Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2005
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, available as unpublished report, no resitrictions, fully adequate for assessment.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Alpha-isomethylionone (also referred to as Meth Ionone Gamma A, and as isoraldeine 95) - a clear, pale yellow to yellow liquid.
Lot number: RO00619567.
Composition: 61.7% alpha-isomethylionone (CASnr 127-51-5), 20.9% n-alphamethylionone (CASnr 7779-30-8), 7.9% n-betamethylionone (CASnr 127-43-5)
The test article was received on 17 September 2004 and stored at room temperature, protected from light.
Information to document or certify the identity, composition, strength and purity of the test article was provided by the Sponsor to the Testing Facility. A Certificate of Analysis is available. Documentation that the method of synthesis is on file with the Sponsor was provided by the Sponsor to the Testing Facility. The expiration date is December 2005.

Test animals

Species:
rat
Strain:
other: Crl:CD®(SD)IGS VAF/Plus®
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina
- Age at study initiation: 62 days
- Weight at study initiation: 213 - 241
- Fasting period before study: no data
- Housing: Rats were individually housed in stainless steel, wire-bottomed cages, except during the cohabitation period. During cohabitation, each pair of male and female rats was housed in the male rat’s cage. All cage sizes and housing conditions were in compliance with the Guide for the Care and Use of Laboratory Animals(8).
- Diet (e.g. ad libitum): Rats were given ad libitum access to Certified Rodent Diet® #5002 (PMI® Nutrition International, Inc., St. Louis, Missouri) in individual feeders.
- Water (e.g. ad libitum): Local water that had been processed by passage through a reverse osmosis membrane (R.O. water) was available to the rats ad libitum from an automatic watering access system and/or individual water bottles attached to the cages. Chlorine was added to the processed water as a bacteriostat.
- Acclimation period: yes


ENVIRONMENTAL CONDITIONS
- Temperature (°C): targeted at 64°F to 79°F (18°C to 26°C);
- Humidity (%): targeted at 30% to 70%
- Air changes (per hr): ten changes per hour of 100% fresh air that had been passed through 99.97% HEPA filters
- Photoperiod (hrs dark / hrs light): An automatically controlled 12-hours light:12-hours dark fluorescent light cycle was maintained. Each dark period began at 1900. The lights were turned on 9 minutes early on 15 December 2004, to facilitate the laboratory schedule.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Suspensions of the test article were prepared daily at the Testing Facility.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Lot/batch no. (if required): 103K0107 and 074K0025
- Purity: Neither the Sponsor nor the Study Director was aware of any potential contaminants likely to have been present in the vehicle that would have interfered with the results of this study. The expiration dates are October 2007 (lot 103K0107) and November 2008 (lot 074K0025).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The results of all concentration and homogeneity analyses were within ±10% of target concentration and ≤5% RSD, respectively. The 3 mg/mL concentration level was determined to be stable for ten days when stored at ambient conditions, protected from light. The results of these analyses are available. Stability at the same conditions covering a range of 0.125 to 1 mg/mL was verified in CR-DDS, Argus Division Protocol TIF00001.
Details on mating procedure:
After acclimation, 140 virgin female rats were placed into cohabitation with 140 breeder male rats, one male rat per female rat. The cohabitation period consisted of a maximum of five days. Female rats with spermatozoa observed in a smear of the vaginal contents and/or a copulatory plug observed in situ were considered to be DG 0 and assigned to individual housing.
Rats were observed for viability at least twice each day of the study and for general appearance weekly during the acclimation period and on DG 0.
Duration of treatment / exposure:
Gestation Days 7-17
Frequency of treatment:
once daily
Duration of test:
21 days
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
3 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
10 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
30 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
25 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dosages were selected on the basis of a dosage-range study (Argus Protocol TIF00001), in which dosages of 0, 1.25, 2.5, 5 and 10 mg/kg/day were administered daily on DGs 7 to 17. In that range-finding study, no adverse clinical observations were considered testarticle related. Although not dosage dependent, body weight gains were increased during the entire dosage period (defined as DGs 7 through 18), the gestation period following the initiation of dosage administration (DGs 7 to 21), and the complete gestation period (DGs 0 to 21), in comparison with the vehicle control group values. During the postdosage period (DGs 18 to 21), body weight gains remained increased in the 1.25, 2.5, 5 and 10 mg/kg/day dosage groups, as compared to the vehicle control group value. Absolute (g/day) and relative (g/kg/day) feed consumption values during the dosage period (DGs 7 to 18) were comparable between the five dosage groups. All rats were pregnant. There were no litters consisting of all resorbed conceptuses, and there were no dead fetuses. All Caesarean-sectioning and litter observations were comparable between the five dosage groups, and no fetal gross alterations were noted. Based on these data, dosages of 0 (Vehicle), 3, 10 and 30 mg/kg/day of alpha-iso-Methylionone were selected for the developmental toxicity study in rats. The 10 mg/kg/day dosage was expected to be a no-observable-adverse-effect-level (NOAEL) for both maternal and embryo-fetal toxicity.
- Rationale for animal assignment (if not random): Upon arrival, rats were assigned to individual housing on the basis of computer-generated
random units. Healthy, mated female rats were assigned to four dosage groups (Groups I through IV), 25 rats per dosage group, by using a computer-generated (weight-ordered) randomization procedure based on body weights recorded on DG 0.

Examinations

Maternal examinations:
Female rats with spermatozoa observed in a smear of the vaginal contents and/or a copulatory plug observed in situ were considered to be DG 0 and assigned to individual housing. Rats were observed for viability at least twice each day of the study and for general appearance weekly during the acclimation period and on DG 0. The rats were also examined for clinical observations, abortions, premature deliveries and deaths before and approximately 60 ± 10 minutes after dosage administration and once daily during the postdosage period.
Body weights were recorded weekly during the acclimation period, on DG 0, and daily during the dosage and postdosage periods. Feed consumption values were recorded on DGs 0, 7, 10, 12, 15, 18 and 21.
Ovaries and uterine content:
All female rats were sacrificed by carbon dioxide asphyxiation on DG 21, Caesarean-sectioned and a gross necropsy of the thoracic, abdominal and pelvic viscera was performed. Uteri of apparently nonpregnant rats were examined while being pressed between glass plates to confirm the absence of implantation sites. Uteri and ovaries of apparently nonpregnant rats were retained in neutral buffered 10% formalin will be discarded when authorized by the Study Director.
The number and distribution of corpora lutea were recorded. The uterus of each rat was excised and examined for pregnancy, number and distribution of implantation sites, live and dead fetuses and early and late resorptions. An early resorption was defined as a conceptus in which organogenesis was not grossly evident. A late resorption was defined as one in which the occurrence of organogenesis was grossly evident. A live fetus was defined as a term fetus that responded to stimuli. Nonresponding term fetuses were considered to be dead (there were no dead fetuses). Dead fetuses and late resorptions were differentiated by the degree of autolysis present; marked to extreme autolysis indicated that the fetus was a late resorption. Placentae were examined for size, color and shape.
Fetal examinations:
Each fetus was removed from the uterus, placed in an individual container and individually identified with a tag noting the study number, litter number, uterine distribution and fixative. Each fetus was subsequently weighed and examined for sex and gross lesions. Live fetuses were sacrificed by an intraperitoneal injection of sodium pentobarbital. Approximately one-half of the fetuses in each litter were examined for soft tissue alterations, using an adaptation of Wilson’s sectioning technique. These fetuses were fixed in Bouin’s solution; sections were retained in alcohol. The remaining fetuses
(approximately one-half of the fetuses in each litter) were eviscerated, cleared, stained with alizarin red S and examined for skeletal alterations. The fetuses were initially fixed in alcohol. Skeletal preparations were retained in glycerin with thymol added as a preservative.
Statistics:
Data generated during the course of this study were recorded either by hand or using the Argus Automated Data Collection and Management System and the Vivarium Temperature and Relative Humidity Monitoring System. All data were tabulated, summarized and/or statistically analyzed using the Argus Automated Data Collection and Management System, the Vivarium Temperature and Relative Humidity Monitoring System, Microsoft® Excel (part of Microsoft® Office 97/2000/XP), Quattro Pro 8 and/or The SAS System (version 6.12).
Averages and percentages were calculated. Litter values were used where appropriate.
Clinical observations and other proportional data were analyzed using the Variance Test for Homogeneity of the Binomial Distribution.
Continuous data (e.g., body weights, body weight changes, feed consumption values and litter averages for percent male fetuses, percent resorbed conceptuses, fetal body weights and fetal anomaly data) were analyzed using Bartlett’s Test of Homogeneity of Variances and the Analysis of Variance, when appropriate [i.e., Bartlett’s Test was not significant (p>0.001)]. If the Analysis of Variance was significant (p≤0.05), Dunnett’s Test was used to identify the statistical significance of the individual groups. If the Analysis of Variance was not appropriate [i.e., Bartlett’s Test was significant
(p≤0.001)], the Kruskal-Wallis Test was used, when less than or equal to 75% ties were present. In cases where the Kruskal-Wallis Test was statistically significant (p≤0.05), Dunn’s Method of Multiple Comparisons was used to identify the statistical significance of the individual groups. If there were greater than 75% ties, Fisher’s Exact Test was used to analyze the data.
Indices:
Count data obtained at Caesarean-sectioning of the dams were evaluated using the procedures described above for the Kruskal-Wallis Test.
Historical control data:
no data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
not applicable

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
>= 30 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
>= 30 mg/kg bw/day
Basis for effect level:
other: developmental toxicity

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
not applicable

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion