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Title 40Chapter ISubchapter RPart 798 → Subpart C


Title 40: Protection of Environment
PART 798—HEALTH EFFECTS TESTING GUIDELINES


Subpart C—Subchronic Exposure


Contents
§798.2250   Dermal toxicity.
§798.2450   Inhalation toxicity.
§798.2650   Oral toxicity.

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§798.2250   Dermal toxicity.

(a) Purpose. In the assessment and evaluation of the toxic characteristics of a chemical, the determination of subchronic dermal toxicity may be carried out after initial information on toxicity has been obtained by acute testing. The subchronic dermal 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 have a 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 dermal route over a limited period of time. It will provide information on 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.

(b) Definitions. (1) Subchronic dermal toxicity is the adverse effects occurring as a result of the repeated daily exposure of experimental animals to a chemical by dermal application for part (approximately 10 percent) of a life span.

(2) Dose in a dermal test is the amount of test substance applied to the skin (applied daily in subchronic tests). Dose is expressed as weight of the substance (g, mg) per unit weight of test animal (e.g., mg/kg).

(3) No-effect level/No-toxic-effect level/No-adverse-effect level/No-observed-effect level 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 test substance given daily per unit weight of test animal (mg/kg).

(4) Cumulative toxicity is the adverse effects of repeated doses occurring as a result of prolonged action on, or increased concentration of the administered test substance or its metabolites in susceptible tissues.

(c) Principle of the test method. The test substance is applied daily to the skin in graduated doses to several groups of experimental animals, one dose level per unit group, for a period of 90 days. During the period of application the animals are observed daily to detect signs of toxicity. Animals which die during the test are necropsied, and at the conclusion of the test the surviving animals are sacrificed and necropsied and appropriate histopathological examinations carried out.

(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 and if toxicity 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 mammalian species shall be used for testing. The rat, rabbit, or guinea pig may be used, although the albino rabbit is preferred. The albino rabbit is preferred because of its size, skin permeability, and extensive data base. Commonly used laboratory strains shall be employed. If another mammalian species is used, the tester shall provide justification/reasoning for its selection.

(ii) Age. Young adult animals shall be used. The following weight ranges at the start of the test are suggested in order to provide animals of a size which facilitates the conduct of the test: rats, 200 to 300 g; rabbits, 2.0 to 3.0 kg; guinea pigs, 350 to 450 g.

(iii) Sex. (A) Equal numbers of animals of each sex with healthy skin shall be used at each dose level.

(B) The females shall be nulliparous and nonpregnant.

(iv) Numbers. (A) At least 20 animals (10 females and 10 males) shall be used at each dose level.

(B) If interim sacrifices are planned, the number shall be increased by the number of animals scheduled to be sacrificed before completion of the study.

(2) Control groups. A concurrent control group is required. This group shall 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 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 posttreatment period of appropriate length, normally not less than 28 days.

(4) Dose level and dose selection. (i) In subchronic toxicity tests, it is desirable to have a dose-response relationship as well as a no-observed-toxic-effect level. Therefore, at least 3 dose levels with a control and, where appropriate, a vehicle control (corresponding to the concentration of vehicle at the highest exposure level) shall be used. Doses should be spaced appropriately to produce test groups with a range of toxic effects. The data shall be sufficient to produce a dose-response curve.

(ii) The highest dose level should result in toxic effects but not produce severe skin irritation or 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) Ideally, the intermediate dose level(s) 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) In the low and intermediate groups and in the controls the incidence of fatalities should be low, to permit a meaningful evaluation of the results.

(5) Exposure conditions. The animals are treated with test substance, ideally for at least 6 hours per day on a 7-day per week basis, for a period of 90 days. However, based primarily on practical considerations, application on a 5-day per week basis is considered to be acceptable.

(6) Observation period. (i) Duration of observation shall be at least 90 days.

(ii) Animals in the satellite group scheduled for followup observations should be kept for at least 28 days further without treatment to detect recovery from, or persistence of, toxic effects.

(7) Preparation of animal skin. (i) Shortly before testing, fur shall be clipped from the dorsal area of the trunk of the test animals. Shaving may be employed, but it should be carried out approximately 24 hours before the test. Repeat clipping or shaving is usually needed at approximately weekly intervals. When clipping or shaving the fur, care should be taken to avoid abrading the skin, which could alter its permeability.

(ii) Not less than 10 percent of the body surface area should be clear for the application of the test substance. The weight of the animal should be taken into account when deciding on the area to be cleared and on the dimensions of any covering used.

(iii) When testing solids, which may be pulverized if appropriate, the test substance should be moistened sufficiently with water or, where necessary, a suitable vehicle to ensure good contact with the skin. When a vehicle is used, the influence of the vehicle on toxicity of and penetration of the skin by the test substance should be taken into account.

(8) Application of the test substance. (i) The test substance shall be applied uniformly over an area which is approximately 10 percent of the total body surface area. With highly toxic substances, the surface area covered may be less, but as much of the area shall be covered with as thin and uniform a film as possible.

(ii) During the exposure period, the test substance shall be held in contact with the skin with a porous gauze dressing and nonirritating tape. The test site shall be further covered in a suitable manner to retain the gauze dressing and test substance and ensure that the animals cannot ingest the test substance. Restrainers may be used to prevent the ingestion of the test substance, but complete immobilization is not a recommended method.

(9) Observation of animals. (i) Each animal shall be observed daily, and if necessary handled to appraise its physical condition.

(ii) Additional observations shall 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 shall be recorded as they are observed, including the time of onset, the degree, and duration.

(iv) Cage-side observations shall 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) Animals shall be weighed weekly. Feed consumption shall also be determined weekly if abnormal body weight changes are observed.

(vi) At the end of the study period, all survivors in the nonsatellite treatment groups shall be sacrificed. Moribund animals shall be removed and sacrificed when noticed.

(10) Clinical examinations. (i) The following examinations shall be made on all animals of each sex in each group:

(A) Certain hematology determinations shall be carried out at least two times during the test period on all groups of animals including concurrent controls: After 30 days of test and just prior to terminal sacrifice at the end of the test period. Hematology determinations which are appropriate to all studies: Hematocrit, hemoglobin concentration, erythrocyte count, total and differential leukocyte count, and a measure of clotting potential such as clotting time, prothrombin time, thromboplastin time, or platelet count.

(B) Certain clinical biochemistry determinations on blood should be carried out at least two times during the test period on all groups of animals including concurrent controls: After 30 days of test and just prior to terminal sacrifice at the end of the test period. Clinical biochemistry test areas which are considered appropriate to all studies: 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. Suggested determinations: Calcium, phosphorus, chloride, sodium, potassium, fasting glucose (with period of fasting appropriate to the species), serum glutamic pyruvic transaminase (now known as serum alanine aminotransferase), serum glutamic oxaloacetic transaminase (now known as serum aspartate aminotransferase), ornithine decarboxylase, gamma glutamyl 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 cholinesterase activity. Additional clinical biochemistry may be employed, where necessary, to extend the investigation of observed effects.

(ii) The following examinations shall be made on high dose and control groups. If changes in the eyes are detected all animals should be examined.

(A) Ophthalmological examination, using an ophthalmoscope or equivalent suitable equipment, shall be made prior to exposure to the test substance and at the termination of the study.

(B) Urinalysis is not recommended on a routine basis, but only when there is an indication based on expected or observed toxicity.

(11) Gross necropsy. (i) All animals shall 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, brain, and gonads shall be weighed wet, as soon as possible after dissection, to avoid drying. In addition, for the rodent, the brain; for the non-rodent, the thyroid with parathyroids also shall be weighed wet.

(iii) The following organs and tissues, or representative samples thereof, shall be preserved in a suitable medium for possible future histopathological examination: All gross lesions; lungs—which should be removed intact, weighed, and treated with a suitable fixative to ensure that lung structure is maintained (perfusion with the fixative is considered to be an effective procedure); nasopharyngeal tissues; brain—including sections of medulla/pons, cerebellar cortex, and cerebral cortex; pituitary; thyroid/parathyroid; thymus; trachea; heart; sternum with bone marrow; salivary glands; liver; spleen; kidneys; adrenals; pancreas; gonads; uterus; accessory genital organs (epididymis, prostate, and, if present, seminal vesicles); aorta; (skin); gall bladder (if present); 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 (zymbal and exorbital lachrymal glands).

(12) Histopathology. The following histopathology shall be performed:

(i) Full histopathology on normal and treated skin and on organs and tissues, listed above, of all animals in the control and high dose groups.

(ii) All gross lesions in all animals.

(iii) Target organs in all animals.

(iv) The tissues listed in parenthesis in paragraph (e)(11)(iii) of this section, if indicated by signs of toxicity or expected target organ involvement.

(v) Lungs of animals (rodents) in the low and intermediate dose groups shall be subjected to histopathological examination for evidence of infection, since this provides a convenient assessment of the state of health of the animals.

(vi) When a satellite group is used, histopathology shall be performed on tissues and organs identified as showing effects in the treated groups.

(f) Data and reporting—(1) Treatment of results. (i) Data shall 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) All observed results, quantitative and incidental, should be evaluated by an appropriate statistical method. Any generally accepted statistical method may be used; the statistical methods should be selected during the design of the study.

(2) Evaluation of results. The findings of a subchronic dermal toxicity study should be evaluated in conjunction with the findings of preceding studies and considered in terms of the observed 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, effect on mortality and any other general or specific toxic effects. A properly conducted subchronic test should provide a satisfactory estimation of a no-effect level.

(3) Test report. In addition to the reporting requirements as specified in the EPA Good Laboratory Practice Standards under 40 CFR part 792, subpart J, the following specific information shall 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.

(ii) Individual animal data. (A) Date of death during the study or whether animals survived to termination.

(B) Date of observation of each abnormal sign and its subsequent course.

(C) Body weight data.

(D) Feed 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.

(g) References. For additional background information on this test guideline the following references should be consulted:

(1) Draize, J.H. “Dermal toxicity,” Appraisal of Chemicals in Food, Drugs and Cosmetics. The Association of Food and Drug Officials of the United States (1959, 3rd printing 1975). pp. 46-59.

(2) 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. 26-35.

(3) 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 auspices of the Committee on Toxicology, National Research Council, National Academy of Sciences, Washington, DC (1977).

(4) World Health Organization. “Part I. Environmental Health Criteria 6,”Principles and Methods for Evaluating the Toxicity of Chemicals. (Geneva: World Health Organization, 1978).

[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19072, May 20, 1987; 53 FR 49149, Dec. 6, 1988; 54 FR 21064, May 16, 1989]

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§798.2450   Inhalation toxicity.

(a) Purpose. In the assessment and evaluation of the toxic characteristics of a gas, volatile substance, or aerosol/particulate, determination of subchronic inhalation toxicity may be carried out after initial information on toxicity has been obtained by acute testing. The subchronic inhalation 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 have a long latency period for development (e.g., carcinogenicity and life shortening). It provides information on health hazards likely to arise from repeated exposures by the inhalation route over a limited period of time. It will provide information on 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. Hazards of inhaled substances are influenced by the inherent toxicity and by physical factors such as volatility and particle size.

(b) Definitions. (1) Subchronic inhalation toxicity is the adverse effects occurring as a result of the repeated daily exposure of experimental animals to a chemical by inhalation for part (approximately 10 percent) of a life span.

(2) Aerodynamic diameter applies to the size of particles of aerosols. It is the diameter of a sphere of unit density which behaves aerodynamically as the particle of the test substance. It is used to compare particles of different size and densities and to predict where in the respiratory tract such particles may be deposited. This term is used in contrast to measured or geometric diameter which is representative of actual diameters which in themselves cannot be related to deposition within the respiratory tract.

(3) The geometric mean diameter or the median diameter is the calculated aerodynamic diameter which divides the particles of an aerosol in half based on the weight of the particles. Fifty percent of the particles by weight will be larger than the median diameter and 50 percent of the particles will be smaller than the median diameter. The median diameter describes the particle size distribution of any aerosol based on the weight and size of the particles.

(4) Inhalable diameter refers to that aerodynamic diameter of a particle which is considered to be inhalable for the organism. It is used to refer to particles which are capable of being inhaled and may be deposited anywhere within the respiratory tract from the trachea to the alveoli. For man, inhalable diameter is considered as 15 micrometers or less.

(5) Dose refers to an exposure level. Exposure is expressed as weight or volume of test substance per volume of air (mg/l), or as parts per million (ppm).

(6) No-effect level/No-toxic-effect level/No-adverse-effect level/No-observed-effect level is the maximum dose used in a test which produces no observed adverse effects. A no-observed-effect level is expressed in terms of weight or volume of test substance given daily per unit volume of air (mg/l or ppm).

(7) Cumulative toxicity is the adverse effects of repeated doses occuring as a result of prolonged action on, or increased concentration of the administered test substance or its metabolites in susceptible tissues.

(c) Principle of the test method. Several groups of experimental animals are exposed daily for a defined period to the test substance in graduated concentrations, one concentration being used 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 test are necropsied and at the conclusion of the test, surviving animals are sacrificed and necropsied and appropriate histopathological examinations carried out.

(d) Test procedures—(1) Animal selection—(i) Species and strain. A mammalian species shall be used for testing. A variety of rodent species may be used, although the rat is the preferred species. Commonly used laboratory strains shall be employed. If another mammalian species is used, the tester shall provide justification/ reasoning for its selection.

(ii) Age. Young adult animals shall be used. At the commencement of the study the weight variation of animals shall not exceed ±20 percent of the mean weight for each sex.

(iii) Sex. (A) Equal numbers of animals of each sex shall be used at each dose level.

(B) Females shall be nulliparous and nonpregnant.

(iv) Numbers. (A) At least 20 rodents (10 females and 10 males) shall be used for each test group. If another mammalian species is selected (e.g. dog, rabbit, or non-human primate), at least 8 animals (4 males and 4 females) shall be used.

(B) If interim sacrifices are planned, the number of animals shall 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 shall be an untreated or sham-treated control group. Except for treatment with the test substance, animals in the control group shall be handled in a manner identical to the test group animals. Where a vehicle is used to help generate an appropriate concentration of the substance in the atmosphere, a vehicle control group shall be used. 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 animals (10 animals per sex) may be treated with the high concentration 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.

(4) Dose levels and dose selection. (i) In subchronic toxicity tests, it is desirable to have a concentration-response relationship as well as a no-observed-toxic-effect level. Therefore, at least 3 concentration levels with a control and, where appropriate, a vehicle control (corresponding to the concentration of vehicle at the highest exposure level) shall be used. Concentrations should be spaced appropriately to produce test groups with a range of toxic effects. The data should be sufficient to produce a concentration-response curve.

(ii) The highest concentration should result in toxic effects but not produce an incidence of fatalities which would prevent a meaningful evaluation.

(iii) The lowest concentration should not produce any evidence of toxicity. Where there is a usable estimation of human exposure the lowest concentration should exceed this.

(iv) Ideally, the intermediate concentration level(s) should produce minimal observable toxic effects. If more than one intermediate concentration level is used, the concentrations should be spaced to produce a gradation of toxic effects.

(v) In the low and intermediate groups and in the controls the incidence of fatalities should be low, to permit a meaningful evaluation of the results.

(vi) In the case of potentially explosive test substances, care should be taken to avoid generating explosive concentrations.

(5) Exposure conditions. The animals should be exposed to the test substance, ideally for 6 hours per day on a 7-day per week basis, for a period of 90 days. However, based primarily on practical considerations, exposure on a 5-day-per-week basis for 6 hours per day is the minimum acceptable exposure period.

(6) Observation period. (i) Duration of observation shall be for at least 90 days.

(ii) Animals in a satellite group scheduled for followup observations should be kept for at least 28 days further without treatment to detect recovery from, or persistence of, toxic effects.

(7) Inhalation exposure. (i) The animals shall be tested in inhalation equipment designed to sustain a minimum dynamic air flow of 12 to 15 air changes per hour and ensure an adequate oxygen content of 19 percent and an evenly distributed exposure atmosphere. Where a chamber is used, its design should minimize crowding of the test animals and maximize their exposure to the test substance. This is best accomplished by individual caging. To ensure stability of a chamber atmosphere, the total “volume” of the test animals shall not exceed 5 percent of the volume of the test chamber. Oronasal or head-only exposure may be used if it is desirable to avoid concurrent exposure by the dermal or oral routes.

(ii) A dynamic inhalation system with a suitable flow control system shall be used. The rate of air flow shall be adjusted to ensure that conditions throughout the exposure chamber are essentially the same. Maintenance of slight negative pressure inside the chamber will prevent leakage of the test substance into surrounding areas.

(iii) The temperature at which the test is performed should be maintained at 22 °C (±2°). Ideally, the relative humidity should be maintained between 40 to 60 percent, but in certain instances (e.g., tests of aerosols, use of water vehicle) this may not be practicable.

(8) Physical measurements. Measurements or monitoring shall be made of the following:

(i) The rate of air flow shall be monitored continuously and recorded at least every 30 minutes.

(ii) The actual concentrations of the test substance shall be measured in the breathing zone. During the exposure period the actual concentrations of the test substance shall be held as constant as practicable, monitored continuously or intermittently depending on the method of analysis, and recorded at least at the beginning, at an intermediate time, and at the end of the exposure period.

(iii) During the development of the generating system, particle size analysis shall be performed to establish the stability of aerosol concentrations with respect to particle size. During exposure, analysis shall be conducted as often as necessary to determine the consistency of particle size distribution.

(iv) Temperature and humidity shall be monitored continuously but shall be recorded at least every 30 minutes.

(9) Feed and water during exposure period. Feed shall be withheld during exposure. Water may also be withheld during exposure.

(10) Observation of animals. (i) Each animal shall be observed daily and, if necessary, handled to appraise its physical condition.

(ii) Additional observations 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 shall be recorded as they are observed including the time of onset, the degree, and duration.

(iv) Cage-side observations should include, but not be limited to, changes in the skin and fur, eyes and mucous membranes, respiratory, circulatory, autonomic and central nervous systems, somatomotor activity and behavior pattern.

(v) Animals shall be weighed weekly. Feed consumption shall also be determined weekly if abnormal body weight changes are observed.

(vi) At the end of the study period all survivors in the nonsatellite treatment groups shall be sacrificed. Moribund animals shall be removed and sacrificed when noticed.

(11) Clinical examinations. (i) The following examinations shall be made on all animals of each sex in each group:

(A) Certain hematology determinations shall be carried out at least two times during the test period on all groups of animals including concurrent controls: After 30 days of test and just prior to terminal sacrifice at the end of the test period. Hematology determinations which are appropriate to all studies: Hematocrit, hemoglobin concentration, erythrocyte count, total and differential leukocyte count, and a measure of clotting potential such as clotting time, prothrombin time, thromboplastin time, or platelet count.

(B) Certain clinical biochemistry determinations on blood should be carried out at least two times during the test period on all groups of animals including concurrent controls: After 30 days of test and just prior to terminal sacrifice at the end of the test period. Clinical biochemistry test areas which are considered appropriate to all studies: 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. Suggested determinations: calcium, phosphorus, chloride, sodium, potassium, fasting glucose (with period of fasting appropriate to the species), serum glutamic-pyruvic transaminase, (now known as serum alanine aminotransferase), serum glutamic-oxaloacetic transaminase (now known as serum aspartate aminotransferase), ornithine decarboxylase, gamma glutamyl 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 cholinesterase activity. Additional clinical biochemistry may be employed, where necessary, to extend the investigation of observed effects.

(ii) The following examinations shall be made on high dose and control groups. If changes in the eyes are detected, all animals shall be examined:

(A) Ophthalmological examination, using an ophthalmoscope or equivalent suitable equipment, shall be made prior to exposure to the test substance and at the termination of the study.

(B) Urinalysis is not recommended on a routine basis, but only when there is an indication based on expected and/or observed toxicity.

(12) Gross pathology. (i) All animals shall 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, brain, and gonads shall be weighed wet, as soon as possible after dissection to avoid drying. In addition, for the rodent, the brain; for the non-rodent, the thyroid with parathyroids also shall be weighed wet.

(iii) The following organs and tissues, or representative samples thereof, shall be preserved in a suitable medium for possible future histopathological examination: All gross lesions; lungs—which should be removed intact, weighed, and treated with a suitable fixative to ensure that lung structure is maintained (perfusion with the fixative is considered to be an effective procedure); nasopharyngeal tissues; brain—including sections of medulla/pons cerebellar cortex and cerebral cortex; pituitary; thyroid/parathyroid; thymus; trachea; heart; sternum with bone marrow; salivary glands; liver; spleen; kidneys; adrenals; pancreas; gonads; uterus; accessory genital organs (epididymis, prostate, and, if present, seminal vesicles); aorta; (skin); gall bladder (if present); 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 (zymbal and exorbital lachrymal glands).

(13) Histopathology. The following histopathology shall be performed:

(i) Full histopathology on the respiratory tract and other organs and tissues, listed above, of all animals in the control and high dose groups.

(ii) All gross lesions in all animals.

(iii) Target organs in all animals.

(iv) The tissues mentioned in brackets (listed above) if indicated by signs of toxicity or target organ involvement.

(v) Lungs of animals (rodents) in the low and intermediate dose groups shall also be subjected to histopathological examination, primarily for evidence of infection since this provides a convenient assessment of the state of health of the animals.

(vi) When a satellite group is used, histopathology shall be performed on tissues and organs identified as showing effects in the treated groups.

(e) Data and reporting—(1) Treatment of results. (i) Data shall 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) All observed results, quantitative and incidental, should be evaluated by an appropriate statistical method. Any generally accepted statistical method may be used; the statistical methods should be selected during the design of the study.

(2) Evaluation of results. The findings of the subchronic inhalation toxicity study should be evaluated in conjunction with the findings of preceding studies and considered in terms of the observed toxic effects and the necropsy and histopathological findings. The evaluation will include the relationship between the concentration of the test substance and duration of exposure, 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. A properly conducted subchronic test should provide a satisfactory estimation of a no-effect level.

(3) Test report. In addition to the reporting requirements as specified under EPA Good Laboratory Practice Standards, 40 CFR part 792, subpart J, the following specific information shall be reported:

(i) Test conditions. (A) Description of exposure apparatus, including design, type, dimensions, source of air, system for generating particulates and aerosols, method of conditioning air, treatment of exhaust air, and the method of housing animals in a test chamber.

(B) The equipment for measuring temperature, humidity, and particulate aerosol concentrations and size shall be described.

(ii) Exposure data. These shall be tabulated and presented with mean values and measure of variability (e.g., standard deviation) and shall include:

(A) Airflow rates through the inhalation equipment.

(B) Temperature and humidity of air.

(C) Nominal concentration (total amount of test substance fed into the inhalation equipment divided by volume of air).

(D) Actual concentration in test breathing zone.

(E) Particle size distribution (e.g., median aerodynamic diameter of particles with standard deviation from the mean).

(iii) 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.

(iv) Individual animal data. (A) Date of death during the study or whether animals survived to termination.

(B) Date of observation of each abnormal sign and its subsequent course.

(C) Body weight data.

(D) Feed 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.

(f) References. For additional background information on this test guideline the following references should be consulted:

(1) Cage, J.C. “Experimental Inhalation Toxicology,” Methods in Toxicology. Ed. G.E. Paget. (Philadelphia: F.A. Davis Co. 1970, pp. 258-277.

(2) Casarett, L.J., Doull, J. “Chapter 9.” Toxicology: The Basic Science of Poisons (New York: Macmillan Publishing Co. Inc. 1975).

(3) MacFarland, H.N. “Respiratory Toxicology,” Essays in Toxicology. Ed. W.J. Hayes. Vol. 7 (New York: Academic Press, 1976) pp. 121-154.

(4) 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 auspices of the Committee on Toxicology, National Research Council, National Academy of Sciences, Washington, DC (1977).

(5) World Health Organization. “Part I. Environmental Health Criteria 6,” Principles and Methods for Evaluating the Toxicity of Chemicals. (Geneva: World Health Organization, 1978).

[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19073, May 20, 1987; 52 FR 26150, July 13, 1987; 53 FR 49150, Dec. 6, 1988; 54 FR 21064, May 16, 1989]

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§798.2650   Oral toxicity.

(a) Purpose. In the assessment and evaluation of the toxic characteristics 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 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 have a 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, and can be of use in selecting dose levels for chronic studies and for establishing safety criteria for human exposure.

(b) Definitions. (1) 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 a life span.

(2) Dose is the amount of test substance administered. Dose is expressed as weight of test substance (g, mg) per unit weight of test animal (e.g., mg/kg), or as weight of test substance per unit weight of food or drinking water.

(3) No-effect level/No-toxic-effect level/No-adverse-effect level/No-observed-effect level 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 test animal (mg/kg). When administered to animals in food or drinking water the no-observed-effect level is expressed as mg/kg of food or mg/ml of water.

(4) Cumulative toxicity is the adverse effects of repeated doses occurring as a result of prolonged action on, or increased concentration of, the administered test substance or its metabolites in susceptible tissue.

(c) 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 histo-pathological examinations carried out.

(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 and if toxicity 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 mammalian species shall be used for testing. A variety of rodent species may be used, although the rat is the preferred species. Commonly used laboratory strains shall be employed. The commonly used nonrodent species is the dog, preferably of a defined breed; the beagle is frequently used. If other mammalian species are used, the tester shall provide justification/reasoning for his or her selection.

(ii) Age—(A) General. Young adult animals shall be employed. At the commencement of the study the weight variation of animals used shall not exceed ±20 percent of the mean weight for each sex.

(B) Rodents. Dosing shall begin as soon as possible after weaning, ideally before the rats are 6, and in any case, not more than 8 weeks old.

(C) Non-rodent. In the case of the dog, dosing shall commence after acclimatization, preferably at 4 to 6 months and not later than 9 months of age.

(iii) Sex. (A) Equal numbers of animals of each sex shall be used at each dose level.

(B) The females shall be nulliparous and nonpregnant.

(iv) Numbers—(A) Rodents. At least 20 animals (10 females and 10 males) shall be used at each dose level.

(B) Non-rodents. At least eight animals (four females and four males) shall be used at each dose level.

(C) If interim sacrifices are planned, the number shall 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 shall 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. (Rodent) 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.

(4) Dose levels and dose selection. (i) In subchronic toxicity tests, it is desirable to have a dose response relationship as well as a no-observed-toxic-effect level. Therefore, at least 3 dose levels with a control and, where appropriate, a vehicle control (corresponding to the concentration of vehicle at the highest exposure level) shall be used. Doses should be spaced appropriately 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 in rodents should result in toxic effects but not produce an incidence of fatalities which would prevent a meaningful evaluation; for non-rodents there should be no fatalities.

(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) Ideally, the intermediate dose level(s) 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) For rodents, 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; for non-rodents, there should be no fatalities.

(5) Exposure conditions. The animals are dosed with the test substance ideally on a 7-day per week basis over a period of 90 days. However, based primarily on practical considerations, dosing in gavage or capsule studies on a 5-day per week basis is considered to be acceptable.

(6) Observation period. (i) Duration of observation shall be for at least 90 days.

(ii) Animals in the satellite group scheduled for followup observations should be kept for at least 28 days further without treatment to detect recovery from, or persistence of, toxic effects.

(7) Administration of the test substance. (i) The test substance may be administered in the diet or in capsules. In addition, for rodents it may also be administered by gavage or in the drinking water.

(ii) All animals shall 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, ideally it should not elicit important 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 administered in the diet either a constant dietary concentration (ppm) or a constant dose level in terms of the animals' body weight shall be used; the alternative used shall be specified.

(v) For a substance administered by gavage or capsule, the dose shall be given at approximately the same time each day, and adjusted at intervals (weekly or bi-weekly) to maintain a constant dose level in terms of animal body weight.

(8) Observation of animals. (i) Each animal shall be observed daily and, if necessary, handled to appraise its physical condition.

(ii) Additional observations shall 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 shall be recorded as they are observed including the time of onset, degree and duration.

(iv) Cage-side observations shall 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 shall be made weekly of feed consumption or water consumption when the test substance is administered in the feed or drinking water, respectively.

(vi) Animals shall be weighed weekly.

(vii) At the end of the 90-day period all survivors in the nonsatellite treatment groups shall be sacrificed. Moribund animals shall be removed and sacrificed when noticed.

(9) Clinical examinations. (i) The following examinations shall be made on all animals of each sex in each group for rodents and all animals when non-rodents are used as test animals.

(A) Certain hematology determinations shall be carried out at least two times during the test period on all groups of animals including concurrent controls: After 30 days of test and just prior to terminal sacrifice at the end of the test period. Hematology determinations which are appropriate to all studies: Hematocrit, hemoglobin concentration, erythrocyte count, total and differential leukocyte count, and a measure of clotting potential such as clotting time, prothrombin time, thromboplastin time, or platelet count.

(B) Certain clinical biochemistry determinations on blood should be carried out at least two times during the test period on all groups of animals including concurrent controls: After 30 days of test and just prior to terminal sacrifice at the end of the test period. Clinical biochemistry test areas which are considered appropriate to all studies: 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. Suggested determinations: Calcium, phosphorus, chloride, sodium, potassium, fasting glucose (with period of fasting appropriate to the species), serum glutamic-pyruvic transaminase (now known as serum alanine aminotransferase), serum glutamic oxaloacetic transaminase (now known as serum aspartate aminotransferase), ornithine decarboxylase, gamma glutamyl 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 cholinesterase activity. Additional clinical biochemistry may be employed, where necessary, to extend the investigation of observed effects.

(ii) The following examinations shall be made on high dose and control groups. If changes in the eyes are detected, all animals should be examined.

(A) Ophthalmological examination, using an ophthalmoscope or equivalent suitable equipment, shall be made prior to the administration of the test substance and at the termination of the study.

(B) Urinalysis is not recommended on a routine basis, but only when there is an indication based on expected and or observed toxicity.

(10) Gross necropsy. (i) All animals shall 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, and gonads shall be weighed wet, as soon as possible after dissection to avoid drying. In addition, for the rodent, the brain; for the non-rodent, the thyroid with parathyroids also shall be weighed wet.

(iii) The following organs and tissues, or representative samples thereof, shall be preserved in a suitable medium for possible future histopathological examination: All gross lesions; lungs—which should be removed intact, weighed, and treated with a suitable fixative to ensure that lung structure is maintained (perfusion with the fixative is considered to be an effective procedure); nasopharyngeal tissues; brain—including sections of medulla/pons, cerebellar cortex, and cerebral cortex; pituitary; thyroid/parathyroid; thymus; trachea; heart; sternum with bone marrow; salivary glands; liver; spleen; kidneys; adrenals; pancreas; gonads; uterus; accessory genital organs (epididymis, prostate, and, if present, seminal vesicles); aorta; (skin); gall bladder (if present); 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 (zymbal and exorbital lachrymal glands); and (rodent-zymbal glands).

(11) Histopathology. The following histopathology shall be performed:

(i) Full histopathology on the organs and tissues, listed above, of all rodents in the control and high dose groups, all non-rodents, and all rodents that died or were killed during the study.

(ii) All gross lesions in all animals.

(iii) Target organs in all animals.

(iv) The tissues mentioned in brackets (listed above) if indicated by signs of toxicity of target organ involvement.

(v) Lungs, liver and kidneys of all animals. Special attention to examination of the lungs of rodents shall be made for evidence of infection since this provides a convenient assessment of the state of health of the animals.

(vi) When a satellite group is used (rodents), histopathology shall be performed on tissues and organs identified as showing effects in the treated groups.

(f) Data and reporting—(1) Treatment of results. (i) Data shall 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) All observed results, quantitative and incidental, should be evaluated by an appropriate statistical method. Any generally accepted 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 will 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. A properly conducted subchronic 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 specified under EPA Good Laboratory Practice Standards, 40 CFR part 792, subpart J, the following specific information shall 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.

(ii) Individual animal data. (A) Date of death during the study or whether animals survived to termination.

(B) Date of observation of each abnormal sign and its subsequent course.

(C) Body weight data.

(D) Feed 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.

(g) References. For additional background information on this test guideline the following references should be consulted:

(1) Boyd, E.M. “Chapter 14—Pilot Studies, 15—Uniposal Clinical Parameters, 16—Uniposal Autopsy Parameters.” Predictive Toxicometrics. (Baltimore: Williams and Wilkins, 1972).

(2) 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. 26-35.

(3) Food Safety Council. “Subchronic Toxicity Studies,” Proposed System for Food Safety Assessment. (Columbia: Food Safety Council, 1978) pp. 83-96.

(4) 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 auspices of the Committee on Toxicology, National Research Council, National Academy of Sciences, Washington, DC (1977).

(5) World Health Organization. “Part I. Environmental Health Criteria 6,” Principles and Methods for Evaluating the Toxicity of Chemicals. (Geneva: World Health Organization, 1978).

[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19074, May 20, 1987; 53 FR 49150, Dec. 6, 1988; 54 FR 21064, May 16, 1989]

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