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HomeMy WebLinkAboutCH-FW-DEQ-0009180 DuPont-18405-1017 - 1 - H-28548: Absorption, Distribution, Metabolism, and Elimination in the Rat Work Request Number 18405 Service Code 1017 Protocol Performing Laboratory: E.I. du Pont de Nemours and Company DuPont Haskell Global Centers for Health & Environmental Sciences P.O. Box 50 Newark, Delaware 19714 U.S.A. Haskell Animal Welfare Committee Number: BT196-P CH-FW-DEQ-0009180 H-28548: Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017 - 2 - TABLE OF CONTENTS Page TABLE OF CONTENTS.................................................................................................................2 INTRODUCTION...........................................................................................................................3 OBJECTIVE....................................................................................................................................3 SPONSOR AND CONTACT INFORMATION.............................................................................3 REGULATORY COMPLIANCE AND TEST GUIDELINES ......................................................3 ANIMAL WELFARE ACT COMPLIANCE..................................................................................4 MATERIALS AND METHODS.....................................................................................................4 A. Test Substance .....................................................................................................................4 B. Test System..........................................................................................................................4 C. Animal Husbandry...............................................................................................................5 D. Pretest Period.......................................................................................................................6 E. Assignment to Groups..........................................................................................................6 F. Dose Preparation, Analysis, and Rates................................................................................6 G. In-Life Phase - Tier 1...........................................................................................................7 H. Quantitation of H-28548......................................................................................................8 I. Quantification and Identification of Metabolites.................................................................8 STATISTICAL ANALYSES..........................................................................................................8 CONTROL OF BIAS ......................................................................................................................8 ADDITIONAL TESTING – TIER 2...............................................................................................8 SAFETY AND HOUSEKEEPING.................................................................................................9 RECORDS AND SAMPLE STORAGE.........................................................................................9 PROPOSED STUDY DATES.........................................................................................................9 REFERENCES ................................................................................................................................9 SIGNATURES...............................................................................Error! Bookmark not defined. CH-FW-DEQ-0009181 H-28548: Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017 - 3 - INTRODUCTION H-28548 is currently under investigation by the sponsor. The data from this study will provide basic information on the absorption, distribution, metabolism, and elimination (ADME) of H-28548 in the rat. OBJECTIVE The objective of this study is to determine the ADME of H-28548 in the rat following a single oral dose of H-28548 in water (Tier 1). Use of a non-radiolabeled test substance for determining a material balance and metabolite identification is justified based on results from an in vitro metabolism experiment with rat hepatocytes and rat oral and rat/monkey intravenous dose kinetic studies, which suggests that H-28548 is not metabolized and is eliminated rapidly.(1,2,3,4) SPONSOR AND CONTACT INFORMATION Sponsor: E.I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A. Sponsor Contact: Jane Bradd Andersen 302-999-2377 Jane-Bradd.Andersen@usa.dupont.com Testing Facility Contact: William J. Fasano, Sr. 302-451-3301 William.J.Fasano@usa.dupont.com Sponsor Approval: found on the Work Authorization Form REGULATORY COMPLIANCE AND TEST GUIDELINES This study will be conducted in compliance with the following good laboratory practice(s), which are compatible with current OECD Good Laboratory Practices: • U.S. EPA TSCA (40 CFR part 792) Good Laboratory Practice Standards This study will be conducted in compliance with the following test guideline: • U.S. EPA, OPPTS 870.7485. Metabolism and Pharmacokinetics, Health Effects Test Guidelines (1998) CH-FW-DEQ-0009182 H-28548: Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017 - 4 - ANIMAL WELFARE ACT COMPLIANCE This study will comply with all applicable sections of the Final Rules of the Animal Welfare Act regulations (9 CFR) and the Guidelines from the Guide for the Care and Use of Laboratory Animals (NRC 1996). The sponsor should make particular note of the following: • The signature of the sponsor and/or the study director ensures that the study described in this protocol does not unnecessarily duplicate previous experiments, and is in compliance with the DuPont Policy on Animal Testing. • Whenever possible, procedures used in this study have been designed to implement a reduction, replacement, and/or refinement in the use of animals in an effort to avoid or minimize discomfort, distress or pain to animals. All methods are described in this study protocol or in written laboratory standard operating procedures. • DuPont Haskell policy is that animals experiencing severe pain or distress that cannot be relieved will be painlessly euthanized, as deemed appropriate by the veterinary staff and study director or appropriate designee. The sponsor will be advised by the study director of all circumstances that could lead to this action in as timely a manner as possible. • Methods of euthanasia used during this study are in conformance with the above referenced regulation and the recommendations of the American Veterinary Medical Association (AVMA), 2007 Guidelines on Euthanasia. • This protocol has been reviewed by the Haskell Animal Welfare Committee and complies with acceptable standards for animal welfare and humane care. • DuPont Haskell is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC) International. MATERIALS AND METHODS A. Test Substance The test substance (CAS registry number 62037-80-3) will be supplied by the sponsor and assigned Haskell number 28548. B. Test System Male and female Crl:CD(SD) rats will be obtained from Charles River Laboratories, Inc. (Raleigh, North Carolina, U.S.A.). The Sprague-Dawley rat was chosen for this study because of the extensive experience with this strain and its suitability with respect to longevity, sensitivity, and low incidence of spontaneous diseases. Furthermore, the Sprague-Dawley rat has been used previously for toxicokinetic and toxicity testing of this chemical. CH-FW-DEQ-0009183 H-28548: Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017 - 5 - At the time of dosing, rats should be sexually mature, and the weight variation should not exceed ±20% of the mean weight by dose group. Each animal will be assigned a unique identification number to be used throughout the study. The last 3 digits of the animal identification number will be marked on the tail of each animal in indelible ink. C. Animal Husbandry 1. Housing During the pretest period, animals will be housed individually in solid bottom caging with bedding. Each cage rack may contain animals of either sex. Animals will be moved to metabolism units or lined solid bottom caging for the in-life phase of the study. 2. Environmental Conditions Animal rooms will be maintained at a temperature of 18-26ºC (64-79ºF) and a relative humidity of 30-70%. Animal rooms will be artificially illuminated (fluorescent light) on an approximate 12 hour light/dark cycle. Unless judged by the study director or the laboratory veterinarian to have significantly affected the results of the study, the relative humidity and temperature ranges in the housing rooms will be recorded but will not be included in the final report. 3. Feed and Water All animals will be provided tap water ad libitum and fed PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002 ad libitum. When housed in metabolism units, feed will be supplied as ground chow. 4. Animal Health and Environmental Monitoring Program As specified in the DuPont Haskell animal health and environmental monitoring program, the following procedures are performed periodically to ensure that contaminant levels are below those that would be expected to impact the scientific integrity of the study: • Water samples are analyzed for total bacterial counts, and the presence of coliforms, lead, and other contaminants. • Samples from freshly washed cages and cage racks are analyzed to ensure adequate sanitation by the cagewashers. Certified animal feed is used, guaranteed by the manufacturer to meet specified nutritional requirements and not to exceed stated maximum concentrations of key contaminants, including specified heavy metals, aflatoxin, chlorinated hydrocarbons, and organophosphates. The presence of these contaminants below the maximum concentration stated by the manufacturer would not be expected to impact the integrity of the study. The animal health and environmental monitoring program is administered by the attending laboratory animal veterinarian. Data are maintained separately from study records and may be included in the final report at the discretion of the study director. CH-FW-DEQ-0009184 H-28548: Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017 - 6 - D. Pretest Period Upon arrival at DuPont Haskell, all rats will be housed in quarantine. The rats will be: • quarantined for at least 6 days. • identified temporarily by cage identification. • weighed at least 3 times during quarantine and once prior to dosing. • observed with respect to weight gain and any gross signs of disease or injury. The animals will be released from quarantine by the laboratory animal veterinarian or designee based on body weights and clinical signs. Animals that are accidentally killed or removed from study during the pretest period will be discarded without necropsy. Animals that are found dead or are sacrificed in extremis during the pretest period will be given a gross examination to check for the presence of disease. Dependant upon these findings, further diagnostic procedures may be employed at the discretion of the study director, a pathologist, or the laboratory animal veterinarian. The results will not be included in the final report unless considered significant to the evaluation of the study. E. Assignment to Groups Animals will be selected for use on study based on adequate body weight gain and freedom from any clinical signs of disease or injury. The weight variation of selected animals should not exceed ± 20% of the mean weight. Each animal will be assigned an animal number and a cage identification number. The animal number and cage identification number will both be included on the cage label. At study start, the animals should be at least 8 weeks old. Animals that have not been assigned to a test group, or which have been removed from study prior to dosing for out-of-range body weights, will be released for other laboratory purposes or be sacrificed and discarded without anatomic pathology evaluation. F. Dose Preparation, Analysis, and Rates The test substance will be prepared for administration by oral gavage. This route was chosen because it is most commonly used for toxicity studies with H-28548. H-28548 will be weighed into a vial and mixed with deionized water (dose vehicle). The dose solution will be prepared at a nominal concentration of 7.5 mg H-28548/mL (adjusted for purity), with a target dose level of 30 mg/kg body weight (bw) and a dose volume of 4 mL/kg bw. The dose level was chosen based on the results of the 28-day daily oral dosing study in rats, where the no-observed-adverse-effect level (NOAEL) was 30 and 300 mg/kg/day for males and females, respectively.(5) CH-FW-DEQ-0009185 H-28548: Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017 - 7 - The dosing solution/suspension may be prepared on the day of use or prior to the day of use and stored refrigerated at 1-10°C. LC/MS/MS will be used to confirm the chemical concentration of H-28548 in the dosing solution/suspension. G. In-Life Phase - Tier 1 1. Material Balance and Tissue Distribution Rats will be housed individually in a metabolism unit and fasted for approximately 16 hours prior to dosing. Food will be returned approximately 2 hours post-dose. Five male and 5 female rats will be administered H-28548 at 30 mg/kg bw. Two male and 2 female rats will each be administered dose vehicle (deionized water at 4 mL/kg bw) for collection of control excreta and tissue samples. Rats will be returned to individual metabolism unit following dosing. Urine and feces will be collected on dry ice predose and at 0-6, 6-12, 12-24, and every 24 hours until 168 hours post dose. Evidence supporting a lack of metabolism of H-28548 in rat hepatocytes and rat oral kinetic studies, precludes the necessity for a radiolabeled form of H-28548 and collection of expired air. At the end of the experiment (168 hours post dose), rats will be killed by CO2 asphyxiation followed by exsanguination. The following tissues (Tier 1) will be collected: liver fat G.I. tract (and contents) kidney spleen whole blood residual carcass After collection, these samples will be stored at approximately -10C. Over the course of the experiment, residual feed will be collected (if necessary) into a single container and stored refrigerated at 1-10°C. Cages will be rinsed with deionized water, which will be collected into a single container. Cage wash will be stored at room temperature and/or refrigerated at 1-10°C. Per the testing guideline, if it is determined that a significant amount of the administered dose (<90%) is unaccounted for in the excreta (urine and/or feces, and cagewash, which is primarily dried excreta) then data on the percent of the total amount of H-28548 in the collected tissues as well as residual carcass will be determined. Analysis of residual feed (if collected) will only occur if a significant amount of the dose is still unaccounted for following the sequential and step-wise analysis of excreta (urine and feces), cagewash, tissues, residual carcass, and residual CH-FW-DEQ-0009186 H-28548: Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017 - 8 - feed (if collected). Total recovery should approximate 100 ± 10% of the amount of H-28548 administered. H. Quantitation of H-28548 H-28548 will be quantitated in urine, feces and cagewash using LC/MS/MS, which may involve direct analysis (urine and cagewash) or solvent extraction (feces), followed by analysis. H-28548 will be quantitated in collected tissues, carcass, and residual feed (if collected) only if the mean recovery of the administered dose in urine and feces, and cagewash, which is primarily dried excreta, is <90%. Details of the methodologies employed will be documented in the study records and presented in the final report. I. Quantification and Identification of Metabolites The profile of H-28548 (and metabolites, if any) will be evaluated in urine and feces samples. Samples may be pooled across animals for a given time interval. Feces samples will be extracted using appropriate solvent(s). Urine and fecal extracts may be filtered if necessary prior to LC/MS analysis. It is expected that H-28548 will not be metabolized by the rat. However, any metabolites that are found to represent greater than 5% of the administered dose will be submitted for structural identification by mass spectroscopy or other suitable methods. The methodology relating to identification of H-28548 and metabolites will be documented in the study records and presented in the final report. STATISTICAL ANALYSES Group data will be represented as a mean ± SD. Other statistical evaluations may be performed at the discretion of the study director and sponsor. CONTROL OF BIAS Effective mixing of samples and avoidance of cross-contamination will be used to control bias during sample collection and analysis. ADDITIONAL TESTING – TIER 2 Additional studies (Tier 2) may be added by amendment to this protocol if required. Studies at the Tier 2 level are designed to answer questions about the disposition of test chemicals based on the existing toxicology data base and/or results of Tier 1 testing. Only those studies that address a specific concern not resolved at Tier 1 will be conducted following a discussion with the Agency and a mutual agreed path forward. CH-FW-DEQ-0009187 H-28548: Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017 - 9 - SAFETY AND HOUSEKEEPING All chemicals used during this study will be handled according to the procedures specified in the MSDS and disposed of according to the Stine-Haskell Waste Disposal Guidelines and the area Safety, Health and Environmental (SHE) manual. RECORDS AND SAMPLE STORAGE All raw data, the protocol, amendments (if any), and the final report will be retained. PROPOSED STUDY DATES Experimental Start: March 2010 Experimental Termination: May 2010 REFERENCES 1. DuPont Haskell (2007). In Vitro Rat Hepatocyte Screen. Unpublished report, DuPont-23460. 2. DuPont Haskell (2008). Repeated Dose Oral Toxicity 7-Day Gavage Study in Rats. Unpublished report, DuPont-24009. 3. DuPont Haskell (2007). Biopersistence and Pharmacokinetic Screen in Rats. Unpublished report, DuPont-24281. 4. DuPont Haskell (2009). Cross-Species Comparison of FRD-902 Plasma Pharmacokinetics in the Rat and Primate Following Intravenous Dosing. Unpublished report, DuPont-17751-1579 RV1. 5. DuPont-Haskell (2008). A 28-Day Oral (Gavage) Toxicity Study of H-28397 in Rats with a 28-Day Recovery. Unpublished report, DuPont-24447. CH-FW-DEQ-0009188 H-28548: Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017 - 10 - CH-FW-DEQ-0009189