United States Prevention, Pesticides EPA712-C-98-199 Environmental Protection and Toxic Substances August 1998 Agency (7101) &EPA Health Effects Test Guidelines OPPTS 870.3100 90-Day Oral Toxicity in Rodents ------- 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 the National Technical Information Service (NTIS) and the guidelines pub- lished 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 procedures that must be performed to meet the data requirements of the U. S. Environ- mental Protection Agency under the Toxic Substances Control Act (15 U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act (7U.S.C. I36,etseq.). Final Guideline Release: This guideline is available from the U.S. Government Printing Office, Washington, DC 20402 on disks or paper copies: call (202) 512-0132. This guideline is also available electronically in PDF (portable document format) from EPA's World Wide Web site (http://www.epa.gov/epahome/research.htm) under the heading "Research- ers and Scientists/Test Methods and Guidelines/OPPTS Harmonized Test Guidelines." ------- OPPTS 870.3100 90-Day oral toxicity in rodents. (a) Scope—(1) Applicability. This guideline is intended to meet test- ing 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 materials used in developing this har- monized OPPTS test guideline are 40 CFR 798.2650 Oral Toxicity; OPP 82-1 90-Day Oral—Two Species, Rodent and Nonrodent; and OECD 408 Subchronic Oral Toxicity—Rodent: 90-Day. (b) Purpose. In the assessment and evaluation of the toxic character- istics of a chemical, 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 deter- mination of the no-observed-effect level (NOEL) and toxic effects associ- ated with continuous or repeated exposure to a test substance for a period of 90 days. This study is not capable of determining those effects that have a long latency period for development (e.g., carcinogenicity and life shortening). Extrapolation from the results of this study to humans is valid only to a limited degree. However, it can useful in providing information on health hazards likely to arise from repeated exposure by the oral route over a limited period of time, such as target organs, the possibilities of accumulation, and can 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 the Toxic Substance Control Act (TSCA) and the definitions in 40 CFR Part 792—Good Lab- oratory Practice Standards apply to this test guideline. The following defi- nitions also apply to this test guideline. Cumulative toxicity is the adverse effects of repeated dosesoccurring as a result of prolonged action on, or increased concentration of, the ad- ministered test substance or its metabolites in susceptible tissue. Dose in a subchronic oral study is the amount of test substance ad- ministered daily via the oral route (gavage, drinking water or diet) for a period of 90 days. Dose is expressed as weight of the test substance (grams, milligrams) per unit body weight (BW) of test animal (milligram per kilogram) or as weight of the test substance in parts per million in food or drinking water per day. No-observed-effect-level (NOEL) is the maximum dose used in a study which produces no adverse effects. The NOEL is usually expressed in terms of the weight of a test substance given daily per unit weight of test animal (milligrams per kilogram per day). Subchronic oral toxicity is the adverse effects occurring as a result of the repeated daily exposure of experimental animals to a chemical by ------- the oral route for a part (approximately 10 percent) of the test animal's life span. Target organ is any organ of a test animal showing evidence of an effect induced by a test substance. (d) Limit test. If a test at one dose level of at least 1,000 mg/kg body weight (expected human exposure may indicate the need for a higher dose level), using the procedures described for this study, produces no observable toxic effects or if toxic effects would not be expected based upon data of structurally related compounds, then a full study using three dose levels might not be necessary. (e) Test procedures—(1) Animal selection—(i) Species and strain. A variety of rodent species may be used, although the rat is the preferred species. Commonly used laboratory strains should be employed. (ii) Age/weight. (A) Testing should be started with young healthy animals as soon as possible after weaning and acclimatization. (B) Dosing of rodents should generally begin no later than 8-9 weeks of age. (C) At the commencement of the study the weight variation of ani- mals used should be within 20 percent of the mean weight for each sex. (iii) Sex. Equal numbers of animals of each sex should be used at each dose level, and the females should be nulliparous and nonpregnant. (iv) Numbers. (A) At least 20 rodents (10 males and 10 females) at each dose level. (B) If interim sacrifices are planned, the number should be increased by the number of animals scheduled to be sacrificed before the completion of the study. (C) To avoid bias, the use of adequate randomization procedures for the proper allocation of animals to test and control groups is required. (D) Each animal should be assigned a unique identification number. Dead animals, their preserved organs and tissues, and microscopic slides should be identified by reference to the animal's unique number. (v) Husbandry. (A) Animals may be group-caged by sex, but the number of animals per cage must not interfere with clear observation of each animal. The biological properties of the test substance or toxic effects (e.g., morbidity, excitability) may indicate a need for individual caging. (B) The temperature of the experimental animal rooms should be at 22 + 3 °C. ------- (C) The relative humidity of the experimental animal rooms should be 50 + 20 percent. (D) Where lighting is artificial, the sequence should be 12 hours light/ 12 hours dark. (E) Control and test animals should be fed from the same batch and lot. The feed should be analyzed to assure adequacy of nutritional require- ments of the species tested and for impurities that might influence the outcome of the test. For feeding, conventional laboratory diets may be used with an unlimited supply of drinking water. (F) The study should not be initiated until animals have been allowed a period of acclimatization/quarantine to environmental conditions, nor should animals from outside sources be placed on test without an adequate period of quarantine. An acclimation period of at least five days is rec- ommended. (2) Control and test substances, (i) Where necessary, the test sub- stance is dissolved or suspended in a suitable vehicle. If a vehicle or dilu- ent is needed, the vehicle should not elicit toxic effects or 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 in oil and then solution in other vehicles. (ii) If possible, one lot of the test substance tested should be used throughout the duration of the study and the research sample should be stored under conditions that maintain its purity and stability. Prior to the initiation of the study, there should be a characterization of the test sub- stance, including the purity of the test compound and, if technically fea- sible, the names and quantities of contaminants and impurities. (iii) If the test or control substance is to be incorporated into feed or another vehicle, the period during which the test substance is stable in such a mixture should be determined prior to the initiation of the study. Its homogeneity and concentration should be determined prior to the initi- ation of the study and periodically during the study. Statistically random- ized samples of the mixture should be analyzed to ensure that proper mix- ing, formulation, and storage procedures are being followed, and that the appropriate concentration of the test or control substance is contained in the mixture. (3) 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. ------- (4) Satellite group. A satellite group of 20 animals (10 animals per sex) may be treated with the high dose level for 90 days and observed for reversibility, persistence, or delayed occurrence of toxic effects for a post-treatment period of appropriate length, normally not less than 28 days. In addition, a control group of 20 animals (10 animals of each sex) should be added to the satellite study. (5) Dose levels and dose selection, (i) In subchronic toxicity tests, it is desirable to determine a dose-response relationship as well as a NOEL. Therefore, at least three dose levels plus a control and, where ap- propriate, a vehicle control (corresponding to the concentration of vehicle at the highest dose level) should be used. Doses should be spaced appro- priately to produce test groups with a range of toxic effects. The data should be sufficient to produce a dose-response curve. (ii) The highest dose level should result in toxic effects but not produce an incidence of fatalities which would prevent a meaningful eval- uation. (iii) The intermediate dose levels should be spaced to produce a gra- dation of toxic effects. (iv) The lowest dose level should produce no evidence of toxicity. (6) Administration of the test substance, (i) If the test substance is administered by gavage, the animals are dosed with the test substance on a 7-day per week basis for a period of at least 90 days. However, based primarily on practical considerations, dosing by gavage on a 5-day per week basis is acceptable. If the test substance is administered in the drinking water, or mixed in the diet, then exposure should be on a 7- day per week basis. (ii) All animals should be dosed by the same method during the entire experimental period. (iii) 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 administered in the diet, either a constant dietary concentration (parts per million) or a constant dose level in terms of body weight should be used; the alter- native used should be specified. (iv) For a substance administered by gavage, the dose should be given at approximately the same time each day, and adjusted at intervals (weekly or biweekly) to maintain a constant dose level in terms of body weight. (7) Observation period, (i) The animals should be observed for a period of 90 days. ------- (ii) Animals in the satellite group (if used) scheduled for follow-up observations should be kept for at least 28 days further without treatment to detect recovery from, or persistence of, toxic effects. (8) Observation of animals, (i) Observations should be made at least twice each day for morbidity and mortality. Appropriate actions should be taken to minimize loss of animals to the study (e.g., necropsy or refrig- eration of those animals found dead and isolation or sacrifice of weak or moribund animals). General clinical observations should be made at least once a day, preferably at the same time each day, taking into consid- eration the peak period of anticipated effects after dosing. The clinical condition of the animal should be recorded. (ii) A careful clinical examination should be made at least once prior to the initiation of treatment (to allow for within subject comparisons) and once weekly during treatment in all animals. These observations should be made outside the home cage, preferably in a standard arena, and at similar times on each occasion. Effort should be made to ensure that vari- ations in the observation conditions are minimal. Observations should be detailed and carefully recorded, preferably using scoring systems, explic- itly defined by the testing laboratory. Signs noted should include, but not be limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, pos- ture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling) or bizarre behavior (e.g., self-mutilation, walking backwards) should be re- corded. (iii) Once, near the end of the exposure period and in any case not earlier than in week 11, assessment of motor activity, grip strength, and sensory reactivity to stimuli of different types (e.g., visual, auditory, and proprioceptive stimuli) should be conducted. Further details of the proce- dures that could be followed are described in the references listed under paragraphs (h)(2), (h)(5), (h)(6), (h)(7), (h)(8), and (h)(ll) of this guide- line. (iv) Functional observations conducted towards the end of the study may be omitted when data on functional observations are available from other studies and the daily clinical observations did not reveal any func- tional deficits. (v) Exceptionally, functional observations may be omitted for groups that otherwise reveal signs of toxicity to an extent that would significantly interfere with functional test performance. (vi) Measurements of food consumption and water consumption, if drinking water is the exposure route, should be made weekly. ------- (vii) Individual weights of animals should be determined shortly be- fore the test substance is administered, weekly thereafter, and at death. (viii) Moribund animals should be removed and sacrificed when no- ticed and the time of death should be recorded as precisely as possible. (ix) At termination, all survivors in the treatment and control groups should be sacrificed. (9) Clinical pathology. Hematology and clinical chemistry examina- tions should be made on all animals, including controls, of each sex in each group. The hematology and clinical chemistry parameters should be examined at terminal sacrifice at the end of the study. Overnight fasting of the animals prior to blood sampling is recommended. Overall, there is a need for a flexible approach in the measures examined, depending on the observed or expected effects from a chemical, and in the frequency of measures, depending on the duration of potential chemical exposures. (i) Hematology. The recommended parameters are red blood cell count, hemoglobin concentration, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin con- centration, white blood cell count, differential leukocyte count, platelet count, and a measure of clotting potential, such as prothrombin time or activated partial thromboplastin time. (ii) Clinical chemistry. (A) Parameters which are considered appro- priate to all studies are electrolyte balance, carbohydrate metabolism, and liver and kidney function. The selection of specific tests will be influenced by observations on the mode of action of the substance and signs of clini- cal toxicity. (B) The recommended clinical chemistry determinations are potas- sium, sodium, glucose, total cholesterol, urea nitrogen, creatinine, total protein and albumin. More than 2 hepatic enzymes, (such as alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, sorbitol dehydrogenase, or gamma glutamyl transpeptidase) should also be meas- ured. Measurements of addtional enzymes (of hepatic or other origin) and bile acids, may also be useful. (C) If a test chemical has an effect on the hematopoietic system, reticulocyte counts and bone marrow cytology may be indicated. (D) Other determinations that should be carried out if the test chemi- cal is known or suspected of affecting related measures include calcium, phosphorus, fasting triglycerides, hormones, methemoglobin, and cholinesterases. (iii) Optionally, the following urinalysis determinations could be per- formed during the last week of the study using timed urine volume collec- ------- tion: appearance, volume, osmolality or specific gravity, pH, protein, glu- cose and blood/blood cells. (10) Ophthalmological examination. Ophthalmological examina- tions using an ophthalmoscope or an equivalent device should be made on all animals prior to the administration of the test substance and on all high dose and control groups at termination. If changes in the eyes are detected, all animals in the other dose groups should be examined. (11) 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) The liver, kidneys, adrenals, testes, epididymides, ovaries, uterus, thymus, spleen, brain, and heart should be trimmed and weighed wet, as soon as possible after dissection. (iii) The following organs and tissues, or representative samples there- of, should be preserved in a suitable medium for possible future histopathological examination: (A) Digestive system—salivary glands, esophagus, stomach, duode- num, jejunum, ileum, cecum, colon, rectum, liver, pancreas, gallbladder (when present). (B) Nervous system—brain (including sections of medulla/pons, cere- bellum and cerebrum), pituitary, peripheral nerve (sciatic or tibial, pref- erably in close proximity to the muscle), spinal cord (three levels: cervical, mid-thoracic and lumbar), eyes (retina, optic nerve). (C) Glandular system—adrenals, parathyroid, thyroid. (D) Respiratory system—trachea, lungs, pharynx, larynx, nose. (E) Cardiovascular/hemopoietic system—aorta, heart, bone marrow (and/or fresh aspirate), lymph nodes (preferably one lymph node covering the route of administration and another one distant from the route of ad- ministration to cover systemic effects), spleen, thymus. (F) Urogenital system—kidneys, urinary bladder, prostate, testes, epididymides, seminal vesicle(s), uterus, ovaries, female mammary gland. (G) Others—all gross lesions and masses, skin. (13) Histopathology. (i) The following histopathology should be per- formed: (A) Full histopathology on the organs and tissues, listed under para- graph (e)(12)(iii) of this guideline, of all rodents in the control and high dose groups, and all rodents that died or were killed during the study. ------- (B) All gross lesions in all animals. (C) Target tissues in all animals. (D) When a satellite group is used, histopathology should be per- formed on tissues and organs identified as showing effects in the treated groups. (ii) If excessive early deaths or other problems occur in the high dose group compromising the significance of the data, the next dose level should be examined for complete histopathology. (iii) An attempt should be made to correlate gross observations with microscopic findings. (iv) Tissues and organs designated for microscopic examination should be fixed in 10 percent buffered formalin or a recognized suitable fixative as soon as necropsy is performed and no less than 48 hours prior to trimming. (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 types of lesions and the percentage of animals displaying each type of lesion. (ii) When applicable, all observed results, qualitative and quantitative, should be evaluated by an appropriate and generally accepted statistical method. Any generally accepted statistical methods may be used; the sta- tistical methods, including significance criteria, should be selected during the design of the study. (2) Evaluation of study results. The findings of a subchronic oral toxicity study should be evaluated in conjunction with the findings of pre- ceding studies and considered in terms of the toxic effects and the ne- cropsy and histopathological findings. The evaluation will include the rela- tionship between the dose of the test substance and the presence or ab- sence, 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. A properly conducted subchronic test should provide a satisfactory estimation of a NOEL. It also can indicate the need for an additional longer-term study and provide information on the selection of dose levels. (3) Test report. In addition to reporting requirements specified under EPA Good Laboratory Practice Standards at 40 CFR part 792, subpart J and 40 CFR part 160, and the OECD principles of GLP (ISBN 92- 64-12367-9), the following specific information should be reported: (i) Test substance characterization should include: 8 ------- (A) Chemical identification. (B) Lot or batch number. (C) Physical properties. (D) Purity/impurities. (ii) Identification and composition of any vehicle used. (iii) Test system should contain data on: (A) Species and strain of animals used and rationale for selection if other than that recommended. (B) Age including body weight data and sex. (C) Test environment including cage conditions, ambient temperature, humidity, and light/dark periods. (D) Identification of animal diet. (E) Acclimation period. (iv) Test procedure should include the following data: (A) Method of randomization used. (B) Full description of experimental design and procedure. (C) Dose regimen including levels, methods, and volume. (v) Test results should include: (A) Group animal data. Tabulation of toxic response data by species, strain, sex and exposure level for: (7) Number of animals exposed. (2) Number of animals showing signs of toxicity. (3) Number of animals dying. (B) Individual animal data. Data should be presented as summary (group mean) as well as for individual animals. (7) Date of death during the study or whether animals survived to termination. (2) Date of observation of each abnormal sign and its subsequent course. Body weight data. (4) Feed and water (if collected) consumption data. 9 ------- (5) Achieved dose (mg/kg/day) as a time-weighted average if the test substance is administered in the diet or drinking water. (6) Results of ophthalmological examination. (7) Results of hematological tests performed. (8) Results of clinical chemistry tests performed. (9) Results of urinalysis, if performed. (10) Necropsy findings, including absolute and relative (to body weight) organ weight data. (11) Detailed description of all histopathological findings. (12) Statistical treatment of results, where appropriate. (g) Quality control. A system should be developed and maintained to assure and document adequate performance of laboratory staff and equipment. The study must be conducted in compliance with GLP regula- tions. (h) References. The following references should be consulted for ad- ditional background information on this test guideline. (1) Boyd, E.M. Chapter 14. Pilot Studies, 15. Uniposal Clinical Pa- rameters, 16. Uniposal Autopsy Parameters. Predictive Toxicometrics. Wil- liams and Wilkins, Baltimore (1972). (2) Crofton K.M., Howard J.L., Moser V.C., Gill M.W., Leiter L.W., Tilson H.A., MacPhail, R.C. Interlaboratory Comparison of Motor Activity Experiments: Implication for Neurotoxicological Assesments. Neurotoxicol. Teratol. 13, 599-609. (1991) (3) Fitzhugh, O.G. Subacute Toxicity, Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. The Association of Food and Drug Officials of the United States (1959, 3rd Printing 1975) pp. 260935. (4) Food Safety Council. Subchronic Toxicity Studies, Proposed Sys- tem for Food Safety Assessment. (Columbia: Food Safety Council, 1978) pp. 830996. (5) Gad S.C. A Neuromuscular Screen for Use in Industrial Toxi- cology. Journal of Toxicology and Environmental Health. 9, 691-704. (1982) (6) International Programme on Chemical Safety. Principles and Methods for the Assessment of Neurotoxicity Associated with Exposure to Chemicals. Environmental Health Criteria Document No. 60. (1986) 10 ------- (7) Meyer O.A., Tilson H.A., Byrd W.C., Riley M.T. A Method for the Routine Assessment of Fore- and Hind-Limb Grip Strength of Rats and Mice. Neurobehav. Toxicol. 1, 233-236. (1979) (8) Moser V.C., McDaniel K.M., Phillips P.M. Rat Strain and Stock Comparisons using a Functional Observational Battery: Baseline Values and Effects of Amitraz. Toxicol. Appl. Pharmacol. 108, 267-283 (1991) (9) National Academy of Sciences. Principles and Procedures for Evaluating the Toxicity of Household Substances, A report prepared by the Committee for the Revision of NAS Publication 1138, under the aus- pices of the Committee on Toxicology, National Research Council, Na- tional Academy of Sciences, Washington, DC (1977). (10) Organization for Economic Cooperation and Development. OECD Guidelines for Testing of Chemicals. Guideline 408: Subchronic Oral Toxicity-Rodent: 90-day Study, Adopted: May 12, 1981. (11) Tupper, D.E., Wallace R.B. Utility of the Neurologic Examina- tion in Rats. Acta. Neurobiol. Exp. 40, 999-1003 (1980). (12) United States Environmental Protection Agency. Office of Test- ing and Evaluation. Proposed Health Effects Test Standards for Toxic Sub- stances Control Act Test Rules. 40 CFR Part 772. Standard for Develop- ment of Test Data. Subpart D. FEDERAL REGISTER Vol. 44, pp. 27350- 27362. (13) Weingand K., Brown G., Hall R. et al. Harmonization of Animal Clinical Pathology Testing in Toxicity and Safety Studies. Fundam. & Appl. Toxicol. 29:198-201. (1996) (14) World Health Organization. Guidelines for Evaluation of Drugs for Use in Man, WHO Technical Report Series No. 563. (Geneva: World Health Organization, 1975). (15) World Health Organization. Part I. Environmental Health Criteria 6, Principles and Methods for Evaluating the Toxicity of Chemicals. (Ge- neva: World Health Organization, 1978). (16) World Health Organization. Principles for Pre-Clinical Testing of Drug Safety, WHO Technical Report Series No. 341. (Geneva: World Health Organization, 1966). 11 ------- |