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Toxicological information

Repeated dose toxicity: dermal

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Administrative data

Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 Feb - 04 Mar 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Version / remarks:
adopted in 1981
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3200 (Repeated Dose Dermal Toxicity -21/28 Days)
Version / remarks:
adopted in 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
National Institute for Quality- and Organizational Development in Healthcare and Medicines, National Institute of Pharmacy, Budapest, Hungary
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
other: Crl:(WI) Wistar
Details on species / strain selection:
The Wistar rat is a standard species and strain used on dermal toxicity studies.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: 291 - 336 g (males), 193 - 249 g (females)
- Housing: up to 5 animals of the same sex per cage during acclimatisation period and individual during treatment in type II polycarbonate cages, deep wood sawdust bedding (Grade 5, Johannes Brandenburg GmbH und Co. KG, Goldenstedt, Germany)
- Diet: ssniff® SM R/M-Z+H "Autoclavable complete diet for rats and mice – breeding and maintenance" (ssniff Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water from municipal supply, ad libitum
- Acclimation period: 12 days (males), 13 days (females)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Type of coverage:
semiocclusive
Vehicle:
water
Details on exposure:
TEST SITE
- Area of exposure: scapulae (shoulders) to the wing of the ileum (hipbone) and half way down the flank on each side of the animal
- % coverage: not less than 10%
- Type of wrap if used: porous gauze dressing (6 - 8 plies), additional non-irritating plastic wrap and restrainers (Lomir jackets with inserts)
- Time intervals for shavings or clippings: 20 and 18 h before the test for males and females, respectively

REMOVAL OF TEST SUBSTANCE
- Washing: lukewarm water
- Time after start of exposure: 6 h

TEST MATERIAL
- Amount applied: not specified
- Constant volume or concentration used: not specified
- For solids, paste formed: yes

VEHICLE
- Lot/batch no.: 2171213 (TEVA Co.)

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily, 6 h/day
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected by the Sponsor in consultation with the Study Director based on available data and information from previous experimental work, including the results of a 14-day repeated-dose dermal range-finding study in the rat, where no clear signs of toxicity were observed at any dose level tested, including the highest dose level of 1000 mg/kg bw/day. In addition, in an acute dermal toxicity study in the rat, the LD50 value was > 2000 mg/kg bw for both sexes, with no treatment-related findings (2013, Doc number M-513347-01-1). In the 90-day dietary study in the rat, no treatment-related findings were observed up to the highest dose level tested of 1000 ppm (equates approximately to 608 mg/kg bw/day in males and 723 mg/kg bw/day in females; 2012). Therefore, the dose levels selected on the current study were 100, 300 and 1000 mg/kg bw/day, where the limit dose of 1000 mg/kg bw/day was expected to be tolerated without causing death or overt suffering, the low dose was expected to be a clear NOAEL and the mid dose provides an approximate 3-fold factor between the dose levels.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations included: morbidity, mortality, ill health

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily following patch removal (general clinical observations), once before the first exposure and than at least weekly (detailed examinations)

DERMAL IRRITATION: Yes
- Time schedule for examinations: at least daily following the patch removal

BODY WEIGHT: Yes
- Time schedule for examinations: the day before the first exposure and then at least weekly

FOOD CONSUMPTION:
Food consumption was determined by re-weighing the non-consumed diet at weekly intervals. The weekly food consumption was calculated.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once pre-treatment and on day 27
- Dose groups that were examined: all dose groups (pre-treatment), control and high dose (day 27)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to necropsy on day 28
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: red blood cell (erythrocyte) count, haemoglobin concentration, haematocrit, mean corpuscular (erythrocyte) volume, mean corpuscular (erythrocyte) haemoglobin, mean corpuscular (erythrocyte) haemoglobin concentration, red cell (erythrocyte) volume, platelet (thrombocyte) count, mean platelet (thrombocyte) volume, reticulocyte count, white blood (leukocyte) count, neutrophils, lymphocyte, monocyte, basophil, eosinophil, large unstained cells, blood smears, activated partial thromboplastin time, prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to necropsy on day 28
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: glucose, total bilirubin, urea, cholesterol, triglycerides, creatinine, phosphorus, sodium, potassium, calcium, chloride, total protein, albumin, albumin/globulin ratio, aspartate aminotransferase activity, alanine aminotransferase activity, alkaline phosphatase activity, gamma glutamyltransferase activity, bile acids

URINALYSIS: Yes
- Time schedule for collection of urine: prior to necropsy on day 28
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes
- Parameters examined: leukocyte, nitrite, pH, protein, glucose, urobilinogen, bilirubin, ketones, blood/erythrocytes, specific gravity, sediment, volume

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: day 23 (last week of treatment)
- Dose groups that were examined: all dose groups
- Battery of functions tested: sensory activity, grip strength, motor activity

OTHER
Prior to necropsy, the oestrous cycle of all females was determined by taking vaginal smears, which were prepared and stained with 1% aqueous methylene blue solution. The smears were examined with a light microscope, in order to provide information regarding the stage of oestrous cycle at the time of sacrifice and assist in histological evaluation of oestrogen sensitive tissues.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Macroscopic examination of the tissues and organs and collection for histopathological examination where abnormalities were recorded. The following organs were collected and weighed: adrenals, brain, epididymides, heart, kidneys, liver, ovaries, pituitary, prostate including seminal vesicles with coagulating glands, spleen, testes, thymus, thyroids with parathyroids, uterus including cervix

HISTOPATHOLOGY: Yes
The following organs and tissues were collected: aorta, adrenals, brain, epididymides, external lachrymal glands, eyes with the optic nerves, femur with marrow, harderian glands, heart, kidneys, large intestine, larynx, liver, lungs with bronchi, lymph nodes, mammary gland (inguinal), nose/nasal cavity, oesophagus, ovaries with oviduct, pancreas, pharynx, pituitary, prostate, salivary glands, sciatic nerve, seminal vesicles with coagulating glands, skeletal muscle (quadriceps), skin and subcutis (inguinal), small intestine, spinal cord, spleen, sternum with marrow, stomach, testes, thymus, thyroid with parathyroids, tongue, trachea, treated skin area, urinary bladder, uterus, vagina

The eyes with the optic nerves and testes with epididymides were retained in modified Davidson’s fixative, all other organs in 10% buffered formalin solution.
Bone marrow smears were stained with Giemsa but not examined as there were no haematology findings. The retained tissues and organs were embedded in paraffin wax, sections were cut at 4-6 μm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope. Full histopathology was performed on the selected list of organs in control and four high dose animals, and in all macroscopic abnormalities in all groups.
Statistics:
Mean and standard deviations values were calculated. The statistical analysis was performed using SPSS PC+4.0 software. The heterogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan’s Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. If the data was not normal distributed, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there was a positive result, the inter-group comparisons were performed using Mann-Whitney U-Test.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Dermal irritation:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, non-treatment-related
Urinalysis findings:
effects observed, non-treatment-related
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Gross pathological findings:
effects observed, non-treatment-related
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
For details on results including tables see the attached document.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no treatment-related effects at highest dose tested

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

For details on results including tables see the attached document.

Applicant's summary and conclusion