Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Equivalent or similar to OECD 414 and indicated in compliance with GLP.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2006

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity 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: clear, yellowish brown liquid
Details on test material:
- Name of test material (as cited in study report): Acetyl cedrene
- Physical state: clear, yellowish brown liquid
- Lot/batch No.:HRF053136
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
other: Sprague Dawley; Crl:CD (SD) IGS BR VAF/Plus
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, North Carolina, USA.
- Weight at study initiation: Females weighed 178 to 219 g; male rats weighed 272 to 378 g
- Housing: Housed individually, All cage sizes and housing conditions were in compliance with the Guidefor the Care and Use ofLaboratory Animals (Institute of Laboratory Animal Resources 1996).
- Diet (e.g. ad libitum): Certified Rodent Diet 5002 (PMI Nutrition International, St. Louis, Missouri) ad libitum
- Water (e.g. ad libitum): Reverse-osmosis deionized water, ad libitum
- Acclimation period: Acclimation began at arrival and was for 5 days until cohabitation period

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 64°F-79°F
- Humidity (%): 30-70%
- Air changes (per hr): At least ten changes per hour of 100% fresh air passed through 99.97% HEPA filters
- Photoperiod (hrs dark / hrs light): 12 hrs dark/ 12 hrs light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
mazola
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:Dosing formulations were prepared daily from bulk materials. The dosage volume was 1 mL/kg, adjusted daily according to individual body weights recorded directly before gavage and administered at approximately the same time each day.


VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil is acceptable according to OECD 414
- Concentration in vehicle: 0, 25, 50, or l00 mg/kg bw/day
- Amount of vehicle (if gavage): Dosage volume of 1 mL/kg.
- Lot/batch no. (if required): APR1902A
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples from each concentration of the dosing suspensions (first and last days of treatment) were analyzed for AC content by International Flavors & Fragrances. Results from the analysis of dosage preparations verified that actual dosages reflected the calculated dosages ±10%. Achieved concentrations of AC were 0, 23.62, 44.95, and 102.87 mg/mL.

Details on mating procedure:
- Impregnation procedure: Co-housed
- If cohoused: Each pair of male and female rats was housed in the male rat's cage
- M/F ratio per cage: 1:1
- Length of cohabitation: Until the day when sperm was found in the vaginal smear or a copulatory plug was found in the vagina (gestation day 0).
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy (gestation day 0)
Duration of treatment / exposure:
Days 7 through 17 of gestation (GDs 7 through 17)
Frequency of treatment:
Once daily
Duration of test:
Until gestation day 21
No. of animals per sex per dose:
0 mg/kg bw/day: 25 female pregnant animals
25 mg/kg bw/day: 23 female pregnant animals
50 mg/kg bw/day: 21 female pregnant animals
100 mg/kg bw/day: 24 female pregnant animals


Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dosages used in the full study were determined from results obtained by a previous two-part dosage-range study in which eight pregnant rats per group were treated on GDs 7 through 17.

Part 1: four groups of rats were administered gavaged volumes of 0.25, 0.50, 1.0, or 2.0 mL/kg/day neat acetyl cedrene (250, 500, 1000, or 2000 mg/kg bw/day based on a specific gravity of 1.0). A fifth group (control) was administered 2 mL/kg of water. All rats at the highest dosage died or were sacrificed in a moribund state; one rat died and another delivered prematurely in the 1000 mg/kg bw/day group; dosage-related reductions in feed consumption and body weight gains occurred in all surviving acetyl-cedrene treated groups. Fetal body weights were reduced in the 500 and 1000 mg/kg groups. Other external alterations were not considered to be due to test substance because incidences were within the historical ranges of the testing facility.

Part 2: Four groups of pregnant rats administered 0 (corn oil vehicle), 50, 100, or 250 mg/kg bw/day in a dosage volume of 1 mL/kg. No
deaths occurred in any group; excess salivation, reduced feed consumption, and body weight gains were observed at 100 and 250 mg/kg bw/day; no cesarean-sectioning or litter parameters were affected by any acetyl cedrene dosage; there were no gross external fetal alterations.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: No


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:Animals were observed daily for clinical signs

BODY WEIGHT: Yes
- Time schedule for examinations: Prior to start of study, during dosing and postdosing period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes - Days 0, 7, 10, 12, 15 and 18 and 21.

- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Maternal absolute and relative feed consumption values (mean of each group) in g/day for Days 0-7, 7-10, 10-12, 12-15, 15-18, 18-21.

- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: Uteri of apparently nonpregnant rats and pregnant rats

OTHER: Gross necropsy of the thoracic, abdominal, and pelvic viscera was performed.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: No
Statistics:
Clinical observation/other proportion data : variance test for homogeneity of the binomial distribution (Snedecor and Cochran 1967);
Continuous data: Bartlett's test (Sokal and Rohlf 1969);
ANOVA: Snedecor and Cochran 1967;
Statistical significance of individual groups: Dunnett's test (Dunnett 1955), Kruskal-Wallis test (Sokal and Rohlf 1969), Dunn's method of multiple comparisons (Dunn 1964), Fisher's exact test (Siegel 1956);
Level of significance p<0.01.
Indices:
See Table 4: Cesarean section and litter data in Results.
Historical control data:
None

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Mortality: No deaths or premature deliveries occurred in the study.

Clinical observations:There was statistically significant salivation in 11/25 (100mg/kg) and 5/25 (50mg/kg) due to administration of compound.

Body weight gain: In the high dosage group (100 mg/kg bw/day), there were significantly lower mean body weights compared to controls. Body weight gains were significantly reduced in transient manner on GDs 7 to 10 in this dosage group. All other body weights and body weight gains were generally comparable among the four dosage groups (Table 1).

Food consumption: Maternal absolute(g/day) (Table 2) and relative (g/kg/day) (Table 3) feed consumption values were significantly decreased on GDs 7 to 12 and 15 to 18 (relative only) and the entire dosage period (calculated as GDs7 to 18) in the 100 mg/kg bw/day dosage group.

Gross pathology: Necropsy of the dams after cesarian section did not reveal any morphological changes attributable to the test substance.

Cesarean section observations: There were no dead fetuses. Litter averages for corpus lutea, implantation, litter size, live fetuses, early and late resorption, fetal weight, % resorbed conceptuses and % live male fetuses were comparable across dose groups and did not significantly differ. No uterine, ovarian, or litter parameters were affected by the test substance in doses as high as 100 mg/kg bw/day (Table 4).

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Basis for effect level:
other: maternal toxicity

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Mortality: 100% live in all dose groups

External, soft tissue, and skeletal malformations and other relevant alterations:
Any fetal gross, soft tissue or skeletal alteration that occured was not dose dependent, occurred in single fetus or was not statistically significant.
Therefore, no fetal abnormalities were attributed to the test substance. (Table 5)

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: No effects were observed at 100 mg/kg bw/day, the highest dosage tested

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

Table 1: Maternal body weight changes

Dosage group (mg/kg bw/day) (a)
  0 (control) 25 50 100
Rats tested 25 25 25 25
Rats pregnant 25 23 21 24
Body weight increases of pregnant rats  (g) (b)      
Days 0-7 32.3 ±7.8 30.8 ± 8.3 33.3 ± 6.4 31.3 ±8.7
Days 7-10 16.4 ±3.9 15.5 ±8.5 12.7 ±6.0 5.8 ± 5.9**
Days 10-12 11.8±4.2 13.4 ±5.1 13.0 ±3.3 13.7 ±5.2
Days 12-15 17.3 ±3,0 17.4 ±4.5 17.5 ±4.0 19.8 ±6.4
Days 15-18 37.9 ±7.3 39.7 ±4.9 40.1 ±6.2 41.7 ±7.6
Days 7-18 83.4 ±11.5 86.1 ± 12.2 3.3 ±9.6 80.9 ± 10.1
Days 18-21 57.7 ± 10.3 60.7 ±9.0 53.8 ±10.6 59.1 ±9.1
Days 7-21 141.1 ± 18.5 146.8 ± 16.2 137.1 ± 11.7 140.0 ±17.0
Days 0-21 173.4 ±22.3 177.6 ± 17.1 170.4 ±11.0 171.3 ± 19.6

(a) Dosage occurred on days 7 through 17 of gestation.

(b) Values are mean ±SD).

**Significantly different from the control value {p < 0.01)

Table 2: Maternal body weight changes

Dosage group (mg/kg bw/day) (a)   
  0 (control) 25 50 100
Rats tested 25 25 25 25
Rats pregnant 25 23 21 24
Maternal feed consumption (g/day) (b)      
Days 0-7 21.9 ±2.0 21.5 ± 1.6 22.0 ± 1.8 21.5 ±1.8
Days 7-10 23.5 ±2.3 22.8 ±2.8 22.4 ± 2.6 17.7 ±2.3**
Days 10-12 23.6 ±2.4 22.5 ±2.6 22.6 ± 2.3 20,2 ±2.2**
Days 12-15 23.8 ± 1.9 23.2 ± 2.6 23.3 ±2.3 22.6 ±2.1
Days 15-18 25.4 ±3.1 26.1 ±2.3 26.7 ± 2.4 27.1 ±2,9
Days 7-18 24.1 ±2.0 23.7 ±2.1 23.8 ± 1.8 22,0 ± 1,9
Days 18-21 26.0 ±2.3 26.6 ± 2.3 25.4 ±2.9 26.5 ± 2.5
Days 7-21 24.5 ± 1.9 24.4 ± 1.9 24.1 ± 1.6 23.0 ± 1.7
Days 0-21 23.6± 1.8 23.4 ± 1.6 23.4 ±1.5 22.5 ± 1.6

(a) Dosage occurred on days 7 through 17 of gestation.

(b) Values are mean ±SD).

**Significantly different from the control value {p < 0.01)

Table 3: Maternal relative feed consumption values

Dosage group (mg/kg bw/day) (a)    
  0 (control) 25 50 100
Rats tested 25 25 25 25
Rats pregnant 25 23 21 24
Maternal feed consumption (g/kg/day) (b)      
Days 0-7 87.9 ±6.5 86.8 ±4.9 88.1 ±5.4 87.0 ±5.2
Days 7-10 85.9 ±6.3 84.3 ± 9.7 82.5 ± 8.2 66.9 ± 7.2**
Days 10-12 82.0 ±5.6 78.8 ±7.7 79.1 ±5.9 73.4 ±6.2**
Days 12-15 79.4 ± 4.6 77.3 ±7.5 77.7 ±6.3 77.6 ±5.9
Days 15-18 77,3 ± 8.6 79.7 ±6.4 81.3 ±7.0 84.3 ±7.2**
Days 7-18 80.6 ±4.6 79.9 ±5.7 80.1 ±4.3 76.0 ±4.5**
Days 18-21 69.5 ±4.6 70.8 ±5.2 68.0 ±7.8 71.4±4.8
Days 7-21 77.7 ±4.1 77.4 ±4.6 76.8 ±3.6 74.8 ±3.5
Days 0-21 76.2 ±4.1 75.6 ±3.8 75.6 ±3.3 74.2 ± 2.7

(a) Dosage occurred on days 7 through 17 of gestation.

(b) Values are mean ±SD).

**Significantly different from the control value {p < 0.01)

Table 4: Cesarean section and litter data

Dosage group (mg/kg bw/day) (a) 
  0 (control) 25 50 100
Rats tested 25 25 25 25
Rats cesarian sectioned GD 21 25 (100%) 23 (92%) 21 (84%) 24 (96%)
Corpora lutea (b) 17.6 ±3.0 18.1 ±3.4 18.0±3.1 17.8 ±3.4
Implantations (b) 15.4 ±2.2 15.2 ±2.0 15.1 ± 1.7 15.4 ± 1,9
Resorptions (b) 1.1 ±2.4 0.7 ± 0.8 0.8 ± 1.1 0.4 ± 0.9
Litter size (b) 14.4 ±2.9 14.5 ±2.1 14,4 ±2.4 15.0 ±1.8
Fetal body wt/litter (b) 5.03 ± 021g 5.09 ± 0.24g 4.97 ± 0.20 5.00 ± 0.36g
No. live fetuses (b) 359 333 302 360
No. dead fetuses (b) 0 0 0 0

(a) Dosage occurred on days 7 through 17 of gestation.

(b) Values are mean ±SD.

Table 5: Fetal alterations observed (includes gross external, soft tissue or skeletal variations or malformations)

Dosage group (mg/kg bw/day) (a)  
0 (control) 25 50 100
No. of litters evaluated 25 23 21 24
No, of fetuses evaluated 359 333 302 360
No. of litters with fetuses with any observed alterations (%)  9 (36.0)  7 (30.4)  4 (19.0)  6 (25.0)
No. of fetuses with any observed alterations (%)  11(3.1)  10(3.0)  5(1,6)  10(2.8)
% fetuses with any alteration per litter (mean   ±SD) 0.4 ± 0.6 0,4 ± 0.7 0.2 ± 0.5 0.4 ± 0.9

(a) Dosage occurred on days 7 through 17 of gestation.

Applicant's summary and conclusion

Conclusions:
Oral gavage administration of Acetyl Cedrene (0, 25, 50, or l00 mg/kg bw/day in mazola corn oil) to female rats during DG 7-17 weeks was associated with the following test material-related maternal observations: clinical signs (excessive salivation at 50 and 100 mg/kg bw/day), significant reductions in absolute and relative feed consumption (GDs 7 to 12) and body weight gains (GDs 7 to 10) at 100 mg/kg bw/day when compared to controls. No gross external, soft tissue, or skeletal fetal alterations (malformations or variations) were attributed to any dose of the test substance. Based on this study, the maternal NOAEL is 50 mg/kg bw/day and the developmental NOAEL is 100 mg/kg bw/day.
Executive summary:

In a pre-natal developmental toxicity study, Methyl Cedryl Ketone was administered to 25 Sprague Dawley; Crl:CD (SD) IGS BR VAF/Plus females/dose by gavage in corn oil at dose levels of 0, 25, 50, or 100 mg/kg bw/day from days 7 through 17 of gestation.

No deaths or premature deliveries occurred in the study. No cesarean-sectioning or litter parameters were affected by dosages of the test substance and necropsy of the dams after cesarean section did not reveal any gross changes attributable to the test substance. Clinical signs included excessive salivation at 50 and 100 mg/kg bw/day, which was attributed to the administration of the substance. Significant reductions in absolute and relative feed consumption (GDs 7 to 12) and body weight gains (GDs 7 to 10) occurred only at 100 mg/kg bw/day when compared to controls. The maternal NOAEL is 50 mg/kg bw/day.

There were no dead fetuses. No gross external, soft tissue, or skeletal fetal alterations (malformations or variations) were attributed to any dose of the test substance. The average number of ossifications sites per fetus per litter did not differ among the groups. The developmental NOAEL is 100 mg/kg bw/day.

The developmental toxicity study in the rat is classified acceptable and satisfies the guideline requirement for a developmental toxicity study (OECD 414) in rats.