Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
09 September 1999 to 29 April 2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: According to OECD Guideline 411 and GLP but incorrect dermal data presentation

Data source

Reference
Reference Type:
other company data
Title:
Unnamed
Year:
2002

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
[3R-(3α,3aβ,7β,8aα)]-1-(2,3,4,7,8,8a-hexahydro-3,6,8,8-tetramethyl-1H-3a,7-methanoazulen-5-yl)ethan-1-one
EC Number:
251-020-3
EC Name:
[3R-(3α,3aβ,7β,8aα)]-1-(2,3,4,7,8,8a-hexahydro-3,6,8,8-tetramethyl-1H-3a,7-methanoazulen-5-yl)ethan-1-one
Cas Number:
32388-55-9
Molecular formula:
C17H26O
IUPAC Name:
1-((3R,3aR,7R,8aS)-3,6,8,8-tetramethyl-2,3,4,7,8,8a-hexahydro-1H-3a,7-methanoazulen-5-yl)ethanone
Test material form:
other: yellow liquid
Details on test material:
- Name of test material (as cited in study report): Acetyl cedrene
- Physical state: yellow liquid
- Lot/batch No.: HRJ-04-2709
- Expiration date of the lot/batch: Received 22/07/1999; Expiration 01/10/2001
- Storage condition of test material: Stored at room temperature protected from light

Test animals

Species:
rat
Strain:
other: Crl:CD(SD) IGS BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Inc., Michigan, USA.
- Age at study initiation: 60-66 days
- Weight at study initiation: Male rats: 255-348 g; Female rats: 175-242 g.
- Housing: Housed individually in suspended, stainless-steel cages
- Diet (e.g. ad libitum): Certified rodent diet (No. 8728C Harlan Teklad) ad libitum. Animals fasted for clinical pathology.
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days before initiation of treatment. Animals collared for 6 hours/day for 3 days to acclimate to collars.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19°C-25°C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12 hrs dark/ 12 hrs light

IN-LIFE DATES: End date: 13/01/2000

Administration / exposure

Type of coverage:
other: Gauze dressing over the application site, secured with nonirritating tape, covered with elastic bandage, and overwrapped with elastic tape
Vehicle:
water
Remarks:
reverse osmosis
Details on exposure:
TEST SITE
- Area of exposure: Dorsal trunk area
- % coverage: 5cm x 5cm
- Type of wrap if used: Gauze dressing over the application site, secured with nonirritating tape, covered with elastic bandage, and overwrapped with elastic tape.
- Time intervals for shavings or clipplings: Fur clipped from dorsal trunk area before 1st dose and as needed after.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): At the end of each exposure, the area was wiped gently with a dosposable paper towel moistened with reverse osmosis water to remove any remaining test material.
- Time after start of exposure: At the end of each exposure period i.e. after 6-7 hrs exposure per day.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50, 150, 300 mg/kg bw/day
- Constant volume or concentration used: No. 0.050mL; 0.150mL; .300mL for 0, 50, 150, 300 mg/kg bw/day respectively.
Dose preparations were dispensed approximately weekly.


VEHICLE
- Amount(s) applied (volume or weight with unit): 0 mg/kg/day as 0.300 mL/kg bw/day

USE OF RESTRAINERS FOR PREVENTING INGESTION: Yes. Flexible plastic collars during exposure period.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
The test material was dosed as supplied, dose analysis was not done.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6-7 hours per day , 7 days per week
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 150, 300 mg/kg bw/day
Basis:
nominal per unit body weight
No. of animals per sex per dose:
30 animals (15 male and 15 female) per dose

Five animals/sex/group were designated as recovery animals and were observed for approximately 4 weeks post-treatment.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:Not provided in this test report
- Rationale for animal assignment (if not random): Randomised
- Rationale for selecting satellite groups: Five animals/sex/group were designated as recovery animals and were observed for approximately 4 weeks
post-treatment.
- Post-exposure recovery period in satellite groups: 4 weeks
- Section schedule rationale (if not random): Randomised

Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were observed twice daily (a.m. and p.m.) for mortality and moribundity. Once daily for 2 weeks and weekly thereafter during treatment, on the day of terminal necropsy (all animals), daily during recovery, and on the day of recovery sacrifice, detailed observations were made for each animal; abnormal findings or an indication of normal was recorded.

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: Dermal irritation will be scored daily immediately before each application (Table 1), on the day of terminal necropsy (all animals), daily during recovery, and on the day of recovery sacrifice.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weight data were recorded on the first day of treatment and weekly thereafter.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Individual food consumption data (grams of food) were recorded weekly for males and females during treatment and recovery.

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

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to treatment and during Weeks 13 and 17 an examination was performed by a veterinarian on all animals using an indirect ophthalmoscope. The eyes were dilated with a mydriatic agent prior to examination.
- Dose groups that were examined: All groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected from each animal during Weeks 13 and 17.
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes; overnight
- How many animals: All animals
- Parameters checked in Table 2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected from each animal during Weeks 13 and 17.
- Animals fasted: Yes; overnight
- How many animals: All animals
- Parameters checked in Table 3 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No




Sacrifice and pathology:
GROSS PATHOLOGY: Yes
A necropsy was done on each animal that was sacrificed or found dead at an unscheduled interval. After Week 13, 10 animals/sex/group (based on survival) were necropsied. After 13 weeks of treatment and 4 weeks of recovery, all surviving recovery animals were necropsied. The necropsy included a macroscopic examination of the extemal features of the carcass; application site; extemal body orifices; the abdominal, thoracic, and cranial cavities; organs; and tissues. Organ weights were also recorded (see Table 4).


HISTOPATHOLOGY: Yes (see Table 5)
Tissues from each animal in the control and high-dose groups and each animal that died or was sacrificed at an unscheduled interval were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically. Macroscopic lesions and the lung, liver, kidney, and skin (treated and untreated) were also examined microscopically from each animal in the low- and mid-dose groups.
Other examinations:
Estrous Cycle.
Beginning on Day 71 and continuing until Day 91, all females had daily vaginal smears done. Beginning on Day 100 and continuing until Day 120, all recovery females had daily vaginal smears done. Vaginal smears were prepared and examined to evaluate the stage of the estrous cycle.

Male Reproductive Assessment.
Male reproductive capacity was evaluated for 10 males/group at the terminal sacrifice. Sperm morphology, motility and total count were assessed.
Statistics:
Variance homogeneity: Levene's test. (Levene, 1960)

Body weights, body weight changes, food consumption, continuous clinical pathology values, organ weight data : ANOVA; Dunnett's t-test (Dunnett, 1964) .

Group comparisons: two tailed t- test (p<0.05)

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
See details on results
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
See details on results
Mortality:
no mortality observed
Description (incidence):
See details on results
Body weight and weight changes:
no effects observed
Description (incidence and severity):
See details on results
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
See details on results
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
See details on results
Haematological findings:
no effects observed
Description (incidence and severity):
See details on results
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
See details on results
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
See details on results
Gross pathological findings:
no effects observed
Description (incidence and severity):
See details on results
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
See details on results
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
See details on results
Details on results:
CLINICAL SIGNS AND MORTALITY
Two females in control group died at day 33 (no adverse reason identified). One female in mid-dose sacrificed at day 63 as moribund (due to spontaneous fibrosarcoma). All other animals survived and no test material-related clinical observations noted.

DERMAL IRRITATION
Dermal irritation observations noted during the treatment phase included erythema, edema, atonia, desquamation, and fissuring for animals treated with the test material. One female given 50 mg/kg bw/day had exfoliation. Generally, incidence and severity increased in a dose-related manner and severity ranged from slight to severe or marked. In general, the onset of observations occurred during Weeks 1 to 2 (the earliest observations were noted on Day 4.). Most observations disappeared or the severity was only slight prior to the terminal sacrifice during Week 14. Erythema, edema, atonia, and desquamation were still noted during recovery in animals treated with test material; however, the severity was only slight, which indicates that animals were improving. Most of the observations noted during the recovery period disappeared during Weeks 14 and 15.

BODY WEIGHT AND WEIGHT GAIN
There were no test material-related effects on mean body weights. Body weight changes were statistically significantly lower during Week 1 for females given 50 mg/kg bw/day, during Week 10 for males given 50 or 300 mg/kg bw/day, and during Week 13 for males given 150 mg/kg bw/day. Body weight changes were higher for females during Weeks 15 and 16, significantly during Week 15 for females given 50 or 150 mg/kg bw/day (133% and 200% of controls, respectively). Throughout the duration of the recovery phase (Weeks 14 to 18), body weight changes for the females in all dose groups were remarkably higher, 187.5 to 250% of the control females while body weight changes for the males were 92 to 96% of the control males. The reason for this difference is probably due to the difference in growth rate for males and females. The sporadic significant differences in body weight gains were considered within normal biologic variation.

FOOD CONSUMPTION
Food consumption was statistically significantly higher (7 to 9%) during Weeks 1, 3, and 7 for females given 50 mg/kg bw/day compared with controls, and during Weeks 1 and 2 for females given 300 mg/kg bw/day compared with controls. During Week 4, food consumption was statistically significantly decreased by 8% for females given 150 or 300 mg/kg bw/day. These changes in food consumption were probably due to normal variation and are not considered to be adverse.

OPHTHALMOSCOPIC EXAMINATION
Animals selected for the study had no lesions at the prestudy examination. One incidence of focal retinal degeneration in the right eye was noted during the Week 13 examination for a male given 150 mg/kg bw/day. This finding was considered due to biological variation and not test material-related. No test material-related ophthalmic observations were noted at the Week 17 examination.


HAEMATOLOGY and CLINICAL CHEMISTRY
Administration of Acetyl Cedrene had no obvious effects on clinical pathology test results. Of uncertain relationship to administration of the Acetyl Cedrene was slightly, but statistically, higher activated partial thromboplastin time for males treated with 300 mg/kg bw/day. The difference in the mean values for the control males and the males treated with 300 mg/kg bw/day was less than 1 second, and females were not similarly affected.

ORGAN WEIGHTS, GROSS PATHOLOGY, HISTOPATHOLOGY: NON-NEOPLASTIC
At the terminal sacrifice, test material-related increases in kidney-to-body weight percentages were noted in Group 3 and 4 males. In the kidneys of the males given 300 mg/kg bw/day, hyaline droplet formation was noted in the tubular epitheum. This finding may be unique to the renal tubular biochemistry of the male rat. By the recovery sacrifice, this finding had resolved. At the terminal sacrifice, mild chronic inflammation and acanthosis/hyperkeratosis was noted in the treated skin from all dose groups and both sexes. Again, these changes had completely resolved by the recovery sacrifice.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
One female in mid-dose sacrificed at day 63 as moribund (due to spontaneous fibrosarcoma).

OTHER FINDINGS
Sperm Analysis Report
Mean percent motility, total sperm count, and morphology were not affected by treatment with Acetyl Cedrene at dosage levels of 50,150, and 300 mg/kg bw/day. No biologically meaningful differences were observed between the study groups.

Estrous Cycle
There were no test material-related effects during the treatment or recovery periods on the estrous cycle of females given 0,50,150, or 300 mg/kg bw/day of Acetyl Cedrene.

Effect levels

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: not appplicable

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Daily dermal administration of Acetyl Cedrene to male and female Crl:CD(SD)IGS BR rats for 13 weeks with a 4-week recovery was associated with test material-related observations in the treated skin of treated animals at all dose levels. However, these findings were resolved by the recovery sacrifice. Based on the findings in this study, the NOAEL is considered to be 300 mg/kg bw/day. The LOAEL (local) is 50mg/kg bw/day.

Executive summary:

In a repeated dose dermal toxicity study (Covance 7018 -100), Methyl Cedryl Ketone was applied to the shaved skin of four groups of Crl:CD(SD)IGS BR male and female rats (15/sex/group) at dose levels of 0, 50, 150, 300 mg/kg bw/day, 6 -7 hours/day for 7 days/week during a 13 week period, with a 4 week recovery period.

There were no test material-related effects in mortality, clinical signs, body weight, food consumption, hematology, clinical chemistry, organ weights, or gross and histologic pathology.

At the terminal sacrifice, test material-related increases in kidney-to-body weight percentages were noted in Group 3 and 4 males. In the kidneys of the males given 300 mg/kg bw/day, hyaline droplet formation was noted in the tubular epitheum. This finding may be unique to the renal tubular biochemistry of the male rat. By the recovery sacrifice, this finding had resolved.

Dermal irritation observations noted during the treatment phase included erythema, edema, atonia, desquamation, and fissuring for animals treated with the test material. In general, the effects were dose dependent and were evident beginning from weeks 1 -2, but were resolved upon sacrifice or were almost fully resolved during weeks 14 -15 of the recovery phase. The NOAEL is 300 mg/kg bw/day. The LOAEL (local) is 50 mg/kg bw/day.