.... United States Environmental Protection Agency Prevention, Pesticides and Toxic Substances (7101) EPA 712–C–96–259 June 1996 

# Health Effects Test Guidelines OPPTS 870.8700 Subchronic Oral Toxicity 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 870.8700 Subchronic oral toxicity 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 795.260 Subchronic Oral Toxicity Test. (b) Purpose. In the assessment and evaluation of the toxic characteristics of a test substance, the determination of subchronic oral toxicity may be carried out after initial information on toxicity has been obtained by acute testing. The subchronic oral study has been designed to permit the determination of the no-observed-effect level and toxic effects associated with continuous or repeated exposure to a test substance for a period of 90 days. The test is not capable of determining those effects that havea long latency period for development (e.g., carcinogenicity and life shortening). It provides information on health hazards likely to arise from repeated exposure by the oral route over a limited period of time. It will provide information on target organs, the possibilities of accumulation, andcan be of use in selecting dose levels for chronic studies and for establishing safety criteria for human exposure. (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 definitions also apply to this test guideline. Cumulative toxicity is the adverse effects of repeated doses occurringas a result of prolonged action on, or increased concentration of, the administered substance or its metabolites in susceptible tissue.

Dose is the amount of test substance administered. Dose is expressed as weight of test substance (grams, milligrams) per unit weight of test animal (e.g. expressed as milligrams per kilogram), or as weight of test substance per unit weight of food or drinking water.No-effect level/No-toxic-effect level/No-adverse-effect level/No-observed-effect level (NOEL) is the maximum dose used in a test which produces no observed adverse effects. A no-observed-effect level is expressed in terms of the weight of a substance given daily per unit weight of testanimal (expressed as milligrams per kilogram). When administered to animals in food or drinking water, the NOEL is expressed as milligrams per kilogram of food or milligrams per milliliter of water. Subchronic oral toxicity is the adverse effects occurring as a result of the repeated daily exposure of experimental animals to a chemical for a part (approximately 10 percent for rats) of a life span.

### 2 (d) Principle of the test method. The test substance is administered orally in graduated daily doses to several groups of experimental animals, one dose level per group, for a period of 90 days. During the period of administration the animals are observed daily to detect signs of toxicity. Animals which die during the period of administration are necropsied. At the conclusion of the test all animals are necropsied and histopathological examinations carried out. (e) Test procedures—(1) Animal selection—(i) Species. Rats and mice should be used. (ii) Age. (A) Young adult animals should be employed. At the commencement of the study the weight variation of animals used should not exceed ± 20 percent of the mean weight for each sex. (B) Dosing should begin as soon as possible after weaning, before the animals are 6 weeks old, and in any case not more than 8 weeks old. (iii) Sex. (A) Equal numbers of animals of each sex should be used at each dose level. (B) The females should be nulliparous and nonpregnant. (iv) Numbers. (A) At least 20 rats and 20 mice (10 females and 10 males of each species) should be used at each dose level. (B) If interim sacrifices are required, the number should be increased by the number of animals scheduled to be sacrificed before the completion of the study. (2) Control groups. A concurrent control group is required. This group should be an untreated or sham-treated control group or, if a vehicle is used in administering the test substance, a vehicle control group. If the toxic properties of the vehicle are not known or cannot be made available, both untreated and vehicle control groups are required. (3) Satellite group. A satellite group of 20 rats and 20 mice (10 females and 10 males of each species) should be treated with the highdose level for 90 days and observed for reversibility, persistence, or delayed occurrence of toxic effects for a post-treatment period of not less than 28 days. (4) Dose levels and dose selection. (i) In subchronic toxicity tests,it is desirable to have a dose response relationship as well as no-observedtoxic-effect level. Therefore, at least three dose levels with a control and, where appropriate, a vehicle control (corresponding to the concentration of vehicle at the highest exposure level) should be used. Doses shouldbe spaced appropriately to produce test groups with a range of toxic effects. The data should be sufficient to produce a dose-response curve.

### 3 (ii) The highest dose level should result in toxic effects but notproduce an incidence of fatalities which would prevent a meaningful evaluation.(iii) The lowest dose level should not produce any evidence of toxicity. Where there is a usable estimation of human exposure the lowest dose level should exceed this. (iv) The intermediate dose levels should produce minimal observable toxic effects. If more than one intermediate dose is used, the dose levels should be spaced to produce a gradation of toxic effects. (v) The incidence of fatalities in low and intermediate dose groups and in the controls should be low to permit a meaningful evaluation of the results. (5) Exposure conditions. The animals should be dosed with the test substance on a 7–day per week basis over a period of 90 days. However, based primarily on practical considerations, dosing by gavage or capsule studies on a 5–day per week basis should be acceptable. (6) Observation period. (i) Duration of observation should be for at least 90 days. (ii) Animals in the satellite group scheduled for followup observationsshould be kept for not less than 28 days without treatment to detect recovery from, or persistence of, toxic effects. (7) Administration of the test substance. (i) The test substance should be administered in the diet or in capsules. Alternatively, it may be administered by gavage or in the drinking water. (ii) All animals should be dosed by the same method during the entire experimental period. (iii) Where necessary, the test substance is dissolved or suspended in a suitable vehicle. If a vehicle or diluent is needed, it should not elicitimportant toxic effects itself nor substantially alter the chemical or toxicological properties of the test substance. It is recommended that wherever possible the usage of an aqueous solution be considered first, followed by consideration of a solution of oil, and then by possible solution in other vehicles. (iv) For substances of low toxicity, it is important to ensure that when administered in the diet the quantities of the test substance involved do not interfere with normal nutrition. When the test substance is administeredin the diet, either a constant dietary concentration (given in parts per million) or a constant dose level in terms of animal body weight should be used; the alternative used should be specified.

### 4 (v) For a substance administered by gavage or capsule, the doseshould be given at similar times each day, and adjusted at intervals (weekly or biweekly) to maintain a constant dose level in terms of animal body weight. (8) Observation of animals. (i) Each animal should be handled and its physical condition appraised at least once each day.(ii) Additional observation should be made daily with appropriate actions taken to minimize loss of animals to the study (e.g., necropsy or refrigeration of those animals found dead and isolation or sacrifice of weak or moribund animals).(iii) Signs of toxicity should be recorded as they are observed including the time of onset, degree, and duration. (iv) Cage-side observations should include, but not be limited to,changes in skin and fur, eyes and mucous membranes, respiratory, circulatory, autonomic and central nervous systems, somatomotor activity, and behavior pattern. (v) Measurements should be made weekly of food consumption or water consumption when the test substance is administered in the food or drinking water, respectively. (vi) Animals should be weighed weekly. (vii) At the end of the 90–day period all survivors in the nonsatellitetreatment group should be sacrificed. Moribund animals should be removed and sacrificed when noticed. (9) Clinical examinations. (i) The following examinations should be made on at least five animals of each sex in each group of rats. (A) Certain hematology determinations should be carried out justprior to terminal sacrifice at the end of the test period. The following hematology determinations should be carried out: hematocrit, hemoglobin concentration, erythrocyte count, total and differential leucocyte count, and a measure of clotting potential such as clotting time, prothrombin time, thromboplastin time, or platelet count. (B) Certain clinical biochemistry determinations should be carried out just prior to terminal sacrifice at the end of the test period. The following clinical biochemical test areas should be carried out: Electrolyte balance, carbohydrate metabolism, and liver and kidney function. The selection of additional tests should be influenced by observations on the mode of action of the substance. Suggested additional determinations include calcium, phosphorus, chloride, sodium, potassium, fasting glucose (with period of fasting appropriate to the species/breed), serum alanine aminotransferase, serum aspartate aminotransferase, ornithine decarboxylase, gamma

### 5glutamyl transpeptidase, urea nitrogen, albumen, blood creatinine, total bilirubin, and total serum protein measurements. Other determinations which may be necessary for an adequate toxicological evaluation include analyses of lipids, hormones, acid/base balance, methemoglobin, and cholinesteraseactivity. Additional clinical biochemistry may be employed where necessary to extend the investigation of observed effects.(ii) The following examinations should be made on at least five animals of each sex in each group. (A) Ophthalmological examination, using an ophthalmoscope or equivalent suitable equipment, should be made prior to the administration of the test substance and at the termination of the study. If changes in the eyes are detected, all animals should be examined. (B) Urinalysis is required only when there is an indication based on expected or observed toxicity. (10) Gross necropsy. (i) All animals should be subjected to a full gross necropsy which includes examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents. (ii) At least the liver, kidneys, adrenals, gonads, and brain should be weighed wet, as soon as possible after dissection to avoid drying.(iii) The following organs and tissues, or representative samples thereof, should be preserved in a suitable medium for possible future histopathological examination—all gross lesions, brain—including sections of medulla/pons, cerebellar cortex and cerebral cortex, pituitary, thyroid/parathyroid, thymus, lungs, trachea, heart, sternum with bone marrow, salivary glands, liver, spleen, kidneys/adrenals, pancreas, gonads, uterus, accessory genital organs (epididymis, prostrate, and, if present, seminal vesicles), aorta, (skin), (nonrodent gall bladder), esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, urinary bladder, representative lymph node, (mammary gland), (thigh musculature), peripheral nerve, (eyes), (femur including articular surface), (spinal cord at three levels— cervical, midthoracic and lumbar), and, (rodent-exorbital lachrymal glands). (11) Histopathology. (i) Full histopathology should be performed on the organs and tissues, listed under paragraphs (e)(10)(ii) and (e)(10)(iii) of this guideline of all animals in the control and high-dose groups, and all animals that died or were killed during the study. (ii) Histopathology should be performed on all gross lesions in all animals.(iii) Histopathology should be performed on target organs in all animals.

### 6 (iv) Histopathology should be performed on the tissues mentioned in parentheses under paragraph (e)(10)(iii) of this guideline if indicated by signs of toxicity or target organ involvement. (v) Histopathology should be performed on lungs, liver, and kidneys of all animals. Special attention to examination of the lungs should be made for evidence of infection since this provides a convenient assessment of the state of health of the animals. (vi) For the satellite group, histopathology should be performed on tissues and organs identified as showing effects in the treated groups. (f) Data and reporting—(1) Treatment of results. (i) Data should be summarized in tabular form, showing for each test group the number of animals at the start of the test, the number of animals showing lesions, the type of lesions, and the percentage of animals displaying each type of lesion.(ii) All observed results, quantitative and incidental, should be evaluated by an appropriate statistical method. Any generally acceptable statistical methods may be used; the statistical methods should be selected during the design of the study. (2) Evaluation of the study results. (i) The findings of a subchronic oral toxicity study should be evaluated in conjunction with the findings of preceding studies and considered in terms of the toxic effects and the necropsy and histopathological findings. The evaluation should include the relationship between the dose of the test substance and the presence or absence, the incidence and severity, of abnormalities, including behavioral and clinical abnormalities, gross lesions, identified target organs, body weight changes, effects on mortality and any other general or specific toxic effects. The test should provide a satisfactory estimation of a no-effect level.(ii) In any study which demonstrates an absence of toxic effects, further investigation to establish absorption and bioavailability of the test substance should be considered. (3) Test report. In addition to the reporting requirements as specifiedin the TSCA Good Laboratory Practice Standards, 40 CFR part 792, subpart J, the following specific information should be reported: (i) Group animal data. Tabulation of toxic response data by species, strain, sex, and exposure level for: (A) Number of animals dying. (B) Number of animals showing signs of toxicity. (C) Number of animals exposed.

### 7 (ii) Individual animal data. (A) Time of death during the study or whether animals survived to termination. (B) Time of observation of each abnormal sign and its subsequent

course. (C) Body weight data. (D) Food consumption data when collected. (E) Hematological tests employed and all results. (F) Clinical biochemistry tests employed and all results. (G) Necropsy findings. (H) Detailed description of all histopathological findings. (I) Statistical treatment of results where appropriate.