.... United States Environmental Protection Agency Prevention, Pesticides and Toxic Substances (7101) EPA 712–C–96–254 June 1996 

# Health Effects Test Guidelines OPPTS 870.8340 Oral and Inhalation Pharmacokinetic Test

‘‘Public Draft’’

### i INTRODUCTION This guideline is one of a series of test guidelines that have been developed by the Office of Prevention, Pesticides and Toxic Substances, United States Environmental Protection Agency for use in the testing of pesticides and toxic substances, and the development of test data that must be submitted to the Agency for review under Federal regulations. The Office of Prevention, Pesticides and Toxic Substances (OPPTS) has developed this guideline through a process of harmonization that blended the testing guidance and requirements that existed in the Office of Pollution Prevention and Toxics (OPPT) and appeared in Title 40, Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the Office of Pesticide Programs (OPP) which appeared in publications of theNational Technical Information Service (NTIS) and the guidelines published by the Organization for Economic Cooperation and Development (OECD). The purpose of harmonizing these guidelines into a single set of OPPTS guidelines is to minimize variations among the testing proceduresthat must be performed to meet the data requirements of the U. S. Environmental Protection Agency under the Toxic Substances Control Act (15 U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act (7 U.S.C. 136, et seq. ).

Public Draft Access Information: This draft guideline is part of a series of related harmonized guidelines that need to be considered as a unit. For copies: These guidelines are available electronically from theEPA Public Access Gopher (gopher.epa.gov) under the heading ‘‘Environmental Test Methods and Guidelines’’ or in paper by contacting the OPP Public Docket at (703) 305–5805 or by e-mail: guidelines@epamail.epa.gov.

To Submit Comments: Interested persons are invited to submit comments. By mail: Public Docket and Freedom of Information Section, Office of Pesticide Programs, Field Operations Division (7506C), Environmental Protection Agency, 401 M St. SW., Washington, DC 20460. In person:bring to: Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Arlington, VA. Comments may also be submitted electronically by sending electronic mail (e-mail) to: guidelines@epamail.epa.gov.

Final Guideline Release: This guideline is available from the U.S. Government Printing Office, Washington, DC 20402 on The Federal Bulletin Board . By modem dial 202–512–1387, telnet and ftp: fedbbs.access.gpo.gov (IP 162.140.64.19), or call 202–512–0132 for disks or paper copies. This guideline is also available electronically in ASCII and PDF (portable document format) from the EPA Public Access Gopher (gopher.epa.gov) under the heading ‘‘Environmental Test Methods and Guidelines.’’

### 1 OPPTS 870.8340 Oral and inhalation pharmacokinetic test. (a) Scope—(1) Applicability. This guideline is intended to meet testing requirements of both the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq. ) and the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601). (2) Background. The source material used in developing this harmonized OPPTS test guideline is OPPT 40 CFR 795.230 Oral and Inhalation Pharmacokinetic Test. (b) Purpose. The purpose of these studies is to determine bioavailability of test substance after oral and inhalation exposure; whetheror not the biotransformation of the test substance is qualitatively and quantitatively the same after oral and inhalation exposure; and whether or not the biotransformation of the test substance is changed qualitatively or quantitatively by repeated dosing. (c) Definitions. The definitions in section 3 of TSCA and in 40 CFR Part 792—Good Laboratory Practice Standards (GLP) apply to this test guideline. The following definition also applies to this test guideline. Bioavailability refers to the rate and extent to which an administeredchemical substance compound is absorbed, i.e. reaches the systemic circulation. (d) Test procedures—(1) Animal selection—(i) Species. The ratshould be used for pharmacokinetics testing because it has been used extensively for metabolic and toxicological studies. (ii) Animals. Adult male and female Fischer 344 rats should be used. The rats should be 7 to 9 weeks old. Prior to testing, the animals are selected at random for each group. Animals showing signs of ill health should not be used. (iii) Animal care. Animals should be housed in environmentally controlled rooms with 10 to 15 air changes per hour. The rooms should be maintained at a temperature of 24 ± 2 ° C and humidity 50 ± 10 percent with a 12-h light/dark cycle per day. The rats should be isolated for at least 7 days prior to use, and their health status should then be evaluated. The animals should be acclimated to the experimental environment for a minimum of 48 h prior to treatment. Certified feed and water should be provided ad libitum. (iv) Numbers. (A) At least eight animals (four males and four females) should be used at each dose level. (B) Females should be nulliparous and nonpregnant. (2) Administration of the test substance—(i) Test substance. The test substance should be at least 99 percent pure. The studies require the

### 2 use of both nonradioactive and 14 C-labeled test substance. Both preparations are needed to investigate the provisions of paragraph (b)(2) of this guideline. The use of 14 C-test substance is recommended for the provisionsin paragraphs (b)(1), (b)(2), and (b)(3) of this guideline in order to facilitate the work, improve the reliability of quantitative determinations, and increase the probability of observing previously unidentified metabolites. (ii) Dosage and treatment—(A) Oral study. At least two doses should be used in the study, a low and a high dose. When administered orally, the high dose should induce some overt toxicity such as weight loss. The low dose should not induce observable effects attributable to the test substance. Oral dosing should be performed by gavage using an appropriate vehicle. (B) Inhalation study. Three concentrations should be used in the study. Upon exposure, the two higher concentrations should ideally induce some overt symptoms of toxicity, although the intermediate concentration may be excluded from this condition. The lowest concentration should not induce observable effects attributable to the test substance. (iii) Determination of bioavailability—(A) Oral studies. ( 1 ) Group A (a minimum of eight animals, four males and four females) should be dosed once orally with the low dose of 14 C-labeled test substance. ( 2 ) Group B (a minimum of eight animals, four males and four females) should be dosed once orally with the high dose of 14 C-labeled test substance. (B) Inhalation studies. ( 1 ) Group C (a minimum of eight animals, four males and four females) should be exposed (6 h) to a mixture of nonradioactive test substance in air at the prescribed low test substance concentration. (2 ) Group D (a minimum of eight animals, four males and four females) should be exposed (6 h) to a mixture of nonradioactive test substance in air at the prescribed intermediate test substance concentration. ( 3 ) Group E (a minimum of eight animals, four males and four females) should be exposed (6 h) to a mixture of nonradioactive test substance in air at the prescribed high concentration. (4 ) Group F (a minimum of eight animals, four males and four females) should be exposed (6 h) to a mixture of 14 C-labeled test substance in air at the prescribed low test substance concentration. ( 5 ) Group G (a minimum of eight animals, four males and four females) should be exposed (6 h) to a mixture of 14 C-labeled test substance in air at the prescribed intermediate test substance concentration.

### 3 ( 6) Group H (a minimum of eight animals, four males and four females) should be exposed (6 h) to a mixture of 14 C-labeled test substance in air at the prescribed high test substance concentration. (C) Collection of excreta. After oral administration (Groups A and B) and inhalation exposure (Groups F through H), the rats should be placed in individual metabolic cages and excreta (urine, feces, and expired air) should be collected from 0 to 24 h and then from 24 to 48 h posttreatment, or until 90 percent of the dose has been excreted, whichever occurs first. (D) Kinetic studies. Groups C through E should be used to determine the concentration of the test substance in blood at 0, 5, 10, 15, and 30min, and at 1, 2, 4, 8, 16, 24, and 48 h after initiation of inhalation exposure. (E) Repeated dosing study. Rats (a minimum of eight animals, four males and four females) should receive a series of single daily oral doses of nonradioactive test substance over a period of at least 7 days, followed at 24 h after the last dose by a single oral dose of 14 C-labeled test substance. Each dose should be at the low-dose level. Urine should be collected from 0 to 24 h and then 24 to 48 h after administering the 14 Clabeled test substance. (3) Observation of animals—(i) Bioavailability—(A) Blood levels. The levels of total 14 C-label should be determined in whole blood, blood plasma, or blood serum of each rat at 0, 4, 8, 16, 24, and 48 h after dosing rats in Groups A–B and F–H. (B) Expired air, urinary and fecal excretion. The quantities of total 14 C-label eliminated in expired air, urine, and feces by each rat in Groups A and B and F through H should be determined in collections made from 0 to 24 h and then 24 to 48 h after dosing and, if necessary, daily thereafter until at least 90 percent of the dose has been excreted or until 7 days after dosing, whichever occurs first. (C) Tissue distribution. The concentration and quantity of 14 C-label in tissue and organs should be determined at the time of sacrifice for each rat in Groups A and B, F through H, and the repeated-dosing group. (ii) Biotransformation after oral and inhalation exposure. Appropriate qualitative and quantitative methods should be used to assay urine specimens collected from each rat in Groups A and B and F through H. Suitable enzymatic steps should be used to distinguish, characterize, and quantify conjugated and unconjugated metabolites of the test substance. (iii) Changes in biotransformation. Appropriate qualitative andquantitative assay methodologies should be used to compare the composition of 14 C-labeled components of urine collected from 0 to 24 h and

### 4 then from 24 to 48 h after dosing rat Group A with those componentsin the urine collected over the same intervals after administering the radioactive dose in the repeated dosing study. (e) Data and reporting—(1) Treatment of results. Data should be summarized in tabular form. (2) Evaluation of results. All observed results should be evaluated by an appropriate statistical method. (3) Test report. In addition to the reporting requirements as specified in 40 CFR part 792, subpart J, the following specific information should be reported: (i) Labeling site of the test substance. (ii) A full description of the sensitivity and precision of all procedures used to produce the data.(iii) Quantity of isotope, together with percent recovery of the administered dose in feces, urine, expired air, and blood for both routes of administration. (iv) Quantity and distribution of 14 C-test substance in bone, brain,fat, gonads, heart, kidney, liver, lung, muscle, spleen, tissue which displayed pathology, and residual carcass. (v) Biotransformation pathways and quantities of test substance andits metabolites in urine, feces, and expired air collected after oral administration (single low and high doses) and inhalation exposure (low, intermediate, and high concentrations). (vi) Biotransformation pathways and quantities of the test substance and its metabolites in urine collected after repeated administration of test substance to rats.(vii) Pharmacokinetic models, if any, developed from the experimental data. (4) Counting efficiency. Data should be made available to the Agency upon request.