Title 40—Protection of Environment--Volume 1
PART 798—HEALTH EFFECTS TESTING GUIDELINES
§ 798.2250   Dermal toxicity.
(a) Purpose. In the assessment and evaluation of the toxic characteristics of a chemical, the determination of subchronic dermal toxicity may be carried out after initial information on toxicity has been obtained by acute testing. The subchronic dermal study has been designed to permit the determination of the no-observed-effect level and toxic effects associated with continuous or repeated exposure to a test substance for a period of 90 days. The test is not capable of determining those effects that have a long latency period for development (e.g., carcinogenicity and life shortening). It provides information on health hazards likely to arise from repeated exposure by the dermal route over a limited period of time. It will provide information on target organs, the possibilities of accumulation, and can be of use in selecting dose levels for chronic studies and for establishing safety criteria for human exposure.
(b) Definitions. (1) Subchronic dermal toxicity is the adverse effects occurring as a result of the repeated daily exposure of experimental animals to a chemical by dermal application for part (approximately 10 percent) of a life span.
(2) Dose in a dermal test is the amount of test substance applied to the skin (applied daily in subchronic tests). Dose is expressed as weight of the substance (g, mg) per unit weight of test animal (e.g., mg/kg).
(3) No-effect level/No-toxic-effect level/No-adverse-effect level/No-observed-effect level is the maximum dose used in a test which produces no observed adverse effects. A no-observed-effect level is expressed in terms of the weight of a test substance given daily per unit weight of test animal (mg/kg).
(4) Cumulative toxicity is the adverse effects of repeated doses occurring as a result of prolonged action on, or increased concentration of the administered test substance or its metabolites in susceptible tissues.
(c) Principle of the test method. The test substance is applied daily to the skin in graduated doses to several groups of experimental animals, one dose level per unit group, for a period of 90 days. During the period of application the animals are observed daily to detect signs of toxicity. Animals which die during the test are necropsied, and at the conclusion of the test the surviving animals are sacrificed and necropsied and appropriate histopathological examinations carried out.
(d) Limit test. If a test at one dose level of at least 1,000 mg/kg body weight (expected human exposure may indicate the need for a higher dose level), using the procedures described for this study, produces no observable toxic effects and if toxicity would not be expected based upon data of structurally related compounds, then a full study using three dose levels might not be necessary.
(e) Test procedures —(1) Animal selection —(i) Species and strain. A mammalian species shall be used for testing. The rat, rabbit, or guinea pig may be used, although the albino rabbit is preferred. The albino rabbit is preferred because of its size, skin permeability, and extensive data base. Commonly used laboratory strains shall be employed. If another mammalian species is used, the tester shall provide justification/reasoning for its selection.
(ii) Age. Young adult animals shall be used. The following weight ranges at the start of the test are suggested in order to provide animals of a size which facilitates the conduct of the test: rats, 200 to 300 g; rabbits, 2.0 to 3.0 kg; guinea pigs, 350 to 450 g.
(iii) Sex. (A) Equal numbers of animals of each sex with healthy skin shall be used at each dose level.
(B) The females shall be nulliparous and nonpregnant.
(iv) Numbers. (A) At least 20 animals (10 females and 10 males) shall be used at each dose level.
(B) If interim sacrifices are planned, the number shall be increased by the number of animals scheduled to be sacrificed before completion of the study.
(2) Control groups. A concurrent control group is required. This group shall be an untreated or sham-treated control group or, if a vehicle is used in administering the test substance, a vehicle control group. If the toxic properties of the vehicle are not known or cannot be made available, both untreated and vehicle control groups are required.
(3) Satellite group. A satellite group of 20 animals (10 animals per sex) may be treated with the high dose level for 90 days and observed for reversibility, persistence, or delayed occurrence, of toxic effects for a posttreatment period of appropriate length, normally not less than 28 days.
(4) Dose level and dose selection. (i) In subchronic toxicity tests, it is desirable to have a dose-response relationship as well as a no-observed-toxic-effect level. Therefore, at least 3 dose levels with a control and, where appropriate, a vehicle control (corresponding to the concentration of vehicle at the highest exposure level) shall be used. Doses should be spaced appropriately to produce test groups with a range of toxic effects. The data shall be sufficient to produce a dose-response curve.
(ii) The highest dose level should result in toxic effects but not produce severe skin irritation or an incidence of fatalities which would prevent a meaningful evaluation.
(iii) The lowest dose level should not produce any evidence of toxicity. Where there is a usable estimation of human exposure, the lowest dose level should exceed this.
(iv) Ideally, the intermediate dose level(s) should produce minimal observable toxic effects. If more than one intermediate dose is used, the dose levels should be spaced to produce a gradation of toxic effects.
(v) In the low and intermediate groups and in the controls the incidence of fatalities should be low, to permit a meaningful evaluation of the results.
(5) Exposure conditions. The animals are treated with test substance, ideally for at least 6 hours per day on a 7-day per week basis, for a period of 90 days. However, based primarily on practical considerations, application on a 5-day per week basis is considered to be acceptable.
(6) Observation period. (i) Duration of observation shall be at least 90 days.
(ii) Animals in the satellite group scheduled for followup observations should be kept for at least 28 days further without treatment to detect recovery from, or persistence of, toxic effects.
(7) Preparation of animal skin. (i) Shortly before testing, fur shall be clipped from the dorsal area of the trunk of the test animals. Shaving may be employed, but it should be carried out approximately 24 hours before the test. Repeat clipping or shaving is usually needed at approximately weekly intervals. When clipping or shaving the fur, care should be taken to avoid abrading the skin, which could alter its permeability.
(ii) Not less than 10 percent of the body surface area should be clear for the application of the test substance. The weight of the animal should be taken into account when deciding on the area to be cleared and on the dimensions of any covering used.
(iii) When testing solids, which may be pulverized if appropriate, the test substance should be moistened sufficiently with water or, where necessary, a suitable vehicle to ensure good contact with the skin. When a vehicle is used, the influence of the vehicle on toxicity of and penetration of the skin by the test substance should be taken into account.
(8) Application of the test substance. (i) The test substance shall be applied uniformly over an area which is approximately 10 percent of the total body surface area. With highly toxic substances, the surface area covered may be less, but as much of the area shall be covered with as thin and uniform a film as possible.
(ii) During the exposure period, the test substance shall be held in contact with the skin with a porous gauze dressing and nonirritating tape. The test site shall be further covered in a suitable manner to retain the gauze dressing and test substance and ensure that the animals cannot ingest the test substance. Restrainers may be used to prevent the ingestion of the test substance, but complete immobilization is not a recommended method.
(9) Observation of animals. (i) Each animal shall be observed daily, and if necessary handled to appraise its physical condition.
(ii) Additional observations shall be made daily with appropriate actions taken to minimize loss of animals to the study (e.g., necropsy or refrigeration of those animals found dead and isolation or sacrifice of weak or moribund animals).
(iii) Signs of toxicity shall be recorded as they are observed, including the time of onset, the degree, and duration.
(iv) Cage-side observations shall include, but not be limited to, changes in skin and fur, eyes and mucous membranes, respiratory, circulatory, autonomic and central nervous systems, somatomotor activity and behavior pattern.
(v) Animals shall be weighed weekly. Feed consumption shall also be determined weekly if abnormal body weight changes are observed.
(vi) At the end of the study period, all survivors in the nonsatellite treatment groups shall be sacrificed. Moribund animals shall be removed and sacrificed when noticed.
(10) Clinical examinations. (i) The following examinations shall be made on all animals of each sex in each group:
(A) Certain hematology determinations shall be carried out at least two times during the test period on all groups of animals including concurrent controls: After 30 days of test and just prior to terminal sacrifice at the end of the test period. Hematology determinations which are appropriate to all studies: Hematocrit, hemoglobin concentration, erythrocyte count, total and differential leukocyte count, and a measure of clotting potential such as clotting time, prothrombin time, thromboplastin time, or platelet count.
(B) Certain clinical biochemistry determinations on blood should be carried out at least two times during the test period on all groups of animals including concurrent controls: After 30 days of test and just prior to terminal sacrifice at the end of the test period. Clinical biochemistry test areas which are considered appropriate to all studies: Electrolyte balance, carbohydrate metabolism, and liver and kidney function. The selection of specific tests will be influenced by observations on the mode of action of the substance. Suggested determinations: Calcium, phosphorus, chloride, sodium, potassium, fasting glucose (with period of fasting appropriate to the species), serum glutamic pyruvic transaminase (now known as serum alanine aminotransferase), serum glutamic oxaloacetic transaminase (now known as serum aspartate aminotransferase), ornithine decarboxylase, gamma glutamyl transpeptidase, urea nitrogen, albumen blood creatinine, total bilirubin, and total serum protein measurements. Other determinations which may be necessary for an adequate toxicological evaluation include: Analyses of lipids, hormones, acid/base balance, methemoglobin, and cholinesterase activity. Additional clinical biochemistry may be employed, where necessary, to extend the investigation of observed effects.
(ii) The following examinations shall be made on high dose and control groups. If changes in the eyes are detected all animals should be examined.
(A) Ophthalmological examination, using an ophthalmoscope or equivalent suitable equipment, shall be made prior to exposure to the test substance and at the termination of the study.
(B) Urinalysis is not recommended on a routine basis, but only when there is an indication based on expected or observed toxicity.
(11) Gross necropsy. (i) All animals shall be subjected to a full gross necropsy which includes examination of the external surface of the body, all orifices, and the cranial, thoracic, and abdominal cavities and their contents.
(ii) The liver, kidneys, adrenals, brain, and gonads shall be weighed wet, as soon as possible after dissection, to avoid drying. In addition, for the rodent, the brain; for the non-rodent, the thyroid with parathyroids also shall be weighed wet.
(iii) The following organs and tissues, or representative samples thereof, shall be preserved in a suitable medium for possible future histopathological examination: All gross lesions; lungs—which should be removed intact, weighed, and treated with a suitable fixative to ensure that lung structure is maintained (perfusion with the fixative is considered to be an effective procedure); nasopharyngeal tissues; brain—including sections of medulla/pons, cerebellar cortex, and cerebral cortex; pituitary; thyroid/parathyroid; thymus; trachea; heart; sternum with bone marrow; salivary glands; liver; spleen; kidneys; adrenals; pancreas; gonads; uterus; accessory genital organs (epididymis, prostate, and, if present, seminal vesicles); aorta; (skin); gall bladder (if present); esophagus; stomach; duodenum; jejunum; ileum; cecum; colon; rectum; urinary bladder; representative lymph node; (mammary gland); (thigh musculature); peripheral nerve; (eyes); (femur—including articular surface); (spinal cord at three levels—cervical, midthoracic, and lumbar); and (zymbal and exorbital lachrymal glands).
(12) Histopathology. The following histopathology shall be performed:
(i) Full histopathology on normal and treated skin and on organs and tissues, listed above, of all animals in the control and high dose groups.
(ii) All gross lesions in all animals.
(iii) Target organs in all animals.
(iv) The tissues listed in parenthesis in paragraph (e)(11)(iii) of this section, if indicated by signs of toxicity or expected target organ involvement.
(v) Lungs of animals (rodents) in the low and intermediate dose groups shall be subjected to histopathological examination for evidence of infection, since this provides a convenient assessment of the state of health of the animals.
(vi) When a satellite group is used, histopathology shall be performed on tissues and organs identified as showing effects in the treated groups.
(f) Data and reporting —(1) Treatment of results. (i) Data shall be summarized in tabular form, showing for each test group the number of animals at the start of the test, the number of animals showing lesions, the types of lesions, and the percentage of animals displaying each type of lesion.
(ii) All observed results, quantitative and incidental, should be evaluated by an appropriate statistical method. Any generally accepted statistical method may be used; the statistical methods should be selected during the design of the study.
(2) Evaluation of results. The findings of a subchronic dermal toxicity study should be evaluated in conjunction with the findings of preceding studies and considered in terms of the observed toxic effects and the necropsy and histopathological findings. The evaluation should include the relationship between the dose of the test substance and the presence or absence, the incidence and severity, of abnormalities, including behavioral and clinical abnormalities, gross lesions, identified target organs, body weight changes, effect on mortality and any other general or specific toxic effects. A properly conducted subchronic test should provide a satisfactory estimation of a no-effect level.
(3) Test report. In addition to the reporting requirements as specified in the EPA Good Laboratory Practice Standards under 40 CFR part 792, subpart J, the following specific information shall be reported.
(i) Group animal data. Tabulation of toxic response data by species, strain, sex and exposure level for:
(A) Number of animals dying.
(B) Number of animals showing signs of toxicity.
(C) Number of animals exposed.
(ii) Individual animal data. (A) Date of death during the study or whether animals survived to termination.
(B) Date of observation of each abnormal sign and its subsequent course.
(C) Body weight data.
(D) Feed consumption data when collected.
(E) Hematological tests employed and all results.
(F) Clinical biochemistry tests employed and all results.
(G) Necropsy findings.
(H) Detailed description of all histopathological findings.
(I) Statistical treatment of results where appropriate.
(g) References. For additional background information on this test guideline the following references should be consulted:
(1) Draize, J.H. “Dermal toxicity,” Appraisal of Chemicals in Food, Drugs and Cosmetics. The Association of Food and Drug Officials of the United States (1959, 3rd printing 1975). pp. 46-59.
(2) Fitzhugh, O.G. “Subacute toxicity,” Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. The Association of Food and Drug Officials of the United States (1959, 3rd printing 1975). pp. 26-35.
(3) National Academy of Sciences. “Principles and Procedures for Evaluating the Toxicity of Household Substances,” a report prepared by the Committee for the Revision of NAS Publication 1138, under the auspices of the Committee on Toxicology, National Research Council, National Academy of Sciences, Washington, DC (1977).
(4) World Health Organization. “Part I. Environmental Health Criteria 6,” Principles and Methods for Evaluating the Toxicity of Chemicals. (Geneva: World Health Organization, 1978).
§ 798.2450   Inhalation toxicity.
(a) Purpose. In the assessment and evaluation of the toxic characteristics of a gas, volatile substance, or aerosol/particulate, determination of subchronic inhalation toxicity may be carried out after initial information on toxicity has been obtained by acute testing. The subchronic inhalation study has been designed to permit the determination of the no-observed-effect level and toxic effects associated with continuous or repeated exposure to a test substance for a period of 90 days. The test is not capable of determining those effects that have a long latency period for development (e.g., carcinogenicity and life shortening). It provides information on health hazards likely to arise from repeated exposures by the inhalation route over a limited period of time. It will provide information on target organs, the possibilities of accumulation, and can be of use in selecting dose levels for chronic studies and for establishing safety criteria for human exposure. Hazards of inhaled substances are influenced by the inherent toxicity and by physical factors such as volatility and particle size.
(b) Definitions. (1) Subchronic inhalation toxicity is the adverse effects occurring as a result of the repeated daily exposure of experimental animals to a chemical by inhalation for part (approximately 10 percent) of a life span.
(2) Aerodynamic diameter applies to the size of particles of aerosols. It is the diameter of a sphere of unit density which behaves aerodynamically as the particle of the test substance. It is used to compare particles of different size and densities and to predict where in the respiratory tract such particles may be deposited. This term is used in contrast to measured or geometric diameter which is representative of actual diameters which in themselves cannot be related to deposition within the respiratory tract.
(3) The geometric mean diameter or the median diameter is the calculated aerodynamic diameter which divides the particles of an aerosol in half based on the weight of the particles. Fifty percent of the particles by weight will be larger than the median diameter and 50 percent of the particles will be smaller than the median diameter. The median diameter describes the particle size distribution of any aerosol based on the weight and size of the particles.
(4) Inhalable diameter refers to that aerodynamic diameter of a particle which is considered to be inhalable for the organism. It is used to refer to particles which are capable of being inhaled and may be deposited anywhere within the respiratory tract from the trachea to the alveoli. For man, inhalable diameter is considered as 15 micrometers or less.
(5) Dose refers to an exposure level. Exposure is expressed as weight or volume of test substance per volume of air (mg/l), or as parts per million (ppm).
(6) No-effect level/No-toxic-effect level/No-adverse-effect level/No-observed-effect level is the maximum dose used in a test which produces no observed adverse effects. A no-observed-effect level is expressed in terms of weight or volume of test substance given daily per unit volume of air (mg/l or ppm).
(7) Cumulative toxicity is the adverse effects of repeated doses occuring as a result of prolonged action on, or increased concentration of the administered test substance or its metabolites in susceptible tissues.
(c) Principle of the test method. Several groups of experimental animals are exposed daily for a defined period to the test substance in graduated concentrations, one concentration being used per group, for a period of 90 days. During the period of administration, the animals are observed daily to detect signs of toxicity. Animals which die during the test are necropsied and at the conclusion of the test, surviving animals are sacrificed and necropsied and appropriate histopathological examinations carried out.
(d) Test procedures —(1) Animal selection —(i) Species and strain. A mammalian species shall be used for testing. A variety of rodent species may be used, although the rat is the preferred species. Commonly used laboratory strains shall be employed. If another mammalian species is used, the tester shall provide justification/ reasoning for its selection.
(ii) Age. Young adult animals shall be used. At the commencement of the study the weight variation of animals shall not exceed ±20 percent of the mean weight for each sex.
(iii) Sex. (A) Equal numbers of animals of each sex shall be used at each dose level.
(B) Females shall be nulliparous and nonpregnant.
(iv) Numbers. (A) At least 20 rodents (10 females and 10 males) shall be used for each test group. If another mammalian species is selected (e.g. dog, rabbit, or non-human primate), at least 8 animals (4 males and 4 females) shall be used.
(B) If interim sacrifices are planned, the number of animals shall be increased by the number of animals scheduled to be sacrificed before the completion of the study.
(2) Control groups. A concurrent control group is required. This group shall be an untreated or sham-treated control group. Except for treatment with the test substance, animals in the control group shall be handled in a manner identical to the test group animals. Where a vehicle is used to help generate an appropriate concentration of the substance in the atmosphere, a vehicle control group shall be used. If the toxic properties of the vehicle are not known or cannot be made available, both untreated and vehicle control groups are required.
(3) Satellite group. A satellite group of 20 animals (10 animals per sex) may be treated with the high concentration level for 90 days and observed for reversibility, persistence, or delayed occurrence of toxic effects for a post-treatment period of appropriate length, normally not less than 28 days.
(4) Dose levels and dose selection. (i) In subchronic toxicity tests, it is desirable to have a concentration-response relationship as well as a no-observed-toxic-effect level. Therefore, at least 3 concentration levels with a control and, where appropriate, a vehicle control (corresponding to the concentration of vehicle at the highest exposure level) shall be used. Concentrations should be spaced appropriately to produce test groups with a range of toxic effects. The data should be sufficient to produce a concentration-response curve.
(ii) The highest concentration should result in toxic effects but not produce an incidence of fatalities which would prevent a meaningful evaluation.
(iii) The lowest concentration should not produce any evidence of toxicity. Where there is a usable estimation of human exposure the lowest concentration should exceed this.
(iv) Ideally, the intermediate concentration level(s) should produce minimal observable toxic effects. If more than one intermediate concentration level is used, the concentrations should be spaced to produce a gradation of toxic effects.
(v) In the low and intermediate groups and in the controls the incidence of fatalities should be low, to permit a meaningful evaluation of the results.
(vi) In the case of potentially explosive test substances, care should be taken to avoid generating explosive concentrations.
(5) Exposure conditions. The animals should be exposed to the test substance, ideally for 6 hours per day on a 7-day per week basis, for a period of 90 days. However, based primarily on practical considerations, exposure on a 5-day-per-week basis for 6 hours per day is the minimum acceptable exposure period.
(6) Observation period. (i) Duration of observation shall be for at least 90 days.
(ii) Animals in a satellite group scheduled for followup observations should be kept for at least 28 days further without treatment to detect recovery from, or persistence of, toxic effects.
(7) Inhalation exposure. (i) The animals shall be tested in inhalation equipment designed to sustain a minimum dynamic air flow of 12 to 15 air changes per hour and ensure an adequate oxygen content of 19 percent and an evenly distributed exposure atmosphere. Where a chamber is used, its design should minimize crowding of the test animals and maximize their exposure to the test substance. This is best accomplished by individual caging. To ensure stability of a chamber atmosphere, the total “volume” of the test animals shall not exceed 5 percent of the volume of the test chamber. Oronasal or head-only exposure may be used if it is desirable to avoid concurrent exposure by the dermal or oral routes.
(ii) A dynamic inhalation system with a suitable flow control system shall be used. The rate of air flow shall be adjusted to ensure that conditions throughout the exposure chamber are essentially the same. Maintenance of slight negative pressure inside the chamber will prevent leakage of the test substance into surrounding areas.
(iii) The temperature at which the test is performed should be maintained at 22 °C (±2°). Ideally, the relative humidity should be maintained between 40 to 60 percent, but in certain instances (e.g., tests of aerosols, use of water vehicle) this may not be practicable.
(8) Physical measurements. Measurements or monitoring shall be made of the following:
(i) The rate of air flow shall be monitored continuously and recorded at least every 30 minutes.
(ii) The actual concentrations of the test substance shall be measured in the breathing zone. During the exposure period the actual concentrations of the test substance shall be held as constant as practicable, monitored continuously or intermittently depending on the method of analysis, and recorded at least at the beginning, at an intermediate time, and at the end of the exposure period.
(iii) During the development of the generating system, particle size analysis shall be performed to establish the stability of aerosol concentrations with respect to particle size. During exposure, analysis shall be conducted as often as necessary to determine the consistency of particle size distribution.
(iv) Temperature and humidity shall be monitored continuously but shall be recorded at least every 30 minutes.
(9) Feed and water during exposure period. Feed shall be withheld during exposure. Water may also be withheld during exposure.
(10) Observation of animals. (i) Each animal shall be observed daily and, if necessary, handled to appraise its physical condition.
(ii) Additional observations should be made daily with appropriate actions taken to minimize loss of animals to the study (e.g., necropsy or refrigeration of those animals found dead and isolation or sacrifice of weak or moribund animals).
(iii) Signs of toxicity shall be recorded as they are observed including the time of onset, the degree, and duration.
(iv) Cage-side observations should include, but not be limited to, changes in the skin and fur, eyes and mucous membranes, respiratory, circulatory, autonomic and central nervous systems, somatomotor activity and behavior pattern.
(v) Animals shall be weighed weekly. Feed consumption shall also be determined weekly if abnormal body weight changes are observed.
(vi) At the end of the study period all survivors in the nonsatellite treatment groups shall be sacrificed. Moribund animals shall be removed and sacrificed when noticed.
(11) Clinical examinations. (i) The following examinations shall be made on all animals of each sex in each group:
(A) Certain hematology determinations shall be carried out at least two times during the test period on all groups of animals including concurrent controls: After 30 days of test and just prior to terminal sacrifice at the end of the test period. Hematology determinations which are appropriate to all studies: Hematocrit, hemoglobin concentration, erythrocyte count, total and differential leukocyte count, and a measure of clotting potential such as clotting time, prothrombin time, thromboplastin time, or platelet count.
(B) Certain clinical biochemistry determinations on blood should be carried out at least two times during the test period on all groups of animals including concurrent controls: After 30 days of test and just prior to terminal sacrifice at the end of the test period. Clinical biochemistry test areas which are considered appropriate to all studies: Electrolyte balance, carbohydrate metabolism, and liver and kidney function. The selection of specific tests will be influenced by observations on the mode of action of the substance. Suggested determinations: calcium, phosphorus, chloride, sodium, potassium, fasting glucose (with period of fasting appropriate to the species), serum glutamic-pyruvic transaminase, (now known as serum alanine aminotransferase), serum glutamic-oxaloacetic transaminase (now known as serum aspartate aminotransferase), ornithine decarboxylase, gamma glutamyl transpeptidase, urea nitrogen, albumen, blood creatinine, total bilirubin, and total serum protein measurements. Other determinations which may be necessary for an adequate toxicological evaluation include: Analyses of lipids, hormones, acid/base balance, methemoglobin, and cholinesterase activity. Additional clinical biochemistry may be employed, where necessary, to extend the investigation of observed effects.
(ii) The following examinations shall be made on high dose and control groups. If changes in the eyes are detected, all animals shall be examined:
(A) Ophthalmological examination, using an ophthalmoscope or equivalent suitable equipment, shall be made prior to exposure to the test substance and at the termination of the study.
(B) Urinalysis is not recommended on a routine basis, but only when there is an indication based on expected and/or observed toxicity.
(12) Gross pathology. (i) All animals shall be subjected to a full gross necropsy which includes examination of the external surface of the body, all orifices and the cranial, thoracic, and abdominal cavities and their contents.
(ii) At least the liver, kidneys, adrenals, brain, and gonads shall be weighed wet, as soon as possible after dissection to avoid drying. In addition, for the rodent, the brain; for the non-rodent, the thyroid with parathyroids also shall be weighed wet.
(iii) The following organs and tissues, or representative samples thereof, shall be preserved in a suitable medium for possible future histopathological examination: All gross lesions; lungs—which should be removed intact, weighed, and treated with a suitable fixative to ensure that lung structure is maintained (perfusion with the fixative is considered to be an effective procedure); nasopharyngeal tissues; brain—including sections of medulla/pons cerebellar cortex and cerebral cortex; pituitary; thyroid/parathyroid; thymus; trachea; heart; sternum with bone marrow; salivary glands; liver; spleen; kidneys; adrenals; pancreas; gonads; uterus; accessory genital organs (epididymis, prostate, and, if present, seminal vesicles); aorta; (skin); gall bladder (if present); esophagus; stomach; duodenum; jejunum; ileum; cecum; colon; rectum; urinary bladder; representative lymph node; (mammary gland); (thigh musculature); peripheral nerve; (eyes); (femur—including articular surface); (spinal cord at three levels—cervical, midthoracic, and lumbar); and (zymbal and exorbital lachrymal glands).
(13) Histopathology. The following histopathology shall be performed:
(i) Full histopathology on the respiratory tract and other organs and tissues, listed above, of all animals in the control and high dose groups.
(ii) All gross lesions in all animals.
(iii) Target organs in all animals.
(iv) The tissues mentioned in brackets (listed above) if indicated by signs of toxicity or target organ involvement.
(v) Lungs of animals (rodents) in the low and intermediate dose groups shall also be subjected to histopathological examination, primarily for evidence of infection since this provides a convenient assessment of the state of health of the animals.
(vi) When a satellite group is used, histopathology shall be performed on tissues and organs identified as showing effects in the treated groups.
(e) Data and reporting —(1) Treatment of results. (i) Data shall be summarized in tabular form, showing for each test group the number of animals at the start of the test, the number of animals showing lesions, the types of lesions, and the percentage of animals displaying each type of lesion.
(ii) All observed results, quantitative and incidental, should be evaluated by an appropriate statistical method. Any generally accepted statistical method may be used; the statistical methods should be selected during the design of the study.
(2) Evaluation of results. The findings of the subchronic inhalation toxicity study should be evaluated in conjunction with the findings of preceding studies and considered in terms of the observed toxic effects and the necropsy and histopathological findings. The evaluation will include the relationship between the concentration of the test substance and duration of exposure, and the presence or absence, the incidence and severity, of abnormalities, including behavioral and clinical abnormalities, gross lesions, identified target organs, body weight changes, effects on mortality and any other general or specific toxic effects. A properly conducted subchronic test should provide a satisfactory estimation of a no-effect level.
(3) Test report. In addition to the reporting requirements as specified under EPA Good Laboratory Practice Standards, 40 CFR part 792, subpart J, the following specific information shall be reported:
(i) Test conditions. (A) Description of exposure apparatus, including design, type, dimensions, source of air, system for generating particulates and aerosols, method of conditioning air, treatment of exhaust air, and the method of housing animals in a test chamber.
(B) The equipment for measuring temperature, humidity, and particulate aerosol concentrations and size shall be described.
(ii) Exposure data. These shall be tabulated and presented with mean values and measure of variability (e.g., standard deviation) and shall include:
(A) Airflow rates through the inhalation equipment.
(B) Temperature and humidity of air.
(C) Nominal concentration (total amount of test substance fed into the inhalation equipment divided by volume of air).
(D) Actual concentration in test breathing zone.
(E) Particle size distribution (e.g., median aerodynamic diameter of particles with standard deviation from the mean).
(iii) Group animal data. Tabulation of toxic response data by species, strain, sex, and exposure level for:
(A) Number of animals dying.
(B) Number of animals showing signs of toxicity.
(C) Number of animals exposed.
(iv) Individual animal data. (A) Date of death during the study or whether animals survived to termination.
(B) Date of observation of each abnormal sign and its subsequent course.
(C) Body weight data.
(D) Feed consumption data when collected.
(E) Hematological tests employed and all results.
(F) Clinical biochemistry tests employed and all results.
(G) Necropsy findings.
(H) Detailed description of all histopathological findings.
(I) Statistical treatment of results where appropriate.
(f) References. For additional background information on this test guideline the following references should be consulted:
(1) Cage, J.C. “Experimental Inhalation Toxicology,” Methods in Toxicology. Ed. G.E. Paget. (Philadelphia: F.A. Davis Co. 1970, pp. 258-277.
(2) Casarett, L.J., Doull, J. “Chapter 9.” Toxicology: The Basic Science of Poisons (New York: Macmillan Publishing Co. Inc. 1975).
(3) MacFarland, H.N. “Respiratory Toxicology,” Essays in Toxicology. Ed. W.J. Hayes. Vol. 7 (New York: Academic Press, 1976) pp. 121-154.
(4) National Academy of Sciences. “Principles and Procedures for Evaluating the Toxicity of Household Substances,” a report prepared by the Committee for the Revision of NAS Publication 1138, under the auspices of the Committee on Toxicology, National Research Council, National Academy of Sciences, Washington, DC (1977).
(5) World Health Organization. “Part I. Environmental Health Criteria 6,” Principles and Methods for Evaluating the Toxicity of Chemicals. (Geneva: World Health Organization, 1978).
§ 798.2650   Oral toxicity.
(a) Purpose. In the assessment and evaluation of the toxic characteristics of a chemical, the determination of subchronic oral toxicity may be carried out after initial information on toxicity has been obtained by acute testing. The subchronic oral study has been designed to permit the determination of the no-observed-effect level and toxic effects associated with continuous or repeated exposure to a test substance for a period of 90 days. The test is not capable of determining those effects that have a long latency period for development (e.g., carcinogenicity and life shortening). It provides information on health hazards likely to arise from repeated exposure by the oral route over a limited period of time. It will provide information on target organs, the possibilities of accumulation, and can be of use in selecting dose levels for chronic studies and for establishing safety criteria for human exposure.
(b) Definitions. (1) Subchronic oral toxicity is the adverse effects occurring as a result of the repeated daily exposure of experimental animals to a chemical by the oral route for a part (approximately 10 percent) of a life span.
(2) Dose is the amount of test substance administered. Dose is expressed as weight of test substance (g, mg) per unit weight of test animal (e.g., mg/kg), or as weight of test substance per unit weight of food or drinking water.
(3) No-effect level/No-toxic-effect level/No-adverse-effect level/No-observed-effect level is the maximum dose used in a test which produces no observed adverse effects. A no-observed-effect level is expressed in terms of the weight of a substance given daily per unit weight of test animal (mg/kg). When administered to animals in food or drinking water the no-observed-effect level is expressed as mg/kg of food or mg/ml of water.
(4) Cumulative toxicity is the adverse effects of repeated doses occurring as a result of prolonged action on, or increased concentration of, the administered test substance or its metabolites in susceptible tissue.
(c) Principle of the test method. The test substance is administered orally in graduated daily doses to several groups of experimental animals, one dose level per group, for a period of 90 days. During the period of administration the animals are observed daily to detect signs of toxicity. Animals which die during the period of administration are necropsied. At the conclusion of the test all animals are necropsied and histo-pathological examinations carried out.
(d) Limit test. If a test at one dose level of at least 1,000 mg/kg body weight (expected human exposure may indicate the need for a higher dose level), using the procedures described for this study, produces no observable toxic effects and if toxicity would not be expected based upon data of structurally related compounds, then a full study using three dose levels might not be necessary.
(e) Test procedures —(1) Animal selection —(i) Species and strain. A mammalian species shall be used for testing. A variety of rodent species may be used, although the rat is the preferred species. Commonly used laboratory strains shall be employed. The commonly used nonrodent species is the dog, preferably of a defined breed; the beagle is frequently used. If other mammalian species are used, the tester shall provide justification/reasoning for his or her selection.
(ii) Age —(A) General. Young adult animals shall be employed. At the commencement of the study the weight variation of animals used shall not exceed ±20 percent of the mean weight for each sex.
(B) Rodents. Dosing shall begin as soon as possible after weaning, ideally before the rats are 6, and in any case, not more than 8 weeks old.
(C) Non-rodent. In the case of the dog, dosing shall commence after acclimatization, preferably at 4 to 6 months and not later than 9 months of age.
(iii) Sex. (A) Equal numbers of animals of each sex shall be used at each dose level.
(B) The females shall be nulliparous and nonpregnant.
(iv) Numbers —(A) Rodents. At least 20 animals (10 females and 10 males) shall be used at each dose level.
(B) Non-rodents. At least eight animals (four females and four males) shall be used at each dose level.
(C) If interim sacrifices are planned, the number shall be increased by the number of animals scheduled to be sacrificed before the completion of the study.
(2) Control groups. A concurrent control group is required. This group shall be an untreated or sham-treated control group or, if a vehicle is used in administering the test substance, a vehicle control group. If the toxic properties of the vehicle are not known or cannot be made available, both untreated and vehicle control groups are required.
(3) Satellite group. (Rodent) A satellite group of 20 animals (10 animals per sex) may be treated with the high dose level for 90 days and observed for reversibility, persistence, or delayed occurrence of toxic effects for a post-treatment period of appropriate length, normally not less than 28 days.
(4) Dose levels and dose selection. (i) In subchronic toxicity tests, it is desirable to have a dose response relationship as well as a no-observed-toxic-effect level. Therefore, at least 3 dose levels with a control and, where appropriate, a vehicle control (corresponding to the concentration of vehicle at the highest exposure level) shall be used. Doses should be spaced appropriately to produce test groups with a range of toxic effects. The data should be sufficient to produce a dose-response curve.
(ii) The highest dose level in rodents should result in toxic effects but not produce an incidence of fatalities which would prevent a meaningful evaluation; for non-rodents there should be no fatalities.
(iii) The lowest dose level should not produce any evidence of toxicity. Where there is a usable estimation of human exposure the lowest dose level should exceed this.
(iv) Ideally, the intermediate dose level(s) should produce minimal observable toxic effects. If more than one intermediate dose is used, the dose levels should be spaced to produce a gradation of toxic effects.
(v) For rodents, the incidence of fatalities in low and intermediate dose groups and in the controls should be low, to permit a meaningful evaluation of the results; for non-rodents, there should be no fatalities.
(5) Exposure conditions. The animals are dosed with the test substance ideally on a 7-day per week basis over a period of 90 days. However, based primarily on practical considerations, dosing in gavage or capsule studies on a 5-day per week basis is considered to be acceptable.
(6) Observation period. (i) Duration of observation shall be for at least 90 days.
(ii) Animals in the satellite group scheduled for followup observations should be kept for at least 28 days further without treatment to detect recovery from, or persistence of, toxic effects.
(7) Administration of the test substance. (i) The test substance may be administered in the diet or in capsules. In addition, for rodents it may also be administered by gavage or in the drinking water.
(ii) All animals shall be dosed by the same method during the entire experimental period.
(iii) Where necessary, the test substance is dissolved or suspended in a suitable vehicle. If a vehicle or diluent is needed, ideally it should not elicit important toxic effects itself nor substantially alter the chemical or toxicological properties of the test substance. It is recommended that wherever possible the usage of an aqueous solution be considered first, followed by consideration of a solution of oil and then by possible solution in other vehicles.
(iv) For substances of low toxicity, it is important to ensure that when administered in the diet the quantities of the test substance involved do not interfere with normal nutrition. When the test substance is administered in the diet either a constant dietary concentration (ppm) or a constant dose level in terms of the animals' body weight shall be used; the alternative used shall be specified.
(v) For a substance administered by gavage or capsule, the dose shall be given at approximately the same time each day, and adjusted at intervals (weekly or bi-weekly) to maintain a constant dose level in terms of animal body weight.
(8) Observation of animals. (i) Each animal shall be observed daily and, if necessary, handled to appraise its physical condition.
(ii) Additional observations shall be made daily with appropriate actions taken to minimize loss of animals to the study (e.g., necropsy or refrigeration of those animals found dead and isolation or sacrifice of weak or moribund animals).
(iii) Signs of toxicity shall be recorded as they are observed including the time of onset, degree and duration.
(iv) Cage-side observations shall include, but not be limited to, changes in skin and fur, eyes and mucous membranes, respiratory, circulatory, autonomic and central nervous systems, somatomotor activity and behavior pattern.
(v) Measurements shall be made weekly of feed consumption or water consumption when the test substance is administered in the feed or drinking water, respectively.
(vi) Animals shall be weighed weekly.
(vii) At the end of the 90-day period all survivors in the nonsatellite treatment groups shall be sacrificed. Moribund animals shall be removed and sacrificed when noticed.
(9) Clinical examinations. (i) The following examinations shall be made on all animals of each sex in each group for rodents and all animals when non-rodents are used as test animals.
(A) Certain hematology determinations shall be carried out at least two times during the test period on all groups of animals including concurrent controls: After 30 days of test and just prior to terminal sacrifice at the end of the test period. Hematology determinations which are appropriate to all studies: Hematocrit, hemoglobin concentration, erythrocyte count, total and differential leukocyte count, and a measure of clotting potential such as clotting time, prothrombin time, thromboplastin time, or platelet count.
(B) Certain clinical biochemistry determinations on blood should be carried out at least two times during the test period on all groups of animals including concurrent controls: After 30 days of test and just prior to terminal sacrifice at the end of the test period. Clinical biochemistry test areas which are considered appropriate to all studies: Electrolyte balance, carbohydrate metabolism, and liver and kidney function. The selection of specific tests will be influenced by observations on the mode of action of the substance. Suggested determinations: Calcium, phosphorus, chloride, sodium, potassium, fasting glucose (with period of fasting appropriate to the species), serum glutamic-pyruvic transaminase (now known as serum alanine aminotransferase), serum glutamic oxaloacetic transaminase (now known as serum aspartate aminotransferase), ornithine decarboxylase, gamma glutamyl transpeptidase, urea nitrogen, albumen, blood creatinine, total bilirubin, and total serum protein measurements. Other determinations which may be necessary for an adequate toxicological evaluation include: Analyses of lipids, hormones, acid/base balance, methemoglobin, and cholinesterase activity. Additional clinical biochemistry may be employed, where necessary, to extend the investigation of observed effects.
(ii) The following examinations shall be made on high dose and control groups. If changes in the eyes are detected, all animals should be examined.
(A) Ophthalmological examination, using an ophthalmoscope or equivalent suitable equipment, shall be made prior to the administration of the test substance and at the termination of the study.
(B) Urinalysis is not recommended on a routine basis, but only when there is an indication based on expected and or observed toxicity.
(10) Gross necropsy. (i) All animals shall be subjected to a full gross necropsy which includes examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents.
(ii) At least the liver, kidneys, adrenals, and gonads shall be weighed wet, as soon as possible after dissection to avoid drying. In addition, for the rodent, the brain; for the non-rodent, the thyroid with parathyroids also shall be weighed wet.
(iii) The following organs and tissues, or representative samples thereof, shall be preserved in a suitable medium for possible future histopathological examination: All gross lesions; lungs—which should be removed intact, weighed, and treated with a suitable fixative to ensure that lung structure is maintained (perfusion with the fixative is considered to be an effective procedure); nasopharyngeal tissues; brain—including sections of medulla/pons, cerebellar cortex, and cerebral cortex; pituitary; thyroid/parathyroid; thymus; trachea; heart; sternum with bone marrow; salivary glands; liver; spleen; kidneys; adrenals; pancreas; gonads; uterus; accessory genital organs (epididymis, prostate, and, if present, seminal vesicles); aorta; (skin); gall bladder (if present); esophagus; stomach; duodenum; jejunum; ileum; cecum; colon; rectum; urinary bladder; representative lymph node; (mammary gland); (thigh musculature); peripheral nerve; (eyes); (femur—including articular surface); (spinal cord at three levels—cervical, midthoracic, and lumbar); and (zymbal and exorbital lachrymal glands); and (rodent-zymbal glands).
(11) Histopathology. The following histopathology shall be performed:
(i) Full histopathology on the organs and tissues, listed above, of all rodents in the control and high dose groups, all non-rodents, and all rodents that died or were killed during the study.
(ii) All gross lesions in all animals.
(iii) Target organs in all animals.
(iv) The tissues mentioned in brackets (listed above) if indicated by signs of toxicity of target organ involvement.
(v) Lungs, liver and kidneys of all animals. Special attention to examination of the lungs of rodents shall be made for evidence of infection since this provides a convenient assessment of the state of health of the animals.
(vi) When a satellite group is used (rodents), histopathology shall be performed on tissues and organs identified as showing effects in the treated groups.
(f) Data and reporting —(1) Treatment of results. (i) Data shall be summarized in tabular form, showing for each test group the number of animals at the start of the test, the number of animals showing lesions, the types of lesions and the percentage of animals displaying each type of lesion.
(ii) All observed results, quantitative and incidental, should be evaluated by an appropriate statistical method. Any generally accepted statistical methods may be used; the statistical methods should be selected during the design of the study.
(2) Evaluation of the study results. (i) The findings of a subchronic oral toxicity study should be evaluated in conjunction with the findings of preceding studies and considered in terms of the toxic effects and the necropsy and histopathological findings. The evaluation will include the relationship between the dose of the test substance and the presence or absence, the incidence and severity, of abnormalities, including behavioral and clinical abnormalities, gross lesions, identified target organs, body weight changes, effects on mortality and any other general or specific toxic effects. A properly conducted subchronic test should provide a satisfactory estimation of a no-effect level.
(ii) In any study which demonstrates an absence of toxic effects, further investigation to establish absorption and bioavailability of the test substance should be considered.
(3) Test report. In addition to the reporting requirements as specified under EPA Good Laboratory Practice Standards, 40 CFR part 792, subpart J, the following specific information shall be reported:
(i) Group animal data. Tabulation of toxic response data by species, strain, sex and exposure level for:
(A) Number of animals dying.
(B) Number of animals showing signs of toxicity.
(C) Number of animals exposed.
(ii) Individual animal data. (A) Date of death during the study or whether animals survived to termination.
(B) Date of observation of each abnormal sign and its subsequent course.
(C) Body weight data.
(D) Feed consumption data when collected.
(E) Hematological tests employed and all results.
(F) Clinical biochemistry tests employed and all results.
(G) Necropsy findings.
(H) Detailed description of all histopathological findings.
(I) Statistical treatment of results where appropriate.
(g) References. For additional background information on this test guideline the following references should be consulted:
(1) Boyd, E.M. “Chapter 14—Pilot Studies, 15—Uniposal Clinical Parameters, 16—Uniposal Autopsy Parameters.” Predictive Toxicometrics. (Baltimore: Williams and Wilkins, 1972).
(2) Fitzhugh, O.G. “Subacute Toxicity,” Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. The Association of Food and Drug Officials of the United States (1959, 3rd Printing 1975) pp. 26-35.
(3) Food Safety Council. “Subchronic Toxicity Studies,” Proposed System for Food Safety Assessment. (Columbia: Food Safety Council, 1978) pp. 83-96.
(4) National Academy of Sciences. “Principles and Procedures for Evaluating the Toxicity of Household Substances,” a report prepared by the Committee for the Revision of NAS Publication 1138, under the auspices of the Committee on Toxicology, National Research Council, National Academy of Sciences, Washington, DC (1977).
(5) World Health Organization. “Part I. Environmental Health Criteria 6,” Principles and Methods for Evaluating the Toxicity of Chemicals. (Geneva: World Health Organization, 1978).
§ 798.3260   Chronic toxicity.
(a) Purpose. The objective of a chronic toxicity study is to determine the effects of a substance in a mammalian species following prolonged and repeated exposure. Under the conditions of the chronic toxicity test, effects which require a long latency period or which are cumulative should become manifest. The application of this guideline should generate data on which to identify the majority of chronic effects and shall serve to define long term dose-response relationships. The design and conduct of chronic toxicity tests should allow for the detection of general toxic effects, including neurological, physiological, biochemical, and hematological effects and exposure-related morphological (pathology) effects.
(b) Test procedures —(1) Animal selection —(i) Species and strain. Testing should be performed with two mammalian species, one a rodent and another a non-rodent. The rat is the preferred rodent species and the dog is the preferred non-rodent species. Commonly used laboratory strains should be employed. If other mammalian species are used, the tester should provide justification/reasoning for their selection.
(ii) Age. (A) Dosing of rats should begin as soon as possible after weaning, ideally before the rats are 6, but in no case more than 8 weeks old.
(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. (A) Equal numbers of animals of each sex should be used at each dose level.
(B) The females should be nulliparous and non-pregnant.
(iv) Numbers. (A) For rodents, at least 40 animals (20 females and 20 males) and for non-rodents (dogs) at least 8 animals (4 females and 4 males) should be used at each dose level.
(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. (i) A concurrent control group is suggested. This group should be an untreated or sham treated control group or, if a vehicle is used in administering the test substance, a vehicle control group. If the toxic properties of the vehicle are not known or cannot be made available, both untreated and vehicle control groups are strongly suggested.
(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. (i) In chronic toxicity tests, it is necessary to have a dose-response relationship as well as a no-observed-toxic-effect level. Therefore, at least three dose levels should be used in addition to the concurrent control group. Dose levels should be spaced to produce a gradation of effects.
(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. The animals are dosed with the test substance ideally on a 7-day per week basis over a period of at least 12 months. However, based primarily on practical considerations, dosing on a 5-day per week basis is considered to be acceptable.
(5) Observation period. Duration of observation should be for at least 12 months, and may be concurrent with or subsequent to dosing. If there is a post-exposure observation period, an interim sacrifice should be performed on no fewer than half of the animals of each sex at each dose level immediately upon termination of exposure.
(6) Administration of the test substance. The three main routes of administration are oral, dermal, and inhalation. The choice of the route of administration depends upon the physical and chemical characteristics of the test substance and the form typifying exposure in humans.
(i) Oral studies. (A) The animals should receive the test substance in their diet, dissolved in drinking water, or given by gavage or capsule for a period of at least 12 months.
(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. (A) The animals are treated by topical application with the test substance, ideally for at least 6 hours per day.
(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. (A) The animals should be tested with inhalation equipment designed to sustain a dynamic air flow of 12 to 15 air changes per hour, ensure an adequate oxygen content of 19 percent and an evenly distributed exposure atmosphere. Where a chamber is used, its design should minimize crowding of the test animals and maximize their exposure to the test substance. This is best accomplished by individual caging. As a general rule to ensure stability of a chamber atmosphere, the total “volume” of the test animals should not exceed 5 percent of the volume of the test chamber. Alternatively, oro-nasal, head-only or whole body individual chamber exposure may be used.
(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. (i) Each animal should be handled and its physical condition appraised at least once each day.
(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. For inhalation studies, measurements or monitoring should be made of the following:
(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. The following examinations should be made on at least 10 rats of each sex per dose and on all non-rodents.
(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. (i) A complete gross examination should be performed on all animals, including those which died during the experiment or were killed in moribund conditions.
(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. (i) The following histopathology should be performed:
(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 —(1) Treatment of results. (i) Data should be summarized in tabular form, showing for each test group the number of animals at the start of the test, the number of animals showing lesions, the types of lesions and the percentage of animals displaying each type of lesion.
(ii) 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. (i) The findings of a chronic toxicity study should be evaluated in conjunction with the findings of preceding studies and considered in terms of the toxic effects, the necropsy and histopathological findings. The evaluation will include the relationship between the dose of the test substance and the presence, incidence and severity of abnormalities (including behavioral and clinical abnormalities), gross lesions, identified target organs, body weight changes, effects on mortality and any other general or specific toxic effects.
(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) In addition to the reporting requirements as specified under 40 CFR part 792 subpart J, the following specific information should be reported:
(A) Group animal data. Tabulation of toxic response data by species, strain, sex and exposure level for:
( 1 ) Number of animals dying.
( 2 ) Number of animals showing signs of toxicity.
( 3 ) Number of animals exposed.
(B) Individual animal data. ( 1 ) Time of death during the study or whether animals survived to termination.
( 2 ) Time of observation of each abnormal sign and its subsequent course.
( 3 ) Body weight data.
( 4 ) Feed and water consumption data, when collected.
( 5 ) Results of ophthalmological examination, when performed.
( 6 ) Hematological tests employed and all results.
( 7 ) Clinical biochemistry tests employed and all results.
( 8 ) Necropsy findings.
( 9 ) Detailed description of all histopathological findings.
( 10 ) Statistical treatment of results, where appropriate.
(ii) In addition, for inhalation studies the following should be reported:
(A) Test conditions. ( 1 ) Description of exposure apparatus including design, type, dimensions, source of air, system for generating particulates and aerosols, method of conditioning air, treatment of exhaust air and the method of housing the animals in a test chamber.
( 2 ) The equipment for measuring temperature, humidity, and particulate aerosol concentrations and size should be described.
(B) Exposure data. These should be tabulated and presented with mean values and a measure of variability (e.g., standard deviation) and should include:
( 1 ) Airflow rates through the inhalation equipment.
( 2 ) Temperature and humidity of air.
( 3 ) Nominal concentration (total amount of test substance fed into the inhalation equipment divided by volume of air).
( 4 ) Actual concentration in test breathing zone.
( 5 ) Particle size distribution (e.g., median aerodynamic diameter of particles with standard deviation from the mean).
(d) References. For additional background information on this test guideline the following references should be consulted:
(1) Benitz, K.F. “Measurement of Chronic Toxicity,” Methods of Toxicology. Ed. G.E. Paget. (Oxford: Blackwell Scientific Publications, 1970) pp. 82-131.
(2) D'Aguanno, W. “Drug Safety Evaluation—Pre-Clinical Considerations,” Industrial Pharmacology: Neuroleptics. Vol. I, Ed. S. Fielding and H. Lal. (Mt. Kisco: Futura Publishing Co. 1974) pp. 317-332.
(3) Fitzhugh, O.G. Third Printing: 1975. “Chronic Oral Toxicity,” Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. The Association of Food and Drug Officials of the United States (1959, 3rd Printing 1975) pp. 36-45.
(4) Goldenthal, E.I., D'Aguanno, W. “Evaluation of Drugs,” Appraisal of the Safety of Chemicals in Foods, Drugs, and Cosmetics. The Association of Food and Drug Officials of the United States (1959, 3rd Printing 1975) pp. 60-67.
(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. (Rockville: National Center for Toxicological Research, 1972).
(7) Page, N.P. “Chronic Toxicity and Carcinogenicity Guidelines,” Journal of Environmental Pathology and Toxicology, 1:161-182 (1977).
(8) Schwartz, E. “Toxicology of Neuroleptic Agents,” Industrial Pharmacology: Neuroleptics Ed. S. Fielding and H. Lal. (Mt. Kisco, Futura Publishing Co., 1974) pp. 203-221.
(9) United States Pharmaceutical Manufacturers Association. Guidelines for the Assessment of Drug and Medical Device Safety in Animals. (1977).
(10) World Health Organization. “Guidelines for Evaluation of Drugs for Use in Man,” WHO Technical Report Series No. 563. (Geneva: World Health Organization, 1975).
(11) World Health Organization. “Part I. Environmental Health Criteria 6,” Principles and Methods for Evaluating the Toxicity of Chemicals. (Geneva: World Health Organization, 1978).
(12) World Health Organization. “Principles for Pre-Clinical Testing of Drug Safety,” WHO Technical Report Series No. 341. (Geneva: World Health Organization, 1966).
§ 798.3300   Oncogenicity.
(a) Purpose. The objective of a long-term oncogenicity study is to observe test animals for a major portion of their life span for the development of neoplastic lesions during or after exposure to various doses of a test substance by an appropriate route of administration.
(b) Test procedures —(1) Animal selection —(i) Species and strain. A compound of unknown activity shall be tested on two mammalian species. Rats and mice are the species of choice because of their relatively short life spans, the limited cost of their maintenance, their widespread use in pharmacological and toxicological studies, their susceptibility to tumor induction, and the availability of inbred or sufficiently characterized strains. Commonly used laboratory strains shall be employed. If other species are used, the tester shall provide justification/reasoning for their selection.
(ii) Age. (A) Dosing of rodents shall begin as soon as possible after weaning, ideally before the animals are 6 weeks old, but in no case more than 8 weeks old.
(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. (A) Animals of each sex shall be used at each dose level.
(B) The females shall be nulliparous and non-pregnant.
(iv) Numbers. (A) For rodents, at least 100 animals (50 females and 50 males) shall be used at each dose level and concurrent control.
(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. (i) A concurrent control group is required. This group shall be an untreated or sham treated control group or, if a vehicle is used in administering the test substance, a vehicle control group. If the toxic properties of the vehicle are not known or cannot be made available, both untreated and vehicle control groups are required.
(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. (i) For risk assessment purposes, at least 3 dose levels shall be used, in addition to the concurrent control group. Dose levels should be spaced to produce a gradation of chronic effects.
(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. The animals are dosed with the test substance ideally on a 7 day per week basis over a period of at least 24 months for rats, and 18 months for mice. However, based primarily on practical considerations, dosing on a 5 day per week basis is considered to be acceptable.
(5) Observations period. It is necessary that the duration of an oncogenicity test comprise the majority of the normal life span of the strain of animals to be used. This time period shall not be less than 24 months for rats and 18 months for mice, and ordinarily not longer than 30 months for rats and 24 months for mice. For longer time periods, and where any other species are used, consultation with the Agency in regard to the duration of the test is advised.
(6) Administration of the test substance. The three main routes of administration are oral, dermal, and inhalation. The choice of the route of administration depends upon the physical and chemical characteristics of the test substance and the form typifying exposure in humans.
(i) Oral studies. (A) The animals shall receive the test substance in their diet, dissolved in drinking water at levels that do not exceed the maximum solubility of the test chemical under testing condition.
(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. (A) The animals are treated by topical application with the test substance, ideally for at least 6 hours per day.
(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. (A) The animals shall be tested with inhalation equipment designed to sustain a minimum dynamic air flow of 12 to 15 air changes per hour, ensure an adequate oxygen content of 19 percent and an evenly distributed exposure atmosphere. Where a chamber is used, its design should minimize crowding of the test animals and maximize their exposure to the test substance. This is best accomplished by individual caging. To ensure stability of a chamber atmosphere, the total “volume” of the test animals shall not exceed 5 percent of the volume of the test chamber. Alternatively, oro-nasal, head-only, or whole-body individual chamber exposure may be used.
(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. (i) Each animal shall be observed daily and if necessary should be handled to appraise its physical condition.
(ii) Additional observations shall be made daily with appropriate actions taken to minimize loss of animals to the study (e.g., necropsy or refrigeration of those animals found dead and isolation or sacrifice of weak or moribund animals).
(iii) 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. For inhalation studies, measurements or monitoring should be made of the following:
(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. At 12 months, 18 months, and at sacrifice, a blood smear shall be obtained from all animals. A differential blood count shall be performed on blood smears from those animals in the highest dosage group and the controls. If these data, or data from the pathological examination indicate a need, then the 12- and 18-month blood smears from other dose levels shall also be examined. Differential blood counts shall be performed for the next lower group(s) if there is a major discrepancy between the highest group and the controls. If clinical observations suggest a deterioration in health of the animals during the study, a differential blood count of the affected animals shall be performed.
(10) Gross necropsy. (i) A complete gross examination shall be performed on all animals, including those which died during the experiment or were killed in moribund conditions.
(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. (i) The following histopathology shall be performed:
(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 —(1) Treatment of results. (i) Data shall be summarized in tabular form, showing for each test group the number of animals at the start of the test, the number of animals showing lesions, the types of lesions and the percentage of animals displaying each type of lesion.
(ii) All observed results, quantitative and incidental, 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. (i) The findings of an oncogenic toxicity study shall be evaluated in conjunction with the findings of preceding studies and considered in terms of the toxic effects, the necropsy and histopathological findings. The evaluation shall include the relationship between the dose of the test substance and the presence, incidence and severity of abnormalities (including behavioral and clinical abnormalities), gross lesions, identified target organs, body weight changes, effects on mortality and any other general or specific toxic effects.
(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. (i) In addition to the reporting requirements as specified under 40 CFR part 792, subpart J the following specific information shall be reported:
(A) Group animal data. Tabulation of toxic response data by species, strain, sex and exposure level for:
( 1 ) Number of animals dying.
( 2 ) Number of animals showing signs of toxicity.
( 3 ) Number of animals exposed.
(B) Individual animal data. ( 1 ) Time of death during the study or whether animals survived to termination.
( 2 ) Time of observation of each abnormal sign and its subsequent course.
( 3 ) Body weight data.
( 4 ) Feed and water consumption data, when collected.
( 5 ) Results of ophthalmological examination, when performed.
( 6 ) Hematological tests employed and all results.
( 7 ) Clinical biochemistry tests employed and all results.
( 8 ) Necropsy findings.
( 9 ) Detailed description of all histopathological findings.
( 10 ) Statistical treatment of results, where appropriate.
( 11 ) Historical control data, if taken into account.
(ii) In addition, for inhalation studies the following shall be reported:
(A) Test conditions. (1) Description of exposure apparatus including design, type, dimensions, source of air, system for generating particulates and aerosols, method of conditioning air, treatment of exhaust air and the method of housing the animals in a test chamber.
( 2 ) The equipment for measuring temperature, humidity, and particulate aerosol concentrations and size shall be described.
(B) Exposure data. These shall be tabulated and presented with mean values and a measure of variability (e.g., standard deviation) and shall include:
( 1 ) Airflow rates through the inhalation equipment.
( 2 ) Temperature and humidity of air.
( 3 ) Nominal concentration (total amount of test substance fed into the inhalation equipment divided by volume of air).
( 4 ) Actual concentration in test breathing zone.
( 5 ) Particle size distribution (e.g., median aerodynamic diameter of particles with standard deviation from the mean).
(d) References. For additional background information on this test guideline the following references should be consulted:
(1) 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).
(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. 20:419-438 (1971).
(3) International Union Against Cancer. “Carcinogenicity Testing,” IUCC Technical Report Series. Vol. 2., Ed. I. Berenblum. (Geneva: International Union Against Cancer, 1969).
(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. (Bethesda: United States National Cancer Institute, 1976).
(7) National Center for Toxicological Research. “Appendix B,” Report of Chronic Studies Task Force Committee. April 13-21 (Rockville: National Center for Toxicological Research, 1972).
(8) Page, N.P. “Chronic Toxicity and Carcinogenicity Guidelines,” Journal of Environmental Pathology and Toxicology. 1:161-182 (1977).
(9) Page, N.P. “Concepts of a Bioassay Program in Environmental Carcinogenesis,” Advances in Modern Toxicology Vol. 3, Ed. Kraybill and Mehlman. (Washington, DC: Hemisphere Publishing Corporation, 1977) pp. 87-171.
(10) Sontag, J.M., Page N.P., Saffiotti, U. Guidelines for Carcinogen Bioassay in Small Rodents. NCI-CS-TR-1. (Bethesda: United States Cancer Institute, Division of Cancer Control and Prevention, Carcinogenesis Bioassay Program, 1976).
(11) United States Pharmaceutical Manufacturers Association. Guidelines for the Assessment of Drug and Medical Device Safety in Animals. (1977).
(12) World Health Organization. “Principles for the Testing and Evaluation of Drugs for Carcinogenicity,” WHO Technical Report Series No. 426. (Geneva: World Health Organization, 1969).
(13) World Health Organization. “Part I. Environmental Health Criteria 6,” Principles and Methods for Evaluating the Toxicity of Chemicals. (Geneva: World Health Organization, 1978).
§ 798.3320   Combined chronic toxicity/oncogenicity.
(a) Purpose. The objective of a combined chronic toxicity/oncogenicity study is to determine the effects of a substance in a mammalian species following prolonged and repeated exposure. The application of this guideline should generate data which identify the majority of chronic and oncogenic effects and determine dose-response relationships. The design and conduct should allow for the detection of neoplastic effects and a determination of oncogenic potential as well as general toxicity, including neurological, physiological, biochemical, and hematological effects and exposure-related morphological (pathology) effects.
(b) Test procedures —(1) Animal selection —(i) Species and strain. Preliminary studies providing data on acute, subchronic, and metabolic responses should have been carried out to permit an appropriate choice of animals (species and strain). As discussed in other guidelines, the mouse and rat have been most widely used for assessment of oncogenic potential, while the rat and dog have been most often studied for chronic toxicity. The rat is the species of choice for combined chronic toxicity and oncogenicity studies. The provisions of this guideline are designed primarily for use with the rat as the test species. If other species are used, the tester should provide justification/reasoning for their selection. The strain selected should be susceptible to the oncogenic or toxic effect of the class of substances being tested, if known, and provided it does not have a spontaneous background too high for meaningful assessment. Commonly used laboratory strains should be employed.
(ii) Age. (A) Dosing of rats should begin as soon as possible after weaning, ideally before the rats are 6 weeks old, but in no case more than 8 weeks old.
(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. (A) Equal numbers of animals of each sex should be used at each dose level.
(B) The females should be nulliparous and nonpregnant.
(iv) Numbers. (A) At least 100 rodents (50 females and 50 males) should be used at each dose level and concurrent control for those groups not intended for early sacrifice. At least 40 rodents (20 females and 20 males) should be used for satellite dose group(s) and the satellite control group. The purpose of the satellite group is to allow for the evaluation of pathology other than neoplasia.
(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. (i) A concurrent control group (50 females and 50 males) and a satellite control group (20 females and 20 males) are recommended. These groups should be untreated or sham treated control groups or, if a vehicle is used in administering the test substance, vehicle control groups. If the toxic properties of the vehicle are not known or cannot be made available, both untreated and vehicle control groups are recommended. Animals in the satellite control group should be sacrificed at the same time the satellite test group is terminated.
(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. (i) For risk assessment purposes, at least three dose levels should be used, in addition to the concurrent control group. Dose levels should be spaced to produce a gradation of effects.
(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. The animals are dosed with the test substance ideally on a 7-day per week basis over a period of at least 24 months for rats, and 18 months for mice and hamsters, except for the animals in the satellite groups which should be dosed for 12 months.
(5) Observation period. It is necessary that the duration of the oncogenicity test comprise the majority of the normal life span of the animals to be used. It has been suggested that the duration of the study should be for the entire lifetime of all animals. However, a few animals may greatly exceed the average lifetime and the duration of the study may be unnecessarily extended and complicate the conduct and evaluation of the study. Rather, a finite period covering the majority of the expected life span of the strain is preferred since the probability is high that, for the great majority of chemicals, induced tumors will occur within such an observation period. The following guidelines are recommended:
(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. The three main routes of administration are oral, dermal, and inhalation. The choice of the route of administration depends upon the physical and chemical characteristics of the test substance and the form typifying exposure in humans.
(i) Oral studies. (A) The animals should receive the test substance in their diet, dissolved in drinking water, or given by gavage or capsule for a period of at least 24 months for rats and 18 months for mice and hamsters.
(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. (A) The animals are treated by topical application with the test substance, ideally for at least 6 hours per day.
(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. (A) The animals should be tested with inhalation equipment designed to sustain a dynamic air flow of 12 to 15 air changes per hour, to ensure an adequate oxygen content of 19 percent and an evenly distributed exposure atmosphere. Where a chamber is used, its design should minimize crowding of the test animals and maximize their exposure to the test substance. This is best accomplished by individual caging. As a general rule, to ensure stability of a chamber atmosphere, the total “volume” of the test animals should not exceed 5 percent of the volume of the test chamber. Alternatively, oro-nasal, head only, or whole body individual chamber exposure may be used.
(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. (i) Each animal should be handled and its physical condition appraised at least once each day.
(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. For inhalation studies, measurements or monitoring should be made of the following:
(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. (i) The following examinations should be made on at least 20 rodents of each sex per dose level:
(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. (i) A complete gross examination should be performed on all animals, including those which died during the experiment or were killed in moribund conditions.
(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. (i) The following histopathology should be performed:
(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 —(1) Treatment of results. (i) Data should be summarized in tabular form, showing for each test group the number of animals at the start of the test, the number of animals showing lesions, the types of lesions and the percentage of animals displaying each type of lesion.
(ii) 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. (i) The findings of a combined chronic toxicity/oncogenicity study should be evaluated in conjunction with the findings of preceding studies and considered in terms of the toxic effects, the necropsy and histopathological findings. The evaluation will include the relationship between the dose of the test substance and the presence, incidence and severity of abnormalities (including behavioral and clinical abnormalities), gross lesions, identified target organs, body weight changes, effects on mortality and any other general or specific toxic effects.
(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. (i) In addition to the reporting requirements as specified under 40 CFR part 792, subpart J the following specific information should be reported:
(A) Group animal data. Tabulation of toxic response data by species, strain, sex and exposure level for:
( 1 ) Number of animals dying.
( 2 ) Number of animals showing signs of toxicity.
( 3 ) Number of animals exposed.
(B) Individual animal data. ( 1 ) Time of death during the study or whether animals survived to termination.
( 2 ) Time of observation of each abnormal sign and its subsequent course.
( 3 ) Body weight data.
( 4 ) Feed and water consumption data, when collected.
( 5 ) Results of ophthalmological examination, when performed.
( 6 ) Hematological tests employed and all results.
( 7 ) Clinical biochemistry tests employed and all results.
( 8 ) Necropsy findings.
( 9 ) Detailed description of all histopathological findings.
( 10 ) Statistical treatment of results where appropriate.
( 11 ) Historical control data, if taken into account.
(ii) In addition, for inhalation studies the following should be reported:
(A) Test conditions. ( 1 ) Description of exposure apparatus including design, type, dimensions, source of air, system for generating particulates and aerosols, method of conditioning air, treatment of exhaust air and the method of housing the animals in a test chamber.
( 2 ) The equipment for measuring temperature, humidity, and particulate aerosol concentrations and size should be described.
(B) Exposure data. These should be tabulated and presented with mean values and a measure of variability (e.g. standard deviation) and should include:
( 1 ) Airflow rates through the inhalation equipment.
( 2 ) Temperature and humidity of air.
( 3 ) Nominal concentration (total amount of test substance fed into the inhalation equipment divided by volume of air).
( 4 ) Actual concentration in test breathing zone.
( 5 ) Particle size distribution (e.g. median aerodynamic diameter of particles with standard deviation from the mean).
(d) References. For additional background information on this test guideline the following references should be consulted:
(1) Benitz, K.F. “Measurement of Chronic Toxicity,” Methods of Toxicology. Ed. G.E. Paget. (Oxford: Blackwell Scientific Publications, 1970) pp. 82-131.
(2) D'Aguanno, W. “Drug Safety Evaluation—Pre-Clinical Considerations,” “ Industrial Pharmacology: Neuroleptics. Vol. I Ed. S. Fielding and H. Lal. (Mt. Kisco, New York: Futura Publishing Co., 1974) pp. 317-332.
(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. The Association of Food and Drug Officials of the United States (1959, 3rd Printing 1975). pp. 36-45.
(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. 20:419-438 (1971).
(6) Goldenthal, E.I., and D'Aguanno, W. “Evaluation of Drugs,” Appraisal of the Safety of Chemicals in Foods, Drugs, and Cosmetics. The Association of Food and Drug Officials of the United States (1959, 3rd printing 1975) pp.60-67.
(7) International Union Against Cancer. “Carcinogenicity Testing,” IUCC Technical Report Series Vol. 2, Ed. I. Berenblum. (Geneva: International Union Against Cancer, 1969).
(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. (Bethesda: United States National Cancer Institute, 1976).
(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. 1:161-182 (1977).
(13) Page, N.P. “Concepts of a Bioassay Program in Environmental Carcinogenesis,” Advances in Modern Toxicology Volume 3, Ed. Kraybill and Mehlman. (Washington, D.C.: Hemisphere Publishing Corp., 1977) pp. 87-171.
(14) Schwartz, E. 1974. “Toxicology of Neuroleptic Agents,” Industrial Pharmacology: Neuroleptics. Ed. S. Fielding and H. Lal. (Mt. Kisco, New York: Futura Publishing Co, 1974) pp. 203-221.
(15) Sontag, J.M., Page, N.P., and Saffiotti, U. Guidelines for Carcinogen Bioassay in Small Rodents. NCI-CS-TR-1 (Bethesda: United States Cancer Institute, Division of Cancer Control and Prevention, Carcinogenesis Bioassay Program, 1976).
(16) United States Pharmaceutical Manufacturers Association. Guidelines for the Assessment of Drug and Medical Device Safety in Animals. (1977).
(17) World Health Organization. “Principles for the Testing and Evaluation of Drugs for Carcinogenicity,” WHO Technical Report Series No. 426. (Geneva: World Health Organization, 1969).
(18) World Health Organization. “Guidelines for Evaluation of Drugs for Use in Man,” WHO Technical Report Series No. 563. (Geneva: World Health Organization, 1975).
(19) World Health Organization. “Part I. Environmental Health Criteria 6,” Principles and Methods for Evaluating the Toxicity of Chemicals. (Geneva: World Health Organization, 1978).
(20) World Health Organization. “Principles for Pre-Clinical Testing of Drug Safety,” WHO Technical Report Series No. 341. (Geneva: World Health Organization, 1966).
§ 798.4100   Dermal sensitization.
(a) Purpose. In the assessment and evaluation of the toxic characteristics of a substance, determination of its potential to provoke skin sensitization reactions is important. Information derived from tests for skin sensitization serves to identify the possible hazard to a population repeatedly exposed to a test substance. While the desirability of skin sensitization testing is recognized, there are some real differences of opinion about the best method to use. The test selected should be a reliable screening procedure which should not fail to identify substances with significant allergenic potential, while at the same time avoiding false negative results.
(b) Definitions. (1) Skin sensitization (allergic contact dermatitis) is an immunologically mediated cutaneous reaction to a substance. In the human, the responses may be characterized by pruritis, erythema, edema, papules, vesicles, bullae, or a combination of these. In other species the reactions may differ and only erythema and edema may be seen.
(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. Following initial exposure(s) to a test substance, the animals are subsequently subjected, after a period of not less than 1 week, to a challenge exposure with the test substance to establish whether a hypersensitive state has been induced. Sensitization is determined by examining the reaction to the challenge exposure and comparing this reaction to that of the initial induction exposure.
(d) Test procedures. (1) Any of the following seven test methods is considered to be acceptable. It is realized, however, that the methods differ in their probability and degree of reaction to sensitizing substances.
(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 —(i) Species and strain. The young adult guinea pig is the preferred species. Commonly used laboratory strains should be employed. If other species are used, the tester should provide justification/reasoning for their selection.
(ii) Number and sex. (A) The number and sex of animals used will depend on the method employed.
(B) The females should be nulliparous and nonpregnant.
(4) Control animals. (i) Periodic use of a positive control substance with an acceptable level of reliability for the test system selected is recommended;
(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. The dose level will depend upon the method selected.
(6) Observation of animals. (i) Skin reactions should be graded and recorded after the challenge exposures at the time specified by the methodology selected. This is usually at 24, 48, and 72, hours. Additional notations should be made as necessary to fully describe unusual responses;
(ii) Regardless of method selected, initial and terminal body weights should be recorded.
(7) Procedures. The procedures to be used are those described by the methodology chosen.
(e) Data and reporting. (1) Data should be summarized in tabular form, showing for each individual animal the skin reaction, results of the induction exposure(s) and the challenge exposure(s) at times indicated by the method chosen. As a minimum, the erythema and edema should be graded and any unusual finding should be recorded.
(2) Evaluation of the results. The evaluation of results will provide information on the proportion of each group that became sensitized and the extent (slight, moderate, severe) of the sensitization reaction in each individual animal.
(3) Test report. In addition to the reporting requirements as specified under 40 CFR part 792, subpart J, the following specific information should be reported:
(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. For additional background information on this test guideline the following references should be consulted:
(1) Buehler, E.V. “Delayed Contact Hypersensitivity in the Guinea Pig,” Archives Dermatology. 91:171 (1965).
(2) Draize, J.H. “Dermal Toxicity,” Food Drug Cosmetic Law Journal. 10:722-732 (1955).
(3) Klecak, G. “Identification of Contact Allergens: Predictive Tests in Animals,” Advances in Modern Toxicology: Dermatology and Pharmacology. Ed. F.N. Marzulli and H.I. Maibach. (Washington, D.C.: Hemisphere Publishing Corp., 1977) 4:305-339).
(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. 28:53-64 (1977).
(5) Magnusson, B., Kligman, A.M. “The Identification of Contact Allergens by Animal Assay,” The Guinea Pig Maximization Test. The Journal of Investigative Dermatology. 52:268-276 (1973).
(6) Maguire, H.C. “The Bioassay of Contact Allergens in the Guinea Pig” Journal of the Society of Cosmetic Chemists. 24:151-162 (1973).
(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. (Basel: Birkhauser Verlag, 1975) Vol. 5/2.
(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, (1975) Vol. 203.
§ 798.4350   Inhalation developmental toxicity study.
(a) Purpose. In the assessment and evaluation of the toxic characteristics of an inhalable material such as a gas, volatile substance, or aerosol/particulate, determination of the potential developmental toxicity is important. The inhalation developmental toxicity study is designed to provide information on the potential hazard to the unborn which may arise from exposure of the mother during pregnancy.
(b) Definitions. (1) Developmental toxicity is the property of a chemical that causes in utero death, structural or functional abnormalities or growth retardation during the period of development.
(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. The test substance is administered in graduated concentrations, for at least that part of the pregnancy covering the major period of organogenesis, to several groups of pregnant experimental animals, one exposure level being used per group. Shortly before the expected date of delivery, the pregnant females are sacrificed, the uteri removed, and the contents examined for embryonic or fetal deaths, and live fetuses.
(d) Limit test. If a test at an exposure of 5 mg/1 (actual concentration of respirable substances) or, where this is not possible due to physical or chemical properties of the test substance, the maximum attainable concentration, produces no observable developmental toxicity, then a full study using three exposure levels might not be necessary.
(e) Test procedures —(1) Animal selection —(i) Species and strain. Testing shall be performed in at least two mamalian species. Commonly used species include the rat, mouse, rabbit, and hamster. If other mamalian species are used, the tester shall provide justification/reasoning for their selection. Commonly used laboratory strains shall be employed. The strain shall not have low fecundity and shall preferably be characterized for its sensitivity to developmental toxins.
(ii) Age. Young adult animals (nulliparous females) shall be used.
(iii) Sex. Pregnant female animals shall be used at each exposure level.
(iv) Number of animals. At least 20 pregnant rats, mice, or hamsters or 12 pregnant rabbits are required at each exposure level. The objective is to ensure that sufficient pups are produced to permit meaningful evaluation of the potential developmental toxicity of the test substance.
(2) Control group. A concurrent control group shall be used. This group shall be exposed to clean, filtered air under conditions identical to those used for the group exposed to the substance of interest. In addition, a vehicle-exposed group may be necessary when the substance under study requires a vehicle for delivery. It is recommended that during preliminary range finding studies, air vs. vehicle exposure be compared. If there is no substantial difference, air exposure itself would be an appropriate control. If vehicle and air exposure yield different results, both vehicle and air exposed control groups are recommended.
(3) Concentration levels and concentration selection. (i) At least three concentration levels with a control and, where appropriate, a vehicle control, shall be used.
(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. The duration of exposure shall be at least six hours daily allowing appropriate additional time for chamber equilibrium.
(5) Observation period. Day 0 in the test is the day on which a vaginal plug and/or sperm are observed. The exposure period shall cover the period of major organogenesis. This may be taken as days 6 to 15 for rat and mouse, 6 to 14 for hamster, or 6 to 18 for rabbit.
(6) Inhalation exposure. (i)(A) The animals shall be tested in inhalation equipment designed to sustain a minimum dynamic air flow of 12 to 15 air changes per hour and ensure an adequate oxygen content of 19 percent and an evenly distributed exposure atmosphere. Where a chamber is used, its design should minimize crowding of the test animals and maximize their exposure to the test substance. This is best accomplished by individual caging. To ensure stability of a chamber atmosphere, the total “volume” of the test animals shall not exceed 5 percent of the volume of the test chamber.
(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. Measurements or monitoring should be made of the following:
(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. Food should be withheld during exposure. Water may or may not be withheld. If it is not withheld it should not come in direct contact with the test atmospheres.
(9) Observation of animals. (i) A gross examination shall be made at least once each day.
(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. (i) At the time of sacrifice or death during the study, the dam shall be examined macroscopically for any structural abnormalities or pathological changes which may have influenced the pregnancy.
(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 —(1) Treatment of results. Data shall be summarized in tabular form, showing for each test group: the number of animals at the start of the test, the number of pregnant animals, the number and percentages of live fetuses and the number of fetuses with any soft tissue or skeletal abnormalities.
(2) Evaluation of results. The findings of a developmental toxicity study shall be evaluated in terms of the observed effects and the exposure levels producing effects. It is necessary to consider the historical developmental toxicity data on the species/strain tested. A properly conducted developmental toxicity study should provide a satisfactory estimation of a no-effect level.
(3) Test report. In addition to the reporting requirements as specified under 40 CFR part 792, subpart J, the following specific information shall be reported:
(i) Test conditions. (A) Description of exposure apparatus including design, type, dimensions, source of air, system for generating particulates and aerosols, methods of conditioning air, and the method of housing the animals in a test chamber when this apparatus is used.
(B) The equipment for measuring temperature, humidity, and particulate aerosol concentrations and size shall be described.
(ii) Exposure data. These shall be tabulated and presented with mean values and a measure of variability (e.g., standard deviation) and shall include:
(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. (A) Toxic response data by concentration.
(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. For additional background information on this test guideline the following references should be consulted:
(1) Department of Health and Welfare. The Testing of Chemicals for Carcinogenicity, Mutagenicity and Teratogenicity. Minister of Health and Welfare (Canada: Department of Health and Welfare, 1975).
(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. WHO Technical Report Series No. 364. (Geneva: World Health Organization, 1967).
§ 798.4700   Reproduction and fertility effects.
(a) Purpose. This guideline for two-generation reproduction testing is designed to provide general information concerning the effects of a test substance on gonadal function, conception, parturition, and the growth and development of the offspring. The study may also provide information about the effects of the test substance on neonatal morbidity, mortality, and preliminary data on teratogenesis and serve as a guide for subsequent tests.
(b) Principle of the test method. The test substance is administered to parental (P) animals prior to their mating, during the resultant pregnancies, and through the weaning of their F 1 offspring. The substance is then administered to selected F 1 offspring during their growth into adulthood, mating, and production of an F 2 generation, up until the F 2 generation is weaned.
(c) Test procedures —(1) Animal selection —(i) Species and strain. The rat is the preferred species. If another mammalian species is used, the tester shall provide justification/reasoning for its selection. Strains with low fecundity shall not be used.
(ii) Age. Parental (P) animals shall be about 5 to 8 weeks old at the start of dosing.
(iii) Sex. (A) For an adequate assessment of fertility, both males and females shall be studied.
(B) The females shall be nulliparous and non-pregnant.
(iv) Number of animals. Each test and control group shall contain at least 20 males and a sufficient number of females to yield at least 20 pregnant females at or near term.
(2) Control groups. (i) A concurrent control group shall be used. This group shall be an untreated or sham treated control group or if a vehicle is used in administering the test substance, a vehicle control group.
(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. (i) At least three dose levels and a concurrent control shall be used.
(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. The animals should be dosed with the test substance, ideally, on a 7 days per week basis.
(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 males saved for mating shall continue from the time they are weaned through the period they are mated with the F 1 females (11 weeks). F 1 males may be sacrificed after the F 1 mating period.
(C) Daily dosing of the P females shall continue through the three week mating period, pregnancy, and to the weaning of the F 1 offspring. Dosing of the F 1 females saved for mating shall continue from the time they are weaned, through the period they are mated with the F 1 males (11 weeks from the time of weaning) pregnancy, and to the weaning of the F 2 offspring.
(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 males selected for mating should be sacrificed at the end of the three week period of the F 1 mating.
(C) F 1 males and females not selected for mating should be sacrified when weaned.
(D) The P females should be sacrificed upon weaning of their F 1 offspring.
(E) F 1 dams and their F 2 offspring are sacrificed when the offspring are weaned.
(5) Administration of the test substance —(i) Oral studies. (A) It is recommended that the test substance be administered in the diet or drinking water.
(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 —(i) Parental. (A) For each mating, each female shall be placed with a single male from the same dose level until pregnancy occurs or 1 week has elapsed. If mating has not occurred after 1 week, the female shall be placed with a different male. Paired matings should be clearly identified.
(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 cross. (A) For mating the F 1 offspring, one male and one female are randomly selected at weaning from each litter for cross mating with another pup of the same dose level but different litter, to produce the F 2 generation.
(B) F 1 males and females not selected for mating are sacrificed upon weaning.
(iii) Special housing. After evidence of copulation, pregnant animals shall be caged separately in delivery or maternity cages. Pregnant animals shall be provided with nesting materials when parturition is near.
(iv) Standardization of litter sizes. (A) On day 4 after birth, the size of each litter should be adjusted by eliminating extra pups by random selection to yield, as nearly as possible, 4 males and 4 females per litter.
(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 litters is conducted in the same manner.
(7) Observation of animals. (i) A gross examination shall be made at least once each day. Pertinent behavioral changes, signs of difficult or prolonged parturition, and all signs of toxicity, including mortality, shall be recorded. These observations shall be reported for each individual animal. Food consumption for all animals shall be monitored weekly except during the mating period.
(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 litters shall be weighed at birth, or soon thereafter, and on days 4, 7, 14, and 21. In all cases, litter weights shall be calculated from the weights of the individual pups.
(8) Gross necropsy. (i) A complete gross examination shall be performed on all adult animals, including those which died during the experiment or were killed in moribund conditions.
(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 animals selected for mating.
(9) Histopathology. Except if carried out in other studies of comparable duration and dose levels the following histopathology shall be performed:
(i) Full histopathology on the organs listed above for all high dose, and control P 1 and F 1 animals selected for mating.
(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 —(1) Treatment of results. Data shall be summarized in tabular form, showing for each test group the number of animals at the start of the test, the number of animals pregnant, the types of change and the percentage of animals displaying each type of change.
(2) Evaluation of study results. (i) An evaluation of test results, including the statistical analysis, based on the clinical findings, the gross necropsy findings, and the microscopic results shall be made and supplied. This should include an evaluation of the relationship, or lack thereof, between the animals' exposure to the test substance and the incidence and severity of all abnormalities.
(ii) In any study which demonstrates an absence of toxic effects, further investigation to establish absorption and bioavailability of the test substance should be considered.
(3) Test report. In addition to the reporting requirements as specified under 40 CFR part 792, subpart J the following specific information shall be reported:
(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 , and F 2 animals.
(vii) Necropsy findings.
(viii) Detailed description of all histopathological findings.
(ix) Statistical treatment of results where appropriate.
(e) References. For additional background information on this test guideline the following references should be consulted:
(1) Clermont, Y., Perry, B. “Quantitative Study of the Cell Population of the Seminiferous Tubules in Immature Rats,” American Journal of Anatomy. 100:241-267 (1957).
(2) Goldenthal, E.I. Guidelines for Reproduction Studies for Safety Evaluation of Drugs for Human Use. Drug Review Branch, Division of Toxicological Evaluation, Bureau of Science, Food and Drug Administration, Washington, DC (1966).
(3) Hasegawa, T., Hayashi, M., Ebling, F.J.G., Henderson, I.W. Fertility and Sterility. (New York: American Elsevier Publishing Co., Inc., 1973).
(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. 9:507-516 (1956).
(5) Roosen-Runge, E.C. “The Process of Spermatogenesis in Mammals,” Biological Review. 37:343-377 (1962).
§ 798.4900   Developmental toxicity study.
(a) Purpose. In the assessment and evaluation of the toxic characteristics of a chemical, determination of the potential developmental toxicity is important. The developmental toxicity study is designed to provide information on the potential hazard to the unborn which may arise from exposure of the mother during pregnancy.
(b) Definitions. (1) Developmental toxicity is the property of a chemical that causes in utero death, structural or functional abnormalities or growth retardation during the period of development.
(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. The test substance is administered in graduated doses for at least part of the pregnancy covering the major period of organogenesis, to several groups of pregnant experimental animals, one dose level being used per group. Shortly before the expected date of delivery, the pregnant females are sacrificed, the uteri removed, and the contents examined for embryonic or fetal deaths, and live fetuses.
(d) Limit test. If a test at an exposure of at least 1000 mg/kg body weight, using the procedures described for this study, produces no observable developmental toxicity, then a full study using three dose levels might not be necessary.
(e) Test procedures —(1) Animal selection —(i) Species and strain. Testing shall be performed in at least 2 mammalian species. Commonly used species include the rat, mouse, rabbit, and hamster. If other mammalian species are used, the tester shall provide justification/reasoning for their selection. Commonly used laboratory strains shall be employed. The strain shall not have low fecundity and shall preferably be characterized for its sensitivity to developmental toxins.
(ii) Age. Young adult animals (nulliparous females) shall be used.
(iii) Sex. Pregnant female animals shall be used at each dose level.
(iv) Number of animals. At least 20 pregnant rats, mice or hamsters or 12 pregnant rabbits are required at each dose level. The objective is to ensure that sufficient pups are produced to permit meaningful evaluation of the potential developmental toxicity of the test substance.
(2) Control group. A concurrent control group shall be used. This group shall be an untreated or sham treated control group, or, if a vehicle is used in administering the test substance, a vehicle control group. Except for treatment with the test substance, animals in the control group(s) shall be handled in an identical manner to test group animals.
(3) Dose levels and dose selection. (i) At least 3 dose levels with a control and, where appropriate, a vehicle control, shall be used.
(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. Day 0 in the test is the day on which a vaginal plug and/or sperm are observed. The dose period shall cover the period of major organogenesis. This may be taken as days 6 to 15 for rat and mouse, 6 to 14 for hamster, or 6 to 18 for rabbit.
(5) Administration of test substance. The test substance or vehicle is usually administered orally, by oral intubation unless the chemical or physical characteristics of the test substance or pattern of human exposure suggest a more appropriate route of administration. The test substance shall be administered approximately the same time each day.
(6) Exposure conditions. The female test animals are treated with the test substance daily throughout the appropriate treatment period. When given by gavage, the dose may be based on the weight of the females at the start of substance administration, or, alternatively, in view of the rapid weight gain which takes place during pregnancy, the animals may be weighed periodically and the dosage based on the most recent weight determination.
(7) Observation of animals. (i) A gross examination shall be made at least once each day.
(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. (i) At the time of sacrifice or death during the study, the dam shall be examined macroscopically for any structural abnormalities or pathological changes which may have influenced the pregnancy.
(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 —(1) Treatment of results. Data shall be summarized in tablular form, showing for each test group: the number of animals at the start of the test, the number of pregnant animals, the number and percentages of live fetuses and the number of fetuses with any soft tissue or skeletal abnormalities.
(2) Evaluation of results. The findings of a developmental toxicity study shall be evaluated in terms of the observed effects and the exposure levels producing effects. It is necessary to consider the historical developmental toxicity data on the species/strain tested. A properly conducted developmental toxicity study should provide a satisfactory estimation of a no-effect level.
(3) Test report. In addition to the reporting requirements as specified under 40 CFR part 792, subpart J the following specific information shall be reported:
(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. For additional background information on this test guideline the following references should be consulted:
(1) Department of Health and Welfare. The Testing of Chemicals for Carcinogenicity, mutagenicity and Teratogenicity. Minister of Health and Welfare (Canada: Department of Health and Welfare, 1975).
(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. WHO Technical Report Series No. 364. (Geneva: World Health Organization, (1967).
§ 798.5195   Mouse biochemical specific locus test.
(a) Purpose. The mouse biochemical specific locus test (MBSL) may be used to detect and quantitate mutations originating in the germ line of a mammalian species.
(b) Definitions. (1) A biochemical specific locus mutation is a genetic change resulting from a DNA lesion causing alterations in proteins that can be detected by electrophoretic methods.
(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. Not applicable.
(d) Test method —(1) Principle. The principle of the MBSL is that heritable damage to the genome can be detected by electrophoretic analysis of proteins in the tissues of the progeny of mice treated with germ cell mutagens.
(2) Description. For technical reasons, males rather than females are generally treated with the test chemical. Treated males are then mated to untreated females to produce F1 progeny. Both blood and kidney samples are taken from progeny for electrophoretic analysis. Up to 33 loci can be examined by starch-gel electrophoresis and broad-range isoelectric focussing. Mutants are identified by variations from the normal electrophoretic pattern. Presumed mutants are bred to confirm the genetic nature of the change.
(3) Animal selection —(i) Species and strain. Mice shall be used as the test species. Although the biochemical specific locus test could be performed in a number of in bred strains, in the most frequently used cross, C57BL/6 females are mated to DBA/2 males to produce (C57BL/6 × DBA/2) F1 progeny for screening.
(ii) Age. Healthy, sexually-mature (at least 8 weeks old) animals shall be used for treatment and breeding.
(iii) Number. A decision on the minimum number of treated animals should take into account possible effects of the test chemical on the fertility of the treated animals. Other considerations should include:
(A) The production of concurrent spontaneous controls.
(B) The use of positive controls.
(C) The power of the test.
(4) Control groups —(i) Concurrent controls. An appropriate number of concurrent control loci shall be analyzed in each experiment. These should be partly derived from matings of untreated animals (from 5 to 20 percent ofthe treated matings), although some data on control loci can be taken from the study of the alleles transmitted from the untreated parent in the experimental cross. However, any laboratory which has had no prior experience with the test shall produce a spontaneous control sample of about 5,000 progeny animals and a positive control (using 100 mg/kg ethylnitrosourea) sample of at least 1,200 offspring.
(ii) Historical controls. Long-term, accumulated spontaneous control data (currently, 1 mutation in 1,200,000 control loci screened) are available for comparative purposes.
(5) Test chemicals —(i) Vehicle. When possible, test chemicals shall be dissolved or suspended in distilled water or buffered isotonic saline. Water-insoluble chemicals shall be dissolved or suspended in appropriate vehicles. The vehicle used shall neither interfere with the test chemical nor produce major toxic effects. Fresh preparations of the test chemical should be employed.
(ii) Dose levels. Usually, only one dose need be tested. This should be the maximum tolerated dose (MTD), the highest dose tolerated without toxic effects. Any temporary sterility induced due to elimination of spermatogonia at this dose must be of only moderate duration, as determined by are turn of males to fertility within 80 days after treatment. For evaluation of dose-response, it is recommended that at least two dose levels be tested.
(iii) Route of administration. Acceptable routes of administration include, but are not limited to, gavage, inhalation, and mixture with food or water, and intraperitoneal or intravenous injections.
(e) Test performance —(1) Treatment and mating. Male DBA/2 mice shall be treated with the test chemical and mated to virgin C57BL/6 females immediately after cessation of treatment. Each treated male shall be mated to new virgin C57BL/6 females each week. Each pairing will continue for a week until the next week's mating is to begin. This mating schedule permits sampling of all post-spermatogonial stages of germ-cell development during the first 7 weeks after exposure. Spermatogonial stem cells are studied thereafter. Repeated mating cycles should be conducted until sufficient offspring have been obtained to meet the power criterion of the assay for spermatogonial stem cells.
(2) Examination of offspring —(i) Birth and weaning. Offspring shall be examined at birth and at weaning for externally detectable changes in morphology and behavior; these could be due to dominant mutations. Such characteristics may include, but are not limited to, variations in coat color, appearance of eyes, size (in which case weighing of variant animals and littermates should be carried out), fur texture, etc. Gross changes in external form and behavior shall also be sought. Scrutiny of such visible characteristics of all animals shall be made during all subsequent manipulations of the animals.
(ii) Tissue sampling. Blood (about 0.1 mL) and one kidney shall be removed from progeny mice under anesthesia. Both tissues are then prepared for analysis by electrophoresis.
(iii) Electrophoresis. The gene products of 6 loci shall be analyzed in the blood sample by broad-range isoelectric focussing and of 27 loci in the kidney sample by starch-gel electrophoresis and enzyme-specific staining. Details on these procedures are included in paragraphs (g)(1) through (g)(3) of this section.
(iv) Mutant identification. Presumptive electrophoretic mutants shall be identified by variation from the normal electrophoretic banding patterns. Reruns of all variant samples shall be performed to confirm the presence of altered banding patterns. Samples from parents of progeny exhibiting banding pattern variations shall be assayed to determine whether the variant was induced by the experimental treatment or was pre-existing. All treatment-induced variants are bred to determine the genetic nature of the change.
(f) Data and reports —(1) Treatment of results. Data shall be presented in tabular form and shall permit independent analysis of cell stage-specific effects, and dose-dependent phenomena. The data shall be recorded and analyzed in such a way that clusters of identical mutations are clearly identified. The individual mutants detected shall be thoroughly described. In addition, concurrent positive control data (if employed) and spontaneous control data shall also be tabulated. These concurrent controls shall be added to, as well as compared with, the historical control data.
(2) Statistical evaluation. Data shall be evaluated by appropriate statistical methods.
(3) Interpretation of results. (i) There are several criteria for determining a positive response, one of which is a statistically significant dose-related increase in the frequency of electrophoretic mutations. Another criterion may be based upon detection of a reproducible and statistically significant positive response for at least one of these test points.
(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. (i) Positive results in the MBSL indicate that, under the test conditions, the test chemical induces heritable gene mutations in a mammalian species.
(ii) Negative results indicate that, under the test conditions, the test chemical does not induce heritable genemutations in a mammalian species.
(5) Test report. In addition to the reporting requirements as specified under 40 CFR part 792, subpart J, and paragraph (h) of this section, the following specific information shall be reported:
(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. For additional background information on this test guideline, the following references should be consulted:
(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 97:113-124 (1981).
(3) Johnson, F.M. and S.E. Lewis. “Mutation rate determinations based on electrophoretic analysis of laboratory mice.” Mutation Research 82:125-135 (1981a).
(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 78:3138-93141 (1981b).
(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 8:867-872 (1986).
(h) Additional requirements. Testing facilities conducting the mouse biochemical specific locus test in accordance with this section shall, in addition to adhering to the provisions of §§ 792.190 and 792.195 of this chapter, obtain, adequately identify, and retain for at least 10 years, acceptable 35-mm photographs (and their negatives) of the stained isoelectric-focussing columns and the stained starch-gels obtained following analyses of blood and kidney preparations, respectively, from mutant mice, their siblings, and their parents.
§ 798.5200   Mouse visible specific locus test.
(a) Purpose. The mouse visible specific locus test (MSLT) may be used to detect and quantitate mutations in the germ line of a mammalian species.
(b) Definitions. (1) A visible specific locus mutation is a genetic change that alters factors responsible for coat color and other visible characteristics of certain mouse strains.
(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. Not applicable.
(d) Test method —(1) Principle. (i) The principle of the MSLT is to cross individuals who differ with respect to the genes present at certain specific loci, so that a genetic alteration involving the standard gene at any one of these loci will produce an offspring detectably different from the standard heterozygote. The genetic change may be detectable by various means, depending on the loci chosen to be marked.
(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. For technical reasons, males rather than females are generally treated with the test agent. Treated males are then mated to females which are genetically homozygous for certain specific visible marker loci. Offspring are examined in the next generation for evidence that a new mutation has arisen.
(3) Animal selection —(i) Species and strain. Mice shall be used as the test species. Male mice shall be either (C 3 H × 101)F 1 or (101 × C 3 H)F 1 hybrids. Females shall be T stock virgins.
(ii) Age. Healthy sexually mature animals shall be used.
(iii) Number. A decision on the minimum number of treated animals should take into account the spontaneous variation of the biological characterization being evaluated. Other considerations should include:
(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. Animals shall be randomized and assigned to treatment and control groups.
(4) Control groups —(i) Concurrent controls. The use of positive or spontaneous controls is left to the discretion of the investigator. However, any laboratory which has had no prior experience with the test shall, at its first attempt, produce a negative control sample of 20,000 and a positive control, using 100 mg/kg 1-ethyl-nitrosourea, in a sample of 5,000 offspring.
(ii) Historical controls. Long term, accumulated spontaneous control data of 43/801,406 are available for comparative purposes.
(5) Test chemicals —(i) Vehicle. When possible, test chemicals should be dissolved or suspended in distilled water or isotonic saline buffered appropriately, if needed, for stability. Water-insoluble chemicals shall be dissolved or suspended in appropriate vehicles. The vehicle used shall neither interfere with the test compound nor produce major toxic effects. Fresh preparations of the test chemical should be employed.
(ii) Dose levels. Usually, only one dose level need be tested. This should be the highest dose tolerated without toxic effects, provided that any temporary sterility induced due to elimination of spermatagonia is of only moderate duration, as determined by a return of males to fertility within 80 days after treatment. For evaluation of dose-response, it is recommended that at least two dose levels be tested.
(iii) Route of administration. Acceptable routes of administration include gavage, inhalation, admixture with food or water, and IP or IV injections.
(e) Test performance —(1) Treatment and mating. Hybrid F 1 (C 3 H × 101 or 101 × C 3 H) male mice shall be treated with the test substance and immediately mated to virgin T stock females. Each treated male shall be mated to a fresh group of 2 to 4 virgin females each week for 7 weeks, after which he shall be returned to the first group of females and rotated through the seven sets of females repeatedly. This mating schedule generally permits sampling of all postspermatagonial stages of germ cell development during the first 7 weeks and rapid accumulation of data for exposed spermatagonial stem cells thereafter. Repeated mating cycles should be conducted until the entire spermatogonial cycle has been evaluated and enough offspring have been obtained to meet the power criterion of the assay.
(2) Examination of offspring. (i) Offspring may be examined at (or soon after) birth but must be examined at about 3 weeks of age at which time the numbers of mutant and nonmutant offspring in each litter shall be recorded.
(ii) Nonmutant progeny should be discarded. Mutant progeny shall be subjected to genetic tests for verification.
(f) Data and report —(1) Treatment of results. Data shall be presented in tabular form and shall permit independent analysis of cell stage specific effects and dose dependent phenomena. The data shall be recorded and analyzed in such a way that clusters of identical mutations are clearly identified. The individual mutants detected shall be thoroughly described. In addition, concurrent positive and negative control data, if they are available, shall be tabulated so that it is possible to differentiate between concurrent (when available) and long-term accumulated mutation frequencies.
(2) Statistical evaluation. Data shall be evaluated by appropriate statistical methods.
(3) Interpretation of results. (i) There are several criteria for determining a positive result, one of which is a statistically significant dose-related increase in the number of specific locus mutations. Another criterion may be based upon detection of a reproducible and statistically significant positive response for at least one of the test points.
(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. (i) Positive results in the MSLT indicate that under the test conditions the test substance induces heritable gene mutations in the test species.
(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. In addition to the reporting requirements as specified under 40 CFR part 792, subpart J, and paragraph (h) of this section, the following specific information shall be reported:
(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. For additional background information on this test guideline the following references should be consulted:
(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, 86:329-354 (1981).
(2) [Reserved]
(h) Additional requirements. Testing facilities conducting the mouse visible specific locus test in accordance with this section shall, in addition to adhering to the provisions of §§ 792.190 and 792.195 of this chapter, obtain, and retain for at least 10 years, acceptable 35-mm color photographs (and their negatives) demonstrating the visible mutations observed in mutant animals and the lack of such mutations in their siblings and parents.
§ 798.5265   The salmonella typhimurium reverse mutation assay.
(a) Purpose. The Salmonella typhimurium histidine (his) reversion system is a microbial assay which measures his − → his = reversion induced by chemicals which cause base changes or frameshift mutations in the genome of this organism.
(b) Definitions. (1) A reverse mutation assay in Salmonella typhimurium detects mutation in a gene of a histidine requiring strain to produce a histidine independent strain of this organism.
(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. These may include, but need not be limited to, sodium azide, 2-nitrofluorene, 9-aminoacridine, 2-aminoanthracene, congo red, benzopurpurin 4B, trypan blue or direct blue 1.
(d) Test method —(1) Principle. Bacteria are exposed to test chemical with and without a metabolic activation system and plated onto minimal medium. After a suitable period of incubation, revertant colonies are counted and compared to the number of spontaneous revertants in an untreated and/or vehicle control culture.
(2) Description. Several methods for performing the test have been described. Among those used are:
(i) The direct plate incorporation method.
(ii) The preincubation method.
(iii) The azo-reduction method.
(3) Strain selection —(i) Designation. At the present time four strains, TA 1535, TA 1537, TA 98 and TA 100 should be used. The use of other strains in addition to these four is left to the discretion of the investigator.
(ii) Preparation and storage. Recognized methods of stock culture preparation and storage should be used. The requirement of histidine for growth should be demonstrated for each strain. Other phenotypic characteristics should be checked using such methods as crystal violet sensitivity and resistance to ampicillin. Spontaneous reversion frequency should be in the range expected either as reported in the literature or as established in the laboratory by historical control values.
(iii) Bacterial growth. Fresh cultures of bacteria should be grown up to the late exponential or early stationary phase of growth (approximately 10 8 -10 9 cells per ml).
(4) Metabolic activation. Bacteria should be exposed to the test substance both in the presence and absence of an appropriate metabolic activation system. For the direct plate incorporation method, the most commonly used system is a cofactor supplemented postmitochondrial fraction prepared from the livers of rodents treated with enzyme inducing agents such as Aroclor 1254. For the azo-reduction method, a cofactor supplemented postmitochondrial fraction prepared from the livers of untreated hamsters is preferred. For this method, the cofactor supplement should contain flavin mononucleotide, exogenous glucose 6-phosphate dehydrogenase, NADH and excess of glucose-6-phosphate.
(5) Control groups —(i) Concurrent controls. Concurrent positive and negative (untreated and/or vehicle) controls shall be included in each experiment. Positive controls shall ensure both strain responsiveness and efficacy of the metabolic activation system.
(ii) Strain specific positive controls. Strain specific positive controls shall be included in the assay. Examples of strain specific positive controls are as follows:
(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. The positive control reference substance for tests including a metabolic activation system should be selected on the basis of the type of activation system used in the test. 2-Aminoanthracene is an example of a positive control compound in plate-incorporation tests using postmitochondrial fractions from the livers of rodents treated with enzyme inducing agents such as Aroclor-1254. Congo red is an example of a positive control compound in the azo-reduction method. Other positive control reference substances may be used.
(iv) Class-specific positive controls. The azo-reduction method should include positive controls from the same class of compounds as the test agent wherever possible.
(6) Test chemicals —(i) Vehicle. Test chemicals and positive control reference substances should be dissolved or suspended in an appropriate vehicle and then further diluted in vehicle for use in the assay.
(ii) Exposure concentrations. (A) The test should initially be performed over a broad range of concentrations. Among the criteria to be taken into consideration for determining the upper limits of test chemical concentration are cytotoxicity and solubility. Cytotoxicity of the test chemical may be altered in the presence of metabolic activation systems. Toxicity may be evidenced by a reduction in the number of spontaneous revertants, a clearing of the background lawn or by the degree of survival of treated cultures. Relatively insoluble compounds should be tested up to the limits of solubility. For freely soluble nontoxic chemicals, the upper test chemical concentration should be determined on a case by case basis.
(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 —(1) Direct plate incorporation method. For this test without metabolic activation, test chemica1 and 0.1 m1 of a fresh bacterial culture should be added to 2.0 ml of overlay agar. For tests with metabolic activation, 0.5 ml of activation mixture containing an adequate amount of postmitochondrial fraction should be added to the agar overlay after the addition of test chemical and bacteria. Contents of each tube shall be mixed and poured over the surface of a selective agar plate. Overlay agar shall be allowed to solidify before incubation. At the end of the incubation period, revertant colonies per plate shall be counted.
(2) Azo-reduction method. (i) For this test with metabolic activation, 0.5 ml of S-9 mix containing 150 ul of S-9 and 0.1 ml of bacterial culture should be added to a test tube kept on ice. One-tenth milliliter of chemical should be added, and the tubes should be incubated with shaking at 30 °C for 30 min. At the end of the incubation period, 2.0 ml of agar should be added to each tube, the contents mixed and poured over the surface of a selective agar plate. Overlay agar shall be allowed to solidify before incubation. At the end of the incubation period, revertant colonies per plate shall be counted.
(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. Other methods may also be appropriate.
(4) Media. An appropriate selective medium with an adequate overlay agar shall be used.
(5) Incubation conditions. All plates within a given experiment shall be incubated for the same time period. This incubation period shall be for 48-72 hours at 37 °C.
(6) Number of cultures. All plating should be done at least in triplicate.
(f) Data and report —(1) Treatment of results. Data shall be presented as number of revertant colonies per plate for each replicate and dose. The numbers of revertant colonies on both negative (untreated and/or vehicle) and positive control plates shall also be presented. Individual plate counts, the mean number of revertant colonies per plate and standard deviation shall be presented for test chemical and positive and negative (untreated and/or vehicle) controls.
(2) Statistical evaluation. Data should be evaluated by appropriate statistical methods.
(3) Interpretation of results. (i) There are several criteria for determining a positive result, one of which is a statistically significant dose-related increase in the number of revertants. Another criterion may be based upon detection of a reproducible and statistically significant positive response for at least one of the test substance concentrations.
(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. (i) Positive results from the S. typhimurium reverse mutation assay indicate that, under the test conditions, the test substance induces point mutations by base changes or frameshifts in the genome of this organism.
(ii) Negative results indicate that under the test conditions the test substance is not mutagenic in S. typhimurium.
(5) Test report. In addition to the reporting recommendations as specified under 40 CFR part 792, subpart J, the following specific information shall be reported:
(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. For additional background information on this test guideline the following references should be consulted:
(1) Ames, B.N., McCann, J., Yamasaki, E. “Methods for detecting carcinogens and mutagens with the Salmonella/ mammalian-microsome mutagenicity test,” Mutation Research 31:347-364 (1975).
(2) de Serres, F.J., Shelby, M.D. “The Salmonella mutagenicity assay: recommendations,” Science 203:563-565 (1979).
(3) Prival, M.J., Mitchell, V.D. “Analysis of a method for testing azo dyes for mutagenic activity in Salmonella typhimurium in the presence of flavin mononucleotide and hamster liver S-9,” Mutation Research 97:103-116 (1982).
(4) Vogel, H.J., Bonner, D.M. “Acetylornithinase of E. coli: partial purification and some properties,” Journal of Biological Chemistry. 218:97-106 (1956).