Registration Dossier

Administrative data

Description of key information

90-day oral rats study – OECD 408 Repeated dose 90-day oral toxicity study in rodents; groups of rats were treated with the test item via incorporation into the diet continuously for 90 days. The actual dose received equated to 0 (controls), 25, 50, 125 or 312 mg/kg/day. Clinical observations, body weight and food intake were frequently monitored and blood and urine samples were taken at intervals for analysis. Full necropsy and tissue examinations were undertaken at termination.

90-day dermal rat study – OECD 411 Subchronic dermal toxicity 90-day study in rodents; groups of rats were treated with the test item via semi-occlusive topical administration at dose levels of 100, 400 or 2000 mg/kg/day for 6 hours/day and up to 90 days. The high dose level animals were terminated on test day 15 due to severe skin reactions. Clinical observations, body weight and food intake were frequently monitored and blood and urine samples were taken at intervals for analysis. Full necropsy and tissue examinations were undertaken at termination.

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:
90 days
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
Actual guideline not stated in the report
Deviations:
not specified
Remarks:
No deviations were recorded
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Wi-AF7 Han (SPF)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Not stated
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Approximately 5 weeks
- Weight at study initiation: Part I - males 101-155g, females 111-139g; Part II - males 120-143g, females 112-132g
- Fasting period before study: Not stated
- Housing: Individually housed in wire cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Not stated

DETAILS OF FOOD AND WATER QUALITY:
- Details of analyses were not given

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Part I - 22-30 oC; Part II - 21-26 oC
- Humidity (%): Part I - 35-55%; Part II - 33-50%
- Air changes (per hr): Not stated
- Photoperiod (hrs dark / hrs light): Not stated

IN-LIFE DATES: Part I - From: August 1982 To: December 1982; Part II - From: January 1983 To: May 1983
Route of administration:
oral: feed
Details on route of administration:
The test material was administered to the animals via incorporation into the diet. Concentrations of the test material in the diet (ppm) were varied on a weekly basis in accordance with the growth and feed consumption of the animals such that the dose levels in terms of mg/kg bw/day were maintained at a constant level throughout the study.
Vehicle:
not specified
Remarks:
The test material was incorporated and mixed directly into the standard feed (Altromin Standard-diat TPF N 1321 fortified)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: NA

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): Altromin Standard-diat TPF N 1321 fortified (ex Altromin, Lage/Lippe)
- Storage temperature of food: Not stated

VEHICLE
- Justification for use and choice of vehicle (if other than water): NA
- Concentration in vehicle: NA
- Amount of vehicle (if gavage): NA
- Lot/batch no. (if required): NA
- Purity: NA
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
In the 3rd, 6th, 9th and 10th weeks of the study, random samples of the feed mixes were analysed. It was determined that the feed contained the requisite quantities of the test material. The concentration determinations yielded levels of 90-105% of the nominal value.
Duration of treatment / exposure:
3 months or 90 days
Frequency of treatment:
Treated feed was available ad libitum
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Part I - Control (Group1)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
Part I - Low dose level (Group 2)
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Remarks:
Part I - Intermediate dose level (Group 3)
Dose / conc.:
312 mg/kg bw/day (actual dose received)
Remarks:
Part I - High dose level (Group 4)
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Part II - Control (Group 1A)
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Remarks:
Part II - Low dose level (Group 2A)
No. of animals per sex per dose:
20 males and 20 females per group
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Not stated
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: Additional 10 males and 10 females per group were included to remain after the treatment period was completed to be assessed for signs of recovery from any treatement-related effects during a 4 week off-treatment period
- Post-exposure recovery period in satellite groups: 4 weeks
- Section schedule rationale (if not random): Random
- Since a NOAEL was not determined in Part 1, a separate study was commenced in male and female rats with a new control group and a new low dietary inclusion concentration equivalent to an achieved dose level of 25 mg/kg/day.
Positive control:
No positive control was required
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: 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: Yes
- Time schedule for examinations: Twice weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Both eyes were examined for all individuals at the autopsy at the end of the treatment period.
- Dose groups that were examined: All treated and control animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 6 and 12/13
- Anaesthetic used for blood collection: Yes (halothane)
- Animals fasted: Yes
- How many animals: 10 males and 10 females per group
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 6 and 12/13
- Animals fasted: Yes
- How many animals: 10 males and 10 females per group
- Parameters checked in table [No.2] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Week 6 and 12/13
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No.3] were examined.

NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations: NA
- Dose groups that were examined: NA
- Battery of functions tested: sensory activity / grip strength / motor activity / other: NA

IMMUNOLOGY: No
- Time schedule for examinations: NA
- How many animals: NA
- Dose groups that were examined: NA

OTHER:
THYROID HORMONES: In Week 7 blood was withdrawn under halothane anaesthesia from 10 males and 10 females per group for the measurement of thyroid hormones (total T3, total T4 and TSH). The same animals were sampled at termination after 3 months treatment and after the recovery period, for the same parameters.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 4)
- Full autopsy on any animals dying during the study and all animals killed at scheduled sacrifices after 3 months, and at the end of the 1 month recovery period

HISTOPATHOLOGY: Yes (see table 5)
- The list of tissues specified in Table 5 were processed and examined for each animal killed at the scheduled sacrifices
Statistics:
Body weight, food and water consumption, organ weights and clinicochemical data - analysed using Dunnett's multiple t-test. Differences in group sizes were taken into account by correcting the critical t-values and also the degrees of freedom.
Haematological data - analysed either by using Dunnett's multiple 2-sided t-test with differences in group sizes or variance in homogeneity taken into account by correcting the critical t-values and the degrees of freedom OR, a 2-sided alternative based on a linear rank test.
Statistical significances were quoted at the 5% or 1% level of probability.
Clinical signs:
no effects observed
Description (incidence and severity):
Non treatment-related minor incidences of hair loss, spontaneous injury and one case of skin lesions were observed.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Five animals from the control or 125 mg/kg/day groups died following the diagnostic blood aspiration. Three animals receiving 125 mg/kg/day group died during halothane anaesthesia.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Transient reduced body weight gain in females treated at 125 mg/kg/day from the 4th week (77.5%), and in females receiving 312 mg/kg/day from the start of the study (94%) when compared with the control females. Recovery in the body weight of females from these treatment groups was evident during the follow-up 4-week off-treatment period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Reduced food intake by females treated at 312 mg/kg/day (89.8%) when compared with the controls.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Increased water intake by males treated at 312 mg/kg/day when compared with the controls.
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Slightly reduced haematocrit, haemoglobin and erythrocyte values were recorded for males and females treated with 312 or 125 mg/kg/day and for females only treated with 50 mg/kg/day. No dependence on dose level was reported for these slight deviations from control values.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Elevated cholesterol levels in males and females treated with 312 mg/kg/day at both week 6 and week 12/13 investigations. Normal values were recored at the end of the recovery period.
Elevated GPT activity in males and females treated with 312 mg/kg/day and females treated with 125 mg/kg/day at both week 6 and week 12/13 invesigations. Normal values were recorded at the end of the recovery period.
Reduced serum albumin levels in females treated with 312 mg/kg/day at both investigations. Normal values were recorded at the end of the recovery period.
Treatment with higher doses resulted in elevation of serum TSH and T3 levels, with the females being more sensitive to this effect (elevated concentrations measured >/= 50 mg/kg/day). Elevated concentrations were seen in males only at 312 mg/kg/day. Apart from a few isolated cases, T4 levels were normal.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Increased absolute and relative thyroid weights were observed in males and females treated at 312 or 125 mg/kg/day, with a slight increase also in males treated at 50 mg/kg/day. The increased thryoid weights were still apparent at the end of the recovery period in rats previously treated at 312 mg/kg/day.
Relative liver weights were increased in females treated at 125 mg/kg/day and in males treated at 312 mg/kg/day. In males and females treated at 312 mg/kg/day adrenal weights were reduced and spleen weights increased. Reduced prostate weights and increased thymus weights (females) were also observed. All these changes proved to be reversible at the end of the follow-up observation period.
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In male and female rats treated at 312, 125 or 50 mg/kg/day, hypertrophy and hyperplasia of the follicular epithelium of the thyroid was observed, with the change rated as slight to moderate in the 50 and 125 mg/kg/day groups and strongly pronounced in the 312 mg/kg/day group. These changes were considered consistent with hormonal stimulation. This change in the thyroid was not observed in those animals treated at 25 mg/kg/day and, whilst the effect did show signs of regression at the end of the 1-month follow-up period, the pathological change was still apparent in animals from these three treatment groups.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Elevated serum TSH and total T3 levels were recorded for males and females treated with 312 mg/kg/day and for females only treated with 125 or 50 mg/kg/day at week 7. At the week 12/13 investigation elevated TSH and T3 levels were only recorded in males treated at 312 mg/kg/day.
Key result
Dose descriptor:
NOEL
Effect level:
> 25 - < 50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
>= 50 - < 125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Remarks on result:
other: The effect on the thyroid is considered an adaptive response to signs of liver enzyme induction at dose levels of >125 mg/kg/day. Reduced body weight gain and food intake was also recorded for females treated at 125 mg/kg/day and above.
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
50 mg/kg bw/day (actual dose received)
System:
endocrine system
Organ:
thyroid gland
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Conclusions:
The principal effect related to treatment with the test material under the conditions of this study was evidence of slight liver enzyme induction at 125 and 312 mg/kg/day and indications of a significant stimulatory effect on the thyroid gland in both sexes indicated by increases in circulatory levels of T3 and TSH, increased thyroid weights and follicular hypertrophy and hyperplasia observed microscopically. None of these findings relating to the liver or thyroid gland were recorded for animals treated at 25 mg/kg/day, therefore, this dose level was designated the no-observed-effect-level.
Executive summary:

Groups of male and female rats were fed the test material in the diet for a 3-month period at varying concentrations in order to maintain a daily intake of 0, 50, 125 or 312 mg/kg/day. At the end of the 3-month treatment period, some animals from each treatment group were maintained off-treatment for a period of 1 month to assess the progression or regression of any treatment-related changes. Since no no-effect-level was established in this initial study, a separate study was commenced in male and female rats with a new control group and a new low dietary inclusion concentration equivalent to an achieved dose level of 25 mg/kg/day.

At the dose level of 50 mg/kg/day and above there were strong indications of a stimulatory effect on the thyroid gland in males and females comprising increases in circulating T3 and TSH levels, increased thyroid weights and follicular hyperplasia and hypertrophy noted histopathologically. The changes showed some dose dependency with the pathological lesions graded as slight to moderate in the groups treated at 50 and 125 mg/kg/day and as strongly pronounced at 312 mg/kg/day. The changes in the thyroid were still apparent in these groups at the end of the follow-up recovery period albeit less pronounced. The increases in circulating GPT levels and increases in liver weight noted in rats treated with 125 or 312 mg/kg/day were considered indicative of a weak hepatic enzyme induction at these dose levels which could, at least in part, be responsible for the thyroid response at these dose levels although there was no significant reduction in circulating T4 levels reported. All changes were shown to be reversible at the end of the follow-up period although examination of the thyroid did not demonstrate complete resolution of the treatment-related changes.

Under the given experimental conditions of this study, the rats showed no treatment-related effects at the dose level of 25 mg/kg/day and this dose is, therefore, designated the no-observed-effect-level. Since the effect on the thyroid, including pathological changes, was dose dependent in degree and was still apparent, to a lesser degree, in the 50 mg/kg/day dose level group at the end of the follow-up off-treatment period, it is postulated that the no-observed-adverse-effect-level (NOAEL) in this study would be >50 but <125 mg/kg/day. This study was assigned a reliability rating of 1: reliable without restrictions.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Key study of regulatory design and conducted to GLP
System:
endocrine system
Organ:
thyroid gland

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

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
Study period:
90 days
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
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
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
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
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
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.
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
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
400 mg/kg bw/day (nominal)
System:
integumentary
Organ:
skin

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.

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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
400 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Key study of regulatory design and conducted to GLP
System:
integumentary
Organ:
other: Skin

Repeated dose toxicity: dermal - local effects

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

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.

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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
400
Study duration:
subchronic
Species:
rat
Quality of whole database:
Key study of regulatory design and conducted to GLP

Additional information

In the 90-day oral toxicity study in rats the only effect of treatment was found to be strong indications of a stimulatory effect on the thyroid gland of males and females comprising increases in circulating T3 and TSH levels, increased thyroid weights with follicular hyperplasia and hypertrophy noted histopathologically. Although dose levels of 50 mg/kg/day and above were implicated, these changes were associated with small increases in circulating GPT levels and minor increases in liver weight at dose levels of 125 and 312 mg/kg/day suggesting involvement of hepatic enzyme induction. However, there were no corresponding reductions in circulating T4 levels.

In the 90-day dermal toxicity study in rats no systemic toxicity was observed despite the significant local effect on the skin at the dosage of 2000 mg/kg/day. Evidence of absorption was presented by the toxicokinetic data from the study in a dose-dependent manner.

Justification for classification or non-classification

The stimulatory response shown in the thyroid in this study was not typical of the consequences of the feedback mechanism resulting from enhanced hepatic clearance of circulating thyroxine following hepatic enzyme induction. The evidence of an enzyme-inducing effect in the liver in this study was weak in comparison with the signs of thyroid stimulation, suggesting the potential for some direct thyroid effect as well as an adaptive response to possible hepatic enzyme induction. Consequently, the oral NOEL in this rat study was determined to be 25 mg/kg/day and the NOAEL to be >50 but <125 mg/kg/day (GHS Category 2 classification range is 10-100 mg/kg/day). In order to confirm the potential for STOT-RE classification GHS Category 2, it is important to establish whether the effect on the thyroid seen in the key study is adaptive or otherwise, is toxicologically relevant and is demonstrative of organ dysfunction. The authors state that the changes seen in liver weight and blood chemistry are not indicative of a hypermetabolic state hence the pronounced, threshold response demonstrated in the thyroid could be a direct effect of treatment. Therefore, it is concluded that the test item should be classified as Category 2 for STOT-RE.

No systemic toxicity was evident in the 90-day dermal toxicity study in rats despite dose dependent absorption data and significant local toxicity at the high dose level (2000 mg/kg/day).