the last 10 animals from the groups 1 and 4, and at the end of the treatment-free period for estradiol and progesterone determination in females for the first 6 animals from the groups 1 and 4 only
- Animals fasted: No
- How many animals: all surviving main group animals
- Parameters examined: testosterone (in males), estradiol and progesterone (in females), thyroid stimulating hormone (TSH) and thyroid hormones (T3 and T4) (in both sexes).

MONITORING OF ESTRUS CYCLE (determined from a fresh vaginal lavage)
- Time schedule:
- on the day of blood collection of hormonal evaluation (week 6, week 13),
- on each morning for the 2 consecutive weeks preceding the day of blood collection of hormonal evaluation in week 13,
- the 3 days preceding blood sampling for hormone analysis at the end of the treatment-free period.
- How many animals: all surviving main group animals (all principal females)

PLASMA LEVELS OF THE TEST ITEM (SATELLITE ANIMALS)
- Time schedule for collection of blood: weeks 1, 6 and 13 (at the end of the treatment period) as follows:
- day 2 predosing: 24 hours after dosing of day 1,
- weeks 6 and 13: 24 hours after dosing
- How many animals: all surviving satellite group animals
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, at the end of the treatment or recovery period
All animals were weighed and necropsied. Descriptions of all macroscopic abnormalities were recorded. All animals surviving to scheduled necropsy and all moribund animals were anesthetized by intraperitoneal injection of sodium pentobarbitone (NARCOREN) and killed by exsanguination.

The following organ weights were recorded on the scheduled dates of necropsy: adrenals, brain, epididymides, heart, kidneys, liver, ovaries, pituitary, spleen, testes, thymus, thyroid including parathyroid gland. The organ to terminal body weight ratios as well as organ to brain weight ratios were determined. The determination of the terminal body weight was performed immediately prior to necropsy.

All preserved tissue samples, as defined under Histopathology, 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:
(i) all preserved tissues of the control and high-dose groups (groups 1 and 4) sacrificed at the end of the treatment period: 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, larynx, 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.
(ii) all macroscopic lesions of all the animals of the low- and intermediate-dose groups (groups 2 and 3) sacrificed on completion of the treatment period
Tissues of rats from the treatment-free period were not examined since no target organs were identified.
Statistics:
Body weight, food consumption, hematology, blood biochemistry, urinalysis and organ weight data:
- 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
Details on results:
CAGE SIDE OBSERVATIONS:
No mortality occurred during the study period.
There were no clinical signs apart from piloerection in 1/10 males receiving 250 mg/kg bw/d and in 3/16 males receiving 1000 mg/kg bw/d, which had always disappeared by week 10. As it was not noted in females and not associated with other findings, this observation was considered to be most probably treatment-related but not to be an adverse effect. There was no treatment-related clinical sign in principal females or in satellite animals.

BODY WEIGHT
The treatment had no effect on body weight or body weight gain, whatever the sex and the treatment period.

FOOD CONSUMPTION
The treatment had no effect on food intake, whatever the sex and the treatment period

OPHTHALMOSCOPIC EXAMINATION
There was no treatment-related finding at ophthalmologic examination.

HAEMATOLOGY / CLINICAL CHEMISTRY
There was no biologically significant treatment-related finding at hematology/biochemistry.
On week 13, low- and high-dose males had moderately lower white blood cell counts when compared to controls (up to -25%), due to significantly lower lymphocyte and monocyte counts. Yet there was a high inter-animal variability (2-fold among controls males) and there was no
difference between the mid-dose and control groups. These changes were therefore considered not to be treatment-related. There was no significant WBC count alteration in females or at the end of the recovery period.

URINALYSIS
There was no biologically significant treatment-related finding at urinalysis.

NEUROBEHAVIOURAL EXAMINATION
Treatment had no effect on neurologic functions in any of the animals in each group and sex.

HORMONAL ASSAYS
The treatment had no influence on T3, T4 and TSH plasmatic levels. There were no biologically significant treatment-related alterations in sexual hormones.

MONITORING OF ESTRUS CYCLE
No disturbance in estrous cycle was recorded during the considered period.

GROSS PATHOLOGY / HISTOPATHOLOGY
Macroscopic examination showed no evidence of treatment related effects.
There was no biologically significant treatment-related change in organ weights and no treatment-related finding in microscopic examination.
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 oral 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 effects.
Critical effects observed:
not specified

CHEMICAL ANALYSIS: The dosage forms stability, homogeneity and achieved concentrations were satisfactory.

TOXICOKINETICS:

When measured on day 2 (24 hours after dosing on Day 1) and on weeks 6 and 13 for groups 2, 3 and 4, which received the test item at 50, 250 and 1000 mg/kg/day, plasma levels of FAT 65'080 were not quantifiable for all animals at any time-point.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
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.
Qualifier:
according to guideline
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
GLP compliance:
yes
Limit test:
no
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
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
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
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
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.


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.
Critical effects observed:
not specified

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Additional information

The substance ETH50 registered represents the form of pure (bulk) non-nano material.  As such, however, it is not usable for its intended use in cosmetic products. In order to transform it into suitable cosmetic ingredient, ETH50 has to be micronized in a water-mill. As a consequence of the extreme hydrophobicity of substance, surfactants need to be added to introduce it in the water phase. After milling, rheology modifiers prevent the nano-particles from agglomerating and stabilize the resulting preparation. Thus, the substance is marketed in a 50 % mixture together with different components; it is not available as pure material in nanoform. The key study for oral repeated dose toxicity was performed with the bulk (non-nano) form of ETH50 (relating to the substance registered), whereas the key study for dermal repeated dose toxicity was performed with a formulation containing micronized ETH50 (relating to the marketed mixture for the use in cosmetics)

Oral:

In the key repeated dose toxicity study under GLP (CIT 28341 TCR, 2005) three groups of 10 male and 10 female Sprague-Dawley rats were treated by daily oral gavage with non-micronized ETH50 as a suspension in the vehicle (0.5 % [w/v]carboxymethylcellulose in purified water), at dose-levels of 0, 50, 250 or 1000 mg/kg/day for 13 weeks. Six additional males and females were added to control and high-dose groups (0 and 1000 mg/kg bw/day) for a 4 -week post-treatment recovery period. Satellite groups of six males and six females were added to the test item-treated groups for toxicokinetic investigations. Satellite animals were discarded without further examination after the last blood sampling. All study animals were checked for mortality, clinical signs and detailed clinical observations were performed. Functional Observation Battery tests were performed and ody weight/ food consumption was recorded. Ophthalmology, hematological and blood biochemical investigations and urinalysis was performed. Plasma levels of the test item were determined on blood samples taken from satellite animals and T3, T4, TSH and sexual hormones (males: testosterone, females: estradiol and progesterone) levels were measured on blood samples taken. Estrous cycle stages of all principal females were determined from vaginal smears. Designated organs were weighed, macroscopic and histological examinations were performed on selected tissues from controls, high-dose group (0 and 1000 mg/kg bw/day) and on macroscopic lesions.

The oral administration of the test substance to rats up to and including 1000 mg/kg bw/day, followed by a 4-week recovery period, did not cause any biologically significant test substance related effects.

The only clinical sign with dose-related frequency was piloerection in males treated at 250 or 1000 mg/kg/day. This finding was infrequent (1/10 at mid-dose, 3/16 at high-dose) and had always disappeared by week 10. As it was not noted in females and not associated with other findings, this observation was considered to be most probably treatment-related but not to be an adverse effect. There was no treatment-related clinical sign in principal females or in satellite animals.

On week 13, low- and high-dose males had moderately lower white blood cell counts when compared to controls (up to -25%), due to significantly lower lymphocyte and monocyte counts. Yet there was a high inter-animal variability (2-fold among controls males) and there was no difference between the mid-dose and control groups. These changes were therefore considered not to be treatment-related. There was no significant WBC count alteration in females or at the end of the recovery period.

Overall, these effects were not considered to be of toxicological significance. Thus, 1000 mg/kg bw/day is considered to be the no-observed-adverse-effect-level (NOAEL) for ETH50 in rats of both sexes.

In the associated 2-weeks range-finding toxicity study (CIT 28340 TSR, 2005) the administration of ETH50 by oral route (gavage) to Sprague-Dawley rats for 14 days was well tolerated when given at 300, 600 or 1000 mg/kg bw/day. Body weight changes and food consumption were unaffected by treatment. No clinical signs were noted during the study. In females at 1000 mg/kg bw/day, the white blood cells count was slightly lower than in the control group. No findings were noted at histopathology.

Dermal:

In the chosen dermal repeated dose toxicity key study according to OECD TG 411 and GLP (CIT 32404 TSR, 2008) a formulation of micronized ETH50 (nano-form; 47.6 % active substance), was administered by daily cutaneous application to Wistar Han rats at the dose-levels of active substance of 150, 500 or 1000 mg/kg bw/day for 13 weeks (corresponding to 315, 1050 or 2100 mg of the test item formulation/kg bw/day, respectively).

Plasma concentrations of ETH50 were detectable (up to 12 ng/ml) but did not show time or dose-related changes, indicating systemic exposure following topical dosing. At the dose-level of 1000 mg/kg bw/day, scabs were noted at the application site, mainly in animals with no protective collar. This finding resolved by the end of the dosing period for almost all the animals. This dosage was associated with clinical signs related to pain, such as abnormal vocalization and/or hyperactivity mainly from week 5, within the 30-minute period after treatment. Lower mean body weight gain was noted in group 5 and 6 males when compared to placebo controls, principally from week 2 and during the whole dosing period; these trends were fully reversed during the non-treatment period. Lower glucose and triglycerides concentrations observed in group 5 and 6 males (100 mg/kg bw/day, with and without protective collar, respectively, when compared to controls at the end of the treatment period were considered to be related to the treatment procedure. Microscopic findings were indicative of irritant-effects related to the mechanical preparation of the application site and repeated treatments at the application site and/or stress. The minimally increased lymphocytolysis recorded in the thymus of a number of females of group 5 and an occasional male and female of group 6 may reflect a minimal non-specific stress-related response associated with the treatment procedure. No change in thymic weight was recorded in females of group 5. Although no histopathological changes were diagnosed in the thymus and adrenals of males of group 5, which showed statistically significant organ weight changes, these alterations were also considered to reflect a non-specific stress-related response. At the dose-levels of 150 and 500 mg/kg/day, no signs of local or systemic toxicity were noted.

Consequently, under the experimental conditions of this study, the No Observed Adverse Effect Level (NOAEL) was 1000 mg/kg bw/day as active ingredient given by cutaneous application to rats of the test item formulation containing 47.6 % active substance during 13 weeks.

In the associated 2-weeks range-finding toxicity study (CIT 28343 TSR, 2007), a micronized (nano-form) ETH50 formulation (purity 49.5 %) was dermally applied to 3 Wistar rats/sex/doselevel at 100 or 1000 mg/kg bw/day corresponding approximately to 50, 500 /kg bw/day mg ETH50. A further treatment group received non-micronized ETH50 (98.8%) at 1000 mg/kg bw/day. Two concurrent groups of each 3 rats/sex either received a placebo formulation or remained untreated.

No signs of dermal or systemic toxicity were observed at any dose-level, except for a slightly low food consumption in females given 1000 mg/kg bw/day micronized ETH50 formulation.

Justification for classification or non-classification

The present data on repeated dose toxicity do not fulfill the criteria laid down in 67/548/EEC and regulation (EU) 1272/2008, and therefore, a non-classification is warranted.