Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 August 1994 to 10 February 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was audited by the Wil Quality Assurance Unit throughout the progression of events and determined to have been conducted in compliance with the United States Environmental Protection Agency (EPA) Toxic Substances Control Act (TSCA) Good Laboratory Practice Regulations (40 CFR Part 792), August 17, 1989, and Health Effects Testing Guidelines for Developmental Toxicity Studies (40 CFR Part 798.4900), May 20, 1987, and the Standard Operating Procedures of WIL Research Laboratories, Inc. The study was conducted in accordance with the protocol and protocol amendments as approved by the sponsor and assured by the QA Unit that the final report accurately describes the conduct and findings of the study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EPA OTS 798.4900 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Isodecyl Benzoate
- Physical state: Liquid, Clear colorless
- Analytical purity: Considered to be 100% Isodecyl Benzoate
- Lot/batch No.: Batch C5-8, 5/15/94
- Stability under test conditions: Considered stable at room temperature
- Storage condition of test material: Dose preparations tested to be stable for 8 days under refridgeration.

Test animals

Species:
rat
Strain:
other: Crl:CD®BR
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Portage, Michigan
- Age at study initiation: approximately 12 weeks old when paired for breeding
- Weight at study initiation: a minimum of 220 g at breeding (220 g to 279 g on day 0 of gestation)
- Fasting period before study: none
- Housing: clean, wire-mesh cages suspended above cage-board
- Diet (e.g. ad libitum):ad libitum, Purina® Certified Rodent Chow® #5002
- Water (e.g. ad libitum):ad libitum, Municipal water delivered by an automatic watering system
- Acclimation period:12 Days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 65° to 71 °F
- Humidity (%): 58% to 82%
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light):12 hrs dark / 12 hrs light

IN-LIFE DATES: From:08-23-1994 To:09-16-1994

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
The selected route of administration was oral because this is the anticipated route of exposure for the general human population. The animal model was selected based on the availability of historical control data and the susceptibility of the species to known developmental toxicants.
PREPARATION OF DOSING SOLUTIONS:An appropriate amount of the vehicle, Ma.zola® corn oil, was dispensed into a properly-labeled storage container for administration to the control group.
An appropriate amount of the test material, Isodecyl Benzoate, was weighed for each group into tared, precalibrated storage containers. A sufficient amount of the vehicle, Mazola® corn oil, was added to bring the volume to the calibration mark. The preparations were placed on the Polytron® PT 6000 laboratory mixer for approximately 5 minutes. A stir bar was added, and the dosing suspensions were stirred on a magnetic stir plate throughout the sampling and dosing procedures. Preparations for all dose groups were formulated three times (August 26, September 2, and September 9, 1994) and were stored refrigerated.

Individual dosages were based on the most recently recorded body weight to provide the correct mg/kg/day dose.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Ma.zola® corn oil was a common supension vehicle for gavage
- Concentration in vehicle: 0, 6, 60, and 200 mg/ml (0, 30, 300, and 1000 mg/kg/day dosage)
- Amount of vehicle (if gavage): A dosage volume of 5 ml/kg was used for all dosage levels.
- Lot/batch no. (if required): NA
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The dosing preparations were analized on three occasions by gaschromatography/flame ionization detection (FID) with splitless injection. Mean analyzed concentrations were between 97.3% and 107% of the target dose concentrations
Details on mating procedure:
- Impregnation procedure:cohoused
- M/F ratio per cage: 1:1
- Length of cohabitation: Not Identified
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol:No
Duration of treatment / exposure:
Gestation days 6-15
Frequency of treatment:
Once daily
Duration of test:
Laparohysterectomies performed on Gestation Day 20
Doses / concentrations
Remarks:
Doses / Concentrations:
Concentration in vehicle: 0, 6, 60, and 200 mg/ml (0, 30, 300, and 1000 mg/kg/day dosage). A dosage volume of 5 ml/kg was used for all dosage levels.
Basis:
analytical conc.
No. of animals per sex per dose:
25 mated females per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected based on the results of a dosage range-finding developmental toxicity study of Isodecyl Benzoate in rats (WIL-152 17).

- Rationale for animal assignment:The bred females were consecutively assigned in a block design to groups containing 25 rats each by the following randomization procedure. The first mated female and the appropriate gestation day 0 designation were recorded, and the female was assigned to group 1, the second mated female was assigned to group 2, and the third to group 3, etc. This process was continued daily until 25 females were placed into each group.

All maternal animals were euthanized by carbon dioxide inhalation on gestation day 20. The thoracic, abdominal and pelvic cavities were opened by a ventral mid-line incision, and the contents were examined. In all instances, the post mortem findings were correlated with the ante mortem comments and any abnormalities were recorded. The uterus and ovaries were excised, and the number of corpora lutea on each ovary was recorded. The trimmed uterus was weighed and opened, and the number and location of all fetuses, early and late resorptions and the total number of implantation sites were recorded. The individual uterine distribution of implantation sites was documented using the following procedure. All implantation sites, including resorptions, were numbered in consecutive order beginning with the left distal to the left proximal uterine horn, noting the position of the cervix, and continuing from the right proximal to the right distal uterine horn. Maternal tissues were preserved in 10% neutral buffered formalin for possible future histopathological examination only as indicated by the gross findings. The carcass of each dam was then discarded.
Uteri with no macroscopic evidence of nidation were excised, opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss as described by Salewski3.


Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily for moribundity and mortality
- Cage side observations checked were included in report Summary Tables 1, 2, and 3; Individual Data in Tables 17 and 18.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were recorded individually from days 0 through 20 of gestation. Observations were recorded before dosing during the dosing period. Animals were also observed for signs of toxicity approximately one hour following treatment throughout the dosing period. All significant findings were recorded at the post-dosing observation periods.


BODY WEIGHT: Yes
- Time schedule for examinations: Individual maternal body weights were recorded on gestation days 0, 6-16 and 20. A group mean body weight was calculated for each of these days. Mean body weight changes were calculated for each corresponding interval and also for days 6-9, 9-12, 12-16, 6-16 and 0-20.


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 was recorded on gestation days 0, 6, 9, 12, 16 and 20. Food intake was reported as g/animal/day and g/kg/day for each corresponding body weight change interval
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: Gross examination of abdominal organs


OTHER:
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes- Gravd uterine weight was collected and net body weight (the day 20 body weight minus the weight of the uterus and contents) and net body weight change (the day 0-20 body weight change minus the weight of the uterus and contents) were calculated and presented for each gravid female at the scheduled laparohysterectomy.
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other :Maternal tissues were preserved in 10% neutral buffered formalin for possible future histopathological examination only as indicated by the gross findings.
Fetal examinations:
- External examinations: Yes: [all per litter ]
- Soft tissue examinations: Yes: [all per litter ] Each fetus was examined viscerally by a modification of the Stuckhardt and Poppe fresh dissection technique to include the heart and major vessels. The sex of each fetus was verified by an internal examination. Fetal kidneys were examined and graded for renal papillae development by a method described by Woo and Hoai.
- Skeletal examinations: Yes: [all per litter] All carcasses were eviscerated and fixed in 100% ethyl alcohol. Following fixation in alcohol, each fetus was macerated in potassium hydroxide and stained with Alizarin Red S by a method similar to that described in Dawson. The skeletal examination was conducted using low power magnification provided by a stereomicroscope. External, visceral and skeletal findings were recorded as developmental variations or malformations.
- Head examinations: Yes: [half per litter] Heads were placed in Bouin’s fixative for subsequent soft-tissue examination by the Wilson sectioning technique. The heads from the remaining one-half of the fetuses were examined by a mid-coronal slice.
Statistics:
All analyses were conducted using two-tailed tests for a minimum significance level of 5% comparing each treated group to the vehicle control group. Each mean was presented with the standard deviation (S.D.) and the number of animals (N) used to calculate the mean. The following statistical tests were performed by a DigitaP MicroVAX® 3400 computer (with appropriate programming) in this laboratory and are referenced on the report tables:
Fetal Sex Ratios (Chi-square test with Yates’ correction factor), Malformations and Variations (Fisher’s Exact test), Early and Late Resorptions, Dead Fetuses, Postimplantation Losses, Mean Litter Proportions of Malformations and Variations (Mann-Whitney U-test), Corpora Lutea, Total Implantations, Viable Fetuses, Fetal Body Weights, Maternal Body Weights and Weight Changes, Maternal Net Body Weight Changes, Gravid Uterine Weights, Maternal Food Consumption (One-way ANOVA with Dunnett’s test), Litter Proportions of Intrauterine Data (Considering the Litter, rather than the Fetus, as the Experimental Unit) (Kruskal-Wallis test).
Indices:
The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as a percentage of the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison and calculating the number of affected fetuses in a litter on a proportional basis.
Historical control data:
Tables of WIL Laboratories Historical Control data for Sprague-Dawley Crl:CD’BR Rats (Summary Values) and data for Sprague-Dawley Crl:CD®BR Rats (Individual Values) are included in the Report Appendicies B and C, respectively.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
A mean body weight loss occurred in the 1000 mg/kg/day group during gestation days 6-9. Food consumption was unaffected at all dose levels.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
The only adverse effects of treatment on the developing fetus were a decrease in the mean fetal body weight and a reduction in the incidence of cervical centrum no. 1 ossified (both of which are indications of developmental retardation in the fetuses) in the 1000 mg/kg/day group.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

All maternal animals survived to the scheduled necropsy; no clinical signs related to compound administration were noted in the treated groups. A mean body weight loss occurred in the 1000 mg/kg/day group during gestation days 6-9. Food consumption was unaffected by treatment throughout the study period in all dose groups. No treatment-related internal findings were observed at necropsy. The only adverse effects of treatment on the developing fetus were a decrease in the mean fetal body weight and a reduction in the incidence of cervical centrum no. 1 ossified (both of which are indications of developmental retardation in the fetuses) in the 1000 mg/kg/day group. No other treatment-related malformations or developmental variations were observed at any dose level.

Applicant's summary and conclusion

Conclusions:
In conclusion, maternal toxicity was exhibited at a dose level of 1000 mg/kg/day by a transient mean body weight loss. Developmental toxicity was exhibited at a dose level of 1000 mg/kg/day by a slight decrease in mean fetal body weight and a reduction in the incidence of cervical centrum no.1 ossified. Based on the results of this study, a dose level of 300 mg/kg/day was considered to be the NOAEL (no observable adverse effect level) for maternal toxicity and developmental toxicity.
Executive summary:

The potential maternal toxicity and developmental toxicity of Isodecyl Benzoate were evaluated. Isodecyl Benzoate in Mazola® corn oil was administered orally by gavage to three groups of 25 bred Sprague-Dawley Crl:CJYBR female rats once daily from gestation days 6 through 15. Dosage levels were 30, 300 and 1000 mg/kg/day administered at a dose volume of 5 mllkg. A concurrent control group composed of 25 bred females received the vehicle, Mazola® corn oil, on a comparable regimen at 5 mllkg. All rats were observed twice daily for appearance and behavior. Body weights and food consumption were recorded at appropriate intervals. A laparohysterectomy was performed on all animals on gestation day 20. The uteri and ovaries were examined and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Mean gravid uterine weights and net body weight changes were calculated for each group. The fetuses were weighed, sexed and examined for external, soft tissue and skeletal malformations and developmental variations.

All maternal animals survived to the scheduled necropsy; no clinical signs related to compound administration were noted in the treated groups. A mean body weight loss occurred in the 1000 mg/kg/day group during gestation days 6-9. Food consumption was unaffected by treatment throughout the study period in all dose groups. No treatment-related internal findings were observed at necropsy. The only adverse effects of treatment on the developing fetus were a decrease in the mean fetal body weight and a reduction in the incidence of cervical centrum no. 1 ossified (both of which are indications of developmental retardation in the fetuses) in the 1000 mg/kg/day group. No other treatment-related malformations or developmental variations were observed at any dose level.

In conclusion, maternal toxicity was exhibited at a dose level of 1000 mg/kg/day by a transient mean body weight loss. Developmental toxicity was exhibited at a dose level of 1000 mg/kg/day by a slight decrease in mean fetal body weight and a reduction in the incidence of cervical centrum no.1 ossified. Based on the results of this study,a dose level of 300 mg/kg/day was considered to be the NOAEL (no observable adverse effect level) for maternal toxicity and developmental toxicity.