40 CFR Part 798 — Health Effects Testing Guidelines

PART 798—HEALTH EFFECTS TESTING GUIDELINES Authority: 15 U.S.C. 2603. Source: 50 FR 39397, Sept. 27, 1985, unless otherwise noted. Subparts A-B [Reserved] Subpart C—Subchronic Exposure § 798.2250 Dermal toxicity. (a) Purpose. (b) Definitions. (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. (d) Limit test. (e) Test procedures Animal selection Species and strain. (ii) Age. (iii) Sex. (B) The females shall be nulliparous and nonpregnant. (iv) Numbers. (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. (3) Satellite group. (4) Dose level and dose selection. (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. (6) Observation period. (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. (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. (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. (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. (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. (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. (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 Treatment of results. (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. (3) Test report. (i) Group animal data. (A) Number of animals dying. (B) Number of animals showing signs of toxicity. (C) Number of animals exposed. (ii) Individual animal data. (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. (1) Draize, J.H. “Dermal toxicity,” Appraisal of Chemicals in Food, Drugs and Cosmetics. (2) Fitzhugh, O.G. “Subacute toxicity,” Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. (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. [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] § 798.2450 Inhalation toxicity. (a) Purpose. (b) Definitions. (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. (d) Test procedures Animal selection Species and strain. (ii) Age. (iii) Sex. (B) Females shall be nulliparous and nonpregnant. (iv) Numbers. (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. (3) Satellite group. (4) Dose levels and dose selection. (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. (6) Observation period. (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. (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. (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. (10) Observation of animals. (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. (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. (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. (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 Treatment of results. (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. (3) Test report. (i) Test conditions. (B) The equipment for measuring temperature, humidity, and particulate aerosol concentrations and size shall be described. (ii) Exposure data. (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. (A) Number of animals dying. (B) Number of animals showing signs of toxicity. (C) Number of animals exposed. (iv) Individual animal data. (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. (1) Cage, J.C. “Experimental Inhalation Toxicology,” Methods in Toxicology. (2) Casarett, L.J., Doull, J. “Chapter 9.” Toxicology: The Basic Science of Poisons (3) MacFarland, H.N. “Respiratory Toxicology,” Essays in Toxicology. (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. [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] § 798.2650 Oral toxicity. (a) Purpose. (b) Definitions. (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. (d) Limit test. (e) Test procedures Animal selection Species and strain. (ii) Age General. (B) Rodents. (C) Non-rodent. (iii) Sex. (B) The females shall be nulliparous and nonpregnant. (iv) Numbers Rodents. (B) Non-rodents. (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. (3) Satellite group. (4) Dose levels and dose selection. (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. (6) Observation period. (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. (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. (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. (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. (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. (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 Treatment of results. (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. (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. (i) Group animal data. (A) Number of animals dying. (B) Number of animals showing signs of toxicity. (C) Number of animals exposed. (ii) Individual animal data. (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. (1) Boyd, E.M. “Chapter 14—Pilot Studies, 15—Uniposal Clinical Parameters, 16—Uniposal Autopsy Parameters.” Predictive Toxicometrics. (2) Fitzhugh, O.G. “Subacute Toxicity,” Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. (3) Food Safety Council. “Subchronic Toxicity Studies,” Proposed System for Food Safety Assessment. (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. [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] Subpart D—Chronic Exposure § 798.3260 Chronic toxicity. (a) Purpose. (b) Test procedures Animal selection Species and strain. (ii) Age. (B) Dosing of dogs should begin between 4 and 6 months of age and in no case later than 9 months of age. (C) At commencement of the study the weight variation of animals used should not exceed ±20 percent of the mean weight for each sex. (iii) Sex. (B) The females should be nulliparous and non-pregnant. (iv) Numbers. (B) If interim sacrifices are planned, the number should be increased by the number of animals scheduled to be sacrificed during the course of the study. (C) The number of animals at the termination of the study must be adequate for a meaningful and valid statistical evaluation of chronic effects. (2) Control groups. (ii) In special circumstances such as in inhalation studies involving aerosols or the use of an emulsifier of uncharacterized biological activity in oral studies, a concurrent negative control group should be utilized. The negative control group should be treated in the same manner as all other test animals except that this control group should not be exposed to either the test substance or any vehicle. (3) Dose levels and dose selections. (ii) The high dose level in rodents should elicit some signs of toxicity without causing excessive lethality; for non-rodents, there should be signs of toxicity but 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 even though this dose level may result in some signs of toxicity. (iv) Ideally, the intermediate dose level(s) should produce minimal observable toxic effects. If more than one intermediate dose is used, the dose level 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. (4) Exposure conditions. (5) Observation period. (6) Administration of the test substance. (i) Oral studies. (B) If the test substance is administered in the drinking water, or mixed in the diet, exposure is continuous. (C) For a diet mixture, the highest concentration should not exceed 5 percent. (ii) Dermal studies. (B) Fur should be clipped from the dorsal area of the trunk of the test animals. Care must be taken to avoid abrading the skin which could alter its permeability. (C) The test substance should be applied uniformly over a shaved 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 should be covered with as thin and uniform a film as possible. (D) During the exposure period, the test substance may be held if necessary, in contact with the skin with a porous gauze dressing and non-irritating tape. The test site should 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. (iii) Inhalation studies. (B) 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. (C) Feed and water should be withheld during each daily 6 hour exposure period. (D) A dynamic inhalation system with a suitable analytical concentration control system should be used. The rate of air flow should be adjusted to ensure that conditions throughout the equipment are essentially the same. Maintenance of slight negative pressure inside the chamber will prevent leakage of the test substance into the surrounding areas. (7) Observation of animals. (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 sacrific of weak or moribund animals). (iii) Clinical signs of toxicity including suspected tumors and mortality should 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 skin and fur, eyes and mucous membranes, respiratory, circulatory, autonomic and central nervous systems, somatomotor activity and behavior pattern. (v) Body weights should be recorded individually for all animals once a week during the first 13 weeks of the test period and at least once every 4 weeks thereafter unless signs of clinical toxicity suggest more frequent weighings to facilitate monitoring of health status. (vi) When the test substance is administered in the feed or drinking water, measurements of feed or water consumption, respectively, should be determined weekly during the first 13 weeks of the study and then at approximately monthly intervals unless health status or body weight changes dictate otherwise. (vii) At the end of the study period all survivors should be sacrificed. Moribund animals should be removed and sacrificed when noticed. (8) Physical measurements. (i) The rate of air flow should be monitored continuously, but should be recorded at intervals of at least once every 30 minutes. (ii) During each exposure period the actual concentrations of the test substance should be held as constant as practicable, monitored continuously and measured at least three times during the test period: at the beginning, at an intermediate time and at the end of the period. (iii) During the development of the generating system, particle size analysis should be performed to establish the stability of aerosol concentrations. During exposure, analysis should be conducted as often as necessary to determine the consistency of particle size distribution and homogeneity of the exposure stream. (iv) Temperature and humidity should be monitored continuously, but should be recorded at intervals of at least once every 30 minutes. (9) Clinical examinations. (i) Certain hematology determinations (e.g., hemoglobin content, packed cell volume, total red blood cells, total white blood cells, platelets, or other measures of clotting potential) should be performed at termination and should be performed at 3 months, 6 months and at approximately 6 month intervals thereafter (for studies extending beyond 12 months) on blood samples collected from all non-rodents and from 10 rats per sex of all groups. These collections should be from the same animals at each interval. If clinical observations suggest a deterioration in health of the animals during the study, a differential blood count of the affected animals should be performed. A differential blood count should be performed on samples from those animals in the highest dosage group and the controls. Differential blood counts should be performed for the next lower group(s) if there is a major discrepancy between the highest group and the controls. If hematological effects were noted in the subchronic test, hematological testing should be performed at 3, 6, 12, 18, and 24 months for a two year study and at 3, 6, and 12 months for a 1-year study. (ii) Certain clinical biochemistry determinations on blood should be carried out at least three times during the test period: just prior to initiation of dosing (base line data), near the middle and at the end of the test period. Blood samples should be drawn for clinical chemistry measurements from all non-rodents and at least ten rodents per sex of all groups; if possible, from the same rodents at each time interval. 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 and signs of clinical toxicity. Suggested chemical 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, blood urea nitrogen, albumen, blood creatinine, creatinine phosphokinase, total cholesterol, 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. (iii) Urine samples from rodents at the same intervals as the hematological examinations under paragraph (b)(9)(i) of this section should be collected for analysis. The following determinations should be made from either individual animals or on a pooled sample/sex/group for rodents: appearance (volume and specific gravity), protein, glucose, ketones, bilirubin, occult blood (semi-quantitatively); and microscopy of sediment (semi-quantitatively). (iv) 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 eyes are detected all animals should be examined. (10) Gross necropsy. (ii) The liver, kidneys, adrenals, brain and gonads should be weighed wet, as soon as possible after dissection to avoid drying. For these organs, at least 10 rodents per sex per group and all non-rodents should be weighed. (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 and tumors; brain—including sections of medulla/pons, cerebellar cortex, and cerebral cortex; pituitary; thyroid/parathyroid; thymus; lungs; trachea; heart; sternum and/or femur with bone marrow; salivary glands; liver; spleen; kidneys; adrenals; esophagus; stomach; duodenum; jejunum; ileum; cecum; colon; rectum; urinary bladder; representative lymph nodes; pancreas; gonads; uterus; accessory genital organs (epididymis, prostate, and, if present, seminal vesicles; female mammary gland; aorta; gall bladder (if present); skin; musculature; peripheral nerve; spinal cord at three levels—cervical, midthoracic, and lumbar; and eyes. In inhalation studies, the entire respiratory tract, including nose, pharynx, larynx, and paranasal sinuses should be examined and preserved. In dermal studies, skin from sites of skin painting should be examined and preserved. (iv) Inflation of lungs and urinary bladder with a fixative is the optimal method for preservation of these tissues. The proper inflation and fixation of the lungs in inhalation studies is considered essential for appropriate and valid histopathological examination. (v) If other clinical examinations are carried out, the information obtained from these procedures should be available before microscopic examination, since they may provide significant guidance to the pathologist. (11) Histopathology. (A) Full histopathology on the organs and tissues, listed above, of all non-rodents, of all rodents in the control and high dose groups and of all rodents that died or were killed during the study. (B) All gross lesions in all animals. (C) Target organs in all animals. (D) Lungs, liver and kidneys of all animals. Special attention to examination of the lungs of rodents should be made for evidence of infection since this provides an assessment of the state of health of the animals. (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) In case the results of an experiment give evidence of substantial alteration of the animals' normal longevity or the induction of effects that might affect a toxic response, the next lower dose level should be examined fully, as described under paragraph (b)(11)(i) of this section. (iv) An attempt should be made to correlate gross observations with microscopic findings. (c) Data and reporting Treatment of results. (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 study results. (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. (A) Group animal data. ( 1 ( 2 ( 3 (B) Individual animal data. 1 ( 2 ( 3 ( 4 ( 5 ( 6 ( 7 ( 8 ( 9 ( 10 (ii) In addition, for inhalation studies the following should be reported: (A) Test conditions. 1 ( 2 (B) Exposure data. ( 1 ( 2 ( 3 ( 4 ( 5 (d) References. (1) Benitz, K.F. “Measurement of Chronic Toxicity,” Methods of Toxicology. (2) D'Aguanno, W. “Drug Safety Evaluation—Pre-Clinical Considerations,” Industrial Pharmacology: Neuroleptics. (3) Fitzhugh, O.G. Third Printing: 1975. “Chronic Oral Toxicity,” Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. (4) Goldenthal, E.I., D'Aguanno, W. “Evaluation of Drugs,” Appraisal of the Safety of Chemicals in Foods, Drugs, and Cosmetics. (5) 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). (6) National Center for Toxicological Research. “Appendix B,” Report of Chronic Studies Task Force Committee, April 13-21, 1972. (7) Page, N.P. “Chronic Toxicity and Carcinogenicity Guidelines,” Journal of Environmental Pathology and Toxicology, (8) Schwartz, E. “Toxicology of Neuroleptic Agents,” Industrial Pharmacology: Neuroleptics (9) United States Pharmaceutical Manufacturers Association. Guidelines for the Assessment of Drug and Medical Device Safety in Animals. (10) World Health Organization. “Guidelines for Evaluation of Drugs for Use in Man,” WHO Technical Report Series No. 563. (11) World Health Organization. “Part I. Environmental Health Criteria 6,” Principles and Methods for Evaluating the Toxicity of Chemicals. (12) World Health Organization. “Principles for Pre-Clinical Testing of Drug Safety,” WHO Technical Report Series No. 341. [50 FR 39397, Sept. 27, 1985, as amended at 54 FR 21064, May 16, 1989] § 798.3300 Oncogenicity. (a) Purpose. (b) Test procedures Animal selection Species and strain. (ii) Age. (B) At commencement of the study, the weight variation of animals used shall not exceed ±20 percent of the mean weight for each sex. (C) Studies using prenatal or neonatal animals may be recommended under special conditions. (iii) Sex. (B) The females shall be nulliparous and non-pregnant. (iv) Numbers. (B) If interim sacrifices are planned the number shall be increased by the number of animals scheduled to be sacrificed during the course of the study. (C) The number of animals at the termination of the study should be adequate for a meaningful and valid statistical evaluation of long term exposure. For a valid interpretation of negative results, it is essential that survival in all groups does not fall below 50 percent at the time of termination. (2) Control groups. (ii) In special circumstances such as in inhalation studies involving aerosols or the use of an emulsifier of uncharacterized biological activity in oral studies, a concurrent negative control group shall be utilized. The negative control group shall be treated in the same manner as all other test animals except that this control group shall not be exposed to either the test substance or any vehicle. (iii) The use of historical control data (i.e., the incidence of tumors and other suspect lesions normally occurring under the same laboratory conditions and in the same strain of animals employed in the test) is desirable for assessing the significance of changes observed in exposed animals. (3) Dose levels and dose selection. (ii) The high dose level should elicit signs of minimal toxicity without substantially altering the normal life span. (iii) The lowest dose should not interfere with normal growth, development and longevity of the animal; and it should not otherwise cause any indication of toxicity. In general, this should not be lower than ten percent of the high dose. (iv) The intermediate dose(s) should be established in a mid-range between the high and low doses, depending upon the toxicokinetic properties of the chemical, if known. (v) The selection of these dose levels should be based on existing data, preferably on the results of subchronic studies. (4) Exposure conditions. (5) Observations period. (6) Administration of the test substance. (i) Oral studies. (B) If the test substance is administered in the drinking water, or mixed in the diet, exposure shall be continuous. (C) For a diet mixture, the highest concentration should not exceed 5 percent. (ii) Dermal studies. (B) Fur should be clipped from the dorsal area of the trunk of the test animals. Care should be taken to avoid abrading the skin which could alter its permeability. (C) The test substance shall be applied uniformly over a shaved 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. (D) During the exposure period, the test substance may be held, if necessary, in contact with the skin with a porous gauze dressing and non-irritating tape. The test site should 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. (iii) Inhalation studies. (B) 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. (C) Feed and water shall be withheld during each daily 6-hour exposure period. (D) 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 equipment are essentially the same. Maintenance of slight negative pressure inside the chamber will prevent leakage of the test substance into the surrounding areas. (7) Observations of animals. (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) Clinical signs and mortality shall be recorded for all animals. Special attention should be paid to tumor development. The day of onset, location, dimensions, appearance and progression of each grossly visible or palpable tumor shall be recorded. (iv) Body weights shall be recorded individually for all animals once a week during the first 13 weeks of the test period and at least once every 4 weeks thereafter unless signs of clinical toxicity suggest more frequent weighings to facilitate monitoring of health status. (v) When the test substance is administered in the feed or drinking water, measurements of feed or water consumption, respectively, shall be determined weekly during the first 13 weeks of the study and then at approximately monthly intervals unless health status or body weight changes dictate otherwise. (vi) At the end of the study period all survivors are sacrificed. Moribund animals shall be removed and sacrificed when noticed. (8) Physical measurements. (i) The rate of air flow shall be monitored continuously and recorded at intervals of at least once every 30 minutes. (ii) During each exposure period the actual concentrations of the test substance shall be held as constant as practicable, monitored continuously and recorded at least three times during the test period: at the beginning, at an intermediate time and at the end of the 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, analyses shall be conducted as often as necessary to determine the consistency of particle size, distribution, and homogeneity of the exposure stream. (iv) Temperature and humidity shall be monitored continuously, but shoud be recorded at intervals of at least once every 30 minutes. (9) Clinical examinations. (10) Gross necropsy. (ii) The following organs and tissues or representative samples thereof, shall be preserved in a suitable medium for possible future histopathological examination: All gross lesions and tumors of all animals shall be preserved; brain—including sections of medulla/pons, cerebellar cortex and cerebral cortex; pituitary; thyroid/parathyroid; thymus; lungs; trachea; heart; spinal cord at three levels—cervical, midthoracic and lumbar; sternum and/or femur with bone marrow; salivary glands; liver; spleen; kidneys; adrenals; esophagus; stomach; duodenum; jejunum; ileum; cecum; colon; rectum; urinary bladder; representative lymph nodes; pancreas; gonads; uterus; accessory genital organs (epididymis, prostate, and, if present, seminal vesicles); mammary gland; skin; musculature; peripheral nerve; and eyes. In inhalation studies, the entire respiratory tract shall be preserved, including nasal cavity, pharynx, larynx and paranasal sinuses. In dermal studies, skin from sites of skin painting shall be examined and preserved. (iii) Inflation of lungs and urinary bladder with a fixative is the optimal method for preservation of these tissues. The proper inflation and fixation of the lungs in inhalation studies is required for appropriate and valid histopathological examination. (iv) If other clinical examinations are carried out, the information obtained from these procedures shall be available before microscopic examination, since they may provide significant guidance to the pathologist. (11) Histopathology. (A) Full histopathology on organs and tissues listed above of all animals in the control and high dose groups and all animals that died or were killed during the study. (B) All gross lesions in all animals. (C) Target organs in all animals. (ii) If a significant difference is observed in hyperplastic, pre-neoplastic or neoplastic lesions between the highest dose and control groups, microscopic examination shall be made on that particular organ or tissue of all animals in the study. (iii) If excessive early deaths or other problems occur in the high dose group, compromising the significance of the data, the next lower dose level shall be examined for complete histopathology. (iv) In case the results of an experiment give evidence of substantial alteration of the animals' normal longevity or the induction of effects that might affect a neoplastic response, the next lower dose level shall be examined fully as described in this section. (v) An attempt shall be made to correlate gross observations with microscopic findings. (c) Data and reporting Treatment of results. (ii) All observed results, quantitative and incidental, shall be evaluated by an appropriate statistical method. Any generally accepted statistical method may be used; the statistical methods shall be selected during the design of the study. (2) Evaluation of study results. (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. (iii) In order for a negative test to be acceptable, it shall meet the following criteria: no more than 10 percent of any group is lost due to autolysis, cannibalism, or management problems; and survival in each group should be no less than 50 percent at 18 months for mice and hamsters and at 24 months for rats. (3) Test report. (A) Group animal data. ( 1 ( 2 ( 3 (B) Individual animal data. 1 ( 2 ( 3 ( 4 ( 5 ( 6 ( 7 ( 8 ( 9 ( 10 ( 11 (ii) In addition, for inhalation studies the following shall be reported: (A) Test conditions. ( 2 (B) Exposure data. ( 1 ( 2 ( 3 ( 4 ( 5 (d) References. (1) Department of Health and Welfare. The Testing of Chemicals (2) Food and Drug Administration Advisory Committee on Protocols for Safety Evaluation: Panel on Carcinogenesis. “Report on Cancer Testing in the Safety of Food Additives and Pesticides,” Toxicology and Applied Pharmacology. (3) International Union Against Cancer. “Carcinogenicity Testing,” IUCC Technical Report Series. (4) Leong, B.K.J., Laskin, S. “Number and Species of Experimental Animals for Inhalation Carcinogenicity Studies” Paper presented at Conference on Target Organ Toxicity, September 1975, Cincinnati, Ohio. (5) 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). (6) National Cancer Institute. Report of the Subtask Group on Carcinogen Testing to the Interagency Collaborative Group on Environmental Carcinogenesis. (7) National Center for Toxicological Research. “Appendix B,” Report of Chronic Studies Task Force Committee. (8) Page, N.P. “Chronic Toxicity and Carcinogenicity Guidelines,” Journal of Environmental Pathology and Toxicology. (9) Page, N.P. “Concepts of a Bioassay Program in Environmental Carcinogenesis,” Advances in Modern Toxicology Vol. 3, (10) Sontag, J.M., Page N.P., Saffiotti, U. Guidelines for Carcinogen Bioassay in Small Rodents. (11) United States Pharmaceutical Manufacturers Association. Guidelines for the Assessment of Drug and Medical Device Safety in Animals. (12) World Health Organization. “Principles for the Testing and Evaluation of Drugs for Carcinogenicity,” WHO Technical Report Series No. 426. (13) World Health Organization. “Part I. Environmental Health Criteria 6,” Principles and Methods for Evaluating the Toxicity of Chemicals. [50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19075, May 20, 1987; 54 FR 21064, May 16, 1989] § 798.3320 Combined chronic toxicity/oncogenicity. (a) Purpose. (b) Test procedures Animal selection Species and strain. (ii) Age. (B) At commencement of the study, the weight variation of animals used should not exceed ±20 percent of the mean weight for each sex. (C) Studies using prenatal or neonatal animals may be recommended under special conditions. (iii) Sex. (B) The females should be nulliparous and nonpregnant. (iv) Numbers. (B) If interim sacrifices are planned, the number of animals should be increased by the number of animals scheduled to be sacrificed during the course of the study. (C) The number of animals at the termination of each phase of the study should be adequate for a meaningful and valid statistical evaluation of long term exposure. For a valid interpretation of negative results, it is essential that survival in all groups not fall below 50 percent at the time of termination. (2) Control groups. (ii) In special circumstances such as inhalation studies involving aerosols or the use of an emulsifier of uncharacterized biological activity in oral studies, a concurrent negative control group should be utilized. The negative control group should be treated in the same manner as all other test animals, except that this control group should not be exposed to the test substance or any vehicle. (iii) The use of historical control data (i.e., the incidence of tumors and other suspect lesions normally occuring under the same laboratory conditions and in the same strain of animals employed in the test) is desirable for assessing the significance of changes observed in exposed animals. (3) Dose levels and dose selection. (ii) The highest dose level in rodents should elicit signs of toxicity without substantially altering the normal life span due to effects other than tumors. (iii) The lowest dose level should produce no evidence of toxicity. Where there is a usable estimation of human exposure, the lowest dose level should exceed this even though this dose level may result in some signs of toxicity. (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. (vi) For chronic toxicological assessment, a high dose treated satellite and a concurrent control satellite group should be included in the study design. The highest dose for satellite animals should be chosen so as to produce frank toxicity, but not excessive lethality, in order to elucidate a chronic toxicological profile of the test substance. If more than one dose level is selected for satellite dose groups, the doses should be spaced to produce a gradation of toxic effects. (4) Exposure conditions. (5) Observation period. (i) Generally, the termination of the study should be at 18 months for mice and hamsters and 24 months for rats; however, for certain strains of animals with greater longevity and/or low spontaneous tumor rate, termination should be at 24 months for mice and hamsters and at 30 months for rats. For longer time periods, and where any other species are used, consultation with the Agency in regard to duration of the test is advised. (ii) However, termination of the study is acceptable when the number of survivors of the lower doses or of the control group reaches 25 percent. In the case where only the high dose group dies prematurely for obvious reasons of toxicity, this should not trigger termination of the study. (iii) The satellite groups and the concurrent satellite control group should be retained in the study for at least 12 months. These groups should be scheduled for sacrifice for an estimation of test-substance-related pathology uncomplicated by geriatric changes. (6) Administration of the test substance. (i) Oral studies. (B) If the test substance is administered in the drinking water, or mixed in the diet, exposure is continuous. (C) For a diet mixture, the highest concentration should not exceed 5 percent. (ii) Dermal studies. (B) Fur should be clipped from the dorsal area of the trunk of the test animals. Care should be taken to avoid abrading the skin which could alter its permeability. (C) The test substance should be applied uniformly over a shaved 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 as possible should be covered with as thin and uniform a film as possible. (D) During the exposure period, the test substance may be held, if necessary, in contact with the skin with a porous gauze dressing and nonirritating tape. The test site should 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. (iii) Inhalation studies. (B) 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. (C) Feed and water should be withheld during each daily 6-hour exposure period. (D) A dynamic inhalation system with a suitable analytical concentration control system should be used. The rate of air flow should be adjusted to ensure that conditions throughout the equipment are essentially the same. Maintenance of slight negative pressure inside the chamber will prevent leakage of the test substance into the surrounding areas. (7) Observation of animals. (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) Clinical signs and mortality should be recorded for all animals. Special attention should be paid to tumor development. The time of onset, location, dimensions, appearance and progression of each grossly visible or palpable tumor should be recorded. (iv) Body weights should be recorded individually for all animals once a week during the first 13 weeks of the test period and at least once every 4 weeks thereafter, unless signs of clinical toxicity suggest more frequent weighings to facilitate monitoring of health status. (v) When the test substance is administered in the feed or drinking water, measurements of feed or water consumption, respectively, should be determined weekly during the first 13 weeks of the study and then at approximately monthly intervals unless health status or body weight changes dictate otherwise. (vi) At the end of the study period, all survivors are sacrificed. Moribund animals should be removed and sacrificed when noticed. (8) Physical measurements. (i) The rate of airflow should be monitored continuously, but should be recorded at intervals of at least once every 30 minutes. (ii) During each exposure period the actual concentrations of the test substance should be held as constant as practicable, monitored continuously and recorded at least three times during the test period: At the beginning, at an intermediate time and at the end of the period. (iii) During the development of the generating system, particle size analysis should be performed to establish the stability of aerosol concentrations. During exposure, analyses should be conducted as often as necessary to determine the consistency of particle size distribution and homogeneity of the exposure stream. (iv) Temperature and humidity should be monitored continuously, but should be recorded at intervals of at least once every 30 minutes. (9) Clinical examinations. (A) Certain hematology determinations (e.g., hemoglobin content, packed cell volume, total red blood cells, total white blood cells, platelets, or other measures of clotting potential) should be performed at termination and should be performed at 3 months, 6 months and at approximately 6-month intervals thereafter (for those groups on test for longer than 12 months) on blood samples collected from 20 rodents per sex of all groups. These collections should be from the same animals at each interval. If clinical observations suggest a deterioration in health of the animals during the study, a differential blood count of the affected animals should be performed. A differential blood count should be performed on samples from animals in the highest dosage group and the controls. Differential blood counts should be performed for the next lower group(s) if there is a major discrepancy between the highest group and the controls. If hematological effects were noted in the subchronic test, hematological testing should be performed at 3, 6, 12, 18 and 24 months for a year study. (B) Certain clinical biochemistry determinations on blood should be carried out at least three times during the test period: Just prior to initiation of dosing (baseline data), near the middle and at the end of the test period. Blood samples should be drawn for clinical measurements from at least ten rodents per sex of all groups; if possible, from the same rodents at each time interval. 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 and signs of clinical toxicity. Suggested chemical 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, blood urea nitrogen, albumen, creatinine phosphokinase, total cholesterol, 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 should be performed on at least 10 rodents of each sex per dose level: (A) Urine samples from the same rodents at the same intervals as hematological examination above, should be collected for analysis. The following determinations should be made from either individual animals or on a pooled sample/sex/group for rodents: appearance (volume and specific gravity), protein, glucose, ketones, bilirubin, occult blood (semi-quantitatively) and microscopy of sediment (semi-quantitatively). (B) 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. (10) Gross necropsy. (ii) The liver, kidneys, adrenals, brain and gonads should be weighed wet, as soon as possible after dissection to avoid drying. For these organs, at least 10 rodents per sex per group should be weighed. (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 and tumors; brain-including sections of medulla/pons, cerebellar cortex, and cerebral cortex; pituitary; thyroid/parathyroid; thymus; lungs; trachea; heart; sternum and/or femur with bone marrow; salivary glands; liver; spleen; kidneys; adrenals; esophagus; stomach; duodenum; jejunum; ileum; cecum; colon; rectum; urinary bladder; representative lymph nodes; pancreas; gonads; uterus; accessory genital organs (epididymis, prostate, and, if present, seminal vesicles); female mammary gland; aorta; gall bladder (if present); skin; musculature; peripheral nerve; spinal cord at three levels—cervical, midthoracic, and lumbar; and eyes. In inhalation studies, the entire respiratory tract, including nose, pharynx, larynx and paranasal sinuses should be examined and preserved. In dermal studies, skin from sites of skin painting should be examined and preserved. (iv) Inflation of lungs and urinary bladder with a fixative is the optimal method for preservation of these tissues. The proper inflation and fixation of the lungs in inhalation studies is considered essential for appropriate and valid histopathological examination. (v) If other clinical examinations are carried out, the information obtained from these procedures should be available before microscopic examination, since they may provide significant guidance to the pathologist. (11) Histopathology. (A) Full histopathology on the organs and tissues, listed above, of all non-rodents, of all rodents in the control and high dose groups and of all rodents that died or were killed during the study. (B) All gross lesions in all animals. (C) Target organs in all animals. (D) Lungs, liver and kidneys of all animals. Special attention to examination of the lungs of rodents should be made for evidence of infection since this provides an assessment of the state of health of the animals. (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) In case the results of the experiment give evidence of substantial alteration of the animals' normal longevity or the induction of effects that might affect a toxic response, the next lower dose level should be examined as described above. (iv) An attempt should be made to correlate gross observations with microscopic findings. (c) Data and reporting Treatment of results. (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 study results. (ii) In any study which demonstrates an absence of toxic effects, further investigation to establish absorption and bioavailablity of the test substance should be considered. (iii) In order for a negative test to be acceptable, it should meet the following criteria: No more than 10 percent of any group is lost due to autolysis, cannibalism, or management problems; and survival in each group is no less than 50 percent at 18 months for mice and hamsters and at 24 months for rats. (3) Test report. (A) Group animal data. ( 1 ( 2 ( 3 (B) Individual animal data. 1 ( 2 ( 3 ( 4 ( 5 ( 6 ( 7 ( 8 ( 9 ( 10 ( 11 (ii) In addition, for inhalation studies the following should be reported: (A) Test conditions. 1 ( 2 (B) Exposure data. ( 1 ( 2 ( 3 ( 4 ( 5 (d) References. (1) Benitz, K.F. “Measurement of Chronic Toxicity,” Methods of Toxicology. (2) D'Aguanno, W. “Drug Safety Evaluation—Pre-Clinical Considerations,” “ Industrial Pharmacology: Neuroleptics. (3) Department of Health and Welfare. The Testing of Chemicals for Carcinogenicity, Mutagenicity, Teratogenicity. Minister of Health and Welfare. (Canada: Department of Health and Welfare, 1975). (4) Fitzhugh, O.G. “Chronic Oral Toxicity,” Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. (5) Food and Drug Administration Advisory Committee on Protocols for Safety Evaluation: Panel on Carcinogenesis. “ Report on Cancer Testing in the Safety of Food Additives and Pesticides,” Toxicology and Applied Pharmacology. (6) Goldenthal, E.I., and D'Aguanno, W. “Evaluation of Drugs,” Appraisal of the Safety of Chemicals in Foods, Drugs, and Cosmetics. (7) International Union Against Cancer. “Carcinogenicity Testing,” IUCC Technical Report Series (8) Leong, B.K.J., and Laskin, S. “Number and Species of Experimental Animals for Inhalation Carcinogenicity Studies,” Paper presented at Conference on Target Organ Toxicity. September, 1975, Cincinnati, Ohio. (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 auspices of the Committee on Toxicology, National Research Council, National Academy of Sciences, Washington, DC (1977). (10) National Cancer Institute. Report of the Subtask Group on Carcinogen Testing to the Interagency Collaborative Group on Environmental Carcinogenesis. (11) National Center for Toxicological. Report of Chronic Studies Task Force Research Committee. “Appendix B, (Rockville: National Center for Toxicological Research, 1972)). (12) Page, N.P. “Chronic Toxicity and Carcinogenicity Guidelines,” Journal Environmental Pathology and Toxicology. (13) Page, N.P. “Concepts of a Bioassay Program in Environmental Carcinogenesis,” Advances in Modern Toxicology (14) Schwartz, E. 1974. “Toxicology of Neuroleptic Agents,” Industrial Pharmacology: Neuroleptics. (15) Sontag, J.M., Page, N.P., and Saffiotti, U. Guidelines for Carcinogen Bioassay in Small Rodents. (16) United States Pharmaceutical Manufacturers Association. Guidelines for the Assessment of Drug and Medical Device Safety in Animals. (17) World Health Organization. “Principles for the Testing and Evaluation of Drugs for Carcinogenicity,” WHO Technical Report Series No. 426. (18) World Health Organization. “Guidelines for Evaluation of Drugs for Use in Man,” WHO Technical Report Series No. 563. (19) World Health Organization. “Part I. Environmental Health Criteria 6,” Principles and Methods for Evaluating the Toxicity of Chemicals. (20) World Health Organization. “Principles for Pre-Clinical Testing of Drug Safety,” WHO Technical Report Series No. 341. [50 FR 39397, Sept. 27, 1985, as amended at 54 FR 21064, May 16, 1989] Subpart E—Specific Organ/Tissue Toxicity § 798.4100 Dermal sensitization. (a) Purpose. (b) Definitions. (2) Induction period is a period of at least 1 week following a sensitization exposure during which a hypersensitive state is developed. (3) Induction exposure is an experimental exposure of a subject to a test substance with the intention of inducing a hypersensitive state. (4) Challenge exposure is an experimental exposure of a previously treated subject to a test substance following an induction period, to determine whether the subject will react in a hypersensitive manner. (c) Principle of the test method. (d) Test procedures. (i) Freund's complete adjuvant test. (ii) Guinea-pig maximization test. (iii) Split adjuvant technique. (iv) Buehler test. (v) Open epicutaneous test. (vi) Mauer optimization test. (vii) Footpad technique in guinea pig. (2) Removal of hair is by clipping, shaving, or possibly by depilation, depending on the test method used. (3) Animal selection Species and strain. (ii) Number and sex. (B) The females should be nulliparous and nonpregnant. (4) Control animals. (ii) Animals may act as their own controls or groups of induced animals can be compared to groups which have received only a challenge exposure. (5) Dose levels. (6) Observation of animals. (ii) Regardless of method selected, initial and terminal body weights should be recorded. (7) Procedures. (e) Data and reporting. (2) Evaluation of the results. (3) Test report. (i) A description of the method used and the commonly accepted name. (ii) Information on the positive control study, including positive control used, method used, and time conducted. (iii) The number and sex of the test animals. (iv) Species and strain. (v) Individual weights of the animals at the start of the test and at the conclusion of the test. (vi) A brief description of the grading system. (vii) Each reading made on each individual animal. (f) References. (1) Buehler, E.V. “Delayed Contact Hypersensitivity in the Guinea Pig,” Archives Dermatology. (2) Draize, J.H. “Dermal Toxicity,” Food Drug Cosmetic Law Journal. (3) Klecak, G. “Identification of Contact Allergens: Predictive Tests in Animals,” Advances in Modern Toxicology: Dermatology and Pharmacology. (4) Klecak, G., Geleick, H., Grey, J.R. “Screening of Fragrance Materials for Allergenicity in the Guinea Pig.-1. Comparison of Four Testing Methods,” Journal of the Society of Cosmetic Chemists. (5) Magnusson, B., Kligman, A.M. “The Identification of Contact Allergens by Animal Assay,” The Guinea Pig Maximization Test. The Journal of Investigative Dermatology. (6) Maguire, H.C. “The Bioassay of Contact Allergens in the Guinea Pig” Journal of the Society of Cosmetic Chemists. (7) Maurer, T., Thomann, P., Weirich, E.G., Hess, R. “The Optimization Test in the Guinea Pig. A Method for the Predictive Evaluation of the Contact Allergenicity of Chemicals,” Agents and Actions. (8) Maurer, T., Thomann, P., Weirich, E.G., Hess, R. “The Optimization Test in the Guinea Pig: A Method for the Predictive Evaluation of the Contact Allergenicity of Chemicals,” International Congress Series Excerpta Medica No. 376, § 798.4350 Inhalation developmental toxicity study. (a) Purpose. (b) Definitions. (2) “Aerodynamic diameter” applies to the behavioral size of particles of aerosols. It is the diameter of a sphere of unit density which behaves aerodynamically like the particles of the test substance. It is used to compare particles of different sizes, shapes, and densities and to predict where in the respiratory tract such particles may be deposited. This term is used in contrast to “optical,” “measured” or “geometric” diameters which are representation of actual diameters which in themselves cannot be related to deposition within the respiratory tract. (3) “Geometric mean diameter” or “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 and its geometeric standard deviation are used to statistically describe 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 deep lung (the alveoli). For man, the inhalable diameter is considered here as 15 micrometers or less. (5) “Concentration” refers to an exposure level. Exposure is expressed as weight or volume of test substance per volume of air (mg/1), or as parts per million (ppm). (6) “No-observed-effect level” is the maximum concentration 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. (c) Principle of the test method. (d) Limit test. (e) Test procedures Animal selection Species and strain. (ii) Age. (iii) Sex. (iv) Number of animals. (2) Control group. (3) Concentration levels and concentration selection. (ii) The vehicle shall neither be developmentally toxic nor have effects on reproduction. (iii) To select the appropriate concentration levels, a pilot or trial study may be advisable. Since pregnant animals have an increased minute ventilation as compared to non-pregnant animals, it is recommended that the trial study be conducted in pregnant animals. Similarly, since presumably the minute ventilation will vary with progression of pregnancy, the animals should be exposed during the same period of gestation as in the main study. In the trial study, the concentration producing embryonic or fetal lethalities or maternal toxicity should be determined. (iv) Unless limited by the physical/chemical nature or biological properties of the substance, the highest concentration level shall induce some overt maternal toxicity such as reduced body weight or body weight gain, but not more than 10 percent maternal deaths. (v) The lowest concentration level should not produce any grossly observable evidence of either maternal or developmental toxicity. (vi) Ideally, the intermediate concentration level(s) shall produce minimal observable toxic effects. If more than one intermediate concentration is used, the concentration levels shall be spaced to produce a gradation of toxic effects. (4) Exposure duration. (5) Observation period. (6) Inhalation exposure. (B) Pregnant animals shall not be subjected to beyond the minimum amount of stress. Since whole-body exposure appears to be the least stressful mode of exposure, it is the method preferred. In general oro-nasal or head-only exposure, which is sometimes used to avoid concurrent exposure by the dermal or oral routes, is not recommended because of the associated stress accompanying the restraining of the animals. However, there may be specific instances where it may be more appropriate than whole-body exposure. The tester shall provide justification/reasoning for its selection. (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. Test material distribution should be established before animals are committed to dosing. Maintenance of slight negative pressure inside the chamber will prevent leakage of the test substance into the surrounding areas. (iii) The temperature at which the test is performed should be maintained at 22 °C (±2°) for rodents or 20 °C (±3°) for rabbits. 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. (7) Physical measurements. (i) The rate of airflow shall be monitored continuously but shall be recorded at least every 30 minutes. (ii) The actual concentration 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 continously or intermittently depending on the method of analysis and measured 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 and be recorded at least every 30 minutes. (8) Food and water during exposure period. (9) Observation of animals. (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 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, eye and mucous membranes, as well as respiratory, autonomic and central nervous systems, somatomotor activity and behavioral pattern. Particular attention should be directed to observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep, and coma. (v) Measurements should be made weekly of food consumption for all animals in the study. (vi) Animals shall be weighed at least weekly. (vii) Females showing signs of abortion or premature delivery shall be sacrificed and subjected to a thorough macroscopic examination. (10) Gross necropsy. (ii) Immediately after sacrifice or death, the uterus shall be removed, weighed, and the contents examined for embryonic or fetal deaths and the number of viable fetuses. Gravid uterine weights should not be obtained from dead animals if autolysis or where decomposition has occurred. The degree of resorption shall be described in order to help estimate the relative time of death. (iii) The number of corpora lutea shall be determined for all species except mice. (iv) The sex of the fetuses shall be determined and they shall be weighed individually, the weights recorded, and the mean fetal weight derived. (v) Following removal, each fetus shall be examined externally. (vi) For rats, mice and hamsters, one-third to one-half of each litter shall be prepared and examined for skeletal anomalies, and the remaining part of each litter shall be prepared and examined for soft tissue anomalies using appropriate methods. (vii) For rabbits, each fetus shall be examined by careful dissection for visceral anomalies and then examined for skeletal anomalies. (f) Data and reporting Treatment of results. (2) Evaluation of results. (3) Test report. (i) Test conditions. (B) The equipment for measuring temperature, humidity, and particulate aerosol concentrations and size shall be described. (ii) Exposure data. (A) Airflow rates through the inhalation equipment. (B) Temperature of air. (C) Nominal concentration—total amount of test substance fed into the inhalation equipment divided by volume of air (no standard deviation). (D) Measured total concentrations (particulate and/or gaseous phases) in test breathing zone. (E) Particle size distribution (e.g., median aerodynamic diameter of particles with geometric standard deviation) including estimates of the percents of inhalable and non-inhalable portions for the test animals. (iii) Animal data. (B) Species and strain. (C) Date of death during the study or whether animals survived to termination. (D) Date of onset and duration of each abnormal sign and its subsequent course. (E) Feed, body weight and uterine weight data. (F) Pregnancy and litter data. (G) Fetal data (live/dead, sex, soft tissue and sketetal defects, resorptions). (g) References. (1) Department of Health and Welfare. The Testing of Chemicals for Carcinogenicity, Mutagenicity and Teratogenicity. (2) 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). (3) World Health Organization. Principles for the Testing of Drugs for Teratogenicity. [50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19076, May 20, 1987; 52 FR 26150, July 13, 1987; 54 FR 21064, May 16, 1989] § 798.4700 Reproduction and fertility effects. (a) Purpose. (b) Principle of the test method. 1 1 2 2 (c) Test procedures Animal selection Species and strain. (ii) Age. (iii) Sex. (B) The females shall be nulliparous and non-pregnant. (iv) Number of animals. (2) Control groups. (ii) If a vehicle is used in administering the test substance, the control group shall receive the vehicle in the highest volume used. (iii) If a vehicle or other additive is used to facilitate dosing, it shall not interfere significantly with absorption of the test substance or produce toxic effects. (3) Dose levels and dose selection. (ii) The highest dose level should induce toxicity but not high levels of mortality in the parental (P) animals. (iii) The lowest dose level should not produce any grossly observable evidence of toxicity. (iv) Ideally the intermediate dose level(s) should produce minimal observable toxic effects. If more than one intermediate dose is used, dose levels should be spaced to produce a gradation of toxic effects. (4) Exposure conditions. (i) Dosing, mating, delivery, and sacrifice schedule. (A) Daily dosing of the parental (P) males and females shall begin when they are 5 to 8 weeks old. For both sexes, dosing shall be continued for at least 10 weeks before the mating period. (B) Dosing of P males shall continue through the 3 week mating period. At the end of the mating period, P males may be sacrificed and examined, or may be retained for possible production of a second litter. If these animals are retained for a second litter, dosing shall be continued. Dosing of the F 1 1 1 1 (C) Daily dosing of the P females shall continue through the three week mating period, pregnancy, and to the weaning of the F 1 1 1 2 (ii) All animals are sacrificed as scheduled. (A) All P males should be sacrificed at the end of the 3-week mating period, or may be retained for possible production of a second litter. If these animals are retained for a second litter, dosing shall be continued. (B) F 1 1 (C) F 1 (D) The P females should be sacrificed upon weaning of their F 1 (E) F 1 2 (5) Administration of the test substance Oral studies. (B) If administered by gavage or capsule, the dosage administered to each animal prior to mating shall be based on the individual animal's body weight and adjusted weekly. During pregnancy the dosage shall be based on the body weight at day 0 and 6 of pregnancy. (ii) If another route of administration is used, the tester should provide justification and reasoning for its selection. (6) Mating procedure Parental. (B) Those pairs that fail to mate should be evaluated to determine the cause of the apparent infertility. This may involve such procedures as additional opportunities to mate with proven fertile males or females, histological examination of the reproductive organs, and examination of the estrus or spermatogenic cycles. (C) Each day, the females shall be examined for presence of sperm or vaginal plugs. Day 0 of pregnancy is defined as the day vaginal plugs or sperm are found. (ii) F 1 1 2 (B) F 1 (iii) Special housing. (iv) Standardization of litter sizes. (B) Whenever the number of male or female pups prevents having 4 of each sex per litter, partial adjustment (for example, 5 males and 3 females) is permitted. Adjustments are not appropriate for litters of less than 8 pups. (C) Elimination of runts only is not appropriate. (D) Adjustments of the F 2 (7) Observation of animals. (ii) The duration of gestation shall be calculated from day 0 of pregnancy. (iii) Each litter should be examined as soon as possible after delivery for the number of pups, stillbirths, live births, sex, and the presence of gross anomalies. Live pups should be counted and litters weighed at birth or soon thereafter, and on days 4, 7, 14, and 21 after parturition. (iv) Physical or behavioral abnormalities observed in the dams of offspring shall be recorded. (v) P males and females shall be weighed on the first day of dosing and weekly thereafter. F 1 (8) Gross necropsy. (ii) Special attention shall be directed to the organs of the reproductive system. (iii) The following organs and tissues, or representative samples thereof, shall be preserved in a suitable medium for possible future histopathological examination: Vagina; uterus; ovaries; testes; epididymides; seminal vesicles; prostate, pituitary gland; and, target organ(s) when previously identified of all P and F 1 (9) Histopathology. (i) Full histopathology on the organs listed above for all high dose, and control P 1 1 (ii) Organs demonstrating pathology in these animals shall then be examined in animals from the other dose groups. (iii) Microscopic examination shall be made of all tissues showing gross pathological changes. (d) Data and reporting Treatment of results. (2) Evaluation of study results. (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. (i) Toxic response data by sex and dose, including fertility, gestation, viability and lactation indices, and length of gestation. (ii) Species and strain. (iii) Date of death during the study or whether animals survived to termination. (iv) Toxic or other effects on reproduction, offspring, or postnatal growth. (v) Date of observation of each abnormal sign and its subsequent course. (vi) Body weight data for P, F 1 2 (vii) Necropsy findings. (viii) Detailed description of all histopathological findings. (ix) Statistical treatment of results where appropriate. (e) References. (1) Clermont, Y., Perry, B. “Quantitative Study of the Cell Population of the Seminiferous Tubules in Immature Rats,” American Journal of Anatomy. (2) Goldenthal, E.I. Guidelines for Reproduction Studies for Safety Evaluation of Drugs for Human Use. (3) Hasegawa, T., Hayashi, M., Ebling, F.J.G., Henderson, I.W. Fertility and Sterility. (4) Oakberg, E.F. “Duration of Spermatogenesis in the Mouse and Timing of Stages of the Cycle of the Seminiferous Epithelium,” American Journal of Anatomy. (5) Roosen-Runge, E.C. “The Process of Spermatogenesis in Mammals,” Biological Review. [50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19077, May 20, 1987] § 798.4900 Developmental toxicity study. (a) Purpose. (b) Definitions. (2) Dose is the amount of test substance administered. Dose is expressed as weight of test substance (g, mg) per unit weight of a test animal (e.g., mg/kg). (3) No-observed-effect level is the maximum concentration in a test which produces no observed adverse effects. A no-observed-effect level is expressed in terms of weight of test substance given daily per unit weight of test animal (mg/kg) (c) Principle of the test method. (d) Limit test. (e) Test procedures Animal selection Species and strain. (ii) Age. (iii) Sex. (iv) Number of animals. (2) Control group. (3) Dose levels and dose selection. (ii) The vehicle shall neither be developmentally toxic nor have effects on reproduction. (iii) To select the appropriate dose levels, a pilot or trial study may be advisable. It is not always necessary to carry out a trial study in pregnant animals. Comparison of the results from a trial study in non-pregnant, and the main study in pregnant animals will demonstrate if the test substance is more toxic in pregnant animals. If a trial study is carried out in pregnant animals, the dose producing embryonic or fetal lethalities or maternal toxicity shall be determined. (iv) Unless limited by the physical/chemical nature or biological properties of the substance, the highest dose level shall induce some overt maternal toxicity such as reduced body weight or body weight gain, but not more than 10 percent maternal deaths. (v) The lowest dose level should not produce any grossly observable evidence of either maternal or developmental toxicity. (vi) Ideally, the intermediate dose level(s) should produce minimal observable toxic effects. If more than one intermediate concentration is used, the concentration levels should be spaced to produce a gradation of toxic effects. (4) Observation period. (5) Administration of test substance. (6) Exposure conditions. (7) Observation of animals. (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, eye and mucous membranes, as well as respiratory, autonomic and central nervous systems, somatomotor activity and behavioral pattern. (v) Measurements should be made weekly of food consumption for all animals in the study. (vi) Animals shall be weighed at least weekly. (vii) Females showing signs of abortion or premature delivery shall be sacrificed and subjected to a thorough macroscopic examination. (8) Gross necropsy. (ii) Immediately after sacrifice or as soon as possible after death, the uterus shall be removed and the contents examined for embryonic or fetal deaths and the number of viable fetuses. The degree of resorption shall be described in order to help estimate the relative time of death of the conceptus. The weight of the gravid uterus should be recorded for dams that are sacrificed. Gravid uterine weights should not be obtained from dead animals if autolysis or decomposition has occurred. (iii) The number of corpora lutea shall be determined for all species except mice. (iv) The sex of the fetuses shall be determined and they shall be weighed individually, the weights recorded, and the mean fetal weight derived. (v) Following removal, each fetus shall be examined externally. (vi) For rats, mice and hamsters, one-third to one-half of each litter shall be prepared and examined for skeletal anomalies, and the remaining part of each litter shall be prepared and examined for soft tissue anomalies using appropriate methods. (vii) For rabbits, each fetus shall be examined by careful dissection for visceral anomalies and then examined for skeletal anomalies. (f) Data and reporting Treatment of results. (2) Evaluation of results. (3) Test report. (i) Toxic response data by concentration. (ii) Species and strain. (iii) Date of death during the study or whether animals survived to termination. (iv) Date of onset and duration of each abnormal sign and its subsequent course. (v) Food, body weight and uterine weight data. (vi) Pregnancy and litter data. (vii) Fetal data (live/dead, sex, soft tissue and skeletal defects, resorptions). (g) References. (1) Department of Health and Welfare. The Testing of Chemicals for Carcinogenicity, mutagenicity and Teratogenicity. (2) 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). (3) World Health Organization. Principles for the Testing of Drugs for Teratogenicity. [50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19077, May 20, 1987] Subpart F—Genetic Toxicity § 798.5195 Mouse biochemical specific locus test. (a) Purpose. (b) Definitions. (2) The germ line is comprised of the cells in the gonads of higher eukaryotes, which are the carriers of the genetic information for the species. (c) Reference substances. (d) Test method Principle. (2) Description. (3) Animal selection Species and strain. (ii) Age. (iii) Number. (A) The production of concurrent spontaneous controls. (B) The use of positive controls. (C) The power of the test. (4) Control groups Concurrent controls. (ii) Historical controls. (5) Test chemicals Vehicle. (ii) Dose levels. (iii) Route of administration. (e) Test performance Treatment and mating. (2) Examination of offspring Birth and weaning. (ii) Tissue sampling. (iii) Electrophoresis. (iv) Mutant identification. (f) Data and reports Treatment of results. (2) Statistical evaluation. (3) Interpretation of results. (ii) A test chemical which does not produce a statistically significant increase in the frequency of electrophoretic mutations over the spontaneous frequency, or a statistically significant and reproducible positive response for at least one of the test points, is considered nonmutagenic in this system, provided that the sample size is sufficient to exclude a biologically significant increase in mutation frequency. (iii) Both biological and statistical significance should be considered together in the evaluation. (4) Test evaluation. (ii) Negative results indicate that, under the test conditions, the test chemical does not induce heritable genemutations in a mammalian species. (5) Test report. (i) Strain, age and weight of animals used; numbers of animals of each sex in experimental and control groups. (ii) Test chemical vehicle, doses used, rationale for dose selection, and toxicity data, if available. (iii) Route and duration of exposure. (iv) Mating schedule. (v) Number of loci screened for both treated and spontaneous data. (vi) Criteria for scoring mutants. (vii) Number of mutants found/locus. (viii) Loci at which mutations were found. (ix) Use of concurrent negative and positive controls. (x) Dose-response relationship, if applicable. (g) References. (1) Personal communication from Susan E. Lewis, Ph.D. to Dr. Michael Cimino, U.S. EPA, OPPT, October 5, 1989. (2) Johnson, F.M., G.T. Roberts, R.K. Sharma, F.Chasalow, R. Zweidinger, A. Morgan, R.W. Hendren, and S.E.Lewis. “The detection of mutants in mice by electrophoresis: Results of a model induction experiment with procarbazine.” Genetics (3) Johnson, F.M. and S.E. Lewis. “Mutation rate determinations based on electrophoretic analysis of laboratory mice.” Mutation Research (4) Johnson, F.M. and S.E. Lewis. “Electrophoretically detected germinal mutations induced by ethylnitrosourea in the mouse.” Proceedings of the National Academy of Sciences (5) Lewis, S.E., C. Felton, L.B. Barnett, W. Generoso, N. Cacheiro, and M.D. Shelby. “Dominant visible and electrophoretically expressed mutations induced in male mice exposed to ethylene oxide by inhalation.” Environmental Mutagenesis (h) Additional requirements. [55 FR 12641, Apr. 5, 1990] § 798.5200 Mouse visible specific locus test. (a) Purpose. (b) Definitions. (2) The germ line is the cells in the gonads of higher eukaryotes which are the carriers of the genetic information for the species. (c) Reference substances. (d) Test method Principle. (ii) Three variations of the method currently exist for detecting newly arising point mutations in mouse germ cells: (A) The visible specific locus test using either 5 or 7 loci. (B) The biochemical specific locus test using up to 20 enzymes. (C) The test for mutations at histocompatibility loci. (iii) Of the three tests, the visible specific locus test has been most widely used in assessing genetic hazard due to environmental agents. It is the method described in this guideline. (2) Description. (3) Animal selection Species and strain. 3 1 3 1 (ii) Age. (iii) Number. (A) The use of either historical or concurrent controls. (B) The power of the test. (C) The minimal rate of induction required. (D) The use of positive controls. (E) The level of significance desired. (iv) Assignment to groups. (4) Control groups Concurrent controls. (ii) Historical controls. (5) Test chemicals Vehicle. (ii) Dose levels. (iii) Route of administration. (e) Test performance Treatment and mating. 1 3 3 (2) Examination of offspring. (ii) Nonmutant progeny should be discarded. Mutant progeny shall be subjected to genetic tests for verification. (f) Data and report Treatment of results. (2) Statistical evaluation. (3) Interpretation of results. (ii) A test substance which does not produce either a statistically significant dose-related increase in the number of specific locus mutations or a statistically significant and reproducible positive response at any one of the test points is considered nonmutagenic in this system. (iii) Both biological and statistical significance should be considered together in the evaluation. (4) Test evaluation. (ii) Negative results indicate that under the test conditions the test substance does not induce heritable gene mutations in the test species. (5) Test report. (i) Strain, age and weight of animals used, number of animals of each sex in experimental and control groups. (ii) Test chemical vehicle, doses used and rationale for dose selection, toxicity data. (iii) Route and duration of exposure. (iv) Mating schedule. (v) Time of examination for mutant progeny. (vi) Criteria for scoring mutants. (vii) Use of concurrent or negative controls. (viii) Dose response relationship, if applicable. (g) References. (1) Russell, L.B., Shelby, P.B., von Halle, E., Sheridan, W., Valcovic, L. The mouse specific locus test with agents other than radiations: interpretation of data and recommendations for future work: A report of the U.S. EPA's Gene-Tox Program,” Mutation Research, (2) [Reserved] (h) Additional requirements. [50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19078, May 20, 1987; 55 FR 12643, Apr. 5, 1990] § 798.5265 The salmonella typhimurium reverse mutation assay. (a) Purpose. Salmonella typhimurium − = (b) Definitions. Salmonella typhimurium (2) Base pair mutagens are agents which cause a base change in the DNA. In a reversion assay, this change may occur at the site of the original mutation or at a second site in the chromosome. (3) Frameshift mutagens are agents which cause the addition or deletion of single or multiple base pairs in the DNA molecule. (c) Reference substances. (d) Test method Principle. (2) Description. (i) The direct plate incorporation method. (ii) The preincubation method. (iii) The azo-reduction method. The procedures described here are for the direct plate incorporation method and the azo-reduction method. (3) Strain selection Designation. (ii) Preparation and storage. (iii) Bacterial growth. 8 9 (4) Metabolic activation. (5) Control groups Concurrent controls. (ii) Strain specific positive controls. (A) Strain TA 1535, TA 100, sodium azide. (B) TA 98, 2-nitrofluorene. (C) TA 1537, 9-aminoacridine. (iii) Positive controls to ensure the efficacy of the activation system. (iv) Class-specific positive controls. (6) Test chemicals Vehicle. (ii) Exposure concentrations. (B) Generally, a maximum of 5 mg/plate for pure substances is considered acceptable. At least 5 different amounts of test substance shall be tested with adequate intervals between test points. (C) When appropriate, a single positive response shall be confirmed by testing over a narrow range of concentrations. (e) Test performance Direct plate incorporation method. (2) Azo-reduction method. (ii) For tests without metabolic activation, 0.5 ml of buffer should be used in place of the 0.5 ml of S-9 mix. All other procedures shall be the same as those used for the test with metabolic activation. (3) Other methods. (4) Media. (5) Incubation conditions. (6) Number of cultures. (f) Data and report Treatment of results. (2) Statistical evaluation. (3) Interpretation of results. (ii) A test substance which does not produce either a statistically significant dose-related increase in the number of revertants or a statistically significant and reproducible positive response at any one of the test points is considered nonmutagenic in this system. (iii) Both biological and statistical significance should be considered together in the evaluation. (4) Test evaluation. S. typhimurium (ii) Negative results indicate that under the test conditions the test substance is not mutagenic in S. typhimurium. (5) Test report. (i) Bacterial strain used. (ii) Metabolic activation system used (source, amount and cofactor); details of preparations of S-9 mix. (iii) Dose levels and rationale for selection of dose. (iv) Positive and negative controls. (v) Individual plate counts, mean number of revertant colonies per plate, standard deviation. (vi) Dose-response relationship, if applicable. (g) References. (1) Ames, B.N., McCann, J., Yamasaki, E. “Methods for detecting carcinogens and mutagens with the Salmonella/ Mutation Research (2) de Serres, F.J., Shelby, M.D. “The Salmonella Science (3) Prival, M.J., Mitchell, V.D. “Analysis of a method for testing azo dyes for mutagenic activity in Salmonella typhimurium Mutation Research (4) Vogel, H.J., Bonner, D.M. “Acetylornithinase of E. coli: Journal of Biological Chemistry. [50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19078, May 20, 1987] § 798.5275 Sex-linked recessive lethal test in drosophila melanogaster. (a) Purpose. Drosophila melanogaster (b) Definitions. (2) Recessive mutation is a change in the genome which is expressed in the homozygous or hemizygous condition. (3) Sex-Linked genes are present on the sex (X or Y) chromosomes. Sex-linked genes in the context of this guideline refer only to those located on the X-chromosome. (c) Reference substances. (d) Test method Principle. D. melanogaster (2) Description. 1 (3) Drosophila stocks. (4) Control groups Concurrent controls. (ii) Positive controls. (iii) Other positive controls. (iv) Negative controls. (5) Test chemicals Vehicle. (ii) Dose levels. (iii) Route of administration. (e) Test performance Treatment and mating. (2) F 1 matings. 1 2 1 (3) Number of matings. (ii) Test results should be confirmed in a separate experiment. (f) Data and report Treatment of results. (2) Statistical evaluation. (3) Interpretation of results. (ii) A test substance which does not produce either a statistically significant dose-related increase in the number of sex-linked recessive lethals or a statistically significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system. (iii) Both biological and statistical significance should be considered together in the evaluation. (4) Test evaluation. D. melanogaster (ii) Negative results indicate that under the test conditions the test substance is not mutagenic in D. melanogaster. (5) Test report. (i) Drosophila 2 2 (ii) Test chemical vehicle, treatment and sampling schedule, exposure levels, toxicity data, negative (vehicle) and positive controls, if appropriate. (iii) Criteria for scoring lethals. (iv) Number of chromosomes tested, number of chromosomes scored, number of chromosomes carrying a lethal mutation. (v) Historical control data, if available. (vi) Dose-response relationship, if applicable. (g) References. (1) Sobels, F.H., Vogel, E. “The capacity of Drosophila Mutation Research (2) Wurgler F.E., Sobels F.H., Vogel E. “ Drosophila Handbook of mutagenicity test procedures. [50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19079, May 20, 1987] § 798.5300 Detection of gene mutations in somatic cells in culture. (a) Purpose. = = = = (b) Definitions. (2) Base pair mutagens are agents which cause a base change in the DNA. (3) Frameshift mutagens are agents which cause the addition or deletion of single or multiple base pairs in the DNA molecule. (4) Phenotypic expression time is a period during which unaltered gene products are depleted from newly mutated cells. (c) Reference substances. (d) Test method Principle. = − de novo = = (2) Description. (3) Cells Type of cells used in the assay. Mycoplasma (ii) Cell growth and maintenance. 2 (4) Metabolic activation. (5) Control groups. (6) Test chemicals Vehicle. (ii) Exposure concentrations. (B) Several concentrations (usually at least 4) of the test substance shall be used. Generally, these shall yield a concentration-related toxic effect. The highest concentration shall produce a low level of survival (approximately 10 percent), and the survival in the lowest concentration shall approximate the negative control. Cytotoxicity shall be determined after treatment with the test substance both in the presence and in the absence of an exogenous metabolic activation system. Relatively insoluble substances should be tested up to their limit of solubility under culture conditions. For freely-soluble nontoxic substances the highest concentration used should be determined on a case-by-case basis. (e) Test performance. (2) At the end of the exposure period, cells shall be washed and cultured to determine viability and to allow for expression of the mutant phenotype. (3) At the end of the expression period, which shall be sufficient to allow near optimal phenotypic expression of induced mutants, cells should be grown in medium with and without selective agent(s) for determination of number of mutants and cloning efficiency, respectively. (4) Results shall be confirmed in an independent experiment. When appropriate, a single positive response should be confirmed by testing over a narrow range of concentrations. (f) Data and report Treatment of results. (2) Statistical evaluation. (3) Interpretation of results. (ii) A test substance which does not produce either a statistically significant concentration-related increase in the mutant frequency or a statistically significant and reproducible positive response at any one of the test points is considered nonmutagenic in this system. (iii) Both biological and statistical significance should be considered together in the evaluation. (4) Test evaluation. (ii) Negative results indicate that, under the test conditions, the test substance does not induce gene mutations in the cultured mammalian cells used. (5) Test report. (i) Cell type used, number of cell cultures, methods used for maintenance of cell cultures. (ii) Rationale for selection of concentrations and number of cultures. (iii) Test conditions: composition of media, CO 2 (iv) Methods used to enumerate numbers of viable and mutant cells. (v) Dose-response relationship, where possible. (g) References. (1) Amacher, D.E., Paillet, S.C., Ray, V. “Point mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells. I. Application to genetic toxicology testing,” Mutation Research, (2) Amacher, D.E., Paillet, S.C., Turner, G.N., Ray, V.A. Salsburg, V.A. “Point mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells. II. Test validation and interpretation,” Mutation Research, (3) Bradley, M.O., Bhuyan B., Francis, M.C., Langenback, R., Peterson, A., Huberman, E. “Mutagenesis by chemical agents in V-79 Chinese hamster cells: a review and analysis of the literature: a report of the Gene-Tox Program,” Mutation Research, (4) Clive, D., Johnson, K.O., Spector, J.F.S., Batson, A.G., Brown, M.M. “Validation and characterization of the L5178Y TK = − Mutation Research, (5) Clive, D., Spector, J.F.S. “Laboratory procedures for assessing specific locus mutations at the TK locus in cultured L5178Y mouse lymphoma cells,” Mutation Research, (6) Hsie, A.W., Casciano, D.A., Couch, D.B., Krahn, D.F., O'Neill, J.P., Whitfield, B.L. “The use of Chinese hamster ovary cells to quantify specific locus mutation and to determine mutagenicity of chemicals: a report of the U.S. EPA's Gene-Tox Program,” Mutation Research, [50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19079, May 20, 1987] § 798.5375 In vitro mammalian cytogenetics. (a) Purpose. (b) Definitions. (2) Chromatid-type aberrations are damage expressed as breakage of single chromatids or breakage and/or reunion between chromatids. (c) Reference substances. (d) Test method Principle. (2) Description. (3) Cells Type of cells used in the assay. Mycoplasma (ii) Cell growth and maintenance. 2 (4) Metabolic activation. (5) Control groups. (6) Test chemicals Vehicle. (ii) Exposure concentrations. (e) Test performance Established cell lines and strains. (2) Human lymphocyte cultures. (3) Treatment with test substance. (4) Number of cultures. (5) Culture harvest time. (ii) For human lymphocyte cultures, the substance to be tested may be added to the cultures at various times after mitogen stimulation so that there is a single harvest time after the initiation of the cell culture. Alternatively, a single treatment may be followed by multiple harvest times. Harvest time should be extended for those chemicals which induce an apparent cell cycle delay. Because the population of human lymphocytes is only partially synchronized, a single treatment, at, or close to, the time when metaphase stages first appear in the culture will include cells in all phases of the division cycle. Therefore, a single harvest at the time of second mitosis may be carried out for screening purposes. (iii) Cell cultures shall be treated with a spindle inhibitor, (e.g., colchicine or Colcemid ®), 1 or 2 hours prior to harvesting. Each culture shall be harvested and processed separately for the preparation of chromosomes. (6) Chromosome preparation. (7) Analysis. (8) Confirmatory tests. (f) Data and report Treatment of results. (2) Statistical evaluation. (3) Interpretation of results. (ii) A test substance which does not produce either a statistically significant dose-related increase in the number of structural chromosomal aberrations or a statistically significant and reproducible positive response at any one of the test points is considered nonmutagenic in this system. (iii) Both biological and statistical significance should be considered together in the evaluation. (4) Test evaluation. (ii) Negative results indicate that under the test conditions the test substance does not induce chromosomal aberrations in cultured mammalian somatic cells. (5) Test report. (i) Cells used, density and passage number at time of treatment, number of cell cultures. (ii) Methods used for maintenance of cell cultures including medium, temperature and CO 2 (iii) Test chemical vehicle, concentration and rationale for the selection of the concentrations used in the assay, duration of treatment. (iv) Details of both the protocol used to prepare the metabolic activation system and of its use in the assay. (v) Identity of spindle inhibitor, its concentration and duration of treatment. (vi) Date of cell harvest. (vii) Positive and negative controls. (viii) Methods used for preparation of slides for microscopic examination. (ix) Number of metaphases analysed. (x) Mitotic index where applicable. (xi) Criteria for scoring aberrations. (xii) Type and number of aberrations, given separately for each treated and control culture, total number of aberrations per group; frequency distribution of number of chromosomes in established cell lines and strains. (xiii) Dose-response relationship, if applicable. (g) References. (1) Ames, B.N., McCann, J., Yamasaki, E. “Methods for detecting carcinogens and mutagens with the Salmonella/ Mutation Research, (2) Evans, H.J. “Cytological methods for detecting chemical mutagens,” Chemical mutagens, principles and methods for their detection, (3) Howard, P.N., Bloom, A.D., Krooth, R.S. “Chromosomal aberrations induced by N-methyl-N′-nitro-N-nitrosoguanidine in mammalian cells,” In Vitro (4) Ishidate, M. Jr., Odashima, S. “Chromosome tests with 134 compounds on Chinese hamster cells in vitro: A screening for chemical carcinogens,” Mutation Research, (5) Preston, R.J., Au, W., Bender, M.A., Brewen, J.G., Carrano, A.V., Heddle, J.A., McFee, A.F., Wolff, S., Wassom, J.S., “Mammalian in vivo and in vitro cytogenetic assays: A report of the Gene-tox Program,” Mutation Research, [50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19079, May 20, 1987] § 798.5385 In vivo mammalian bone marrow cytogenetics tests: Chromosomal analysis. (a) Purpose. in vivo (b) Definitions. (2) Chromatid-type aberrations are damage expressed as breakage of single chromatids or breakage and/or reunion between chromatids. (c) Reference substances. (d) Test method Principle. (2) Description. (3) Animal selection Species and strain. (ii) Age. (iii) Number and sex. (iv) Assignment to groups. (4) Control groups Concurrent controls. (ii) Positive controls. (5) Test chemicals Vehicle. (ii) Dose levels. (iii) Route of administration. (iv) Treatment schedule. (e) Test performance Generally the test may be performed in two assays. (ii) If a repeated treatment schedule is used at the selected dose(s), samples shall be taken 6 and 24 hours after the last treatment; other sampling times may be used if justified. Where the additional dose levels are tested in a subsequent experiment, samples shall be taken at the predetermined most sensitive interval or, if this is not established, at 6 hours after the last treatment. (2) Administration of spindle inhibitor. (3) Preparation of slides. (4) Analysis. (f) Data and report Treatment of results. (2) Statistical evaluation. (3) Interpretation of results. (ii) A test substance which does not produce either a statistically significant dose-related increase in the number of chromosomal aberrations or abnormal metaphase figures or a statistically significant and reproducible positive response at any one of the test points is considered nonmutagenic in this system. (iii) Both biological and statistical significance should be considered together in the evaluation. (4) Test evaluation. in vivo (ii) Negative results indicate that under the test conditions, the test substance does not induce chromosomal aberrations in the bone marrow of the test species. (5) Test report. (i) Species, strain, age, weight, number and sex of animals in each treatment and control group. (ii) Test chemical vehicle, dose levels used, rationale for dose selection. (iii) Route of administration, treatment and sampling schedules, toxicity data, negative and positive controls. (iv) Identity of spindle-inhibitor, its concentration and duration of treatment. (v) Details of the protocol used for chromosome preparation, number of cells scored per animal, type and number of aberrations given separately for each treated and control animal. (vi) Mitotic index, where applicable. (vii) Criteria for scoring aberrations. (viii) Number and frequency of aberrant cells per animal in each treatment and control groups. (ix) Total number of aberrations per group. (x) Number of cells with aberrations per group. (xi) Dose-response relationship, if applicable. (g) References. (1) Adler, I.D., Ramarao, G., Epstein, S.S. “In vivo cytogenetic effects of trimethyl-phosphate and of TEPA on bone marrow cells of male rats,” Mutation Research, (2) Evans, H.J. “Cytological methods for detecting chemical mutagens,” Chemical Mutagens: Principles and Methods for Their Detection, (3) Kilian, J.D., Moreland, F.E. Benge, M.C., Legator, M.S., Whorton, E.B. Jr. “A collaborative study to measure intralaboratory variation with the in vivo bone morrow metaphase procedure,” Handbook of mutagenicity test procedures. (4) Preston, J.R., Au, W., Bender, M.A., Brewen, J.G., Carrano, A.V. Heddle, J.A., McFee, A.F., Wolff, S., Wassom, J. “Mammalian in vivo and vitro cytogenetics assays: Report of the Gene-Tox Program,” Mutation Research, 87:143-188 (1981). [50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19080, May 20, 1987] § 798.5395 In vivo mammalian bone marrow cytogenetics tests: Micronucleus assay. (a) Purpose. in vivo (b) Definition. (c) Reference substances. (d) Test method Principle. (ii) Micronuclei may also be detected in other test systems: (A) Tissue culture. (B) Plants. (C) Blood smears. (D) Fetal tissues. (E) Meiotic cells. (F) Hepatic cells. (iii) The present guideline is based on the mammalian bone marrow assay. (2) Description. (3) Animal selection Species and strain. (ii) Age. (iii) Number and sex. (iv) Assignment to groups. (4) Control groups Concurrent controls. (ii) Positive controls. in vivo (5) Test chemicals Vehicle. (ii) Dose levels. (iii) Route of administration. (iv) Treatment schedule. (e) Test performance Treatment and sampling times. (ii) If a repeated treatment schedule is used, samples shall be taken at least three times, starting not earlier than 12 hours after the last treatment and at appropriate intervals following the first sample, but not extending beyond 72 hours. (iii) Bone marrow shall be obtained immediately after sacrifice. Cells shall be prepared, put on slides, spread as a smear and stained. (2) Analysis. (f) Data and report Treatment of results. (2) Statistical evaluation. (3) Interpretation of results. (ii) A test substance which does not produce either a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistically significant and reproducible positive response at any one of the test points is considered nonmutagenic in this system. (iii) Both biological and statistical significance should be considered together in the evaluation. (4) Test evaluation. (ii) Negative results indicate that under the test conditions the test substance does not produce micronuclei in the bone marrow of the test species. (5) Test report. (i) Species, strain, age, weight, number and sex of animals in each treatment and control group. (ii) Test chemical vehicle, dose levels used, rationale for dose selection. (iii) Rationale for and description of treatment and sampling schedules, toxicity data, negative and positive controls. (iv) Details of the protocol used for slide preparation. (v) Criteria for identifying micronucleated erythrocytes. (vi) Dose-response relationship, if applicable. (g) References. (1) Cihak, R. “Evaluation of benzidine by the micronucleus test,” Mutation Research, (2) Cole, R.J., Taylor, N., Cole, J., Arlett, C.F. “Short-term tests for transplacentally active carcinogens. 1. Micronucleus formation in fetal and maternal mouse erythroblasts,” Mutation Research, (3) Kliesch, U., Danford, N., Adler, I.D. “Micronucleus test and bone-marrow chromosome analysis. A comparison of 2 methods in vivo for evaluating chemically induced chromosomal alterations,” Mutation Research, (4) Matter, B., Schmid, W. “Trenimon-induced chromosomal damage in bone-marrow cells of six mammalian species, evaluated by the micronucleus test,” Mutation Research, (5) Schmid, W. “The micronucleus test,” Mutation Research, 31:9-15 (1975). (6) Schmid, W. “The micronucleus test for cytogenetic analysis,” Chemical Mutagens, Principles and Methods for their Detection. (7) Heddle, J.A., Hite, M., Kurkhart, B., Mavournin, K., MacGregor, J.T., Newell, G.W., Salamone, M.F. “The induction of micronuclei as a measure of genotoxicity. A report of the U.S. Environmental Protection Agency Gene-Tox Program,” Mutation Research, [50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19080, May 20, 1987; 52 FR 26150, July 13, 1987; 52 FR 34654, Sept. 14, 1987] § 798.5450 Rodent dominant lethal assay. (a) Purpose. (b) Definition. (c) Reference substances. (d) Test method Principle. (2) Description. (ii) Individual males are mated sequentially to virgin females at appropriate intervals. The number of matings following treatment is governed by the treatment schedule and should ensure that germ cell maturation is adequately covered. Females are sacrificed in the second half of pregnancy and the uterine contents examined to determine the total number of implants and the number of live and dead embryos. (3) Animal selection Species. (ii) Age. (iii) Number. (iv) Assignment to groups. (4) Control groups Concurrent controls. (ii) Positive controls. (5) Test chemicals Vehicle. (ii) Dose levels. (iii) Route of administration. (e) Test performance. (2) The number of matings following treatment should be governed by the treatment schedule and should ensure that germ cell maturation is adequately covered. (3) Females should be sacrificed in the second half of pregnancy and uterine contents examined to determine the number of implants and live and dead embryos. The ovaries may be examined to determine the number of corpora lutea. (f) Data and report Treatment of results. (2) Statistical evaluation. (3) Interpretation of results. (ii) A test substance which does not produce either a statistically significant dose-related increase in the number of dominant lethals or a statistically significant and reproducible positive response at any one of the test points is considered nonmutagenic in this system. (iii) Both biological and statistical significance should be considered together in the evaluation. (4) Test evaluation. (ii) A negative result suggests that under the conditions of the test the test substance may not be genotoxic in the germ cells of the treated sex of the test species. (5) Test report. (i) Species, strain, age and weights of animals used, number of animals of each sex in experimental and control groups. (ii) Test substance, vehicle used, dose levels and rationale for dosage selection, negative (vehicle) and positive controls, experimental observations, including signs of toxicity. (iii) Route and duration of exposure. (iv) Mating schedule. (v) Methods used to determine that mating has occurred (where applicable). (vi) Criteria for scoring dominant lethals including the number of early and late embryonic deaths. (vii) Dose-response relationship, if applicable. (g) References. (1) Brewen, J.G., Payne, H.S., Jones, K.P., Preston, R.J. “Studies on chemically induced dominant lethality. I. The cytogenetic basis of MMS-induced dominant lethality in post-meiotic germ cells” Mutation Research, (2) Ehling, U.H., Machemer, L., Buselmaier, E., Dycka, D., Frohberg, H., Kratochvilova, J., Lang, R., Lorke, D., Muller, D., Pheh, J., Rohrborn, G., Roll, R., Schulze-Schencking, M., Wiemann, H. “Standard protocol for the dominant lethal test on male mice. Set up by the Work Group “Dominant lethal mutations of the ad hoc Committee Chemogenetics,” Archives of Toxicology, [50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19081, May 20, 1987] § 798.5460 Rodent heritable translocation assays. (a) Purpose. (b) Definitions. (2) Diakinesis and metaphase I are stages of meiotic prophase scored cytologically for the presence of multivalent chromosome association characteristic of translocation carriers. (c) Reference substances. (d) Test method Principle. (i) Basis for fertility screening. (A) Translocations between non-homologous chromosomes in which at least one of the breaks occurs close to one end of a chromosome. (B) Those that carry multiple translocations. The majority of male translocation heterozygotes are semisterile—they carry one or (rarely) two translocations. The degree of semisterility is dependent upon the proportions of balanced and unbalanced (duplication-deficiency) gametes produced in the ejaculate as a function of meiotic segregation. Balanced and unbalanced sperm are equally capable of fertilizing an egg. Balanced sperm lead to viable progeny. Unbalanced sperm result in early embryonic lethality. (ii) Basis for cytological screening. (2) Description. 1 (3) Animal selection Species. (ii) Age. (iii) Number. ( 1 ( 2 ( 3 ( 4 ( 5 (B) [Reserved] (iv) Assignment to groups. (4) Control groups Concurrent controls. (ii) Historical controls. (5) Test chemicals Vehicle. (ii) Dose levels. (iii) Route of administration. (e) Test performance Treatment and mating. (2) Testing for translocation heterozygosity. 1 (i) Determination of sterility or semisterility Conventional method. (B) Sequential method. (ii) Cytological analysis. (f) Data and report Treatment of results. (ii) These data shall be presented for both treated and control groups. Historical or concurrent controls shall be specified, as well as the randomization procedure used for concurrent controls. (2) Statistical evaluation. (3) Interpretation of results. (ii) A test substance which does not produce either a statistically significant dose-related increase in the number of heritable translocations or a statistically significant and reproducible positive response at any one of the test points is considered nonmutagenic in this system. (iii) Both biological and statistical significance should be considered together in the evaluation. (4) Test evaluation. (ii) Negative results indicate that under the test conditions the test substance does not cause heritable chromosomal damage in the test species. (5) Test report. (i) Species, strain, age, weight and number of animals of each sex in each group. (ii) Test chemical vehicle, route and schedule of administration, toxicity data. (iii) Dosing regimen, doses tested and rationale for dosage selection. (iv) Mating schedule, number of females mated to each male. (v) The use of historical or concurrent controls. (vi) Screening procedure including the decision criteria used and the method by which they were determined. (vii) Dose-response relationship, if applicable. (g) References. (1) Generoso, W.M., Bishop, J.B., Goslee, D.G., Newell, G.W., Sheu, G-J, von Halle, E. “Heritable translocation test in mice,” Mutation Research, (2) [Reserved] [50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19081, May 20, 1987] § 798.5500 Differential growth inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA damage or repair tests.” (a) Purpose. per se. (b) Definition. (c) Reference substances. (d) Test method Principle. (2) Description. (i) Tests performed on solid medium (diffusion tests). (ii) Tests performed in liquid culture (suspension tests). (3) Strain selection Designation. Escherichia coli polA Bacillus subtilis rec (ii) Preparation and storage. (4) Bacterial growth. (5) Metabolic activation. (6) Control groups Concurrent controls. (ii) Negative controls. (iii) Genotype specific controls. polA rec (iv) Positive controls to ensure the efficacy of the activation system. (v) Other positive controls. (7) Test chemicals Vehicle. (ii) Exposure concentrations. (e) Test performance Diffusion assay Disc diffusion assays. (A) A single strain of bacteria may be added to an agar overlay or spread on the surface of the agar and the test chemical placed on a filter disc on the surface of the agar or; (B) DNA repair proficient and DNA repair deficient bacteria may be streaked in a line on the surface of the agar of the same plate and a disc saturated with test chemical placed on the surface of the agar in contact with the streaks. (ii) Well diffusion assays. (2) Suspension assays. (ii) Nonturbid suspensions of bacteria may be exposed to serial dilutions of the test agent and a minimal inhibitory concentration for each strain determined, as evidenced by the presence or absence of visible growth after a period of incubation. (iii) Paired bacterial suspensions (usually with some initial turbidity) may be treated with a single dose of the chemical. Positive results are indicated by a differential inhibition in the rate of increase of turbidity of the paired cultures. (3) Number of cultures. (4) Incubation conditions. (f) Data and report Treatment of results Diffusion assays. 2 (ii) Liquid suspension assays. (B) Results can also be expressed as the concentrations required to effect a predetermined survival rate (e.g., D 37 (C) Similarly, results can be expressed as minimal inhibitory concentration or as minimal lethal dose. The former is determined by the absence of visible growth in liquid medium and the latter is determined by plating dilutions onto semisolid media. (iii) In all tests, concentrations must be given as the final concentrations during the treatment. Raw data, prior to transformation, should be provided. These should include actual quantities measured, e.g., neat numbers. For measurement of diffusion, the diameters of the discs and/or well should be indicated and the measurements should indicate whether the diameter of the discs and/or well was subtracted. Moreover, mention should be made as to whether the test chemical gave a sharp, diffuse, or double-zone of growth inhibition. If it is the latter, the investigator should indicate whether the inner or the outer zone was measured. (iv) Viability data should be given as the actual plate counts with an indication of the dilution used and the volume plated or as derived titers (cells per ml). Transformed data alone in the absence of experimental data are not acceptable (i.e, ratios, differences, survival fraction). (2) Statistical evaluation. (3) Interpretation of results. (ii) A test substance which does not produce either a statistically significant dose-related preferential inhibition or killing of the repair deficient strain or a statistically significant and reproducible positive response at any one of the test points is considered not to interact with the genetic material of the organisms used in assay. (iii) Both biological and statistical significance should be considered together in the evaluation. (4) Test evaluation. per se (5) Test report. (i) Bacterial strains used. (ii) Phase of bacterial cell growth at time of use in the assay. (iii) Media composition. (iv) Details of both the protocol used to prepare the metabolic activation system and its use in the assay. (v) Treatment protocol, including doses used and rationale for dose selection, positive and negative controls. (vi) Method used for determination of degree of cell kill. (vii) Dose-response relationship, if applicable. (g) References. (1) Ames, B.N., McCann, J., Yamasaki, E. “Methods for detecting carcinogens and mutagens with the Salmonella Mutation Research, (2) Kada, T., Sadie, Y., Tutikawa, K. “In vitro and host-mediated “rec-assay” procedures for screening chemical mutagens; and phloxine, a mutagenic red dye detected,” Mutation Research, (3) Leifer, Z., Kada, T., Mandel, M., Zeiger, E., Stafford, R., Rosenkranz, H.S. “An evaluation of bacterial DNA repair tests for predicting genotoxicity and carcinogenicity: A report of the U.S. EPA's Gene-Tox Program,” Mutation Research, (4) Slater, E.E., Anderson, M.D., Rosenkranz, H.S. “Rapid detection of mutagens and carcinogens.” Cancer Research, § 798.5955 Heritable translocation test in drosophila melanogaster. (a) Purpose. Drosophila 1 2 (b) Definitions (2) Reciprocal translocations are chromosomal translocations resulting from reciprocal exchanges between two or more chromosomes. (3) Heritable translocations are reciprocal translocations transmitted from parent to the succeeding progeny. (c) Reference substances. (d) Test method Principle. 2 (2) Description. 1 2 3 (i) Illustrative example. 1 2 1 2 1 2 1 2 (ii) [Reserved] (3) Drosophila stocks. Drosophila (4) Control groups. (ii) Negative (vehicle) controls should be included. The size of the negative (vehicle) control group should be determined by the availability of appropriate laboratory historical control data. (iii) If the historical control data are of sufficient numbers, concurrent controls may not be necessary. (5) Test chemicals Vehicle. (ii) Dose levels. (iii) Route of administration. (e) Test performance P1 mating. (ii) Mass matings may be performed because the control rate for translocations in the available literature is very low (near 0) and clustered events are extremely rare. Mated females may be aged for 2 weeks in order to recover an enhanced incidence of translocation due to the storage effect. The females are then allowed to lay eggs and F 1 (2) F 1 1 1 1 1 (3) Scoring the F 2 generation. 2 1 3 (4) Number of replicate experiments. (f) Data and report Treatment of results. 1 (2) Statistical evaluation. (3) Interpretation of results. (ii) A test substance which does not produce either a statistically significant dose-related increase in the number of heritable translocations or a statistically significant and reproducible positive response at any one of the test points is considered nonmutagenic in this system. (iii) Both biological and statistical significance should be considered together in the evaluation. (4) Test evaluation. Drosophila (ii) Negative results indicate that under the test conditions the test substance does not cause chromosomal damage in D. melanogaster. (5) Test report. (i) Drosophila 2 (ii) Test chemical vehicle, treatment and mating schedule, exposure levels, toxicity data, dose and route of exposure. (iii) Positive and negative (vehicle) controls. (iv) Historical control data, if available. (v) Number of chromosomes scored. (vi) Criteria for scoring mutant chromosomes. (vii) Dose-response relationship, if applicable. (g) References. (1) Wurgler, F.E., Sobels, F.H., Vogel, E. “ Drosophila Handbook of mutagenicity test procedures. (2) [Reserved] Subpart G—Neurotoxicity § 798.6050 Functional observational battery. (a) Purpose. (b) Definitions. (2) A toxic effect is an adverse change in the structure or function of an experimental animal as a result of exposure to a chemical substance. (c) Principle of the test method. (d) Test procedures Animal selection Species and strain. (ii) Age. (iii) Sex. (B) The females shall be nulliparous and nonpregnant. (2) Number of animals. (3) Control groups. (ii) Concurrent or historic data from the laboratory performing the testing shall provide evidence of the ability of the procedures used to detect major neurotoxic endpoints such as limb weakness or paralysis (e.g., acrylamide), CNS stimulation (e.g., β, β′-iminodiproprionitrile) autonomatic signs (e.g., physostigmine). (iii) A satellite group may be treated with the high dose level for the duration of exposure and observed for reversibility, persistence, or delayed occurrence of toxic effects for a post-treatment period of appropriate duration, normally not less than 28 days. (4) Dose levels and dose selection. 1/2 (i) The highest dose shall produce (A) clear behavioral effects or (B) life-threatening toxicity. (ii) The data from the lower doses must show either (A) graded dose-dependent effects at 2 dose levels or (B) no effects at 2 dose levels, respectively. (5) Duration and frequency of exposure. (6) Route of exposure. (7) Combined protocol. (8) Study conduct. (ii) The following is a minimal list of observations that shall be noted: (A) Any unusual responses with respect to body position, activity level, coordination of movement, and gait. (B) Any unusual or bizarre behavior including, but not limited to, headflicking, head searching, compulsive biting or licking, self-mutilation, circling, and walking backwards. (C) The presence of: ( 1 ( 2 ( 3 ( 4 ( 5 ( 6 ( 7 ( 8 ( 9 ( 10 (D) Forelimb/hindlimb grip strength. The procedure described by Meyer et al. (1979), under paragraph (f)(9) of this section is recommended. (E) Sensory function. A simple assessment of sensory function (vision, audition, pain perception) shall be made. Marshall et al. (1971) under paragraph (f)(8) of this section have described a neurologic exam for this purpose; these procedures are also discussed by Deuel (1977), under paragraph (f)(4) of this section. Irwin (1968) under paragraph (f)(7) of this section described a number of reflex tests intended to detect gross sensory deficits, including the visual placing response, Preyer reflex, and tail pinch. Many procedures have been developed for assessing pain perception (e.g., Ankier, 1974 under paragraph (f)(1) of this section; D'Amour and Smith 1941 under paragraph (f)(3) of this section; Evans 1971 under paragraph (f)(6) of this section). (e) Data reporting and evaluation. (1) Description of system and test methods. (ii) Positive control data from the laboratory performing the test that demonstrate the sensitivity of the procedures being used. Historic data may be used if all aspects of the experimental protocol are the same, including personnel. (2) Results. (i) In tabular form, data for each animal must be provided showing: (A) Its identification number. (B) Its body weight and score on each sign at each observation time, the time and cause of death (if appropriate). (ii) Summary data for each group must include: (A) The number of animals at the start of the test. (B) The number of animals showing each observation score at each observation time. (C) The percentage of animals showing each abnormal sign at each observation time. (D) The mean and standard deviation for each continuous endpoint at each observation time. (3) Evaluation of data. (f) References. (1) Ankier, S.I. “New hot plate tests to quantify antinociceptic and narcotic antagonist activities,” European Journal of Pharmacology, (2) Coughenour, L.L., McLean, J.R. and Parker, R.B. “A new device for the rapid measurement of impaired motor function in mice,” Pharmacology, Biochemistry and Behavior, 6: (3) D'Amour, F.E., Smith, D.L. “A method for determining loss of pain sensation,” Journal of Pharmacology and Experimental Therapeutics, 72: (4) Deuel, R.K. “Determining sensory deficits in animals,” Methods in Psychobiology (5) Edwards, P.M., Parker, V.H. “A simple, sensitive and objective method for early assessment of acrylamide neuropathy in rats,” Toxicology and Applied Pharmacology, 40: (6) Evans, W.O. “A new technique for the investigation of some analgesic drugs on reflexive behavior in the rat,” Psychopharmacologia, 2: (7) Irwin, S. “Comprehensive observational assessment: Ia. A systematic quantitative procedure for assessing the behavioral and physiologic state of the mouse,” Psychopharmacologia, 13: (8) Marshall, J.F., Turner, B.H., Teitlbaum, P. “Sensory neglect produced by lateral hypothalamic damage,” Science, 174: (9) Meyer, O.A., Tilson, H.A., Byrd, W.C., Riley, M.T. “A method for the routine assessment of fore- and hindlimb grip strength of rats and mice,” Neurobehavioral Toxicology, 1: [50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19082, May 20, 1987] § 798.6200 Motor activity. (a) Purpose General. (2) Acute Motor Activity Test. (3) Subchronic Motor Activity Test. (b) Definitions. (2) Motor activity is any movement of the experimental animal. (3) A toxic effect is an adverse change in the structure or function of an experimental animal as a result of exposure to a chemical substance. (c) Principle of the test method. (d) Test procedures Animal selection Species and strain. (ii) Age. (iii) Sex. (B) The females shall be nulliparous and nonpregnant. (2) Number of animals. (3) Control groups. (ii) Positive control data are required to demonstrate the sensitivity and reliability of the activity measuring device and testing procedure. These data should demonstrate the ability to detect increases or decreases in activity and to generate a dose-effect curve or its equivalent using three values of the dose or equivalent independent variable. A single administration of the dose (or equivalent) is sufficient. It is recommended that chemical exposure be used to collect positive control data. Positive control data shall be collected at the time of the test study unless the laboratory can demonstrate the adequacy of historical data for this purpose. (iii) A satellite group 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. 1/2 (i) The highest dose shall produce (A) clear effects on motor activity or (B) life-threatening toxicity. (ii) The data from the lower doses must show either (A) graded dose-dependent effects at 2 dose levels or (B) no effects at 2 dose levels, respectively. (5) Duration of testing. (6) Route of administration. (7) Combined protocol. (8) Study conduct General. (ii) Acute. (iii) Subchronic. (e) Data reporting and evaluation. (1) Description of system and test methods. (ii) Procedures for calibrating and assuring the equivalence of devices and balancing treatment groups. (2) Results. (i) In tabular form, data must be provided showing for each animal: (A) Its identification number. (B) Body weight, total session activity counts, and intrasession subtotals for each date measured. (ii) Group summary data should also be reported. (3) Evaluation of data. (f) References. (1) Dixon, W.J., Massey, E.J. Introduction to Statistical Analysis (2) Finger, F.W. “Measuring behavioral activity,” Methods in Psychobiology (3) Jensen, D.R. “Some simultaneous multivariate procedures using Hotelling's T 2 Biometrics, (4) Kinnard, E.J. and Watzman, N. “Techniques utilized in the evaluation of psychotropic drugs on animals activity,” Journal of Pharmaceutical Sciences, 55:995-1012 (1966). (5) Neter, J. and Wasserman, W. Applied Linear Statistical Models. (6) Reiter, L.E. “Use of activity measures in behavioral toxicology,” Environmental Health Perspectives, (7) Reiter, L.W. and MacPhail, R.C. “Motor Activity: A survey of methods with potential use in toxicity testing,” Neurobehavioral Toxicology, (8) Robbins, T.W. “A critique of the methods available for the measurement of spontaneous motor activity,” Handbook of Psychopharmacology. (9) Sokal, R.P. and Rohlf, E.J. Biometry. [50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19082, May 20, 1987] § 798.6400 Neuropathology. (a) Purpose. (b) Definition. (c) Principle of the test method. (d) Test procedure Animal selection Species and strain. (ii) Age. (iii) Sex. (2) Number of animals. (3) Control groups. (ii) A satellite group of animals may be treated with the high 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. 1/2 (i) The highest dose shall produce (A) clear behavioral effects or (B) life-threatening toxicity. (ii) The data from the lower doses must show either (A) graded dose-dependent effects at two dose levels or (B) no effects at two dose levels, respectively. (5) Duration of testing. (6) Route of administration. (7) Combined protocol. (8) Study conduct Observation of animals. (ii) Sacrifice of animals General. (B) Perfusion technique. in situ (C) Removal of brain and cord. (D) Sampling. 3 6 1 4 3 6 1 4 3 6 1 4 (iii) Specimen storage. (iv) Histopathology examination. Fixation. (B) Dehydration. (C) Clearing and embedding. (D) Sectioning. (E) Histopathological techniques. ( 1 General staining. ( 2 Special stains. ( 3 Alternative technique. ( 4 Electron microscopy. (F) Examination 1 General. ( 2 Electron microscopy. ( i Neuronal body. ( ii Neuronal processes. ( iii Supporting cells. (e) Data collection, reporting, and evaluation. (1) Description of test system and test methods. (2) Results. (i) Description of signs and lesions for each animal. (ii) Counts and incidence of lesions, by test group. (A) The number of animals used in each group, the number of animals displaying specific neurologic signs, and the number of animals in which any lesion was found; (B) The number of animals affected by each different type of lesion, the average grade of each type of lesion, and the frequency of each different type and/or location of lesion. (iii) Evaluation of data. (B) The evaluation of dose-response, if existent, for various groups shall be given, and a description of statistical method must be presented. The evaluation of neuropathology data should include, where applicable, an assessment in conjunction with other neurotoxicity studies performed (eg. electrophysiological, behavioral, neurochemical). (f) References. (1) AFIP. Manual of Histologic Staining Methods. (2) Chang, L.W. A Color Atlas and Manual for Applied Histochemistry. (3) Hayat, M.A. “Vol. 1. Biological applications,” Principles and techniques of electron microscopy. (4) Palay S.L., Chan-Palay, V. Cerebellar Cortex: Cytology and Organization. (5) Ralis, H.M., Beesley, R.A., Ralis, Z.A. Techniques in Neurohistology. (6) Spencer, P.S., Schaumburg, H.H. (eds). Experimental and Clinical Neurotoxicology. (7) Zeman, W., JRM Innes, J.R.M. Craigie's Neuroanatomy of the Rat. [50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19082, May 20, 1987] § 798.6500 Schedule-controlled operant behavior. (a) Purpose. (2) This guideline defines procedures for conducting studies of schedule-controlled operant behavior, one way of evaluating functional neurotoxic effects (Dews, 1972 under paragraph (f)(1) of this section; NAS 1975, 1977, 1982 under paragraphs (f)(4), (5) and (6) of this section). Our purpose is to evaluate the effects of acute and repeated exposures on the rate and pattern of responding under schedules of reinforcement. Operant behavior tests may be used to evaluate many other aspects of behavior (Laties, 1978 under paragraph (f)(3) of this section). Additional tests may be necessary to completely assess the behavioral effects of any substance. Behavioral evaluation should be used in conjunction with neuropathologic evaluation and the evaluation of other toxic effects. (b) Definitions Neurotoxicity. (2) Operant, operant behavior, operant conditioning. (3) Schedule of reinforcement. (c) Principle of the test method. (d) Test procedures Experimental design. (2) Animal selection Species. (B) Under some circumstances other species may be recommended. (ii) Age. (iii) Sex. (B) Virgin females should be used. (iv) Experimental history. (3) Number of animals. (4) Control groups Untreated controls. (ii) Positive controls. (5) Dose levels and dose selection. (i) The highest dose shall produce: (A) Clear behavioral effects; or (B) life-threatening toxicity. (ii) The data from the lower doses must show either: (A) Graded dose-dependent effects at 2 dose levels; or (B) no effects at 2 dose levels, respectively. (6) Duration of exposure. (7) Route of Administration. (8) Study conduct Apparatus. (ii) Chamber assignment. (iii) Deprivation and training. (B) Subjects must be trained until they display demonstrable stability in performance across days prior to exposure. One simple and useful criterion is a minimum number of sessions on the schedule and no systematic trend during the 5 days before exposure. (C) Cumulative records of cumulative responding over time for each animal should be presented to demonstrate that the pattern of responding is representative of that generated by the schedule of reinforcement. (iv) Time, frequency, and duration of testing Time of testing. (B) Frequency of testing. (C) Duration of testing. 1 ( 2 (v) Schedule selection. (e) Data reporting and evaluation. (1) Description of system, test methods, experimental design, and control data. (ii) A description of the experimental design including counterbalancing procedures, and the stability criterion. (iii) A description and statistical evaluation of positive control and other control data, including standard measures of central tendency, variability, coefficient of variation of response rates, and the slope of the dose-effect curve. (2) Results. (ii) A description and statistical evaluation of the test results: With particular reference to the overall statistical procedures (e.g., parametric or nonparametric) dose-effect curve, and calculation of slope. Presentation of calculations is encouraged. (f) References. (1) Dews, P.B. “Assessing the Effects of Drugs,” Methods in Psychobiology, (2) Ferster, C.B. Skinner, B.F. Schedules of Reinforcement. (New York: Appleton-Century-Crofts, 1957). (3) Laties, V.G. “How Operant Conditioning can Contribute to Behavioral Toxicology,” Environmental Health Perspectives, 28: 29-35 (1978). (4) National Academy of Science. Principles for Evaluating Chemicals in the Environment. (5) National Academy of Science. Principles and Procedures for Evaluating the Toxicity of Household Substances. (6) National Academy of Science. “Strategies to determine needs and priorities for toxicity testing,” Appendix 3B. Reference Protocol Guidelines For Neurobehavioral Toxicity Tests. § 798.6560 Subchronic delayed neuro-toxicity of organophosphorus substances. (a) Purpose. (b) Definitions. (c) Principle of the test method. Gallus gallus domesticus (d) Test procedures Animal selection. (2) Number of animals. (3) Control group General. (ii) Reference substances. (4) Housing and feeding conditions. (5) Dose levels. (6) Route of administration. (7) Study conduct General. (ii) Pathology Gross necropsy. (B) Histopathology. in situ, (e) Data reporting and evaluation Test report. (i) Toxic response data by group with a description of clinical manifestations of nervous system damage; where a grading system is used the criteria should be defined. (ii) For each animal, time of death during the study or whether it survived to termination. (iii) The day of observation of each abnormal sign and its subsequent course. (iv) Body weight data. (v) Necropsy findings for each animal, when performed. (vi) A detailed description of all histopathological findings. (vii) Statistical treatment of results, where appropriate. (2) Treatment of results. (ii) All observed results 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. (3) Evaluation of results. (f) References. (1) Abou-Donia, M.B. “Organophosphorus ester-induced delayed neurotoxicity” Annual Review of Pharmacology and Toxicology, 21:511-548 (1981). (2) Abou-Donia, M.B., Pressing, S.H. “Delayed neurotoxicity from continuous low-dose oral administration of leptophos to hens.” Toxicology and Applied Pharmacology, (3) Baron, R.L. (ed). “Pesticide Induced Delayed Neurotoxicity,” Proceedings of a Conference, February 19-20, 1976, Washington, DC. U.S. Environmental Protection Agency. EPA Report No. 600/1-76-025, Washington, DC (1976). (4) Cavanaugh, J.B. “Peripheral neuropathy caused by chemical agents” Critical Reviews of Toxicity, (5) Johannsen, F.R., Wright, P.L., Gordon, D.E., Levinskas, G.L., Radue, R.W., Graham, P.R. “Evaluation of delayed neurotoxicity and dose-response relationship of phosphate esters in the adult hen,” Toxicology and Applied Pharmacology, (6) Johnson, M.K. “Organophosphorus esters causing delayed neurotoxic effects: mechanism of action and structure/activity studies,” Archives of Toxicology,

Related documents

• 50 CFR Part 600 — Magnuson-Stevens Act Provisions#512148
• 50 CFR Part 622 — Fisheries of the Caribbean, Gulf of America, and South Atlantic#512149
• 50 CFR Part 635 — Atlantic Highly Migratory Species#512150
• 50 CFR Part 648 — Fisheries of the Northeastern United States#512151
• 50 CFR Part 660 — Fisheries off West Coast States#512152