Teratology_Sample_Study_Final.pdf

1 ATTACHMENT 6

SAMPLE STUDY ASSIGNMENT #2

STUDY TITLE: PRENATAL DEVELOPMENTAL TOXICOLOGY STUDY IN HARLAN

SPRAGUE DAWLEY RATS FOR DIMORPHOLINODIETHYL ETHER

(CAS # 6425-39-4) ADMINISTERED BY GAVAGE

1) Introduction

This study is designed to evaluate the developmental toxicity of dimorpholinodiethyl ether in Harlan Sprague Rats. This compound is a blowing agent for the production of flexible, molded, and moisture-cured foams and coatings.

2) Project Management

This project shall consist of 3 Phases.

Phase 1: Pre-Start Chemistry Phase 2: Range-finding Study Phase 3: Definitive Study

3) Objectives

Conduct a developmental toxicity range-finding study for this chemical using doses specified by the NTP.

Conduct a definitive developmental toxicity study for this chemical using doses specified by the NTP.

4) Test Material

Test material shall be provided by the NTP. NTP will also provide methods for bulk analysis, formulation of the test material in an appropriate vehicle for gavage dosing and methods for analysis of the test material in the dosing formulation (for the purposes of responding to this RFP, assume the vehicle is distilled water).

5) Pre-Start Chemistry

Receive the test material and perform bulk chemical re-analysis (identity and purity confirmation) according to the procedures provided. Results shall be reported to the COR by telephone and e-mail as soon as they are available.

Prepare and analyze dose formulations according to the instructions provided by the NTP.

Conduct dose formulation analysis method performance evaluation.

Provide a pre-start chemistry report. The pre-start chemistry report shall include:

1) a summary of the NTP-provided information on the source of the bulk chemical, storage requirements, identity and purity data,

2) confirmatory data on identity, and purity (including representative chromatograms and spectra of the test article and reference material);

provide copies of the SOPs for the bulk chemical reanalysis methods,

3) data on preparation, handling, and analysis of dose formulations for homogeneity and concentration verification as well as SOPs, 2 ATTACHMENT 6

4) dose formulation analysis method performance evaluation procedures and validation data, including representative chromatograms or spectra,

5) health and safety procedures to be followed when using dimorpholinodiethyl ether.

Animals shall not be dosed until this report has been received by the NTP.

6) Test Animals

Species and Strain: Female Harlan Sprague Dawley Rats from an NTP designated Harlan Laboratories facility. For costing purposes, assume that time mated rats shall be obtained.

Diet and Water: Water and Feed (NIH-07) to be provided ad libitum.

7) Prenatal Developmental Toxicity Dose Range-finding Study Design:

The dose range-finding study shall use pregnant animals that are 11-12 weeks old (approximately 200-225 g when mated) and approximately Gestation Day 2 (GD-2) upon receipt. There shall be 14 animals per group with 4 dosed groups and a control group. Dams shall be randomly assigned to groups by stratified body weight so that each group has comparable body weights (within + 10% of the mean body weight at study initiation). The doses shall be 0, 50, 200, 500 and 1000 mg/kg/day and administered by gavage daily from GD 6 (day of implantation) to GD 20 (vehicle = deionized water). The dosing volume shall be 5 ml/kg/day, based on the most recent body weight. Cesarean section shall be performed on GD 21. (For costing purposes, assume 11 females/group are pregnant.)

Observations: Each dam shall be observed at least twice daily (once before 10 am and once after 2 pm). Mortality, moribundity, pertinent behavioral changes, and signs of overt toxicity shall be recorded at this cageside observation. In addition, thorough physical examinations shall be conducted 1-3 hours after dosing.

Maternal body weights shall be collected daily starting on GD 3 and the most recent body weight shall be used to calculate appropriate dosing volumes for the dams.

Food consumption shall be measured at 3-day intervals starting on GD 3. On the day of cesarean section the dams shall be weighed.

At cesarean section, the gravid uterus shall be weighed and an examination of the uterine contents shall be performed to determine the number of live and dead fetuses as well as the number of early and late resorptions. Individual fetal weights and the number of male and female fetuses shall be determined. Corpora lutea shall be counted and external fetal observations (including evaluation for cleft palate) shall be recorded. The uteri of apparently non-pregnant females shall be opened and immersed in 0.5% ammonium sulfide solution for approximately 10 min under a chemical hood. The uteri shall be examined for implantation sites, which appear as dark residues (nidations) and shall be recorded as early resorptions.

Biological Sample collection: Use 3 randomly selected pregnant dams on GD 18 in each dosed group and control to provide tissue for possible later analysis. Collect maternal plasma (approximately 3.0 ml), amniotic fluid (pooled by litter, 3 ATTACHMENT 6 approximately of 250 ul), and fetuses (pooled by sex by litter). Fetal samples shall be identified so they may be linked to the dam. Samples shall be stored at -20ºC and shipped to an NTP designated location as instructed by the COR.

Data and Reporting: Data tables shall be available to the COR for review within 4 business days of the necropsy. A draft report shall be provided within 6 weeks of the necropsy.

8) Definitive Developmental Toxicity Study Design:

The definitive study shall use time-mated animals, 11-12 weeks of age and approximately GD-2 upon receipt from the supplier. There shall be three dosed groups and one control group with doses of 0, 250, 500 and 1000 mg/kg/day. The dosing volume shall be 5 ml/kg/day, and the vehicle shall be deionized water. Each test and control group shall contain a sufficient number of animals to yield approximately 20 animals with implantation sites at necropsy. (For costing purposes assume 25 time-mated females per group, but include information in the technical proposal regarding the number of animals recommended per group to obtain at least 20 pregnant per group). Animals shall be weighed on GD 3 and randomly assigned to dose groups in a manner that results in comparable mean body weight values among groups (within + 10%). Test material shall be administered daily via gavage from Gestation Day 6 to Gestation Day 20. Cesarean section shall be performed on GD 21.

Observation of animals: Each dam shall be observed at least twice daily (once before 10 am and once after 2 pm). Mortality, moribundity, pertinent behavioral changes, and signs of overt toxicity shall be recorded at this cageside observation.

In addition, thorough physical examinations shall be conducted 1-3 hours after dosing. Animals shall be weighed daily beginning on GD 3. The most recent body weight shall be used to calculate the appropriate formulation volume administered to the dams. Food consumption shall be measured at 3-day intervals, starting on GD 3.

Dams shall be euthanized on the day of cesarean-section (GD21). At this time, the dam shall be weighed and examined for any structural abnormalities or any gross changes that may have influenced the pregnancy. The liver shall also be weighed.

At cesarean section, the gravid uterus shall be weighed and an examination of the uterine contents to determine the number of live and dead fetuses as well as the number of early and late resorptions shall be made as in the range-finding study.

The number of corpora lutea shall be determined.

Fetal Observations: The sex and weight of each fetus shall be recorded. Each fetus shall be examined for external variations and malformations including an examination for cleft palate. All fetuses shall be examined for skeletal and soft tissue variations and malformations. For skeletal evaluation, both bone and cartilage shall be examined using a double stain technique (Alcian Blue/Alizarin Red S). The Staples technique shall be used for visceral evaluations.

Approximately one-half of the fetuses shall be decapitated prior to dissection. Fetal heads shall be fixed and decalcified in Bouin’s solution, and subsequently examined for soft tissue craniofacial alterations.

4 ATTACHMENT 6

Data and reporting: Maternal and fetal data, including the incidence and severity of all findings shall be reported. In evaluations of study data, the litter shall be considered the basic unit of analysis. When appropriate, historical control data shall be used to enhance interpretation of study results. Both individual and summary data shall be presented.

Maternal data to be reported shall include: maternal survival, number pregnant and number aborting; clinical observations; maternal body weight and body weight change data, including weight change adjusted for gravid uterine weight; food consumption at 3 day intervals starting on GD6 including the interval GD6-21;

necropsy findings including gravid uterine weight.

Developmental endpoints by dose for litters with implants shall include: corpora lutea counts; implantation data; number and percent of live and dead fetuses and resorptions (early and late); pre-and post implantation loss calculations.

Developmental endpoints for litters with live fetuses shall include: number and percent of live offspring; sex ratio; fetal body weight data by sex and with sexes combined.

External, soft tissue, and skeletal malformation and variation data shall be reported.

The total number and percent of fetuses and litters with any external, soft tissue, or skeletal alteration, as well as the types and incidences of individual anomalies, shall be reported.

Report: The report shall include sections for materials and methods, results, data tables, discussion, summary and references. Within 12 weeks of the final cesarean section, an audited draft final report shall be provided to the COR. The draft final report shall be considered, in the estimation of the contractor, a final report subject only to review and approval by the COR. If the review results in changes, a new version of the draft final report reflecting the changes is required. Upon approval of the draft, the final report shall be sent to the NTP as a hard copy and in electronic format on a CD-R CD or DVD. All final reports shall be dated and signed by the individual(s) responsible and the QAU. Reports are to be saved or scanned at 300 dpi (black and white) to a single searchable PDF file. The file should have no password protection and no document property restrictions.

Protocol Changes: The COR shall be consulted and agree in advance to any changes in the protocol. The written protocol amendment and justification shall be signed by both the contract lab Study Director and the COR, and maintained with the original approved protocol.

9) Performance/Delivery Requirements

Use Chart in General SOW