Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
90 days
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2006

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Version / remarks:
May 12, 1981
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Remarks:
HanRCC (SPF)
Details on species / strain selection:
The species and strain is recognised by the international guidelines as a recommended test system
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Internationally recognised supplier
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 weeks (males) and 13 weeks (females)
- Weight at study initiation: Males: 205.3-255.0g; Females: 181.7-210.4g
- Fasting period before study: No
- Housing: Individually in Makrolon type-3 cages with standard softwood bedding (Lignocel)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY:
Food - Pelleted standard Provimi Kliba 3433 (batch nos. 4/05 and 39/05) rat maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst/Switzerland). The feed batch was analised for contaminants.
Water - Community tap water from Fullinsdorf in water bottles. None of the contaminants analised in the water was considered to have been present at a concentration tha would have affected the validity of the results

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3 oC
- Humidity (%): 30-70%
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 8th July 2005 To: 9th November 2005

Administration / exposure

Type of coverage:
semiocclusive
Vehicle:
other: Microwax 16%; Butyrospermum parkii (Shea butter) 12%; C12-15 Alkyl benzoate 28%; Caprylic/Capric triglyceride 28%; Dibutyl adipate 16%
Remarks:
Identity reference: SW-06-03; Described as a cream
Details on exposure:
TEST SITE
- Area of exposure: Dorsal area 5cm x 5cm
- % coverage: 10%
- Type of wrap if used: Surgical gauze and tape
- Time intervals for shavings or clipplings: Daily

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Daily with lukewarm tap water
- Time after start of exposure: 6 hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Weight in grams
- Concentration (if solution): NA
- Constant volume or concentration used: no


VEHICLE
- Justification for use and choice of vehicle (if other than water): Material used as supplied
- Amount(s) applied (volume or weight with unit): Weight in grams
- Concentration (if solution): NA
- Lot/batch no. (if required):
- Purity:

USE OF RESTRAINERS FOR PREVENTING INGESTION: no; semiocclusive dressings applied
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
One sample for analytical purposes was collected from the vehicle and each of the two or three formulations administered at the beginning of the application period, during the course of the study (week 5) and at the end. A reference item was used as analytical standard. Only the relevant contents were investigated. The dose forumation analysis was performed by an HPLC analytical method.
Duration of treatment / exposure:
90-91 days
Frequency of treatment:
6 hours exposure daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 other: Vehicle control
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
400 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Group 1 - Control - 10 males and 10 females Main Group A; 5 males and 5 females Satellite Group B; 9 males and 9 females Satellite Group C
Group 2 - 100 mg/kg/day - 10 males and 10 females Main Group A; 9 males and 9 females Satellite Group C
Group 3 - 400 mg/kg/day - 10 males and 10 females Main Group A; 9 males and 9 females Satellite Group C
Group 4 - 2000 mg/kg/day - 10 males and 10 females Main Group A; 5 males and 5 females Satellite Group B; 9 males and 9 females Satellite Group C

A: Main study animals
B: Animals for 4-week recovery
C: Animals for toxicokinetics and hormone analysis
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The highest dose level of 2000 mg/kg was the maximum feasible dose which could be applied. The mid and low does levels were selected to identify any dose related effects.
- Rationale for animal assignment (if not random): Random but to equalise grop mean body weights at start
- Rationale for selecting satellite groups: 5 males and 5 females included in Groups 1 and 4 to assess potential recovery from any effects; 9 males and 9 males per group included to provide blood samples for toxicokinetics and hormone analysis
- Post-exposure recovery period in satellite groups: Groups 1 and 4 only
- Section schedule rationale (if not random):
Positive control:
No positive control was included

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: Once daily
- Observations made checked in table [1]

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean weekly diet consumption calculated as g food/week: Yes

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: Yes

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pretest, week 13 and week 17
- Dose groups that were examined: All animals at pretest; Control and Group 3 animals in main group and recovery period subgroups in week 13; Control and Group 3 aniamls in recovery period subgroup in week 17.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 13 and 17
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: Week 13: Groups 1 and 4 - 15 males and 15 females; Groups 2 and 3 - 10 males and 10 females; Week 17: Groups 1 and 4 - 5 males and 5 females (Sub groups A and B)
- Parameters checked in table [No.2] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 13 and 17
- Animals fasted: Yes
- How many animals: Week 13: Groups 1 and 4 - 15 males and 15 females; Groups 2 and 3 - 10 males and 10 females; Week 17: Groups 1 and 4 - 5 males and 5 females (Sub groups A and B)
- Parameters checked in table [No.3] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Week 13 and 17
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No.4] were examined.

NEUROBEHAVIOURAL EXAMINATION: No data

OTHER:
HORMONE ANALYSIS:
- Approximately 1 mL blood samples taken sublingually. Serum was separated and stored deep frozen until analysed for total T3, T4 and TSH
- All animals from Group 4 sampled on Day 13; All Sub group C animals sampled on Day 42; Remaining Sub grou C animals on Day 90
TOXICOKINETICS:
- On Days 1 and 90 approximate 0.6 mL blood samples were taken from all Sub group C animals (3 animals/sampling time point) at 0.5, 1, 2, 4, 8 and 24 hours after dermal administration of the test item.
- The blood samples were collected sublingually under light anaesthesia into heparinised tubes and the plasma separated by centrifugation. Samples were stored deep frozen prior to analysis by a validated LC-MS/MS method.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes [see table 5]
All animals from Sub groups A and B were subject to autopsy where all macroscopic abnormalities were recorded and selected tissues/organs were weighed [see table 5]

HISTOPATHOLOGY: Yes [see table 6]
Samples of the listed tissues from all animals in Sub groups A and B and Group 4 animals in Sub group C were fixed in neutral phosphate buffered 4% formaldehyde solution [see table 6]
Statistics:
The following statistical methods were used to analyse the body weight, organ weights and ratios:
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test was applied to the macroscopic findings.
The following statistical methods were used for analysis of clinical laboratory data:
- Quantitative data were analysed by a one-way analysis of variance (ANOVA) when the variances were considered homogeneous according to Bartlett. Treated groups were then compared to the control groups using Dunnett's test if the ANVA was significant at the 5% level and by Dunn's test in the case of a significant Kruskal-Wallis test (p

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
Group 1 - Very slight and transient signs of irritation - scabs (9/48 animals); erythema (patchy 17/48); general 32/48); scaling (28/48); epidermal lesions (8/24 females)
Group 2 - Very slight to well-defined general erythema (30/48); very slight to slight scaling (23/38); very slight patchy erythema (11/38); very slight scabs (10/38); very slight epidermal lesions (8/38)
Group 3 - Very slight to moderate/severe general erythema (36/37); very slight to well-defined patchy erythema (14/37); very slight to slight scaling (35/37); slight scabs (11/37); slight epidermal lesions (8/37); slight to well-defined general oedema (3/37); wounds in two females; fissures and watery discharge in one female
Group 4 - Severe signs of irritation leading to premature cessation of treatment - Very slight to moderate/severe general erythema (all animals); scabs (38/48); scaling (43/48); general oedema (17/48); epidermal lesions (41/48); wound formation (44/48); necrosis (2/48); Fissures and watery discharge in two males and three females.
Mortality:
mortality observed, treatment-related
Description (incidence):
All animals from Group 4 were killed prematurely on test day 15 due to the severity of the dermal reaction.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A significant decrease in body weight (-5.4% in males and -6.1% in females) ws noted on test day 8 in the animals treated with 2000 mg/kg/day (Group 4).
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:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
The daily administration of 400 mg/kg/day produced no treatment-related pathological findings. When given at 2000 mg/kg/day, application was stopped after 11 days of treatment because of severe skin reactions. Histopathology of tissues from this group was confined to the thyroid glands and there were no treatment-related changes seen in this organ.
Other effects:
no effects observed
Description (incidence and severity):
No differences in serum levels of TSH, total T3 and total T4 were observed at Day 90 in males and females treated at 100 or 400 mg/kg/day when compared with the controls.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
>= 400 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
dermal irritation

Target system / organ toxicity

Key result
Critical effects observed:
no
Lowest effective dose / conc.:
400 mg/kg bw/day (nominal)
System:
integumentary
Organ:
skin

Any other information on results incl. tables

Toxicokinetics:

In the plasma samples taken on days 1 and 90/91 after the start of treatment both the parent test item and its two major metabolites were quantified.

Mean AUCs of the test item were dose-proportional on days 1 and 90/91 and no apparent sex-related differences were observed. AUCs measured on day 90/91 remained in the same range as on day 1 suggesting no potential for accumulation.

The plasma levels of both metabolites showed apparent sex differences.

Applicant's summary and conclusion

Conclusions:
Based primarily on the local dermal response results in this study, the dose level of 400 mg/kg/day was established as the no-observed-adverse-effect-level (NOAEL) and 100 mg/kg/day was established as the no-observed-effect-level (NOEL).
Executive summary:

Topical administration of the test material at a dose of 100, 400 or 2000 mg/kg/day by a daily 6 -hour semi-occlusive application during 90 or 91 days (100 and 400 mg/kg/day) or 11 days (2000 mg/kg/day), resulted in no systemic clinical signs of toxicity. A slight decrease in bodyweight was observed in Group 4 at week 2 and Group 4 had to be terminated early on test day 15 due to severe skin reactions. Signs of local dermal reaction were recorded for many of the animals treated with the test substance comprising transient signs of irritation such as erythema, scab formation, scaling and epidermal lesions. The formation of wounds, general oedema, necrosis fissures and watery discharge were observed in the group treated at 2000 mg/kg/day. The severity as well as the incidence of general erythema, scaling, scabs and epidermal lesions aggravated with increasing dosage and, in general, the males appeared to be less sensitive than the females.

There were no other treatment related changes observed in any parameters measured.

The test item was found to be absorbed in a dose-dependent manner and metabolised into its two major metabolites after dermal application.

Based on the results of this study, 400 mg/kg/day of the test material was established as the no-observed-adverse-effect level (NOAEL) and 100 mg/kg/day of the test material was established as the no-observed-effect-level (NOEL). This study was assigned a reliability rating of 1: reliable without restrictions.