Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 Feb 2008 - 7 Jan 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3200 (Repeated Dose Dermal Toxicity -21/28 Days)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent

Test animals

Species:
rat
Strain:
other: Wistar Hanover IGS [CRL:WI(Han)]
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Incorporated, Raleigh, North Carolina, USA
- Age at study initiation: 8 weeks
- Weight at study initiation: 213 - 248 g (males) and 164 - 191 g (females)
- Fasting period before study: No
- Housing: Individually housed in stainless steel wire mesh cages (66.5 square inches) with Deotized Animal Cage Board under the cage.
- Diet: Purina Laboratory Rodent Chow No. 5002 in "meal" form provided ad libitum, except when fasted prior to blood sampling.
- Water: Tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 26
- Humidity (%): 30 - 70
- Air changes (per hr): At least 11.0 changes per hour
- Photoperiod (hrs dark / hrs light): Approximately 12/12 hrs

IN-LIFE DATES: From: 12 Mar 2008 To: 14& 15 April 2008

Administration / exposure

Type of coverage:
semiocclusive
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SITE
- Area of exposure: app. 7.6 x 7.6 cm (3 x 3 inches)
- Type of wrap if used: The test material was applied to a gauze pad, which was placed on a piece of plastic. The gauze was secured with Micropore Tape and the torso of the animal was then wrapped with an elastic bandage.
- Time intervals for shavings or clipplings: On study day -3, the hair was removed from the dorsal and lateral areas of the trunk of each rat using electric clippers. During the dosing period, the animals were shaved, as necessary, due to hair growth.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The dose site was wiped with deionized water-dampened and dry gauze to remove as much test substance residue as feasible without damaging the skin.
- Time after start of exposure: 6 hrs

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0, 100, 300 and 1000 mg/kg bw
- Constant volume or concentration used: No
- For solids, paste formed: Yes. Deionized water was used to moisten the gauze pad for the control and test substance treated animals immediately prior to application.

USE OF RESTRAINERS FOR PREVENTING INGESTION: No.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
6 h/day for 28 days (21 doses)
Frequency of treatment:
5 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300, 1000 mg/kg bw/day
Basis:
nominal per unit body weight
No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
- Dose selection rationale: No rationale was given for the dose selection. However, the substance was tested up to the recommended concentration for limit tests, according to OECD guideline 410.
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily, except in the weekend once daily
- Cage side observations included: Mortality, clinical signs

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the first exposure and once a week thereafter

DERMAL IRRITATION: No

BODY WEIGHT: Yes
- Time schedule for examinations: Once before the beginning of the study, weekly during the study and just before scheduled necropsy.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. Determined weekly.

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. The eyes of each rat were checked for a pupillary reflex with a penlight or a transilluminator with Finnoff®. The conjunctiva, cornea, and lens were examined using a slit lamp microscope. The vitreous humor, retina, choroid, and optic disc were examined with an indirect ophthalmoscope and a condensing lens.
- Time schedule for examinations: Following the acclimation period but prior to initiation of dosing and on day 22.
- Dose groups that were examined: All dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 23 of the study
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes, overnight
- How many animals: 10/sex/dose
- Parameters examined: Morphology of erythrocytes, erythrocyte count, hemoglobin, hematocrit, leucocyte count (WBC), platelet count, blood clotting parameters (thromboplastin time, clotting time, prothrombin time), leucocyte differential time, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular volume, reticulocyte count, red blood cell distribution width, hemoglobin distribution width.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 23 of the study
- Animals fasted: Fasted overnight
- How many animals: 10/sex/dose
- Parameters examined:
Enzymes: Alkaline phosphatase, creatine phosphokinase, lactic acid dehydrogenase, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase .
Electrolytes: Calcium, chloride, potassium, sodium, phosphorus.
Other: Albumin, creatinine, urea-nitrogen, total cholesterol, globulins, glucose, protein (total), bilirubin (total), triglycerides, Uric Acid, A/G ratio.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. A complete gross necropsy examination was performed on all animals. The necropsy consisted of a systematic gross examination of each animal's general physical condition, body orifices, external and internal organs and tissues.

The following organs/tissues were collected and examined microscopically: adrenal glands, aorta, bone marrow, bone (sternum), brain (cerebrum, cerebellum,medulla), clitoral gland, epididymides, esophagus, eyes, exorbital lacrimal glands, harderian gland, head (nasal cavity), heart, intestine (duodenum, jejunum, ileum, caecum, colon, rectum, remaining intestine), kidneys, larynx, liver, lungs, lymph nodes (cervical), lymph nodes (mesenteric), mammary gland, optic nerves, ovaries, oral structure, pituitary gland, prepuital gland, prostate, salivary glands, sciatic nerve, seminal vesicles (incl. coagulation glands), skeletal muscle, skin (treated), skin (untreated), spinal cord (cervical, thoracal, lumbar), spleen, stomach (forestomach and glandular stomach), testes, thymus, thyroid glands (with parathyroids), tongue, tooth, trachea, urinary bladder, uterus (with cervix), vagina, zymbal's glands, organs and tissues with macroscopic findings.

HISTOPATHOLOGY: Yes. Gross lesions from animals in all dose groups and representative sections of the tissues were processed, embedded in paraffin, sectioned, mounted, and stained with hematoxylin and eosin (H&E) for examination under the light microscope. Histopathologic evaluation was conducted on all protocol-required tissues from the control and high-dose group animals.

The following tissues were examined in the control and 1000 mg/kg bw groups:
adrenal glands, aorta, bone marrow, bone (sternum), brain (cerebrum, cerebellum,medulla), epididymides, esophagus, eyes, harderian gland, head (nasal cavity), heart, intestine (incl. Fever's patches) (duodenum, jejunum, ileum, caecum, colon, rectum, remaining intestine), kidneys, larynx, liver, lungs, lymph nodes (cervical), lymph nodes (mesenteric), mammary gland, optic nerves, ovaries, oral structure, pituitary gland, prostate, salivary glands, sciatic nerve, seminal vesicles (incl. coagulation glands), skin (treated), skin (untreated), spinal cord (cervical, thoracal, lumbar), spleen, stomach (forestomach and glandular stomach), testes, thymus, thyroid glands (with parathyroids), tongue, tooth, trachea, urinary bladder, uterus, organs and tissues with macroscopic findings.
Other examinations:
The following organs were weighed: liver, heart, spleen, thymus, epididymes, kidneys, ovary, testes, uterus, adrenal gland and brain.
Statistics:
Statistical significance was determined at p<0.05 for all tests with the exception of Bartlett's test, in which a probability value of p<0.001 was used. All tests were two-tailed, except for gross and histopathologic lesion evaluations, which were one-tailed. Continuous data was analyzed by Bartlett's test for homogeneity. If the data were homogeneous, an ANOVA was performed followed by Dunnett's t-test on parameters showing a significant effect by ANOVA. If the data were non-homogeneous, a Kruskal-Wallis ANOVA was performed, followed by the Mann-Whitney U-test to identify statistical significance between groups (pair-wise comparisons). Frequency data that were examined statistically were evaluated using the Chi-Square and/or Fisher’s Exact tests.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
3 females died (control, mid dose and high dose groups); the deaths were considered to be incidental; skin lesions and scabs that were observed in one control male and in some females in the control and treatment groups were due to wrapping the animals with elastic tape.
Dermal irritation:
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
3 females died (control, mid dose and high dose groups); the deaths were considered to be incidental; skin lesions and scabs that were observed in one control male and in some females in the control and treatment groups were due to wrapping the animals with elastic tape.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
in all female dose groups: body weight was reduced on day 21; however considered not to be treatment related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
effects observed, treatment-related
Description (incidence and severity):
Retinal degeneration was noted in males in the control and high dose groups and in females of the control, low and mid dose groups; the effects were not considered to be treatment-related
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
effects on the values of several parameters (e.g. eosinophils, absolute count of large unstained cells, hematocrit, mean cell hemoglobin concentration, reticulocytes) in males and females; the changes were not considered to be treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
effects on the values of several parameters (e.g. uric acid, potassium, sodium levels and albumin) in males and females; the changes were not reflected in histopathological findings and are not considered to be treatment-related.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
at 1000 mg/kg bw/day in females: significant increase in the absolute (28%) and relative (31%) ovary weights; the increase was not interpreted to be a treatment-related effect
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Incidental cases of skin lesions (crusty zones) and reduction in size of the thymus were observed, but not in significant numbers; the effects were not considered to be treatment-related.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Hepatic and splenic extramedullary hematopoiesis was observed in treated and control animals in similar numbers and probably happened during terminal bleeding before study termination and not considered to be treatment-related.
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY

Three females died during the study, one in the control group (day 24), one in the 300 mg/kg bw/day group (day 1) and one in the 1000 mg/kg bw/day group (day 24). The deaths were considered incidental and were probably due to the wrapping that holds the test substance in place. Urine staining that was observed in some females in the treatment groups was due to the dosing procedure. Skin lesions and scabs that were observed in one control male and some females in the control and treatment groups were also due to wrapping the animals with elastic tape. There were no clinical signs that could be attributed to the administration of the test substance.

BODY WEIGHT AND WEIGHT GAIN

On day 21, the body weight of the females in the 100, 300 and 1000 mg/kg bw groups was statistically significantly reduced compared to the control group. This effect was not observed at any other time point, nor in the male dose groups and was therefore considered not to be treatment-related.

FOOD CONSUMPTION

There were no effects on the food consumption of the rats in this study.

OPHTHALMOSCOPIC EXAMINATION

Retinal degeneration was noted in 1, 0, 0 and 1 males in the control, 100, 300 and 1000 mg/kg bw/day groups, respectively, and in 3, 1, 1 and 0 females in the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. There were no test-substance-related clinical ophthalmic lesions observed in this study.

HAEMATOLOGY

A slight statistical decrease in the percentage of eosinophils, without a corresponding change in the absolute counts, was observed in the 1000 mg/kg bw/day males. In the same dose group, the absolute count of large unstained cells was significantly increased, while a similar parameter value in 300 mg/kg bw/day males was not statistically significant. There was a minor, but statistically significant decrease in the hematocrit with an increase in the mean cell hemoglobin concentration of 300 mg/kg bw/day males only. In females, statistical changes were limited to a decrease in the level of reticulocytes of 100 mg/kg bw/day animals.The parameter values fell within the historical control range. None of the statistical differences noted were attributed to any biological significance and are not considered to be treatment-related.

CLINICAL CHEMISTRY

In all dose groups for males and females a statistically significant decrease in uric acid values was observed. In females dosed 1000 mg/kg bw/day there was a significant decrease in the potassium and sodium levels. Females in the 300 and 1000 mg/kg bw/day groups also had a significant increase in the albumin value. The changes were not reflected in histopathological findings and are not considered to be treatment-related. In males dosed with 300 mg/kg bw/day, incidental changes in the values of certain parameters were observed, but not considered to be treatment-related.

ORGAN WEIGHTS

There were no statistical changes in the organ weights of the male animals evaluated.

In females dosed 1000 mg/kg bw/day, there was a significant increase in the absolute (28%) and relative (31%) ovary weights. There were no microscopic changes observed and all stages of the ovarian estrus cycle were noted (i.e., preantral and antral follicles and corpora lutea). In addition, there was no effect on ovary weights in the 28-day oral feeding study (M-087601-01-1). There were a few outliers with relatively high weights, contributing to a high mean value. The largest ovary weight in this present study (0.197 g) is within the upper range of ovarian weights in a previous subacute dermal study (06-S22-GH). Although the weight variability in this study cannot be explained and the biological significance of the increased ovarian weights was equivocal, the increase in ovarian weight was not interpreted to be a treatment-related effect.

GROSS PATHOLOGY

Incidental cases of skin lesions (crusty zones) and reduction in size of the thymus were observed, but not in significant numbers. The gross pathology necropsy observations were not considered to be treatment-related.

HISTOPATHOLOGY: NON-NEOPLASTIC

Hepatic and splenic extramedullary hematopoiesis was observed in treated and control animals in similar numbers and probably happened during terminal bleeding before study termination. Hepatic microgranuloma was present in several control and treated animals of both sexes, and in females, acute necrosis (in some case encapsulated) was observed. The changes were attributed to the tight wrapping round the abdomen of the rats to protect the exposure site. There were no significant differences in microscopic skin changes between the control and treated group.
Due to the significant increase in relative and absolute ovary weights, the ovary sections were evaluated multiple times. No morphologic changes were observed: all follicular transitional stages (primordial and antral follicles and corpora lutea stages) were present from both groups and there were no cystic or inflammatory changes. There were no similar changes to ovary weight or morphology in the subacute oral study with the test substance.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: No treatment-related effects

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion