3-Cyclohexene-1-methanol, α,4-dimethyl-α-(4-methyl-3-penten-1-yl)-

Repeated dose toxicity: dermal

Administrative data

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Version / remarks:

(1981)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(R*,R*)-α,4-dimethyl-α-(4-methyl-3-pentenyl)cyclohex-3-ene-1-methanol

EC Number:

208-205-9

EC Name:

(R*,R*)-α,4-dimethyl-α-(4-methyl-3-pentenyl)cyclohex-3-ene-1-methanol

Cas Number:

515-69-5

Molecular formula:

C15H26O

IUPAC Name:

6-methyl-2-(4-methylcyclohex-3-en-1-yl)hept-5-en-2-ol

Test material form:

other: Liquid

Details on test material:

- Name of the test substance used in the study report: alpha-Bisabolol

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Male and female Wistar rats Chbb:THOM (SPF) were supplied by Dr. Karl Thomae GmbH, Biberach/Riss, FRG. Only animals free from clinical signs of disease were used for the study.

- Reason for species selection: Rats were selected since this rodent species is recommended in the respective test guidelines. Wistar rats were selected since there is extensive experience available in the laboratory with this strain of rats.

- Age at study initiation: 9 - 11 weeks

- Weight at study initiation: The males were in the range of 274 - 297 g (group mean: 286 g) and the females were in the range of 209 - 232 g (group mean: 223 g).

- Housing: Singly housing in type DK III stainless steel wire mesh cages (floor area about 800 cm²). Underneath the cages, waste trays were fixed containing absorbent material (type 3/4 dustfree embedding).

- Diet: The food used was ground Kliba maintenance diet rat/mouse/hamster, 343 meal, supplied by Klingentalmuehle AG, Kaiseraugst, Switzerland. Food was available ad libitum.

- Water: drinking water (from water bottles) were available ad libitum.

- Acclimation period: 6 and 9-days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24°C
- Humidity: 30 - 70%
- Photoperiod: 12 hours light and 12 hours darkness.

Administration / exposure

Type of coverage:

semiocclusive

Vehicle:

olive oil

Details on exposure:

The first shearing of the fur was carried out 2 days before the first administration of the test substance. Thereafter, the fur was clipped when necessary, but at least once a week.
One day prior to the administration period the animals with clipped skin areas meeting the requirements of the test guidelines were distributed according to weight among the individual test groups animals, separated by sex.

Each day, the test substance was administered uniformly to the clipped dorsal skin (dorsal and dorsolateral parts of the trunk; at least 10% of the body surface) using 3 cc syringes for about 4 weeks. The treated skin was covered for 6 hours after administration using a semiocclusive dressing, consisting of 4 layers of porous gauze dressing and an elastic dressing. After removal of the dressing, the treated skin was washed with lukewarm water. Control animals received the vehicle, only. At the end of the administration period all surviving animals were sacrificed after a fasting period (withdrawal of food) for about 16 - 20 hours.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in the vehicle was tested over a period up to 7 days at room temperature.
Homogeneity analyses of the test substance preparations were performed in samples of the highest and lowest concentration drawn from bottom, middle and top of the solution at the start of the administration period. These samples also served for concentration control analyses. Additional
concentration control analyses were performed with samples from the mid concentration drawn from the middle of the solution at the start of the administration period.

Stability analyses: The stability of the test substance in the vehicle over 7 days at room temperature was verified analytically.

Homogeneity analyses: The homogeneity of the test substance in the vehicle was verified
Concentration control analyses: The correctness of the concentrations was proven; the analytically obtained values corresponded to about 97-103% of the target concentrations

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

6 hours/day; 7 days/week

Doses / concentrations

Remarks:

Doses / Concentrations:
50, 200, 1000 mg/kg bw/d
Basis:

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Due to the expected severe skin damage (as judged from several pretests), a 4-week administration as undiluted test substance or as solution > 25% was not possible. As the test substance is soluble in olive oil, this was selected as suitable vehicle.

Pretests:
Concentration: 100%; Dose: 1000 mg/kg bw/d; Administrations: 5; Findings:erosions, crusts, hemorrhages, erythema, scales, edema (from day 3 onward)
Concentration: 50% in olive oil; Dose: 1000 mg/kg bw/d; Administrations: 5; Findings: erosions, erythema, scales, edema (from day 3 onward)
Concentration: 50% in Lutrol; Dose: 1000 mg/kg bw/d; Administrations: 5; Findings: erosions, erythema, scales, edema (from day 3 onward)
Concentration: 25% in olive oil; Dose: 1000 mg/kg bw/d; Administrations: 5; Findings: erythema, scales, (day 5)
Concentration: 10% in olive oil; Dose: 400 mg/kg bw/d; Administrations: 5; Findings: nothing abnormal detected

Positive control:

not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) from Mondays to Fridays and once a day (in the morning) on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS + DERMAL IRRITATION (if dermal study): Yes
Comprehensive clinical examinations including detailed examination of the skin were carried out each day before, and about 30 minutes after treatment.

BODY WEIGHT: Yes
Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period the body weight was determined on day 0 (start of aclministration period) and thereafter at weekly intervals. The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as body weight change.

FOOD CONSUMPTION:
Food consumption was determined weekly over a period of 7 days and calculated as mean food consumption in grams per animal and day.

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
Water consumption was observed daily by visual inspection of the water bottles for any overt changes in volume.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
Blood was taken from the retroorbital venous plexus in the morning from fasted, not anesthetized animals.
Parameters:
- leukocytes
- erythrocytes
- hemoglobin
- hematocrit
- mean corpuscular volume
- mean corpuscular hemoglobin
- mean corpuscular hemoglobin concentration
- platelets
- differential blood count
- prothrombin time

CLINICAL CHEMISTRY: Yes
Blood was taken from the retroorbital venous plexus in the morning from fasted, not anesthetized animals.
Parameters:
- alanine aminotransferase
- aspartate aminotransferase
- alkaline phosphatase
- serum-y-glutamyltransferase
- sodium
- potassium
- chloride
- inorganic phosphate
- calcium
- urea
- creatinine
- glucose
- total bilirubin
- total protein
- albumin
- globulins
- triglycerides
- cholesterol
- magnesium


URINALYSIS: Yes
Individual animais were transferred to metabolism cages (withdrawal of food and water) and urine was collected overnight.
Parameters:
- volume
- color
- turbidity
- nitrite
- pH
- protein
- glucose
- ketones
- urobilinogen
- bilirubin
- blood
- specific gravity
- sediment

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The animals were sacrificed by decapitation under CO2 anesthesia. The exsanguinated animais were necropsied and assessed by gross pathology

ORGAN WEIGHTS: Yes
1. anesthetized animals
2. liver
3. kidneys
4. adrenal glands
5. testes
6. epididymides
7. ovaries
8. brain
9. thymus
10. heart
11. spleen

HISTOPATHOLOGY: Yes
Organs or tissues were fixed in 4% formaldehyde solution and stained with Hematoxylin-Eosin
- All gross lesions (all animals affected)
- Liver (control, high dose group)
- Kidneys (control, high dose group)
- Lungs (control, high dose group)
- Skin, treated (control, high dose group)
- Skin, untreated (control, high dose group)
- Testes (control, high dose group)

Skin, treated from animals of low, mid dose group was embedded in paraplast

Statistics:

Food consumption/efficiency, body weight/change, Clinical pathol. (excl. differential blood count): non-parametric one-way analysis using Kruskal-Wallis test (two-sided), Mann-Whithey U test (two-sided) as post test.
Organ weight parameters: non-parametric one-way analysis using Kruskal-Wallis test (two-sided), Wilcoxon test as post test.
Urinalysis (excl. volume, color, turbidity): Fisher´s exact test

Results and discussion

Results of examinations

Details on results:

CLINICAL SIGNS AND MORTALITY
During the entire study period no animal died prematurely.
Treated skin; immediately after removing of the dressing on several days:
Minimal erythema in one high dose male, 5 high dose females, each 3 mid and low dose females, and one control female;
Moderate erythema in 2 high dose females;
Focal scale formation in 1 high dose female;
Multifocal scale formation in 1 high dose male, 5 high dose females, 3 mid dose females, and 4 low dose females;
Diffuse scale formation in 4 high dose females.

Several findings persisted until the next observation before treatment on the following day:
Minimal erythema in 1 high dose male, 5 high dose females, 2 mid dose females, and 3 low dose females;
Moderate erythema in 2 high dose females;
Multifocal scale formation in 1 high dose male, 5 high dose females, 3 mid dose females, and 3 low dose females;
Diffuse scale formation in 4 high dose females.
The findings mentioned above were observed only on several days of the study, but usually did not persist until necropsy.


BODY WEIGHT AND WEIGHT GAIN
Body weight of the treated animals did not differ statistically significant from controls.
Body weight change was slightly and transiently impaired in high dose males and females, statistically significant in high dose males on day 7, only.


FOOD CONSUMPTION
No substance-related findings were observed.

FOOD EFFICIENCY
Food efficiency was impaired statistically significant in high dose males on day 7. Also in high dose females, the values were considerably lower than controls, although not statistically significant. This was assessed as being the consequence of the impairment in body weight change.

WATER CONSUMPTION
There were no overt changes in volume.

OPHTHALMOSCOPIC EXAMINATION

HAEMATOLOGY
At the end of the study decreased leukocytes and lymphocytes were found in the peripheral blood of the high dose males.
The other hematology examinations and clotting analyses revealed no treatment-related changes.

CLINICAL CHEMISTRY
Statistically significantly decreased calcium and glucose concentrations were measuredi in the sera of the high dose males.
The other clinical chemistry examinations revealed no treatment-related changes.

URINALYSIS
There are no treatment-related changes in the urinalytical parameters measured.

ORGAN WEIGHTS

Absolute weights:
In males of the high dose group, the mean terminal body weight was significantly (p < 0.05) reduced (- 5.4%).
In females of the high dose group, the mean liver weight was significantly (p < 0.05) decreased (- 9.9%) . The mean terminal body weight was also decreased (- 3.7%), however, this was not significant.
There were no further significant changes of the mean absolute weights when compared with the control group.

Relative weights (related to terminal body weight):
In male rats of the high dose group, the mean weight of the testes was significantly (p. < 0.05) increased (+ 26.4%).
There were no other significant changes of the mean relative weights when compared with the control group.

GROSS PATHOLOGY
A white focus with a diameter of 3 mm was recorded as a spontaneous lesion from the forestomach's wall of a male rat of the low dose group. Also of spontaneous origin is a small black focus that was noted in the glandular stomach of a female rat of the high dose group. The lesion was so small that it could not be demonstrated in the histological slide.
Other spontaneous gross lesions were noted in the mesenterc and the axillary lymph nodes (dark red discoloration) of a male rat (low dose group) and in one ovary (cyst) of a control female.
No gross lesion were noted in the treated skin or in any of the other organs.

HISTOPATHOLOGY: NON-NEOPLASTIC
Gross lesions:
A horny cyst in the submucosa was the correlate for the grossly described white focus in the mucosa of the forestomach of a male (low dose group).
The small black focus in the mucosa of the glandular stomach of a top dose female rat could not be identified histopathologically. However, a treatment
related relationship can be excluded as no such observation was made in any of the other treated rats.
The small cyst noted in the ovary of a control female during necropsy was confirmed by histopathology.
The grossly recorded dark red discoloration of the mediastinal and axillary lymph nodes of a male rat (low dose group) turned out as moderate or severe blood resorption. Although the origin of the blood remains uncertain, the
spontaneous nature of the lesion is evident.

Other organs:
Few microscopic findings were observed in the liver, kidneys, lungs, and testes of male and/or female rats of the control group or high dose group. They were either single observations or they were biologically equally
distributed over the control and treatment groups as well by their number as according to the graded severity.
However, in the liver of male and female rats of the high dose group, as well the number of animals affected as the grade of severity for peripheral fatty infiltration was lower than in the control group.
No histologic findings were recorded from the treated and the untreated skin.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

dermal irritation

food efficiency

2

Dose descriptor:

LOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

dermal irritation

food efficiency

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The no observed effect level (NOEL) under the conditions of this study was 200 mg/kg body weight/day.