Title 40—Protection of Environment--Volume 1
PART 799—IDENTIFICATION OF SPECIFIC CHEMICAL SUBSTANCE AND MIXTURE TESTING REQUIREMENTS
§ 799.9110   TSCA acute oral toxicity.
(a) Scope. This section is intended to meet the testing requirements under section 4 of the Toxic Substances Control Act (TSCA). In the assessment and evaluation of the toxic characteristics of a substance, determination of acute oral toxicity is usually an initial step. It provides information on health hazards likely to arise from short-term exposure by the oral route. Data from an acute study may serve as a basis for classification and labeling. It is traditionally a step in establishing a dosage regimen in subchronic and other studies and may provide initial information on the mode of toxic action of a substance. An evaluation of acute toxicity data should include the relationship, if any, between the exposure of animals to the test substance and the incidence and severity of all abnormalities, including behavioral and clinical abnormalities, the reversibility of observed abnormalities, gross lesions, body weight changes, effects on mortality, and any other toxic effects.
(b) Source. The source material used in developing this TSCA test guideline is the Office of Prevention, Pesticides, and Toxic Substances (OPPTS) harmonized test guideline 870.1100 (August 1998, final guideline). This source is available at the address in paragraph (f) of this section.
(c) Definitions. The following definitions apply to this section.
Acute oral toxicity is the adverse effects occurring within a short period of time after oral administration of either a single dose of a substance or multiple doses given within a 24-hour period.
Dosage is a general term comprising the dose, its frequency, and the duration of dosing.
Dose is the amount of test substance administered. Dose is expressed as weight of test substance (milligrams, grams) per unit weight of test animal (e.g., milligrams per kilogram).
Dose-effect is the relationship between the dose and the magnitude of a defined biological effect either in an individual or in a population sample.
Dose-response is the relationship between the dose and the proportion of a population sample showing a defined effect.
LD 50 (median lethal dose) is a statistically derived estimate of single dose of a substance that can be expected to cause death in 50% of animals when administered by the oral route. The LD 50 value is expressed in terms of weight of test substance per unit weight of test animal (milligrams per kilogram).
(d) Alternative approaches to the determination of acute toxicity. (1) EPA will accept the following procedures to reduce the number of animals used to evaluate acute effects of chemical exposure while preserving its ability to make reasoned judgments about safety:
(i) Estimation of acute oral toxicity. When further study is warranted, EPA generally supports limiting such tests to those using the lowest number of animals feasible. EPA will accept three alternative Organization for Economic Cooperation and Development (OECD) test methods in place of the “traditional” acute oral toxicity test. The three OECD alternatives are the following:
(A) The up and down procedure as described in OECD Guideline 425 referenced in paragraph (f)(4) of this section.
(B) The acute toxic class method as described in OECD Guideline 423 and referenced in paragraph (f)(6) of this section.
(C) The fixed dose method as described in OECD Guideline 420 and referenced in paragraph (f)(5) of this section.
(ii) Limit test. When data on structurally related chemicals are inadequate, a limit test may be considered. If rodents are used, a limit dose of at least 2,000 mg per kilogram of body weight may be administered to a single group of five males and five females using the procedures described in paragraph (e) of this section. If no lethality is demonstrated, no further testing for acute oral toxicity is needed. (Under current policy and regulations for pesticide products, precautionary statements may still be required unless there are data to indicate the LD 50 is greater than 5,000 mg/kg.) If compound-related mortality is produced in the limit test, further study may need to be considered.
(2) [Reserved]
(e) Conventional acute toxicity test —(1) Principle of the test method. The test substance is administered orally by gavage in graduated doses to several groups of experimental animals, one dose being used per group. The doses chosen may be based on the results of a range finding test. Subsequently, observations of effects and deaths are made. Animals that die during the test are necropsied, and at the conclusion of the test the surviving animals are sacrificed and necropsied. This section is directed primarily to studies in rodent species but may be adapted for studies in nonrodents. Animals showing severe and enduring signs of distress and pain may need to be humanely sacrificed. Dosing test substances in a way known to cause marked pain and distress due to corrosive or irritating properties need not be carried out.
(2) Substance to be tested. Test, control, and reference substances are described in 40 CFR Part 792—Good Laboratory Practice Standards.
(3) Test procedures —(i) Preparations. Healthy young adult animals are acclimatized to the laboratory conditions for at least 5 days prior to the test before the test animals are randomized and assigned to the treatment groups.
(ii) Animal selection —(A) Species and strain. Although several mammalian test species may be used, the rat is the preferred species. Commonly used laboratory strains must be employed. If another species is used, the tester must provide justification and reasoning for its selection.
(B) Age. Young adult rats between 8- and 12-weeks-old at the beginning of dosing should be used. Rabbits should be at least 12 weeks of age at study initiation. The weight variation of animals used in a test must be within 20% of the mean weight for each sex.
(C) Number and sex of animals. ( 1 ) At least five experimentally naive rodents are used at each dose level. They should all be of the same sex. After completion of the study in one sex, at least one group of five animals of the other sex is dosed to establish that animals of this sex are not markedly more sensitive to the test substance. The use of fewer animals may be justified in individual circumstances. Where adequate information is available to demonstrate that animals of the sex tested are markedly more sensitive, testing in animals of the other sex may be dispensed with. An acceptable option would be to test at least one group of five animals per sex at one or more dose levels to definitively determine the more sensitive sex prior to conducting the main study.
( 2 ) The females must be nulliparous and nonpregnant.
( 3 ) In acute toxicity tests with animals of a higher order than rodents, the use of smaller numbers should be considered.
(D) Assignment of animals. Each animal must be assigned a unique identification number. A system to assign animals to test groups and control groups randomly is required.
(E) Housing. Animals may be group-caged by sex, but the number of animals per cage must not interfere with clear observation of each animal. The biological properties of the test substance or toxic effects (e.g., morbidity, excitability) may indicate a need for individual caging.
( 1 ) The temperature of the experimental animal rooms should be at 22 ±3 °C for rodents.
( 2 ) The relative humidity of the experimental animal rooms should be 30 to 70%.
( 3 ) Where lighting is artificial, the sequence should be 12-hours light/12-hours dark.
( 4 ) For feeding, conventional laboratory diets may be used with an unlimited supply of drinking water.
(iii) Dose levels and dose selection. (A) Three dose levels must be used, spaced appropriately to produce test groups with a range of toxic effects and mortality rates. The data collected must be sufficient to produce a dose-response curve and permit an acceptable estimation of the LD 50 . Range finding studies using single animals may help to estimate the positioning of dose groups so that no more than three dose levels will be necessary.
(B) Limit test. This test has been defined and described in paragraph (d)(1)(ii) of this section.
(C) Vehicle. Where necessary, the test substance is dissolved or suspended in a suitable vehicle. If a vehicle or diluent is needed, it should not elicit toxic effects itself nor substantially alter the chemical or toxicological properties of the test substance. It is recommended that wherever possible the use of an aqueous solution be considered first, followed by consideration of a solution in oil (e.g., corn oil), and then by consideration of possible solution in other vehicles. Toxic characteristics of nonaqueous vehicles should be known, and, if not known, should be determined before the test.
(D) Volume. The maximum volume of liquid that can be administered at one time depends on the size of the test animal. In rodents, the volume should not exceed 1 mL/100 g body weight, except when an aqueous solution is used in which case 2 mL/100 g may be administered. Either constant volume or constant concentration administration is acceptable when dosing, provided the following guidance is employed. When possible, the liquid test material should be dosed neat. Otherwise, it may be diluted, using the highest concentration possible, although volumes less than 0.5 mL per animal would not be required. Lower dose volumes are acceptable if they can be accurately administered. Solid materials should be suspended or dissolved in the minimum amount of vehicle and dosed at the highest concentration possible.
(iv) Exposure and exposure duration. (A) Animals must be fasted prior to test substance administration. For the rat, feed should be withheld overnight; for other rodents with higher metabolic rates a shorter period of fasting is appropriate.
(B) The test substance must be administered in a single dose by gavage, using a stomach tube or suitable intubation cannula.
(C) If a single dose is not possible, the dose may be given in smaller fractions over a period not exceeding 24 hours. Where a dose is administered in fractions, it may be necessary to provide the animals with food and water, depending on the length of the dosing period.
(D) After the substance has been administered, feed may be withheld for an additional 3-4 hours.
(v) Observation period. Although 14 days is recommended as a minimum observation period, the duration of observation should not be fixed rigidly. It should be determined by the toxic reactions, rate of onset, and length of recovery period, and may thus be extended when considered necessary. The time at which signs of toxicity appear, their duration, and the time to death are important, especially if there is a tendency for deaths to be delayed.
(vi) Observation of animals. (A) A careful clinical examination must be made at least once each day.
(B) Additional observations must be made daily, especially in the early days of the study. Appropriate actions should be taken to minimize loss of animals to the study (e.g., necropsy or refrigeration of those animals found dead and isolation of weak or moribund animals).
(C) Observations must be detailed and carefully recorded, preferably using explicitly defined scales. Observations should include, but not be limited to, evaluation of skin and fur, eyes and mucous membranes, respiratory and circulatory effects, autonomic effects such as salivation, central nervous system effects, including tremors and convulsions, changes in the level of activity, gait and posture, reactivity to handling or sensory stimuli, altered strength, and stereotypies or bizarre behavior (e.g., self-mutilation, walking backwards).
(D) Individual weights of animals must be determined shortly before the test substance is administered, weekly thereafter, and at death. Changes in weights should be calculated and recorded when survival exceeds 1 day.
(E) The time of death should be recorded as precisely as possible.
(vii) Gross pathology. (A) At the end of the test, surviving animals must be weighed and sacrificed.
(B) A gross necropsy must be performed on all animals under test. All gross pathology changes should be recorded.
(C) If necropsy cannot be performed immediately after a dead animal is discovered, the animal should be refrigerated (not frozen) at temperatures low enough to minimize autolysis. Necropsies should be performed as soon as practicable, normally within a day or two.
(viii) Additional evaluation. Microscopic examination of organs showing evidence of gross pathology in animals surviving 24 hours or more should also be considered because it may yield useful information.
(ix) Data and reporting —(A) Treatment of results. Data must be summarized in tabular form, showing for each test group the number of animals at the start of the test, body weights, time of death of individual animals at different dose levels, number of animals displaying other signs of toxicity, description of toxic effects, and necropsy findings. Any methods used for calculation of the LD 50 or any other parameters should be specified and referenced. Methods for parameter estimation are described in the references listed in paragraphs (f)(1), (f)(2), and (f)(3) of this section.
(B) Evaluation of results. An evaluation should include the relationship, if any, between exposure of the animals to the test substance and the incidence and severity of all abnormalities, including behavioral and clinical abnormalities, gross lesions, body weight changes, effects on mortality, and any other toxic effects. The LD 50 value should always be considered in conjunction with the observed toxic effects and any necropsy findings. The LD 50 value is a relatively coarse measurement, useful only as a reference value for classification and labeling purposes, and for an expression of the lethal potential of the test substance by the ingestion route. Reference should always be made to the experimental animal species in which the LD 50 value was obtained.
(C) Test report. In addition to the reporting requirements specified under EPA Good Laboratory Practice Standards at 40 CFR part 792, subpart J, the following specific information must be reported. The test report shall include:
( 1 ) Species, strain, sex, and source of test animals.
( 2 ) Method of randomization in assigning animals to test and control groups.
( 3 ) Rationale for selection of species, if other than that recommended.
( 4 ) Tabulation of individual and test group data by sex and dose level (e.g., number of animals exposed, number of animals showing signs of toxicity and number of animals that died or were sacrificed during the test).
( i ) Description of toxic effects, including their time of onset, duration, reversibility, and relationship to dose.
( ii ) Body weights.
( iii ) Time of dosing and time of death after dosing.
( iv ) Dose-response curves for mortality and other toxic effects (when permitted by the method of determination).
( v ) Gross pathology findings.
( vi ) Histopathology findings and any additional clinical chemistry evaluations, if performed.
( 5 ) Description of any pretest conditioning, including diet, quarantine and treatment for disease.
( 6 ) Description of caging conditions including: Number (or change in number) of animals per cage, bedding material, ambient temperature and humidity, photoperiod, and identification of diet of test animals.
( 7 ) Manufacturer, source, purity, and lot number of test substance.
( 8 ) Relevant properties of substance tested including physical state and pH (if applicable).
( 9 ) Identification and composition of any vehicles (e.g., diluents, suspending agents, and emulsifiers) or other materials used in administering the test substance.
( 10 ) A list of references cited in the body of the report. References to any published literature used in developing the test protocol, performing the testing, making and interpreting observations, and compiling and evaluating the results.
(f) References. For additional background information on this test guideline, the following references should be consulted. These references are available at the addresses in § 700.17(b)(1) and (2) of this chapter.
(1) Chanter, D.O. and Heywood, R. The LD 50 Test: Some Considerations of Precision. Toxicology Letters 10:303-307 (1982).
(2) Finney, D.J. Chapter 3—Estimation of the median effective dose and Chapter 4—Maximum likelihood estimation, Probit Analysis, 3rd ed. Cambridge, London (1971).
(3) Finney, D.J. The Median Lethal Dose and Its Estimation. Archives of Toxicology 56:215-218 (1985).
(4) Organization for Economic Cooperation and Development. OECD Guidelines for the Testing of Chemicals. OECD Guideline 425: Acute Oral Toxicity: Up-and-Down Procedure, Approved: June 1998.
(5) Organization for Economic Cooperation and Development. OECD Guidelines for Testing of Chemicals. Guideline 420: Acute Oral Toxicity—Fixed Dose Method, Adopted: July 17, 1992.
(6) Organization for Economic Cooperation and Development. OECD Guidelines for Testing of Chemicals. Guideline 423: Acute Oral Toxicity—Acute Toxic Class Method, Adopted: March 22, 1996.
(7) Organization for Economic Cooperation and Development. OECD Guidelines for Testing of Chemicals. Guideline 401: Acute Oral Toxicity, Adopted: February 24, 1987.
§ 799.9120   TSCA acute dermal toxicity.
(a) Scope. This section is intended to meet the testing requirements under section 4 of the Toxic Substances Control Act (TSCA). In the assessment and evaluation of the toxic characteristics of a substance, determination of acute dermal toxicity is useful where exposure by the dermal route is likely. It provides information on health hazards likely to arise from short-term exposure by the dermal route. Data from an acute study may serve as a basis for classification and labeling. It is an initial step in establishing a dosage regimen in subchronic and other studies and may provide information on dermal absorption and the mode of toxic action of a substance by this route. An evaluation of acute toxicity data should include the relationship, if any, between the exposure of animals to the test substance and the incidence and severity of all abnormalities, including behavioral and clinical abnormalities, the reversibility of observed abnormalities, gross lesions, body weight changes, effects on mortality, and any other toxic effects.
(b) Source. The source material used in developing this TSCA test guideline is the Office of Prevention, Pesticides, and Toxic Substances (OPPTS) harmonized test guideline 870.1200 (August 1998, final guideline). This source is available at the address in paragraph (f) of this section.
(c) Definitions. The following definitions apply to this section.
Acute dermal toxicity is the adverse effects occurring within a short time of dermal application of a single dose of a substance or multiple doses given within a 24-hour period.
Dosage is a general term comprising the dose, its frequency and the duration of dosing.
Dose is the amount of test substance applied. Dose is expressed as weight of test substance (grams, milligrams) per unit weight of test animal (e.g., milligrams per kilogram).
Dose-effect is the relationship between the dose and the magnitude of a defined biological effect either in an individual or in a population sample.
Dose-response is the relationship between the dose and the proportion of a population sample showing a defined effect.
LD 50 (median lethal dose), dermal, is a statistically derived estimate of a single dose of a substance that can be expected to cause death in 50% of treated animals when applied to the skin. The LD 50 value is expressed in terms of weight of test substance per unit weight of test animal (milligrams per kilogram).
(d) Approaches to the determination of acute toxicity. (1) EPA recommends the following means to reduce the number of animals used to evaluate acute effects of chemical exposure while preserving its ability to make reasonable judgments about safety:
(i) Using data from substantially similar mixtures. In order to minimize the need for animal testing, the Agency encourages the review of existing acute toxicity information on mixtures that are substantially similar to the mixture under investigation. In certain cases it may be possible to glean enough information to make preliminary hazard evaluations that may reduce the need for further animal testing.
(ii) Limit test. When data on structurally related chemicals are inadequate, a limit test may be considered. If rodents are used, a limit dose of at least 2,000 mg/kg bodyweight may be administered to a single group of five males and five females using the procedures described in paragraph (e) of this section. If no lethality is demonstrated, no further testing for acute dermal toxicity is needed. If compound-related mortality is produced, further study may need to be considered.
(2) [Reserved]
(e) Conventional acute toxicity test —(1) Principle of the test method. The test substance is applied dermally in graduated doses to several groups of experimental animals, one dose being used per group. The doses chosen may be based on the results of a range finding test. Subsequently, observations of effects and deaths are made. Animals that die during the test are necropsied, and at the conclusion of the test the surviving animals are sacrificed and necropsied. This section is directed primarily to studies in either rats, rabbits, or guinea pigs but may be adapted for studies in other species. Animals showing severe and enduring signs of distress and pain may need to be humanely sacrificed. Dosing test substances in a way known to cause marked pain and distress due to corrosive or irritating properties need not be carried out.
(2) Substance to be tested. Test, control, and reference substances are discussed in 40 CFR Part 792—Good Laboratory Practice Standards.
(3) Test procedures —(i) Preparations. Healthy young adult animals are acclimatized to the laboratory conditions for at least 5 days prior to the test before the test animals are randomized and assigned to the treatment groups.
(ii) Animal selection —(A) Species and strain. The rat, rabbit, or guinea pig may be used. The albino rabbit is preferred because of its size, ease of handling, skin permeability, and extensive data base. Commonly used laboratory strains must be employed. If a species other than rats, rabbits, or guinea pigs is used, the tester must provide justification and reasoning for its selection.
(B) Age. Young adult animals, rats between 8- and 12-weeks-old, rabbits at least 12-weeks-old, and guinea pigs between 5- and 6-weeks-old at the beginning of dosing should be used. The weight variation of animals used in a test must be within 20% of the mean weight for each sex.
(C) Number and sex of animals. ( 1 ) At least five experimentally naive animals with healthy intact skin are used at each dose level. They should all be of the same sex. After completion of the study in one sex, at least one group of five animals of the other sex is dosed to establish that animals of this sex are not markedly more sensitive to the test substance. The use of fewer animals may be justified in individual circumstances. Where adequate information is available to demonstrate that animals of the sex tested are markedly more sensitive, testing in animals of the other sex may be dispensed with. An acceptable option would be to test at least one group of five animals per sex at one or more dose levels to definitively determine the more sensitive sex prior to conducting the main study.
( 2 ) The females must be nulliparous and nonpregnant.
( 3 ) In acute toxicity tests with animals of a higher order than those mentioned above, the use of smaller numbers should be considered.
(D) Assignment of animals. Each animal must be assigned a unique identification number. A system to randomly assign animals to test groups and control groups is required.
(E) Housing. Animals should be housed in individual cages.
( 1 ) The temperature of the experimental animal rooms should be at 22 ±3 °C for rodents, 20 ±3 °C for rabbits.
( 2 ) The relative humidity of the experimental animal rooms should be 30 to 70%.
( 3 ) Where lighting is artificial, the sequence should be 12-hours light/12-hours dark.
( 4 ) For feeding, conventional laboratory diets may be used with an unlimited supply of drinking water.
(iii) Dose levels and dose selection. (A) Three dose levels must be used and spaced appropriately to produce test groups with a range of toxic effects and mortality rates. The data must be sufficient to produce a dose-response curve and permit an acceptable estimation of the median lethal dose. Range finding studies using single animals may help to estimate the positioning of the dose groups so that no more than three dose levels will be necessary.
(B) Limit test. This test is described in paragraph (d)(2)(ii) of this section.
(C) Vehicle. Solids should be pulverized when possible. The test substance should be moistened sufficiently with water or, where necessary, a suitable vehicle to ensure good contact with skin. If a vehicle or diluent is needed, it should not elicit toxic effects itself nor substantially alter the chemical or toxicological properties of the test substance. In addition, the influence of the vehicle on penetration of skin by the test substance should be taken into account. It is recommended that wherever possible the use of an aqueous solution be considered first, followed by consideration of a solution in oil (e.g., corn oil), and then by consideration of possible solution in other vehicles. For nonaqueous vehicles the toxic characteristics of the vehicle should be known, and if not known should be determined before the test. Acceptable alternative vehicles include gum arabic, ethanol and water, carboxymethyl cellulose, glycerol, propylene glycol, PEG vegetable oil, and mineral oil as long as the vehicle is not irritating and the inability to use water or saline is justified in the report.
(iv) Exposure and exposure duration. The test substance must be administered over a period of 24 hours.
(v) Preparation of animal skin. Fur must be clipped from the dorsal area of the trunk of the test animals. Shaving may be employed, but it should be carried out at least 24 hours before dosing. Care must be taken to avoid abrading the skin, which would alter its permeability.
(vi) Application of test substance. (A) The test substance must be applied uniformly over a shaved or clipped area which is approximately 10% of the body surface area. The area starting at the scapulae (shoulders) to the wing of the ileum (hip bone) and half way down the flank on each side of the animal should be shaved or clipped. Liquid test materials should be undiluted if possible. 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 practical. The test material is not removed until 24 hours after application. In the case where less than 10% of the surface area is covered an approximation of the exposed areas should be determined.
(B) The test substance must be held in contact with the skin with a porous gauze dressing (<8 ply) and nonirritating tape throughout a 24-hour exposure period. The test site must 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. Although a semiocclusive dressing is preferred, an occlusive dressing will also be acceptable.
(C) At the end of the exposure period, residual test substance should be removed where practicable using water or an appropriate solvent.
(vii) Observation period. Although 14 days is recommended as a minimum observation period, the duration of observation should not be fixed rigidly. It should be determined by the toxic reactions, rate of onset, and length of recovery period, and may thus be extended when considered necessary. The time at which signs of toxicity appear, their duration, and the time to death are important, especially if there is a tendency for deaths to be delayed.
(viii) Observation of animals. (A) A careful clinical examination must be made at least once each day.
(B) Additional observations must be made daily, especially in the early days of the study. Appropriate actions should be taken to minimize loss of animals to the study (e.g., necropsy or refrigeration of those animals found dead and isolation of weak or moribund animals).
(C) Observations must be detailed and carefully recorded, preferably using explicitly defined scales. Observations should include, but not be limited to, evaluation of skin and fur, eyes and mucous membranes, respiratory and circulatory effects, autonomic effects such as salivation, central nervous system effects, including tremors and convulsions, changes in the level of activity, gait and posture, reactivity to handling or sensory stimuli, altered strength, and stereotypies or bizarre behavior ( e.g., self-mutilation, walking backwards).
(D) Individual weights of animals must be determined shortly before the test substance is administered, weekly thereafter, and at death. Changes in weights should be calculated and recorded when survival exceeds one day.
(E) The time of death should be recorded as precisely as possible.
(ix) Gross pathology. (A) At the end of the test, surviving animals must be weighed and sacrificed.
(B) A gross necropsy must be performed on all animals under test. All gross pathology changes should be recorded.
(C) If necropsy cannot be performed immediately after a dead animal is discovered, the animal should be refrigerated (not frozen) at temperatures low enough to minimize autolysis. Necropsies should be performed as soon as practicable, normally within a day or two.
(x) Additional evaluations. Microscopic examination of organs showing evidence of gross pathology in animals surviving 24 hours or more should also be considered because it may yield useful information.
(xi) Data and reporting —(A) Treatment of results. Data must be summarized in tabular form, showing for each test group the number of animals at the start of the test, body weights, time of death of individual animals at different dose levels, number of animals displaying other signs of toxicity, description of toxic effects and necropsy findings. Any methods used for calculation of the LD 50 or any other parameters should be specified and referenced. Methods for parameter estimation are described in the references listed in paragraphs (f)(1), (f)(2), and (f)(3) of this section.
(B) Evaluation of results. An evaluation should include the relationship, if any, between exposure of the animals to the test substance and the incidence and severity of all abnormalities, including behavioral and clinical abnormalities, gross lesions, body weight changes, effects on mortality, and any other toxic effects. The LD 50 value should always be considered in conjunction with the observed toxic effects and any necropsy findings. The LD 50 value is a relatively coarse measurement, useful only as a reference value for classification and labeling purposes, and for an expression of the lethal potential of the test substance by the dermal route. Reference should always be made to the experimental animal species in which the LD 50 value was obtained.
(C) Test report. In addition to the reporting requirements specified under EPA Good Laboratory Practice Standards at 40 CFR part 792, subpart J, the following specific information must be reported. The test report must include:
( 1 ) Species, strain, sex, and source of test animals.
( 2 ) Method of randomization in assigning animals to test and control groups.
( 3 ) Rationale for selection of species, if other than that recommended.
( 4 ) Tabulation of individual and test group data by sex and dose level ( e.g., number of animals exposed, number of animals showing signs of toxicity and number of animals that died or were sacrificed during the test).
( i ) Description of toxic effects, including their time of onset, duration, reversibility, and relationship to dose.
( ii ) Body weights.
( iii ) Time of dosing and time of death after dosing.
( iv ) Dose-response curves for mortality and other toxic effects (when permitted by the method of determination).
( v ) Gross pathology findings.
( vi ) Histopathology findings and any additional clinical chemistry evaluations, if performed.
( 5 ) Description of any pre-test conditioning, including diet, quarantine and treatment for disease.
( 6 ) Description of caging conditions including: Number (or change in number) of animals per cage, bedding material, ambient temperature and humidity, photoperiod, and identification of diet of test animals.
( 7 ) Manufacturer, source, purity, and lot number of test substance.
( 8 ) Relevant properties of substance tested including physical state and pH (if applicable).
( 9 ) Identification and composition of any vehicles (e.g., diluents, suspending agents, and emulsifiers) or other materials used in administering the test substance.
( 10 ) A list of references cited in the body of the report. References to any published literature used in developing the test protocol, performing the testing, making and interpreting observations, and compiling and evaluating the results.
(f) References. For additional background information on this test guideline, the following references should be consulted. These references are available at the addresses in § 700.17(b)(1) and (2) of this chapter.
(1) Chanter, D.O. and Heywood, R., The LD 50 Test: Some Considerations of Precision, Toxicology Letters 10:303-307 (1982).
(2) Finney, D.J. Chapter 3—Estimation of the median effective dose and Chapter 4-Maximum likelihood estimation, Probit Analysis, 3rd ed. Cambridge, London (1971).
(3) Finney, D.J. The Median Lethal Dose and Its Estimation. Archives of Toxicology 56:215-218 (1985).
(4) Organization for Economic Cooperation and Development. OECD Guideline for the Testing of Chemicals. OECD Guideline 425: Acute Oral Toxicity: Up-and-Down Procedure. Adopted: September 21, 1998.
(5) Organization for Economic Cooperation and Development. OECD Guidelines for Testing of Chemicals. Guideline 420: Acute Oral Toxicity—Fixed Dose Method. Adopted: July 17, 1992.
(6) Organization for Economic Cooperation and Development. OECD Guidelines for Testing of Chemicals. Guideline 423: Acute Oral Toxicity—Acute Toxic Class Method. Adopted: March 22, 1996
(7) Organization for Economic Cooperation and Development. OECD Guidelines for Testing of Chemicals. Guideline 402: Acute Dermal Toxicity. Adopted: February 24, 1987.
§ 799.9130   TSCA acute inhalation toxicity.
(a) Scope. This section is intended to meet testing requirements under section 4 of the Toxic Substances Control Act (TSCA). Determination of acute toxicity is usually an initial step in the assessment and evaluation of the toxic characteristics of a substance that may be inhaled such as a gas, volatile substance, or aerosol/particle. It provides information on health hazards likely to arise from short-term exposure by the inhalation route. Data from an acute study may serve as a basis for classification and labeling. It is traditionally a step in establishing a dosage regimen in subchronic and other studies and may provide initial information on the mode of toxic action of a substance. An evaluation of acute toxicity data should include the relationship, if any, between the animals' exposure to the test substance and the incidence and severity of all abnormalities, including behavioral and clinical abnormalities, the reversibility of observed abnormalities, gross lesions, body weight changes, effects on mortality, and any other toxic effects.
(b) Source. The source material used in developing this TSCA test guideline is the harmonized Office of Prevention, Pesticides, and Toxic Substances (OPPTS) test guideline 870.1300 (August 1998, final guideline). These sources are available at the address in paragraph (g) of this section.
(c) Definitions. The definitions in section 3 of TSCA and the definitions in 40 CFR Part 792—Good Laboratory Practice Standards apply to this section. The following definitions also apply to this section.
Acute inhalation toxicity is the adverse effect caused by a substance following a single uninterrupted exposure by inhalation over a short period of time (24 hours or less) to a substance capable of being inhaled.
Aerodynamic equivalent diameter is defined as the diameter of a unit-density sphere having the same terminal settling velocity as the particle in question, whatever its size, shape, and density. It is used to predict where in the respiratory tract such particles may be deposited.
Concentration is expressed as weight of the test substance per unit volume of air, e.g., milligrams per liter.
Geometric standard deviation (GSD) is a dimensionless number equal to the ratio between the mass median aerodynamic diameter (MMAD) and either 84% or 16% of the diameter size distribution ( e.g., MMAD = 2 m; 84% = 4 m; GSD = 4/2 = 2.0.) The MMAD, together with the GSD, describe the particle size distribution of an aerosol. Use of the GSD may not be valid for non-lognormally distributed aerosols. (If the size distribution deviates from the lognormal, it shall be noted).
Inhalable diameter refers to that aerodynamic diameter of a particle which is considered to be inhalable for the organism under study. It is used to refer to particles which are capable of being inhaled and deposited anywhere within the respiratory tract .
LC 50 (median lethal concentration) is a statistically derived estimate of a concentration of a substance that can be expected to cause death during exposure or within a fixed time after exposure in 50% of animals exposed for a specified time. The LC 50 value is a relatively coarse measurement useful only for classification and labeling purposes and an expression of the lethal potential of the test substance following inhalation. The LC 50 value is expressed as weight of test substance per unit volume of air (milligrams per liter) or parts per million. For clarity, the exposure duration and test animal species should also be specified, e.g., 4 hours LC 50 in F344.
Mass median aerodynamic diameter (MMAD) is the median aero-dynamic diameter and, along with the geometric standard deviation, is used to describe the particle size distribution of any aerosol statistically, based on the weight and size of the particles. Fifty percent of the particles by weight will be smaller than the median diameter and 50% of the particles will be larger.
(d) Approaches to the determination of acute toxicity. (1) EPA recommends the following means to reduce the number of animals used to evaluate acute effects of chemical exposure while preserving its ability to make reasonable judgments about safety:
(i) Using data from substantially similar mixtures. In order to minimize the need for animal testing, the Agency encourages the review of existing acute toxicity information on mixtures that are substantially similar to mixtures under investigation. In certain cases, it may be possible to get enough information to make preliminary hazard evaluations that may reduce the need for further animal testing.
(ii) Limit test. When data on structurally related chemicals are inadequate, a limit test may be considered. In the limit test, a single group of five males and five females is exposed to 2 mg/L for 4 hours, or where this is not possible due to physical or chemical properties of the test substance, the maximum attainable concentration where a particle size distribution having an MMAD between 1 and 4 µm cannot be maintained, using the procedures described under paragraph (e) of this section. For fibers, the bivariate distribution of length and diameter must ensure inhalability. For gases and vapors, the concentrations need not be greater than 50,000 ppm or 50% of the lower explosive limit, whichever is lower. If a test at an aerosol or particulate exposure of 2 mg/L (actual concentration of respirable substance) for 4 hours or, where this is not feasible, the maximum attainable concentration, using the procedures described for this study, produces no observable toxic effects, then a full study using three concentrations will not be necessary. Similarly, if a test at a gas or vapor exposure of 50,000 ppm or 50% of the lower explosive limit, whichever is lower, produces no observable toxic effects, then a full study using three concentrations will not be necessary.
(2) [Reserved]
(e) Conventional acute toxicity test —(1) Principle of the test method. Several groups of experimental animals are exposed to the test substance in graduated concentrations for a defined period, one concentration being used per group. When a vehicle other than water is used to help generate an appropriate concentration of the substance in the atmosphere, a vehicle control group should be used when historical data are not available or adequate to determine the acute inhalation toxicity of the vehicle. Subsequently, observations of effects and death are made. Animals that die during the test are necropsied and at the conclusion of the test surviving animals are sacrificed and necropsied. This guideline is directed primarily to studies in rodent species but may be adapted for studies in non-rodents. Animals showing severe and enduring signs of distress and pain may need to be sacrificed. Dosing test substances in a way known to cause marked pain and distress due to corrosive or irritating properties need not be carried out.
(2) Substance to be tested. Test, control, and reference substances are discussed under EPA Good Laboratory Practice Standards at 40 CFR part 792, subpart f.
(3) Test procedures —(i) Preparation. Healthy young adult animals are acclimatized to the laboratory conditions for at least 5 days prior to the test. Before the test, animals are randomized and assigned to the required number of groups.
(ii) Animal selection —(A) Species and strain. ( 1 ) Although several mammalian test species may be used, the preferred species is the rat. Commonly used laboratory strains should be employed. If another mammalian species is used, the investigator should provide justification and reasoning for the selection.
( 2) Health Status. Body weight and feed consumption are not sufficient indicators of the health status of animals prior to initiating an inhalation toxicity study. Prior to initiating the study, animals must be monitored for known viral and bacterial respiratory pathogens determined by conventional microbiological assays (e.g., serology). The animals must be free from pathogens at the start of exposure.
(B) Age. Young adult rats between 8-12 weeks old at the beginning of dosing, should be used. The weight variation in animals or between groups used in a test should not exceed ±20% of the mean weight of each sex.
(C) Number of animals and sex. ( 1 ) At least five experimentally naive animals are used at each concentration and they must be of one sex. After completion of the study in one sex, at least one group of five animals of the other sex is exposed to establish that animals of this sex are not markedly more sensitive to the test substance. The use of fewer animals may be justified in individual circumstances. Where adequate information is available to demonstrate that animals of the sex tested are markedly more sensitive, testing in animals of the other sex is not required. An acceptable option would be to test at least one group of five animals per sex at one or more dose levels to definitively determine the more sensitive sex prior to conducting the main study.
( 2 ) Females must be nulliparous and nonpregnant.
( 3 ) In acute toxicity tests with animals of a higher order than rodents, the use of fewer animals per concentration group should be considered.
(D) Assignment of animals. ( 1 ) Each animal must be assigned a unique identification number. A system to assign animals to test groups and control groups randomly is required.
( 2 ) Control groups. A concurrent untreated control group is not necessary. Where a vehicle other than water is used to generate an appropriate concentration of the test substance in the atmosphere and historical data are not available or adequate to determine the acute toxicity of the vehicle, a vehicle control group must be used. The vehicle control group must be a sham-treated group. Except for treatment with the test substance, animals in the vehicle control group must be handled in a manner identical to the test-group animals.
(E) Housing. The animals may be group-caged by sex, but the number of animals per cage must not interfere with clear observation of each animal. The biological properties of the test substance or toxic effects (e.g., morbidity, excitability) may indicate a need for individual caging. Animals must be housed individually in inhalation chambers during exposure to aerosols.
( 1 ) Before and after exposure, the temperature of the animal room should be 22 ±3 °C and the relative humidity 30-70%.
( 2 ) Where lighting is artificial, the sequence should be 12 hours light/12 hours dark.
( 3 ) For feeding, conventional laboratory diets may be used with an unlimited supply of drinking water.
(F) Inhalation equipment. ( 1 ) Animals can be exposed to the substance by either a nose-only procedure or in a whole-body exposure chamber. Maintenance of slight negative pressure inside the chamber will prevent leakage of the test substance into the surrounding areas. The nose-only exposure procedure is recommended for studies of aerosols to minimize exposures confounding resultant from test substance ingestion due to test animal fur licking following exposures. Animals must be acclimated to the nose-only exposure chamber prior to study and heat stress minimized during testing.
( 2 ) Inhalation chambers. The animals must be tested in inhalation equipment designed to sustain a dynamic airflow for nose-only exposures of at least 300 ml/minute/animal or an airflow for whole-body exposures of at least 12 to 15 air changes per hour and ensure an adequate oxygen content of at least 19% and an evenly distributed exposure atmosphere. Where a whole-body chamber is used, its design must minimize crowding by providing individual caging. As a general rule, to ensure stability of a chamber atmosphere, the total “volume” of the test animals should not exceed 5% of the volume of the test chamber.
( 3 ) Environmental conditions. The temperature at which the test is performed must be maintained at 22 °C (±2 °C). Ideally, the relative humidity should be maintained between 40% and 60%, but in certain instances (e.g., tests using water as a vehicle), this may not be practical.
(G) Physical measurements. Measurements or monitoring must be made of the following:
( 1 ) Chemical purity of the test material must be analyzed. If the test substance is present in a mixture, the mass and composition of the entire mixture, as well as the principal compound, must be measured. If there is some difficulty in measuring chamber analytical concentration due to precipitation, nonhomogeneous mixtures, volatile components, or other factors, additional analyses of components may be necessary.
( 2 ) The rate of air flow should be monitored continuously, and must be recorded at least every 30 minutes during the exposure period.
( 3 ) The actual concentrations of the test substance must be measured in the breathing zone. During the exposure period, the actual concentrations of the test substance must be held as constant as practicable, monitored continuously or intermittently depending on the method of analysis, and recorded at least three times ( i.e., at the beginning, at an intermediate time, and at the end) during the exposure period. Chamber concentration may be measured using gravimetric or analytical methods as appropriate. If trial run measurements are reasonably consistent (±10% for liquid aerosol, gas, or vapor; ±20% for dry aerosol), then a minimum of two measurements are sufficient. If measurements are not consistent, then a minimum of four measurements should be taken.
( 4 ) During the development of the generating system, particle size analysis must 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. The MMAD particle size range should be between 1-4 µm. The particle size of hygroscopic materials must be small enough when dry to assure that the size of the swollen particle will still be within the 1-4 µm MMAD range. Characterization for fibers must include the bivariate distribution of length and diameter; this distribution must ensure inhalability. Measurements of aerodynamic particle size in the animal's breathing zone must be measured during a trial run. If MMAD values for each exposure level are within 10% of each other, then a minimum of two measurements during the exposures should be sufficient. If pretest measurements are not within 10% of each other, then a minimum of four measurements should be taken.
( 5 ) Temperature and humidity must be monitored continuously, and must be recorded at least every 30 minutes.
(iii) Exposure duration and concentration levels. (A) Exposure duration. Shortly before exposure, the animals are weighed and then exposed to the test target concentration in the designated apparatus for 4 hour exposure period after equilibration of the chamber concentrations. The target concentration is defined by an average of 5% for gases and vapors and 15% for particles and aerosols. The animals are weighed again at the conclusion of the exposure period to determine body weight change. Other durations may be needed to meet specific requirements. Food must be withheld during exposure. Water may also be withheld in certain circumstances.
(B) Exposure concentration levels. At least three concentration levels and a vehicle control group, if required (see paragraph (e)(3)(ii)(D)( 2 ) of this section), must be used. The concentration levels should be spaced appropriately to produce a concentration-response curve and permit an estimation of the median lethal concentration (LC 50 ). The concentrations can either be linearly or logarithmically spaced depending on the anticipated steepness of the concentration-response curve. A rationale for concentration selection should be provided to indicate that the selected concentrations will maximally support detection of concentration-response relationship. The high concentration should be clearly toxic or a limit concentration, but should not result in an incidence of fatalities that would preclude a meaningful evaluation of the data. The lowest concentration should define a no-observed-effects level (NOEL). Range-finding studies using single animals may help to estimate the positioning of the test groups so that no more than three concentration levels will be necessary.
(C) When the physical and chemical properties of the test substance show a low flash point or the test substance is otherwise known or thought to be explosive, care must be taken to avoid exposure level concentrations that could result in an exposure chamber explosion during the test.
(iv) Observation period. The observation period must be at least 14 days. However, the duration of observation should not be fixed rigidly. It should be determined by the toxic reactions, rate of onset, and length of recovery period, and thus may be extended when considered necessary. The time at which signs of toxicity appear, the duration of the signs observed, and the time of death must be recorded and are important, especially if there is a tendency for delayed effects.
(v) Observation of animals. (A) A careful clinical examination must be made at least once each day.
(B) 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 of weak or moribund animals.
(C) Observations must be detailed and carefully recorded, preferably using explicitly defined scales. Observations should include, but not be limited to, evaluation of skin and fur, eyes and mucous membranes, respiratory and circulatory effects, autonomic effects such as salivation, central nervous system effects, including tremors and convulsions, changes in the level of activity, gait and posture, reactivity to handling or sensory stimuli, altered strength, and stereotypies or bizarre behavior (e.g., self mutilation, walking backwards).
(D) Individual weights of animals must be determined pre-exposure and post-exposure, weekly after exposure, and at death. Changes in weights should be calculated and recorded when survival exceeds 1 day.
(E) The time of death should be recorded as precisely as possible.
(vi) Gross pathology. (A) At the end of the test, surviving animals must be weighed, sacrificed and a gross necropsy must be performed on all animals under test, with particular reference to any changes in the respiratory tract. All gross pathology changes must be recorded.
( 1 ) The gross necropsy must include examination of orifices and the cranial, thoracic, and abdominal cavities, and contents.
( 2 ) At least the lungs, liver, kidneys, adrenals, brain, and gonads should be weighed wet, as soon as possible after dissection to avoid drying.
( 3 ) Optionally, the following organs and tissues, or representative samples thereof, may be preserved in a suitable medium for possible future histopathological examination: All gross lesions; brain-including sections of medulla/pons; cerebellar cortex and cerebral cortex; pituitary; thyroid/parathyroid; thymus; heart; sternum with bone marrow; salivary glands; liver; spleen; kidneys; adrenals; pancreas; gonads; accessory genital organs (epididymis, prostrate, and, if present, seminal vesicles); aorta; skin; gall bladder (if present); esophagus; stomach; duodenum; jejunum; ileum; cecum; colon; rectum; urinary bladder; representative lymph nodes; thigh musculature; peripheral nerve; spinal cord at three levels cervical, midthoracic, and lumbar; and eyes. Respiratory tract tissues should be perfusion preserved in a suitable medium.
(B) If necropsy cannot be performed immediately after a dead animal is discovered during the observation period, the animal should be refrigerated (not frozen) at temperatures low enough to minimize autolysis. Necropsies should be performed as soon as possible after death (normally within 24 to 48 hours).
(vii) Additional evaluations. In animals surviving 24 hours or more, microscopic examination of organs showing evidence of gross pathology should be considered since it may yield useful information on the nature of acute toxic effects.
(f) Data and reporting —(1) Treatment of results. Data must be summarized in tabular form showing for each test group the number of animals at the start of the test, body weights, time of death of individual animals at different exposure levels, number of animals displaying other signs of toxicity, description of toxic effects and necropsy findings. The method used for calculation of the LC 50 or any other parameters must be specified and referenced. Some acceptable methods for parameter estimation are described in the references described in paragraphs (g)(1), (g)(2), and (g)(3) of this section.
(2) Evaluation of results. The LC 50 value should be considered in conjunction with the observed toxic effects and the necropsy findings. The evaluation should include the relationship, if any, between exposure of animals to the test substance and the incidence and severity of all abnormalities including behavioral and clinical abnormalities, gross lesions, body weight changes, mortality, and other toxic effects.
(3) Test report. In addition to the reporting requirements specified under EPA Good Laboratory Practice Standards at 40 CFR part 792, subpart J, the following specific information must be reported. The test report shall include:
(i) Test conditions. (A) Description of exposure apparatus including design, type, dimensions.
(B) Source of air, system for generating the test article as particle, aerosol, gas, or vapor.
(C) Method for conditioning air, equipment for measuring temperature, humidity, particle size or particulate aerosol concentration size, and actual concentration.
(D) Treatment of exhaust air and the method of housing the animals in a test chamber when this is used.
(ii) Exposure data. The exposure data must be tabulated and presented with mean values and a measure of variability (e.g., standard deviation) and should include:
(A) Chemical purity of the test material.
(B) Airflow rates through the inhalation equipment.
(C) Temperature and humidity of the air.
(D) Nominal concentration (total amount of test substance fed into the inhalation equipment divided by volume of air).
(E) Actual (analytical or gravimetric) concentration in test breathing zone.
(F) Particle size distribution (calculated MMAD and GSD) and the bivariate distribution of fiber length and diameter, where appropriate.
(G) Explanation as to why the desired chamber concentration and/or particle size could not be achieved (if applicable), and the efforts taken to comply with these aspects of this section.
(iii) Species, strain, sex, and source of test animals.
(iv) Method of randomization in assigning animals to test and control groups.
(v) Rationale for selection of species, if other than that recommended.
(vi) Results. Tabulation of individual and test group data by sex and exposure concentration level (e.g., number of animals exposed, number of animals showing signs of toxicity and number of animals that died or were sacrificed during the test).
(A) Description of toxic effects including time of onset, duration, reversibility, and relationship to the exposure concentration levels.
(B) Pre-exposure and post-exposure body weight change in animals, and weight change during the observation period.
(C) Time of dosing and time of death during or following exposure.
(D) Concentration-response curves for mortality and other toxic effects (when permitted by the method of determination).
(E) Gross pathology necropsy findings in the test animals and vehicle control animals, if included. Data must be tabulated to show the counts and incidence of gross alterations observed for each group tested and the number of animals affected by each type of lesion along with the location and frequency of each type of lesion.
(F) Histopathology findings and any additional evaluations (e.g., clinical chemistry), if performed.
(vii) Description of any pretest conditioning, including diet, quarantine and treatment for disease.
(viii) Description of caging conditions, including: number (or change in number) of animals per cage, bedding material, ambient temperature and humidity, photoperiod, and identification of diet of test animals.
(ix) Manufacturer (source), lot number, and purity of test substance.
(x) Identification and composition of any vehicles (e.g., diluents, suspending agents, and emulsifiers) or other materials , if used in administering the test substance.
(xi) A list of references cited in the body of the report. References to any published literature used in developing the test protocol, performing the testing, making and interpreting observations, and compiling and evaluating the results.
(g) References. For additional background information on this test guideline, the following references should be consulted. These references are available at the addresses in § 700.17(b)(1) and (2) of this chapter.
(1) Chanter, D.O. and Heywood, R. The L D50 test: some considerations of precision. Toxicology Letters 10:303 307 (1982).
(2) Finney, D.G. Chapter 3 Estimation of the median effective dose, Chapter 4 Maximum likelihood estimation. Probit Analysis. 3rd Ed. (Cambridge, London. (1971).
(3) Finney, D.J. The Median Lethal Dose and Its Estimation, Archives of Toxicology 56:215 218 (1985).
(4) Organization for Economic Cooperation and Development. OECD Guidelines for the Testing of Chemicals. Final Draft OECD Guideline 425: Acute Oral Toxicity: Up-and-Down Procedure to be adopted in the Tenth Addendum to the OECD Guidelines for the Testing of Chemicals.
(5) Organization for Economic Cooperation and Development. OECD Guidelines for Testing of Chemicals. Guideline 403: Acute Inhalation Toxicity. Adopted: May 12, 1981.
(6) Organization for Economic Cooperation and Development. OECD Guidelines for Testing of Chemicals. Guideline 420: Acute Oral Toxicity Fixed Dose Method. Adopted: July 17, 1992.
(7) Organization for Economic Cooperation and Development. OECD Guidelines for Testing of Chemicals. Guideline 423: Acute Oral Toxicity Acute Toxic Class Method. Adopted: March 22, 1996.
(8) U. S. EPA. Interim Policy for Particle Size and Limit Concentration Issues in Inhalation Toxicity Studies. 2/1/94. Health Effects Division, Office of Pesticide Programs.
§ 799.9135   TSCA acute inhalation toxicity with histopathology.
(a) Scope. This section is intended to meet the testing requirements under section 4 of the Toxic Substances Control Act (TSCA). In the assessment and evaluation of the potential human health effects of chemical substances, it is appropriate to test for acute inhalation toxic effects. The goals of this test are to characterize the exposure-response relationship for sensitive endpoints following acute exposure and to characterize toxicologic response following acute high exposures. The latter is of particular concern in relation to spills and other accidental releases. This testing is designed to determine the gross pathology and histopathology resulting from acute inhalation exposure to a substance. Because toxic effects on the respiratory tract are of particular concern following inhalation exposure, several indicators of respiratory toxicity consisting of histopathology on fixed tissue and evaluation of cellular and biochemical parameters in bronchoalveolar lavage fluid should be employed. The respiratory histopathology consists of specialized techniques to preserve tissues of the respiratory tract in order to allow detailed microscopic examination to identify adverse effects of chemical substances on this organ system. The bronchoalveolar lavage is designed to be a rapid screening test to provide an early indicator of pulmonary toxicity by examining biochemical and cytologic endpoints of material from the lungs of animals exposed to potentially toxic chemical substances. These acute tests are designed to assess the relationship, if any, between the animals' exposure to the test substance and to demonstrate relationship between the animals' exposure and the incidence and severity of observed abnormalities, including gross or histopathologic lesions, body weight changes, effects on mortality, and any other toxic effects. These acute tests are not intended to provide a complete evaluation of the toxicologic effects of a substance, and additional functional and morphological evaluations may be necessary to assess completely the potential effects produced by a chemical substance. Additional tests may include longer-term exposures, or more in-depth evaluation of specific organ systems as indicated by signs of toxicity following acute exposure.
(b) Source. This a new section developed by the United States Environmental Protection Agency.
(c) Definitions. The following definitions apply to this section.
Aerodynamic diameter (d ae ) refers to the size of particles. It is the diameter of a sphere of unit density that behaves aerodynamically (has the same settling velocity in air) as the particle of the test substance. It is used to compare particles of different size, shape, and density, and to predict where in the respiratory tract such particles may be primarily deposited.
Exposure response is the relationship between the exposure concentration and the measured toxic response, whether expressed as a group mean ±standard deviation) in the case of a continuous variable or as incidence in the case of a quantal variable. This definition should not preclude the exploration of other dose metrics in establishing this relationship.
Geometric standard deviation (GSD) is a dimensionless number equal to the ratio between the mass median aerodynamic diameter (MMAD) and either 84% or 16% of the diameter size distribution (e.g., MMAD = 2 µm; 84% = 4 µm; GSD = 4/2 = 2.0.) The MMAD, together with the GSD, describe the particle size distribution of an aerosol. Use of the GSD may not be valid for non-lognormally distributed aerosols. (If the size distribution deviates from the lognormal, it shall be noted).
Inhalability is the ratio of the number concentration of particles of a certain aerodynamic diameter, d ae , that are inspired through the nose or mouth to the number concentration of the same d ae present in the inspired volume of ambient air. In humans, inhalability can exceed 15 µm d ae , whereas inhalability dramatically decreases for particles above 4 µm d ae in small laboratory animals.
Lower respiratory tract consists of those structures of the respiratory tract below the larynx.
Mass geometric mean aerodynamic diameter or the mass median aerodynamic diameter (MMAD) is the calculated aerodynamic diameter that divides the particles of an aerosol (a gaseous suspension of fine liquid or solid particles) in half, based on the weight of the particles. By weight, 50% of the particles will be larger than the MMAD and 50% of the particles will be smaller than the MMAD.
Particle regional deposition is the fraction of inhaled particles that deposits in the specific region of the respiratory tract. The major mechanisms of particle deposition in the respiratory tract include impaction, sedimentation, diffusion, interception, and electrostatic precipitation. The deposition mechanism that is dominant for a given region depends on the respiratory tract architecture and ventilation rate of the species and the aerosol particle size and distribution. The respiratory tract in both humans and various experimental mammals can be divided into three regions on the basis of structure, size, and function:
(1) The extrathoracic region or upper respiratory tract that includes the nose, mouth, nasopharynx, oropharynx, laryngopharynx, and larynx.
(2) The tracheobronchial region that includes the trachea, bronchi, and bronchioles (including the terminal bronchioles).
(3) The alveolar region that includes the respiratory bronchioles (if present in the species), alveolar ducts, alveolar sacs, and alveoli.
Respiratory effects are any adverse effects on the structure or functions of the respiratory system related to exposure to a chemical substance.
Target organ is any organ found to be a target of toxicity in the 4-hour (hr) high concentration group as a result of:
(1) The initial histopathologic examination (respiratory tract, liver, kidney, gross lesions); or
(2) The retrospective histopathologic examination of archived organs triggered by their identification as targets of toxicity in a 90-day study.
Toxic effects are any adverse changes (a change that is statistically and biologically significant) in the structure or function of an experimental animal as a result of exposure to a chemical substance.
Upper respiratory tract consists of those structures of the respiratory tract above and including the larynx.
(d) Principle of the test method. The test substance shall be administered to several groups of experimental animals; one concentration level and duration being used per group. Bronchoalveolar lavage shall be used to evaluate early effects on the respiratory system by examining changes in the content of the lavage fluid of the lung. At 24 hrs following exposure, the animals shall be sacrificed and necropsied, and tissue samples from the respiratory tract and other major organs will be prepared for microscopic examination. The exposure levels at which significant toxic effects on the respiratory organ system are produced are compared to those levels that produce other toxic effects. As triggered by the results of the 4-hr test, additional exposure periods of 1 hr and 8 hrs will be required to determine the effect of exposure time on the toxicity observed. A 1-hr exposure study can be elected as an option to provide data suitable for risk assessment for very short duration exposures as may occur from chemical releases. In the absence of adequate toxicological data for 1-hr exposure, the Agency will extrapolate to shorter-term exposures from the 4-hr data on the basis of concentration alone. This is a conservative method of extrapolation, consistent with general Agency methods for deriving criteria for short-term exposure from longer-term studies (a concentration x time extrapolation would result in higher concentration for a shorter duration).
(e) Test procedures —(1) Animal selection —(i) Species. In general, the laboratory rat and mouse should be used. Under some circumstances, other species, such as the hamster or guinea pig, may be more appropriate, and if these or other species are used, justification should be provided.
(ii) Strain. If rats and mice are used, the use of the F344 rat and the B6C3F1 mouse is preferred to facilitate comparison with existing data.
(iii) Age. Young adults shall be used. The weight variation of animals used in a test should not exceed ±20% of the mean weight for each species.
(iv) Sex. Equal numbers of animals of each sex shall be used for each concentration level. The females shall be nulliparous and nonpregnant.
(v) Health status. Body weight and feed consumption are not sufficient indicators of the health status of animals prior to initiating an inhalation toxicity study. Prior to initiating the study, animals shall be monitored for known viral and bacterial respiratory pathogens determined by conventional microbiological assays (e.g., serology). The animals shall be free from pathogens at the start of exposure.
(2) Number of animals. At least five males and five females shall be used in each concentration/duration and control group. Animals shall be randomly assigned to treatment and control groups.
(3) Control groups. The control group shall be a sham-treated 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 4- and 8-hr exposure studies are conducted concurrently, a concurrent 8-hr sham-exposed control group may serve as the control group for both the 4-hr and the 8-hr exposure studies, provided there is adequate historical control data showing no changes in histopathology or bronchoalveolar lavage of controls exposed for 4 and 8 hrs. Similarly, if the optional 1-hr exposure study is conducted concurrently with the 4- and/or 8-hr study, the concurrent control group for those studies may also be used for the 1-hr study, provided adequate historical control data show no changes in histopathology or bronchoalveolar lavage between controls exposed for these time periods.
(4) Concentration level and concentration selection. For the 4-hr study, at least three concentrations shall be used in addition to the control group. Ideally, the data generated from the test should be sufficient to produce an exposure-response curve. The concentrations can either be linearly or logarithmically spaced depending on the anticipated steepness of the concentration-response curve. A rationale for concentration selection should be provided to indicate that the selected concentrations will maximally support detection of concentration-response relationship. The high concentration should be clearly toxic or a limit concentration, but should not result in an incidence of fatalities that would preclude a meaningful evaluation of the data. The lowest concentration should define a no-observed-adverse-effects level (NOAEL).
(i) Limit concentration. For aerosols and particles, the high concentrations need not be greater than 2 mg/L, or concentrations that cannot maintain a particle size distribution having an MMAD between 1 and 4 µm (i.e., a particle size that permits inhalability and deposition throughout the respiratory tract). For fibers, the bivariate distribution of length and diameter must ensure inhalability. For gases and vapors, the concentrations need not be greater than 50,000 ppm or 50% of the lower explosive limit, whichever is lower. If a test at an aerosol or particulate exposure of 2 mg/L (actual concentration of respirable substance) for 4 hrs or, where this is not feasible, the maximum attainable concentration, using the procedures described for this study, produces no observable toxic effects, then a full study using three concentrations will not be necessary. Similarly, if a test at a gas or vapor exposure of 50,000 ppm or 50% of the lower explosive limit, whichever is lower, produces no observable toxic effects, then a full study using three concentrations will not be necessary.
(ii) 8-hr study and optional 1-hr study. If the 8-hr study is triggered, three concentrations shall be tested. These concentrations should allow for the determination of an effect level and a NOAEL. If the option to perform a 1-hr study is elected, three concentrations shall be selected and tested in a similar manner.
(5) Inhalation exposure. Animals can be exposed to the substance by either a nose-only procedure or in a whole-body exposure chamber.
(i) Inhalation chambers. The animals shall be tested in inhalation equipment designed to sustain a dynamic airflow for nose-only exposures of at least 300 ml/minute/animal or an airflow for whole-body exposures of at least 12 to 15 air changes per hr and ensure an adequate oxygen content of at least 19% and an evenly distributed exposure atmosphere. Where a whole-body chamber is used, its design shall minimize crowding by providing individual caging. As a general rule, to ensure stability of a chamber atmosphere, the total “volume” of the test animals should not exceed 5% of the volume of the test chamber.
(ii) Environmental conditions. The temperature at which the test is performed shall be maintained at 22 °C (±2 °C). Ideally, the relative humidity should be maintained between 40% and 60%, but in certain instances (e.g., tests using water as a vehicle), this may not be practical.
(iii) Exposure periodicity. For acute testing, the exposure design shall enable 4 hrs of exposure to the target concentrations, as defined by an average of ±5% for gases and vapors and ±15% for particles and aerosols. If triggered by the results of the 4-hr exposure, additional testing shall be conducted in a comparable manner using an 8-hr exposure period.
(6) Physical measurements. Measurements or monitoring shall be made of the following:
(i) Chemical purity of the test material shall be analyzed.
(ii) The rate of airflow shall be monitored continuously, but shall be recorded at least every 30 minutes.
(iii) 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 practical, 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. Well-established and published monitoring methods should be used where available. If no standard methods are available, then accuracy and precision information must be supplied.
(iv) During the development of the generating system, appropriate particle size analysis shall be performed to establish the stability of the aerosol. During exposure, analysis should be conducted as often as necessary to determine the consistency of particle size distribution. The particle size distribution shall have an MMAD between 1 and 4 µm. The particle size of hygroscopic materials shall be small enough when dry to assure that the size of the particle at saturation will still have an MMAD between 1 and 4 µm. Characterization for fibers shall include the bivariate distribution of length and diameter; this distribution must ensure inhalability.
(v) If the test substance is present in a mixture, the mass and composition of the entire mixture, as well as the principal compound, shall be measured.
(vi) Temperature and humidity shall be monitored continuously, but shall be recorded at least every 30 minutes.
(7) Food and water during exposure period. Food shall be withheld during exposure. Water may also be withheld in certain cases.
(8) Observation period. The bronchoalveolar lavage and respiratory pathology shall be conducted 24 hrs following exposure to allow expression of signs of toxicity. There is concern that some latency time will be required to allow migration of cells and macromolecules into the lungs following exposure, and that some pathology may require macromolecular synthesis or degradation before cell damage develops.
(9) Gross pathology. (i) All animals shall be subjected to a full gross necropsy which includes examination of orifices and the cranial, thoracic, and abdominal cavities and their contents.
(ii) At least the lungs, liver, kidneys, adrenals, brain, and gonads shall be weighed wet, as soon as possible after dissection to avoid drying.
(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; brain-including sections of medulla/pons; cerebellar cortex and cerebral cortex; pituitary; thyroid/parathyroid; thymus; heart; sternum with bone marrow; salivary glands; liver; spleen; kidneys; adrenals; pancreas; gonads; accessory genital organs (epididymis, prostrate, and, if present, seminal vesicles); aorta; skin; gall bladder (if present); esophagus; stomach; duodenum; jejunum; ileum; cecum; colon; rectum; urinary bladder; representative lymph nodes; thigh musculature; peripheral nerve; spinal cord at three levels cervical, midthoracic, and lumbar; and eyes. Respiratory tract tissues shall also be preserved in a suitable medium.
(10) Histopathology. The following histopathology shall be performed:
(i) Full histopathology shall be performed on the respiratory tract, liver and kidney of all animals in the control and high concentration groups. The histopathology of the respiratory tract is described under paragraph (e)(11) of this section.
(ii) All gross lesions which differ from controls in frequency, distribution, type, or severity in all concentration groups.
(iii) Target organs in all animals, as indicated by the observations in the high concentration group in this study. Histopathologic examination of target organs in animals at all concentration levels (rather than only to the extent necessary to define the NOAEL) can support the application of exposure-response analyses such as the benchmark concentration approach.
(iv) Archived organs identified as targets of toxicity from results of the 90-day study (if a 90-day study is required for this substance) should be elevated in high concentration animals of the 4-hr acute study to determine if they are also targets of acute toxicity.
(11) Respiratory tract histopathology. (i) Representative sections of the respiratory tract shall be examined histologically. These shall include the trachea, major conducting airways, alveolar region, terminal and respiratory bronchioles (if present), alveolar ducts and sacs, and interstitial tissues.
(ii) Care shall be taken that the method used to kill the animal does not result in damage to the tissues of the upper or lower respiratory tract. The lungs shall be infused with a fixative while in an inflated state of fixed pressure.
(iii) The upper respiratory tract shall be examined for histopathologic lesions. This examination shall use a minimum of four sections located as specified under paragraphs (e)(11)(iii)(A) through (e)(11)(iii)(D) of this section. An evaluation of the nasal vestibule shall be conducted. The method described by the reference under paragraph (h)(11) of this section should be given consideration. The use of additional sections shall be left to the discretion of the study pathologist, but consideration should be given to additional sections as recommended in the reference under paragraph (h)(8) of this section to ensure adequate evaluation of the entire upper respiratory tract, particularly the nasopharyngeal meatus. The following transverse sections shall be examined:
(A) Immediately posterior to the upper incisor teeth.
(B) At the incisor papilla.
(C) At the second palatal ridge.
(D) At the level of the first upper molar teeth.
(iv) The laryngeal mucosa shall be examined for histopathologic changes. Sections of the larynx to be examined include the epithelium covering the base of the epiglottis, the ventral pouch, and the medial surfaces of the vocal processes of the arytenoid cartilages.
(12) Bronchoalveolar lavage. (i) Animals can be exposed to the substance by either a nose-only procedure or in a whole-body exposure chamber.
(ii) Care should be taken that the method used to kill the animal results in minimum changes in the fluid of the lungs of the test animals.
(iii) At the appropriate time, the test animals shall be killed and the heart-lung including trachea removed in bloc. Alternatively, lungs can be lavaged in situ. If the study will not be compromised, one lobe of the lungs may be used for lung lavage while the other is fixed for histologic evaluation. The lungs should be lavaged using physiological saline. The lavages shall consist of two washes, each of which consists of approximately 80% (e.g., 5 ml in rats and 1 ml in mice) of the total lung volume. Additional washes merely tend to reduce the concentrations of the material collected. The lung lavage fluid shall be stored on ice at 5 °C until assayed.
(iv) The following parameters shall be determined in the lavage fluid as indicators of cellular damage in the lungs: total protein, cell count, and percent leukocytes. In addition, a phagocytosis assay shall be performed to determine macrophage activity. Assay methods described in the references under paragraphs (h)(1) and (h)(3) of this section may be used.
(13) Combined protocol. The tests described may be combined with any other toxicity study, as long as none of the requirements of either are violated by the combination.
(f) Triggered testing. If no adverse effects are seen in the 4-hr study as compared with controls, no further testing is necessary. If the 4-hr study shows positive effects in histopathology or the bronchoalveolar lavage, an 8-hr study shall be conducted. Only those tissues showing positive results in the 4-hr study must be pursued in the follow-up 8-hr study. Similarly, if the option to perform a 1-hr study is exercised, only those tissues showing positive results in the 4-hr study shall be pursued.
(g) Data reporting and evaluation. The final test report shall include the following information:
(1) Description of equipment and test methods. A description of the general design of the experiment and any equipment used shall be provided.
(i) Description of exposure apparatus, including design, type, dimensions, source of air, system for generating particles, aerosols, gasses, and vapors, method of conditioning air, treatment of exhaust air, and the method of housing animals in a test chamber.
(ii) Description of the equipment for measuring temperature, humidity, and particulate aerosol concentration and size.
(iii) Exposure data shall be tabulated and presented with mean values and measure of variability (e.g., standard deviation) and should include:
(A) Chemical purity of the test material.
(B) Airflow rates through the inhalation equipment.
(C) Temperature and humidity of air.
(D) Nominal concentration (total amount of test substance fed into the inhalation equipment divided by the volume of air).
(E) Actual concentration in test breathing zone.
(F) Particle size distribution (e.g., MMAD with GSD) and the bivariate distribution of fiber length and diameter, where appropriate.
(2) Results —(i) General group animal data. The following information shall be arranged by test group exposure level.
(A) Number of animals exposed.
(B) Number of animals dying.
(C) Number of animals showing overt signs of toxicity.
(D) Pre- and post-exposure body weight change in animals, and weight change during the observation period.
(ii) Counts and incidence of gross alterations observed at necropsy in the test and control groups. Data shall be tabulated to show:
(A) The number of animals used in each group and the number of animals in which any gross lesions were found.
(B) The number of animals affected by each different type of lesion, and the locations and frequency of each type of lesion.
(iii) Counts and incidence of general histologic alterations in the test group. Data shall be tabulated to show:
(A) The number of animals used in each group and the number of animals in which any histopathologic lesions were found.
(B) The number of animals affected by each different type of lesion, and the locations, frequency, and average grade of each type of lesion.
(iv) Counts and incidence of respiratory histopathologic alterations by the test group. Data shall be tabulated to show:
(A) The number of animals used in each group and the number of animals in which any histopathologic lesions were found.
(B) The number of animals affected by each different type of lesion, and the locations, frequency, and average grade of each type of lesion.
(v) Results of the bronchoalveolar lavage study. Data shall be tabulated to show:
(A) The amount of administered lavage fluid and recovered lavage fluid for each test animal.
(B) The magnitude of change of biochemical and cytologic indices in lavage fluids at each test concentration for each animal.
(C) Results shall be quantified as amount of constituent/mL of lavage fluid. This assumes that the amount of lavage fluid recovered is a representative sample of the total lavage fluid.
(3) Evaluation of data. The findings from this acute study should be evaluated in the context of preceding and/or concurrent toxicity studies and any correlated functional findings. The evaluation shall include the relationship between the concentrations of the test substance and the presence or absence, incidence, and severity of any effects. The evaluation should include appropriate statistical analyses, for example, parametric tests for continuous data and non-parametric tests for the remainder. Choice of analyses should consider tests appropriate to the experimental design, including repeated measures. The report must include concentration-response curves for the bronchoalveolar lavage and tables reporting observations at each concentration level for necropsy findings and gross, general, and respiratory system histopathology.
(h) Reference. For additional background information on this test guideline, the following references should be consulted. These references are available at the addresses in § 700.17(b)(1) and (2) of this chapter.
(1) Burleson, G.R., Fuller, L.B., Ménache, M.G., and Graham, J.A. Poly (I): poly (C)-enhanced alveolar peritoneal macrophage phagocytosis: Quantification by a new method utilizing fluorescent beads. Proceedings of the Society of Experimental Biology and Medicine. 184:468-476 (1987).
(2) Gardner, D.E., Crapo, J.D., and McClellan, R.O. (Eds.) Toxicology of the Lung. (Raven Press, New York, 1993) pp. i-xii, 1-30.
(3) Gilmour, G.I., and Selgrade, M.K. A comparison of the pulmonary defenses against streptococcal infection in rats and mice following O3 exposure: Differences in disease susceptibility and neutrophil recruitment. Toxicology and Applied Pharmacology. 123:211-218 (1993).
(4) Henderson, R.F., Benson, J.M., Hahn, F.F., Hobbs, C.H., Jones, R.K., Mauderly, J.L., McClellan, R.O., and Pickrell, J.A. New approaches for the evaluation of pulmonary toxicity: Bronchoalveolar lavage fluid analysis. Fundamental and Applied Toxicology. 5:451-458 (1985).
(5) Henderson, R.F. Use of bronchoalveolar lavage to detect lung damage. Environmental Health Perspectives. 56:115-129 (1984).
(6) Henderson, R.F., Rebar, A.H., Pickrell, J.A., and Newton, G.J. Early damage indicators in the lung. III. Biochemical and cytological response of the lung to inhaled metal salts. Toxicology and Applied Pharmacology. 50:123-136 (1979).
(7) McClellan, R.O. and Henderson, R.F. (Eds.) Second edition. Concepts in Inhalation Toxicology. (Taylor and Francis, Washington, DC, 1995) pp.i-xxiv, 1-24, 441-470.
(8) Mery, S., Gross, E.A., Joyner, D.R., Godo, M., and Morgan, K.T. Nasal Diagrams: A Tool for Recording the Distribution of Nasal Lesions in Rats and Mice. Toxicologic Pathology. 22:353-372 (1994).
(9) Phalen, R.F. (Ed) Methods in Inhalation Toxicology. (CRC Press, Boca Raton, FL, 1997) pp. i-xii, 1-12.
(10) Renne, R.A., Gideon, K.M., Miller, R.A., Mellick, P.W., and Grumbein, S.L. Histologic methods and interspecies variations in the laryngeal histology of F344/N rats and B6C3F1 mice. Toxicology and Pathology. 20:44-51 (1992).
(11) Young, J.T. Histopathologic examination of the rat nasal cavity. Fundamental and Applied Toxicology. 1:309-312 (1981).
§ 799.9305   TSCA Repeated dose 28-day oral toxicity study in rodents.
(a) Scope —(1) Applicability. This section is intended to meet testing requirements of the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601).
(2) Source. The source material used in developing this TSCA test guideline is the Office of Prevention, Pesticides and Toxic Substances (OPPTS) harmonized test guideline 870.3050 (July 2000, final guidelines). This source is available at the address in paragraph (h) of this section.
(b) Purpose. (1) In the assessment and evaluation of the toxic characteristics of a chemical, the determination of oral toxicity using repeated doses may be carried out after initial information on toxicity has been obtained by acute testing. This study provides information on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. The method comprises the basic repeated dose toxicity study that may be used for chemicals on which a 90-day study is not warranted ( e.g., when the production volume does not exceed certain limits) or as a preliminary to a long term study. The duration of exposure should normally be 28 days although a 14-day study may be appropriate in certain circumstances; justification for use of a 14-day exposure period should be provided.
(2) This section places emphasis on neurological effects as a specific endpoint, and the need for careful clinical observations of the animals, so as to obtain as much information as possible, is stressed. The method should identify chemicals with neurotoxic potential, which may warrant further in-depth investigation of this aspect. In addition, the method may give an indication of immunological effects and reproductive organ toxicity.
(c) Definitions. The definitions in section 3 of TSCA and in 40 CFR Part 792—Good Laboratory Practice Standards apply to this section. The following definitions also apply to this section.
Dosage is a general term comprising of dose, its frequency and the duration of dosing.
Dose is the amount of test substance administered. Dose is expressed as weight (g, mg) or as weight of test substance per unit weight of test animal (e.g., mg/kg), or as constant dietary concentrations (parts per million (ppm)).
No-observed-effects level (NOEL) is the maximum dose used in a study which produces no adverse effects. The NOEL is usually expressed in terms of the weight of a test substance given daily per unit weight of test animals (milligrams per kilograms per day).
(d) Principle of the test. The test substance is orally administered daily in graduated doses to several groups of experimental animals, one dose level per group for a period of 28 days. During the period of administration the animals are observed closely, each day for signs of toxicity. Animals which die or are sacrificed during the test are necropsied and at the conclusion of the test surviving animals are sacrificed and necropsied.
(e) Description of the method —(1) Selection of animal species. The preferred rodent species is the rat, although other rodent species may be used. Commonly used laboratory strains of young healthy adult animals should be employed. The females should be nulliparous and non-pregnant. Dosing should begin as soon as possible after weaning and, in any case, before the animals are 9 weeks old. At the commencement of the study the weight variation of animals used should be minimal and not exceed ±20% of the mean weight of each sex. Where a repeated dose oral study is conducted as a preliminary to a long term study, preferably animals from the same strain and source should be used in both studies.
(2) Housing and feeding conditions. The temperature in the experimental animal room should be 22 °C (±3 °C). Although the relative humidity should be at least 30% and preferably not to exceed 70% other than during room cleaning, the aim should be 50-60%. Lighting should be artificial, the sequence being 12 hours light, 12 hours dark. For feeding, conventional laboratory diets may be used with an unlimited supply of drinking water. The choice of diet may be influenced by the need to ensure a suitable admixture of a test substance when administered by this method. Animals may be housed individually, or be caged in small groups of the same sex; for group caging, no more than five animals should be housed per cage.
(3) Preparation of animals. Healthy young adult animals must be randomly assigned to the control and treatment groups. Cages should be arranged in such a way that possible effects due to cage placement are minimized. The animals are identified uniquely and kept in their cages for at least 5 days prior to the start of the study to allow for acclimatization to the laboratory conditions.
(4) Preparation of doses. (i) The test compound must be administered by gavage or via the diet or drinking water. The method of oral administration is dependent on the purpose of the study, and the physical/chemical properties of the test material.
(ii) Where necessary, the test substance is dissolved or suspended in a suitable vehicle. It is recommended that, wherever possible, the use of an aqueous solution/suspension be considered first, followed by consideration of a solution/emulsion in oil (e.g., corn oil) and then by possible solution in other vehicles. For vehicles other than water the toxic characteristics of the vehicle must be known. The stability of the test substance in the vehicle should be determined.
(f) Procedure —(1) Number and sex of animals. At least 10 animals (five female and five male) should be used at each dose level. If interim sacrifices are planned, the number should be increased by the number of animals scheduled to be sacrificed before the completion of the study. Consideration should be given to an additional satellite group of 10 animals (five per sex) in the control and in the top dose group for observation of reversibility, persistence, or delayed occurrence of toxic effects, for at least 14 days post treatment.
(2) Dosage. (i) Generally, at least three test groups and a control group should be used, but if from assessment of other data, no effects would be expected at a dose of 1000 mg/kg bodyweight/per day, a limit test may be performed. If there are no suitable data available, a range finding study may be performed to aid the determination of the doses to be used. Except for treatment with the test substance, animals in the control group should be handled in an identical manner to the test group subjects. If a vehicle is used in administering the test substance, the control group should receive the vehicle in the highest volume used.
(ii) Dose levels should be selected taking into account any existing toxicity and (toxico-) kinetic data available for the test compound or related materials. The highest dose level should be chosen with the aim of inducing toxic effects but not death or severe suffering. Thereafter, a descending sequence of dose levels should be selected with a view to demonstrating any dosage related response and NOEL at the lowest dose level. Two to four fold intervals are frequently optimal for setting the descending dose levels and addition of a fourth test group is often preferable to using very large intervals (e.g., more than a factor of 10) between dosages.
(3) Limit test. If a test at one dose level of at least 1000 mg/kg body weight/day or, for dietary or drinking water administration, an equivalent percentage in the diet, or drinking water (based upon body weight determinations), using the procedures described for this study, produces no observable toxic effects and if toxicity would not be expected based upon data from structurally related compounds, then a full study using three dose levels may not be considered necessary. The limit test applies except when human exposure indicates the need for a higher dose level to be used.
(4) Administration of doses. (i) The animals are dosed with the test substance daily 7 days each week for a period of 28 days; use of a 5-day per week dosing regime or a 14-day exposure period needs to be justified. When the test substance is administered by gavage, this should be done in a single dose to the animals using a stomach tube or a suitable intubation cannula. The maximum volume of liquid that can be administered at one time depends on the size of the test animal. The volume should not exceed 1ml/100g body weight, except in the case of aqueous solutions where 2ml/100g body weight may be used. Except for irritating or corrosive substances which will normally reveal exacerbated effects with higher concentrations, variability in test volume should be minimized by adjusting the concentration to ensure a constant volume at all dose levels.
(ii) For substances administered via the diet or drinking water it is important to ensure that the quantities of the test substance involved do not interfere with normal nutrition or water balance. When the test substance is administered in the diet either a constant dietary concentration (parts per million (ppm)) or a constant dose level in terms of the animals' body weight may be used; the alternative used must be specified. For a substance administered by gavage, the dose should be given at similar times each day, and adjusted as necessary to maintain a constant dose level in terms of animal body weight. Where a repeated dose study is used as a preliminary to a long term study, a similar diet should be used in both studies.
(5) Observations. (i) The observation period should be 28 days, unless the study duration is 14 days (see paragraph (b)(1) of this section). Animals in a satellite group scheduled for follow-up observations should be kept for at least a further 14 days without treatment to detect delayed occurrence, or persistence of, or recovery from toxic effects.
(ii) General clinical observations should be made at least once a day, preferably at the same time(s) each day and considering the peak period of anticipated effects after dosing. The health condition of the animals should be recorded. At least twice daily, all animals are observed for morbidity and mortality.
(iii) Once before the first exposure (to allow for within-subject comparisons), and at least once a week thereafter, detailed clinical observations should be made in all animals. These observations should be made outside the home cage in a standard arena and preferably at the same time, each time. They should be carefully recorded, preferably using scoring systems, explicitly defined by the testing laboratory. Effort should be made to ensure that variations in the test conditions are minimal and that observations are preferably conducted by observers unaware of the treatment. Signs noted should include, but not be limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling) or bizarre behaviour (e.g., self-mutilation, walking backwards) should also be recorded.
(iv) In the fourth exposure week sensory reactivity to stimuli of different types (see paragraph (h)(2) of this section) (e.g., auditory, visual and proprioceptive stimuli), assessment of grip strength and motor activity assessment should be conducted. Further details of the procedures that could be followed are given in the respective references. However, alternative procedures than those referenced could also be used. Examples of procedures for observation are described in the references in paragraphs (h)(1), (h)(2), (h)(3), (h)(4), and (h)(5) of this section.
(v) Functional observations conducted in the fourth exposure week may be omitted when the study is conducted as a preliminary study to a subsequent subchronic (90-day) study. In that case, the functional observations should be included in this follow-up study. On the other hand, the availability of data on functional observations from the repeated dose study may enhance the ability to select dose levels for a subsequent subchronic study.
(vi) Exceptionally, functional observations may also be omitted for groups that otherwise reveal signs of toxicity to an extent that would significantly interfere with the functional test performance.
(6) Body weight and food/water consumption. All animals should be weighed at least once a week. Measurements of food consumption should be made at least weekly. If the test substance is administered via the drinking water, water consumption should also be measured at least weekly.
(7) Hematology. (i) The following hematological examinations should be made at the end of the test period: hematocrit, hemoglobin concentration, erythrocyte count, total and differential leukocyte count, platelet count and a measure of blood clotting time/potential.
(ii) Blood samples should be taken from a named site just prior to or as part of the procedure for sacrificing the animals, and stored under appropriate conditions.
(8) Clinical Biochemistry. (i) Clinical biochemistry determinations to investigate major toxic effects in tissues and, specifically, effects on kidney and liver, should be performed on blood samples obtained of all animals just prior to or as part of the procedure for sacrificing the animals (apart from those found moribund and/or intercurrently sacrificed). Overnight fasting of the animals prior to blood sampling is recommended. 1 Investigations of plasma or serum shall include sodium, potassium, glucose, total cholesterol, urea, creatinine, total protein and albumin, at least two enzymes indicative of hepatocellular effects (such as alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma glutamyl transpeptidase, and sorbitol dehydrogenase). Measurements of additional enzymes (of hepatic or other origin) and bile acids may provide useful information under certain circumstances.
1 For a number of measurements in serum and plasma, most notably for glucose, overnight fasting would be preferable. The major reason for this preference is that the increased variability which would inevitably result from non-fasting, would tend to mask more subtle effects and make interpretation difficult. On the other hand, however, overnight fasting may interfere with the general metabolism of the animals and, particularly in feeding studies, may disturb the daily exposure to the test substance. If overnight fasting is adopted, clinical biochemical determinations should be performed after the conduct of functional observations in week 4 of the study.
(ii) Optionally, the following urinalysis determinations could be performed during the last week of the study using timed urine volume collection; appearance, volume, osmolality or specific gravity, pH, protein, glucose and blood and blood cells.
(iii) In addition, studies to investigate serum markers of general tissue damage should be considered. Other determinations that should be carried out if the known properties of the test substance may, or are suspected to, affect related metabolic profiles include calcium, phosphate, fasting triglycerides, specific hormones, methemoglobin and cholinesterase. These must to be identified for chemicals in certain classes or on a case-by-case basis.
(iv) Overall, there is a need for a flexible approach, depending on the species and the observed and/or expected effect with a given compound.
(v) If historical baseline data are inadequate, consideration should be given to determination of hematological and clinical biochemistry variables before dosing commences.
(9) Pathology —(i) Gross necropsy. (A) All animals in the study must be subjected to a full, detailed gross necropsy which includes careful examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents. The liver, kidneys, adrenals, testes, epididymides, thymus, spleen, brain and heart of all animals (apart from those found moribund and/or intercurrently sacrificed) should be trimmed of any adherent tissue, as appropriate, and their wet weight taken as soon as possible after dissection to avoid drying.
(B) The following tissues should be preserved in the most appropriate fixation medium for both the type of tissue and the intended subsequent histopathological examination: all gross lesions, brain (representative regions including cerebrum, cerebellum and pons), spinal cord, stomach, small and large intestines (including Peyer's patches), liver, kidneys, adrenals, spleen, heart, thymus, thyroid, trachea and lungs (preserved by inflation with fixative and then immersion), ovaries, uterus, testes, epididymides, accessory sex organs (e.g., prostate, seminal vesicles), urinary bladder, lymph nodes (preferably one lymph node covering the route of administration and another one distant from the route of administration to cover systemic effects), peripheral nerve (sciatic or tibial) preferably in close proximity to the muscle, and a section of bone marrow (or, alternatively, a fresh mounted bone marrow aspirate). The clinical and other findings may suggest the need to examine additional tissues. Also any organs considered likely to be target organs based on the known properties of the test substance should be preserved.
(ii) Histopathology. (A) Full histopathology should be carried out on the preserved organs and tissues of all animals in the control and high dose groups. These examinations should be extended to animals of all other dosage groups, if treatment-related changes are observed in the high dose group.
(B) All gross lesions must be examined.
(C) When a satellite group is used, histopathology should be performed on tissues and organs identified as showing effects in the treated groups.
(g) Data and reporting —(1) Data. (i) Individual data should be provided. Additionally, all 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 found dead during the test or sacrificed for humane reasons and the time of any death or humane sacrifice, the number showing signs of toxicity, a description of the signs of toxicity observed, including time of onset, duration, and severity of any toxic effects, the number of animals showing lesions, the type of lesions and the percentage of animals displaying each type of lesion.
(ii) When possible, numerical results should be evaluated by an appropriate and generally acceptable statistical method. The statistical methods should be selected during the design of the study.
(2) Test report. The test report must include the following information:
(i) Test substance:
(A) Physical nature, purity and physicochemical properties.
(B) Identification data.
(ii) Vehicle (if appropriate): Justification for choice of vehicle, if other than water.
(iii) Test animals:
(A) Species/strain used.
(B) Number, age and sex of animals.
(C) Source, housing conditions, diet, etc.
(D) Individual weights of animals at the start of the test.
(iv) Test conditions:
(A) Rationale for dose level selection.
(B) Details of test substance formulation/diet preparation, achieved concentration, stability and homogeneity of the preparation.
(C) Details of the administration of the test substance.
(D) Conversion from diet/drinking water test substance concentration (parts per million (ppm)) to the actual dose (mg/kg body weight/day), if applicable.
(E) Details of food and water quality.
(v) Results:
(A) Body weight/body weight changes.
(B) Food consumption, and water consumption, if applicable.
(C) Toxic response data by sex and dose level, including signs of toxicity.
(D) Nature, severity and duration of clinical observations (whether reversible or not).
(E) Sensory activity, grip strength and motor activity assessments.
(F) Hematological tests with relevant base-line values.
(G) Clinical biochemistry tests with relevant base-line values.
(H) Body weight at sacrificing and organ weight data.
(I) Necropsy findings.
(J) A detailed description of all histopathological findings.
(K) Absorption data if available.
(L) Statistical treatment of results, where appropriate.
(vi) Discussion of results.
(vii) Conclusions.
(h) References. For additional background information on this test guideline, the following references should be consulted. These references are available at the addresses in § 700.17(b)(1) and (2) of this chapter.
(1) Tupper, D.E., Wallace, R.B. (1980). Utility of the Neurologic Examination in Rats. Acta Neurobiological Exposure, 40:999-1003.
(2) Gad, S.C. (1982). A Neuromuscular Screen for Use in Industrial Toxicology. Journal of Toxicology and Environmental Health, 9:691-704.
(3) Moser, V.C., McDaniel, K.M., Phillips, P.M. (1991). Rat Strain and Stock Comparisons Using a Functional Observational Battery: Baseline Values and Effects of Amitraz. Toxicology and Applied Pharmacology, 108:267-283.
(4) Meyer O.A., Tilson H.A., Byrd W.C., Riley M.T. (1979). A Method forthe Routine Assessment of Fore- and Hindlimb Grip Strength of Rats and Mice. Neurobehavioral Toxicology, 1:233-236.
(5) Crofton K.M., Howard J.L., Moser V.C., Gill M.W., Reiter L.W., Tilson H.A., MacPhail R.C. (1991). Interlaboratory Comparison of Motor Activity Experiments: Implication for Neurotoxicological Assessments. Neurotoxicology and Teratology, 13:599-609.
§ 799.9310   TSCA 90-day oral toxicity in rodents.
(a) Scope. This section is intended to meet the testing requirements under section 4 of the Toxic Substances Control Act (TSCA). 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-effects level (NOEL) and toxic effects associated with continuous or repeated exposure to a test substance for a period of 90 days. This study is not capable of determining those effects that have a long latency period for development (e.g., carcinogenicity and life shortening). Extrapolation from the results of this study to humans is valid only to a limited degree. However, it can useful in providing information on health hazards likely to arise from repeated exposure by the oral route over a limited period of time, such as target organs, the possibilities of accumulation, and can be of use in selecting dose levels for chronic studies and for establishing safety criteria for human exposure.
(b) Source. The source material used in developing this TSCA test guideline is the Office of Prevention, Pesticides, and Toxic Substances (OPPTS) harmonized test guideline 870.3100 (August 1998, final guideline). This source is available at the address in paragraph (h) of this section.
(c) Definitions. The following definitions apply to this section.
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.
Dose in a subchronic oral study is the amount of test substance administered daily via the oral route (gavage, drinking water or diet) for a period of 90 days. Dose is expressed as weight of the test substance (grams, milligrams) per unit body weight of test animal (milligram per kilogram) or as weight of the test substance in parts per million in food or drinking water per day.
No-observed-effects level (NOEL) is the maximum dose used in a study which produces no adverse effects. The NOEL is usually expressed in terms of the weight of a test substance given daily per unit weight of test animal (milligrams per kilogram per day).
Subchronic oral toxicity is the adverse effects occurring as a result of the repeated daily exposure of experimental animals to a chemical by the oral route for a part (approximately 10%) of the test animal's life span.
Target organ is any organ of a test animal showing evidence of an effect induced by a test substance.
(d) Limit test. If a test at one dose level of at least 1,000 mg/kg body weight (expected human exposure may indicate the need for a higher dose level), using the procedures described for this study, produces no observable toxic effects or if toxic effects would not be expected based upon data of structurally related compounds, then a full study using three dose levels might not be necessary.
(e) Test procedures —(1) Animal selection —(i) Species and strain. A variety of rodent species may be used, although the rat is the preferred species. Commonly used laboratory strains must be employed.
(ii) Age/weight. (A) Testing should be started with young healthy animals as soon as possible after weaning and acclimatization.
(B) Dosing of rodents should generally begin no later than 8-9 weeks of age.
(C) At the commencement of the study the weight variation of animals used must be within 20% of the mean weight for each sex.
(iii) Sex. Equal numbers of animals of each sex must be used at each dose level, and the females shall be nulliparous and nonpregnant.
(iv) Numbers. (A) At least 20 rodents (10 males and 10 females) at each dose level.
(B) If interim sacrifices are planned, the number must be increased by the number of animals scheduled to be sacrificed before the completion of the study.
(C) To avoid bias, the use of adequate randomization procedures for the proper allocation of animals to test and control groups is required.
(D) Each animal must be assigned a unique identification number. Dead animals, their preserved organs and tissues, and microscopic slides must be identified by reference to the animal's unique number.
(v) Husbandry. (A) Animals may be group-caged by sex, but the number of animals per cage must not interfere with clear observation of each animal. The biological properties of the test substance or toxic effects (e.g., morbidity, excitability) may indicate a need for individual caging.
(B) The temperature of the experimental animal rooms should be at 22 ±3 °C.
(C) The relative humidity of the experimental animal rooms should be 50 ±20%.
(D) Where lighting is artificial, the sequence should be 12 hours light/12 hours dark.
(E) Control and test animals must be fed from the same batch and lot. The feed should be analyzed to assure adequacy of nutritional requirements of the species tested and for impurities that might influence the outcome of the test. For feeding, conventional laboratory diets may be used with an unlimited supply of drinking water.
(F) The study should not be initiated until animals have been allowed a period of acclimatization/quarantine to environmental conditions, nor should animals from outside sources be placed on test without an adequate period of quarantine. An acclimation period of at least five days is recommended.
(2) Control and test substances. (i) Where necessary, the test substance is dissolved or suspended in a suitable vehicle. If a vehicle or diluent is needed, the vehicle should not elicit toxic effects or substantially alter the chemical or toxicological properties of the test substance. It is recommended that wherever possible the usage of an aqueous solution be considered first, followed by consideration of a solution in oil and then solution in other vehicles.
(ii) If possible, one lot of the test substance tested should be used throughout the duration of the study and the research sample should be stored under conditions that maintain its purity and stability. Prior to the initiation of the study, there should be a characterization of the test substance, including the purity of the test compound and, if technically feasible, the names and quantities of contaminants and impurities.
(iii) If the test or control substance is to be incorporated into feed or another vehicle, the period during which the test substance is stable in such a mixture should be determined prior to the initiation of the study. Its homogeneity and concentration should be determined prior to the initiation of the study and periodically during the study. Statistically randomized samples of the mixture should be analyzed to ensure that proper mixing, formulation, and storage procedures are being followed, and that the appropriate concentration of the test or control substance is contained in the mixture.
(3) Control groups. A concurrent control group is required. This group must be an untreated or sham-treated control group or, if a vehicle is used in administering the test substance, a vehicle control group. If the toxic properties of the vehicle are not known or cannot be made available, both untreated and vehicle control groups are required.
(4) Satellite group. A satellite group of 20 animals (10 animals per sex) may be treated with the high dose level for 90 days and observed for reversibility, persistence, or delayed occurrence of toxic effects for a post-treatment period of appropriate length, normally not less than 28 days. In addition, a control group of 20 animals (10 animals of each sex) should be added to the satellite study.
(5) Dose levels and dose selection. (i) In subchronic toxicity tests, it is desirable to determine a dose-response relationship as well as a NOEL. Therefore, at least three dose levels plus a control and, where appropriate, a vehicle control (corresponding to the concentration of vehicle at the highest dose level) must 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 should result in toxic effects but not produce an incidence of fatalities which would prevent a meaningful evaluation.
(iii) The intermediate dose levels should be spaced to produce a gradation of toxic effects.
(iv) The lowest dose level should produce no evidence of toxicity.
(6) Administration of the test substance. (i) If the test substance is administered by gavage, the animals are dosed with the test substance on a 7-day per week basis for a period of at least 90 days. However, based primarily on practical considerations, dosing by gavage on a 5-day per week basis is acceptable. If the test substance is administered in the drinking water, or mixed in the diet, then exposure should be on a 7-day per week basis.
(ii) All animals must be dosed by the same method during the entire experimental period.
(iii) For substances of low toxicity, it is important to ensure that when administered in the diet the quantities of the test substance involved do not interfere with normal nutrition. When the test substance is administered in the diet, either a constant dietary concentration (parts per million) or a constant dose level in terms of body weight should be used; the alternative used should be specified.
(iv) For a substance administered by gavage, the dose should be given at approximately the same time each day, and adjusted at intervals (weekly or biweekly) to maintain a constant dose level in terms of body weight.
(7) Observation period. (i) The animals must be observed for a period of 90 days.
(ii) Animals in the satellite group (if used) scheduled for follow-up observations should be kept for at least 28 days further without treatment to detect recovery from, or persistence of, toxic effects.
(8) Observation of animals. (i) Observations must be made at least twice each day for morbidity and mortality. Appropriate actions should be taken to minimize loss of animals to the study (e.g., necropsy or refrigeration of those animals found dead and isolation or sacrifice of weak or moribund animals). General clinical observations should be made at least once a day, preferably at the same time each day, taking into consideration the peak period of anticipated effects after dosing. The clinical condition of the animal should be recorded.
(ii) A careful clinical examination must be made at least once weekly. Observations should be detailed and carefully recorded, preferably using explicity defined scales. Observations should include, but not be limited to, evaluation of skin and fur, eyes and mucous membranes, respiratory and circulatory effects, autonomic effects such as salivation, central nervous system effects, including tremors and convulsions, changes in the level of activity, gait and posture, reactivity to handling or sensory stimuli, altered strength, and stereotypes or bizarre behavior (e.g., self-mutilation, walking backwards).
(iii) Signs of toxicity should be recorded as they are observed including the time of onset, degree and duration.
(iv) Measurements of food consumption and water consumption, if drinking water is the exposure route, must be made weekly.
(v) Individual weights of animals must be determined shortly before the test substance is administered, weekly thereafter, and at death.
(vi) Moribund animals should be removed and sacrificed when noticed and the time of death should be recorded as precisely as possible.
(vii) At termination, all survivors in the treatment and control groups must be sacrificed.
(9) Clinical pathology. Hematology and clinical chemistry examinations must be made on all animals, including controls, of each sex in each group. The hematology and clinical chemistry parameters should be examined at terminal sacrifice at the end of the study. Overnight fasting of the animals prior to blood sampling is recommended. Overall, there is a need for a flexible approach in the measures examined, depending on the observed or expected effects from a chemical, and in the frequency of measures, depending on the duration of potential chemical exposures.
(i) Hematology. The recommended parameters are red blood cell count, hemoglobin concentration, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration, white blood cell count, differential leukocyte count, platelet count, and a measure of clotting potential, such as prothrombin time or activated partial thromboplastin time.
(ii) Clinical chemistry. (A) Parameters which are considered appropriate to all studies are electrolyte balance, carbohydrate metabolism, and liver and kidney function. The selection of specific tests will be influenced by observations on the mode of action of the substance and signs of clinical toxicity.
(B) The recommended clinical chemistry determinations are potassium, sodium, glucose, total cholesterol, urea nitrogen, creatinine, total protein and albumin. More than 2 hepatic enzymes, (such as alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, sorbitol dehydrogenase, or gamma glutamyl transpeptidase) should also be measured. Measurements of addtional enzymes (of hepatic or other origin) and bile acids, may also be useful.
(C) If a test chemical has an effect on the hematopoietic system, reticulocyte counts and bone marrow cytology may be indicated.
(D) Other determinations that should be carried out if the test chemical is known or suspected of affecting related measures include calcium, phosphorus, fasting triglycerides, hormones, methemoglobin, and cholinesterases.
(iii) Optionally, the following urinalysis determinations could be performed during the last week of the study using timed urine volume collection: appearance, volume, osmolality or specific gravity, pH, protein, glucose and blood/blood cells.
(10) Ophthalmological examination. Ophthalmological examinations using an ophthalmoscope or an equivalent device must be made on all animals prior to the administration of the test substance and on all high dose and control groups at termination. If changes in the eyes are detected, all animals in the other dose groups must be examined.
(11) Gross necropsy. (i) All animals must be subjected to a full gross necropsy which includes examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents.
(ii) The liver, kidneys, adrenals, testes, epididymides, ovaries, uterus, thymus, spleen, brain, and heart must be trimmed and weighed wet, as soon as possible after dissection.
(iii) The following organs and tissues, or representative samples thereof, should be preserved in a suitable medium for possible future histopathological examination:
(A) Digestive system—salivary glands, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, liver, pancreas, gallbladder (when present).
(B) Nervous system—brain (including sections of medulla/pons, cerebellum and cerebrum), pituitary, peripheral nerve (sciatic or tibial, preferably in close proximity to the muscle), spinal cord (three levels: cervical, mid-thoracic and lumbar), eyes (retina, optic nerve).
(C) Glandular system—adrenals, parathyroid, thyroid.
(D) Respiratory system—trachea, lungs, pharynx, larynx, nose.
(E) Cardiovascular/hemopoietic system—aorta, heart, bone marrow (and/or fresh aspirate), lymph nodes (preferably one lymph node covering the route of administration and another one distant from the route of administration to cover systemic effects), spleen, thymus.
(F) Urogenital system—kidneys, urinary bladder, prostate, testes, epididymides, seminal vesicle(s), uterus, ovaries, female mammary gland.
(G) Others—all gross lesions and masses, skin.
(12) Histopathology. (i) The following histopathology must be performed:
(A) Full histopathology on the organs and tissues, listed in paragraph (e)(11)(iii) of this section, of all rodents in the control and high dose groups, and all rodents that died or were sacrificed during the study.
(B) All gross lesions in all animals.
(C) Target tissues in all animals.
(D) When a satellite group is used, histopathology should be performed on tissues and organs identified as showing effects in the treated groups.
(ii) If excessive early deaths or other problems occur in the high dose group compromising the significance of the data, the next dose level should be examined for complete histopathology.
(iii) An attempt should be made to correlate gross observations with microscopic findings.
(iv) Tissues and organs designated for microscopic examination should be fixed in 10% buffered formalin or a recognized suitable fixative as soon as necropsy is performed and no less than 48 hours prior to trimming.
(f) Data and reporting —(1) Treatment of results. (i) Data must be summarized in tabular form, showing for each test group the number of animals at the start of the test, the number of animals showing lesions, the types of lesions and the percentage of animals displaying each type of lesion.
(ii) When applicable, all observed results, qualitative and quantitative, should be evaluated by an appropriate and generally accepted statistical method. Any generally accepted statistical methods may be used; the statistical methods, including significance criteria, should be selected during the design of the study.
(2) Evaluation of study results. The findings of a subchronic oral toxicity study should be evaluated in conjunction with the findings of preceding studies and considered in terms of the toxic effects and the necropsy and histopathological findings. The evaluation must 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 NOEL. It also can indicate the need for an additional longer-term study and provide information on the selection of dose levels.
(3) Test report. In addition to reporting requirements specified under EPA Good Laboratory Practice Standards at 40 CFR part 792, subpart J, the following specific information must be reported:
(i) Test substance characterization should include:
(A) Chemical identification.
(B) Lot or batch number.
(C) Physical properties.
(D) Purity/impurities.
(ii) Identification and composition of any vehicle used.
(iii) Test system should contain data on:
(A) Species and strain of animals used and rationale for selection if other than that recommended.
(B) Age including body weight data and sex.
(C) Test environment including cage conditions, ambient temperature, humidity, and light/dark periods.
(D) Identification of animal diet.
(E) Acclimation period.
(iv) Test procedure should include the following data:
(A) Method of randomization used.
(B) Full description of experimental design and procedure.
(C) Dose regimen including levels, methods, and volume.
(v) Test results should include:
(A) Group animal data. Tabulation of toxic response data by species, strain, sex and exposure level for:
( 1 ) Number of animals exposed.
( 2 ) Number of animals showing signs of toxicity.
( 3 ) Number of animals dying.
(B) Individual animal data. Data should be presented as summary (group mean) as well as for individual animals.
( 1 ) Date of death during the study or whether animals survived to termination.
( 2 ) Date of observation of each abnormal sign and its subsequent course.
( 3 ) Body weight data.
( 4 ) Feed and water (if collected) consumption data.
(5) Achieved dose (mg/kg/day) as a time-weighted average if the test substance is administered in the diet or drinking water.
( 6 ) Results of ophthalmological examination.
( 7 ) Results of hematological tests performed.
( 8 ) Results of clinical chemistry tests performed.
( 9 ) Results of urinalysis, if performed.
( 10 ) Necropsy findings, including absolute and relative (to body weight) organ weight data.
( 11 ) Detailed description of all histopathological findings.
( 12 ) Statistical treatment of results, where appropriate.
(g) Quality control. A system must be developed and maintained to assure and document adequate performance of laboratory equipment. The study must be conducted in compliance with 40 CFR Part 792—Good Laboratory Practice Standards.
(h) References. For additional background information on this test guideline, the following references should be consulted. These references are available at the addresses in § 700.17(b)(1) and (2) of this chapter.
(1) Boyd, E.M. Chapter 14. Pilot Studies, 15. Uniposal Clinical Parameters, 16. Uniposal Autopsy Parameters. Predictive Toxicometrics. Williams and Wilkins, Baltimore (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) Organization for Economic Cooperation and Development. OECD uidelines for Testing of Chemicals. Guideline 408: Subchronic Oral Toxicity-Rodent: 90-day Study, Adopted: May 12, 1981.
(4) Weingand K., Brown G., Hall R. et al. Harmonization of Animal Clinical Pathology Testing in Toxicity and Safety Studies. Fundam. & Appl. Toxicol. 29:198-201. (1996)
§ 799.9325   TSCA 90-day dermal toxicity.
(a) Scope. This section is intended to meet the testing requirements under section 4 of the Toxic Substances Control Act (TSCA). 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-effects level (NOEL) and toxic effects associated with continuous or repeated exposure to a test substance for a period of 90 days. This study is not capable of determining those effects that have a long latency period for development (e.g., carcinogenicity and life shortening). Extrapolation from the results of this study to humans is valid only to a limited degree. It can, however, provide useful information on the degree of percutaneous absorption, 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) Source. The source material used in developing this TSCA test guideline is the Office of Prevention, Pesticides, and Toxic Substances (OPPTS) harmonized test guideline 870.3250 (August 1998, final guideline). This source is available at the address in paragraph (h) of this section.
(c) Definitions. The following definitions also apply to this section.
Cumulative toxicity is the adverse effect of repeated doses occurring as a result of prolonged action or increased concentration of the administered test substance or its metabolites in susceptible tissues.
Dose in a subchronic dermal study is the amount of test substance applied daily to the skin for 90 days. Dose is expressed as weight of the test substance (grams, milligrams), per unit body weight of test animal (milligrams per kilogram), or as weight of the test substance per unit of surface area (milligrams per square centimeter) per day.
No-observed-effects level (NOEL) is the maximum dose used in a study which produces no adverse effects. The NOEL is expressed in terms of the weight of a test substance given daily per unit weight of test animal (milligrams per kilogram per day).
Subchronic dermal toxicity is the adverse effects occurring as a result of the repeated daily exposure of experimental animals to a chemical by the dermal route for a part of the test animal's life span.
Target organ is any organ of a test animal showing evidence of an effect induced by a test substance.
(d) Limit test. If a test at one dose level of at least 1,000 mg/kg body weight (expected human exposure may indicate the need for a higher dose level), using the procedures described for this section, produces no observable toxic effects or if toxic effects would not be expected based upon data on structurally related compounds, a full study using three dose levels might not be necessary.
(e) Test procedures —(1) Animal selection —(i) Species and strain. A mammalian species must be used for testing. The rat, rabbit, or guinea pig may be used. Commonly used laboratory strains must be employed. If other mammalian species are used, the tester must provide justification/reasoning for their selection. When a subchronic dermal study is conducted as a preliminary to a chronic dermal study, the same species and strain must be used in both studies.
(ii) Age/weight. (A) Testing should be started with young healthy animals as soon as possible after weaning and acclimatization.
(B) Dosing should generally begin in guinea pigs between 5-6 weeks of age, in rats between 8-9 weeks of age, and in rabbits at least 12 weeks old.
(C) At the commencement of the study, the weight variation of animals used must be within 20% of the mean weight for each sex.
(iii) Sex. Equal numbers of animals of each sex with healthy skin must be used at each dose level. The females shall be nulliparous and nonpregnant except for specially designed studies.
(iv) Numbers. (A) At least 20 animals (10 animals per sex) must be used at each dose level.
(B) If interim sacrifices are planned, the number must be increased by the number of animals scheduled to be sacrificed before completion of the study.
(C) To avoid bias, the use of adequate randomization procedures for the proper allocation of animals to test and control groups is required.
(D) Each animal must be assigned a unique identification number. Dead animals, their preserved organs and tissues, and microscopic slides must be identified by reference to the animal's unique number.
(v) Husbandry. (A) Animals should be housed in individual cages.
(B) The temperature of the experimental animal rooms should be at 22 ±3 °C
(C) The relative humidity of the experimental animal rooms should be 50 ±20%.
(D) Where lighting is artificial, the sequence should be 12 hours light/12 hours dark.
(E) Control and test animals must be fed from the same batch and lot. The feed should be analyzed to assure adequacy of nutritional requirements of the species tested and for impurities that might influence the outcome of the test. For feeding, conventional laboratory diets may be used with an unlimited supply of drinking water.
(F) The study should not be initiated until animals have been allowed a period of acclimatization/quarantine to environmental conditions, nor should animals from outside sources be placed on test without an adequate period of quarantine. An acclimation period of at least five days is recommended.
(2) Control and test substances. (i) Where necessary, the test substance is dissolved or suspended in a suitable vehicle. If a vehicle or diluent is needed, the vehicle should not elicit toxic effects or substantially alter the chemical or toxicological properties of the test substance. It is recommended that, whenever possible, the usage of an aqueous solution be considered first, followed by consideration of a solution of oil and then solution of other vehicles.
(ii) One lot of the test substance should be used, if possible, throughout the duration of the study, and the research sample should be stored under conditions that maintain its purity and stability. Prior to the initiation of the study, there should be a characterization of the test substance, including the purity of the test compound and if technically feasible, the name and quantities of unknown contaminants and impurities.
(iii) If the test substance is dissolved or suspended in a vehicle, the period during which the test substance is stable in such a mixture should be determined prior to the initiation of the study. Its homogeneity and concentration should be determined prior to the initiation of the study and periodically during the study. Statistically randomized samples of the mixture should be analyzed to ensure that proper mixing, formulation, and storage procedures are being followed, and that the appropriate concentration of the test or control substance is contained in the mixture.
(3) Control groups. A concurrent control group is required. This group must be an untreated or sham-treated control group or, if a vehicle is used in the application of the test substance, a vehicle control group. If the toxic properties of the vehicle are not known or not available, both untreated/sham-treated and vehicle control groups are required.
(4) Satellite group. A satellite group of 20 animals (10 animals per sex) may be treated with the high dose level for 90 days and observed for reversibility, persistence, or delayed occurrence of toxic effects for a post-treatment period of appropriate length, normally not less than 28 days. In addition a control group of 20 animals (10 animals per sex) should be added to the satellite study.
(5) Dose levels and dose selection. (i) In subchronic toxicity tests, it is desirable to determine a dose-response relationship as well as a NOEL. Therefore, at least three dose levels plus a control and, where appropriate, a vehicle control (corresponding to the concentration of vehicle at the highest dose level) group 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 should elicit signs of toxicity but not produce severe skin irritation or an incidence of fatality which would prevent a meaningful evaluation. If application of the test substance produces severe skin irritation, the concentration may be reduced, although this may result in a reduction in, or absence of, other toxic effects at the high dose level. If the skin has been badly damaged early in the study, it may be necessary to terminate the study and undertake a new one at lower concentrations.
(iii) The intermediate dose levels should be spaced to produce a gradation of toxic effects.
(iv) The lowest dose level should not produce any evidence of toxic effects.
(6) Preparation of animal skin. Shortly before testing, fur must be clipped from not less than 10% of the body surface area for application of the test substance. In order to dose approximately 10% of the body surface, the area starting at the scapulae (shoulders) to the wing of the ileum (hipbone) and half way down the flank on each side of the animal should be shaved. Shaving should be carried out approximately 24 hours before dosing. Repeated 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.
(7) Preparation of test substance. (i) Liquid test substances are generally used undiluted, except as indicated in paragraph (e)(5)(ii) of this section.
(ii) Solids should be pulverized when possible. The substance should be moistened sufficiently with water or, when 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.
(iii) The volume of application should be kept constant, e.g., less than 300 µL for the rat; different concentrations of test solution shall be prepared for different dose levels.
(8) Administration of test substance. (i) The duration of exposure should be at least for 90 days.
(ii) Ideally, the animals should be treated with test substance for at least 6 hours per day on a 7-day per week basis. However, based on practical considerations, application on a 5-day per week basis is acceptable. Dosing should be conducted at approximately the same time each day.
(iii) The test substance must be applied uniformly over the treatment site.
(iv) The surface area covered may be less for highly toxic substances. As much of the area should be covered with as thin and uniform a film as possible.
(v) During the exposure period, the test substance must be held in contact with the skin with a porous gauze dressing (less than or equal to 8 ply). The test site must be further covered with nonirritating tape to retain the gauze dressing and the test substance and to 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 recommended. The test substance may be wiped from the skin after the six-hour exposure period to prevent ingestion.
(9) Observation of animals. (i) Observations must be made at least twice each day for morbidity and mortality. Appropriate actions should be taken to minimize loss of animals to the study (e.g., necropsy or refrigeration of those animals found dead and isolation or sacrifice of weak or moribund animals). General clinical observations must be made at least once a day, preferably at the same time each day, taking into consideration the peak period of anticipated effects after dosing. The clinical condition of the animal should be recorded.
(ii) A careful clinical examination must be made at least once weekly. Observations should be detailed and carefully recorded, preferably using explicity defined scales. Observations should include, but not be limited to, evaluation of skin and fur, eyes and mucous membranes, respiratory and circulatory effects, autonomic effects such as salivation, central nervous system effects, including tremors and convulsions, changes in the level of activity, gait and posture, reactivity to handling or sensory stimuli, altered strength, and stereotypes or bizarre behavior (e.g., self-mutilation, walking backwards).
(iii) Signs of toxicity should be recorded as they are observed including the time of onset, degree and duration.
(iv) Individual weights of animals must be determined shortly before the test substance is administered, weekly thereafter, and at death.
(v) Food consumption must also be determined weekly if abnormal body weight changes are observed.
(vi) Moribund animals should be removed and sacrificed when noticed and the time of death should be recorded as precisely as possible.
(vii) At termination, all survivors in the control and treatment groups must be sacrificed.
(10) Clinical pathology. Hematology and clinical chemistry examinations must be made on all animals, including controls, of each sex in each group. The hematology and clinical chemistry parameters should be examined at terminal sacrifice at the end of the study. Overnight fasting of the animals prior to blood sampling is recommended. Overall, there is a need for a flexible approach in the measures examined, depending on the observed or expected effects from a chemical, and in the frequency of measures, depending on the duration of potential chemical exposures.
(i) Hematology. The recommended parameters are red blood cell count, hemoglobin concentration, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration, white blood cell count, differential leukocyte count, platelet count, and a measure of clotting potential, such as prothrombin time or activated partial thromboplastin time.
(ii) Clinical chemistry. (A) Parameters which are considered appropriate to all studies are electrolyte balance, carbohydrate metabolism, and liver and kidney function. The selection of specific tests will be influenced by observations on the mode of action of the substance and signs of clinical toxicity.
(B) The recommended clinical chemistry determinations are potassium, sodium, glucose, total cholesterol, urea nitrogen, creatinine, total protein and albumin. More than 2 hepatic enzymes, (such as alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, sorbitol dehydrogenase, or gamma glutamyl transpeptidase) should also be measured. Measurements of additional enzymes (of hepatic or other origin) and bile acids, may also be useful.
(C) If a test chemical has an effect on the hematopoietic system, reticulocyte counts and bone marrow cytology may be indicated.
(D) Other determinations that should be carried out if the test chemical is known or suspected of affecting related measures include calcium, phosphorus, fasting triglycerides, hormones, methemoglobin, and cholinesterases.
(iii) Optionally, the following urinalysis determinations could be performed during the last week of the study using timed urine volume collection: appearance, volume, osmolality or specific gravity, pH, protein, glucose and blood/blood cells.
(11) Ophthalmological examination. Using an ophthalmoscope or an equivalent device, ophthalmological examinations must be made on all animals prior to the administration of the test substance and on all high dose and control groups at termination. If changes in the eyes are detected, all animals in the other dose groups must be examined.
(12) Gross necropsy. (i) All animals must 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, brain, kidneys, spleen, adrenals, testes, epididymides, uterus, ovaries, thymus and heart must be trimmed and weighed wet, as soon as possible after dissection.
(iii) The following organs and tissues, or representative samples thereof, must be preserved in a suitable medium for possible future histopathological examination:
(A) Digestive system—salivary glands, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, liver, pancreas, gallbladder (when present).
(B) Nervous system—brain (multiple sections, including cerebrum, cerebellum and medulla/pons), pituitary, peripheral nerve (sciatic or tibial, preferably in close proximity to the muscle), spinal cord (three levels, cervical, mid-thoracic and lumbar), eyes (retina, optic nerve).
(C) Glandular system—adrenals, parathyroid, thyroid.
(D) Respiratory system—trachea, lungs, pharynx, larynx, nose.
(E) Cardiovascular/Hematopoietic system—aorta, heart, bone marrow (and/or fresh aspirate), lymph nodes (preferably one lymph node covering the route of administration and another one distant from the route of administration to cover systemic effects), spleen, thymus.
(F) Urogenital system—kidneys, urinary bladder, prostate, testes, epididymides, seminal vesicle(s), uterus, ovaries, female mammary gland.
(G) Other—all gross lesions and masses, skin (both treated and adjacent untreated areas).
(13) Histopathology. (i) The following histopathology must be performed:
(A) Full histopathology on the organs and tissues, listed in paragraph (e)(12)(iii) of this section, of all animals in the control and high dose groups and all animals that died or were sacrificed during the study.
(B) All gross lesions in all animals.
(C) Target organs in all animals.
(D) When a satellite group is used, histopathology must be performed on tissues and organs identified as showing toxic effects in the treated groups.
(ii) If excessive early deaths or other problems occur in the high dose group compromising the significance of the data, the next dose level must be examined for complete histopathology.
(iii) An attempt should be made to correlate gross observations with microscopic findings.
(iv) Tissues and organs designated for microscopic examination should be fixed in 10% buffered formalin or a recognized suitable fixative as soon as necropsy is performed and no less than 48 hours prior to trimming.
(f) Data and reporting —(1) Treatment of results. (i) Data must be summarized in tabular form, showing for each test group, number of animals at the start of the test, the number of animals showing lesions, the types of lesions and the percentage of animals displaying each type of lesion.
(ii) When applicable, all observed results, qualitative and quantitative, should be evaluated by an appropriate and generally acceptable statistical method. Any generally accepted statistical method should be used; the statistical methods including significance criteria should be selected during the design of the study.
(2) Evaluation of study 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 toxic effects and the necropsy and histopathological findings. The evaluation should include the relationship between the dose of the test substance, 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 90-day subchronic dermal study should provide information on the effects of repeated application of a substance and a satisfactory estimation of a NOEL. It also can indicate the need for an additional longer-term study and provide information on the selection of dose levels.
(3) Test report. In addition to reporting requirements specified under EPA Good Laboratory Practice Standards at 40 CFR part 792, subpart J, the following specific information must be reported:
(i) Test substance characterization should include:
(A) Chemical identification.
(B) Lot or batch numbers.
(C) Physical properties.
(D) Purity/impurities.
(ii) Identification and composition of any vehicle if used.
(iii) Test system should contain data on:
(A) Species and strain of animals used and rationale for selection if other than that recommended.
(B) Age including body weight data and sex.
(C) Test environment including cage conditions, ambient temperature, humidity, and light/dark periods.
(D) Identification of animal diet.
(E) Acclimation period.
(iv) Test procedure should include the following data:
(A) Method of randomization used.
(B) Full description of experimental design and procedure.
(C) Dose regime including levels, method, and volume.
(v) Test results should include:
(A) Group animal data. Tabulation of toxic response data by species, strain, sex and exposure level for:
( 1 ) Number of animals exposed.
( 2 ) Number of animals showing signs of toxicity.
( 3 ) Number of animals dying.
(B) Individual animal data. Data should be presented as summary (group mean) as well as for individual animals.
( 1 ) Date of death during the study or whether animals survived to termination.
( 2 ) Date of observation of each abnormal sign and its subsequent course.
( 3 ) Body weight data.
( 4 ) Feed consumption data, when collected.
( 5 ) Results of ophthalmological examination.
( 6 ) Results of hematological tests performed.
( 7 ) Results of clinical chemistry tests performed.
( 8 ) Results of urinalysis, when performed.
( 9 ) Results of observations made.
( 10 ) Necropsy findings, including absolute and relative (to body weight) organ weight data.
( 11 ) Detailed description of all histopathological findings.
( 12 ) Statistical treatment of results, where appropriate.
(g) Quality control. A system must be developed and maintained to assure and document adequate performance of laboratory equipment. The study must be conducted in compliance with the Good Laboratory Practice (GLP) regulations.
(h) References. For additional background information on this test guideline, the following references should be consulted. These references are available at the addresses in § 700.17(b)(1) and (2) of this chapter.
(1) Organization for Economic Cooperation and Development. Guidelines for Testing of Chemicals, Section 4-Health Effects, Part 411 Subchronic Toxicity Studies, Paris, 1981.
(2) Weingand K, Brown G, Hall R et al. (1996). Harmonization of Animal Clinical Pathology Testing in Toxicity and Safety Studies. Fundam. & Appl. Toxicol. 29:198-201.
§ 799.9346   TSCA 90-day inhalation toxicity.
(a) Scope. This section is intended to meet the testing requirements under section 4 of TSCA. 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 (NOEL) and toxic effects associated with continuous or repeated exposure to a test substance for a period of 90 days. This study is not capable of determining those effects that have a long latency period for development (e.g., carcinogenicity and life shortening). Extrapolation from the results of this study to humans is valid only to a limited degree. It can, however, provide useful 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 and the possibilities of accumulation, and can be of use in selecting concentration levels for chronic studies and 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) Source. The source material used in developing this TSCA test guideline is the OPPTS harmonized test guideline 870.3465 (June 1996 Public Draft). This source is available at the address in paragraph (h) of this section.
(c) Definitions. The following definitions apply to this section.
Aerodynamic equivalent diameter is defined as the diameter of a unit density sphere having the same terminal settling velocity as the particle in question, whatever its size, shape, and density. It is used to predict where in the respiratory tract such particles may be deposited.
Concentration in a subchronic inhalation study is the amount of test substance administered via inhalation for a period of 90-days. Concentration is expressed as weight of the test substance per unit volume of air (milligrams per liter or parts per million).
Cumulative toxicity is the adverse effects of repeated exposures occurring as a result of prolonged action on, or increased concentration of the administered test substance or its metabolites in susceptible tissues.
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
Mass median aerodynamic diameter (MMAD) is the median aerodynamic diameter and along with the geometric standard deviation (GSD) is used to describe the particle size distribution of any aerosol statistically based on the weight and size of the particles. Fifty percent of the particles by weight will be smaller than the median diameter and 50% of the particles will be larger.
No-observed-effect-level (NOEL) is the maximum concentration used in a study which produces no adverse effects.
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%) of a life span.
(d) Limit test. If exposure at a concentration of 1 mg/L (expected human exposure may indicate the need for a higher concentration), or where this is not possible due to physical or chemical properties of the test substance, the maximum attainable concentration produces no observable toxic effects, then a full study using three concentrations 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 should be employed. If another mammalian species is used, the tester shall provide justification/reasoning for its selection.
(ii) Age/weight. Testing should be started with young healthy animals as soon as possible after weaning and acclimatization.
(B) Dosing of rodents should generally begin no later than 8 weeks of age.
(C) At the commencement of the study the weight variation of animals used shall not exceed ±20% of the mean weight for each sex.
(iii) Sex. (A) Equal numbers of animals of each sex shall be used at each concentration.
(B) Females shall be nulliparous and nonpregnant.
(iv) Numbers. (A) At least 20 animals (10 females and 10 males) should be used for each test group.
(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.
(C) To avoid bias, the use of adequate randomization procedures for the proper allocation of animals to test and control groups is required.
(D) Each animal shall be assigned a unique identification number. Dead animals, their preserved organs and tissues, and microscopic slides shall be identified by reference to the animal's unique number.
(v) Husbandry. (A) Animals may be group-caged by sex, but the number of animals per cage must not interfere with clear observation of each animal. The biological properties of the test substance or toxic effects (e.g., morbidity, excitability) may indicate a need for individual caging. Animals must be housed individually in inhalation chambers during exposure to aerosols.
(B) The temperature of the experimental animal rooms should be at 22 ±3 °C.
(C) The relative humidity of the experimental animal rooms should be 30-70%.
(D) Where lighting is artificial, the sequence should be 12 h light/12 h dark.
(E) Control and test animals should be fed from the same batch and lot. The feed should be analyzed to assure adequacy of nutritional requirements of the species tested and for impurities that might influence the outcome of the rest. For feeding, conventional laboratory diets may be used with an unlimited supply of drinking water.
(F) The study should not be initiated until animals have been allowed a period of acclimatization/quarantine to environmental conditions, nor should animals from outside sources be placed on test without an adequate period of quarantine. An acclimatization period of at least 5 days is recommended.
(2) Control and test substances. (i) Whenever it is necessary to formulate the test substance with a vehicle for aerosol generation, the vehicle ideally should not elicit toxic effects or substantially alter the chemical or toxicological properties of the test substance.
(ii) One lot of the test substance should be used, if possible throughout the duration of the study, and the research sample should be stored under conditions that maintain its purity and stability. Prior to the initiation of the study, there should be a characterization of the test substance, including the purity of the test substance and, if technically feasible, the name and quantities of unknown contaminants and impurities.
(3) 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 other than water is used to generate a substance, a vehicle control group should 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.
(4) 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. In addition, a control group of 20 animals (10 animals of each sex) should be added to the satellite study.
(5) Concentration levels and concentration selection. (i) In subchronic toxicity tests, it is desirable to have a concentration-response relationship as well as a NOEL. Therefore, at least three concentration levels plus 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 intermediate concentrations should be spaced to produce a gradation of toxic effects.
(iv) The lowest concentration should produce no evidence of toxicity.
(v) In the case of potentially explosive test substances, care should be taken to avoid generating explosive concentrations.
(6) Administration of the test substance. Animals should be exposed to the test substance for 6 h per day on a 7-day per week basis for a period of at least 90 days. Based primarily on practical considerations, exposure for 6 h per day on a 5-day per week basis is acceptable.
(7) Observation period. The animals should be observed for a period of 90 days. Animals in the satellite group (if used) scheduled for follow-up observations should be kept for at least 28 days further without treatment to assess reversibility.
(8) Exposure specifications. (i) The animals shall be tested in dynamic inhalation equipment designed to sustain a minimum airflow of 10 air changes per hr, an adequate oxygen content of at least 19%, and uniform conditions throughout the exposure chamber. Maintenance of slight negative pressure inside the chamber will prevent leakage of the test substance into the surrounding areas. It is not normally necessary to measure chamber oxygen concentration if airflow is adequate.
(ii) The selection of a dynamic inhalation chamber should be appropriate for the test substance and test system. Where a whole body chamber is used to expose animals to an aerosol, individual housing must be used to minimize crowding of the test animals and maximize their exposure to the test substance. To ensure stability of a chamber atmosphere, the total volume occupied by the test animals shall not exceed 5% of the volume of the test chamber. It is recommended, but not required, that nose-only or head-only exposure be used for aerosol studies in order to minimize oral exposures due to animals licking compound off their fur. Heat stress should be minimized.
(iii) The temperature at which the test is performed should be maintained at 22 ±2 °C. The relative humidity should be maintained between 40 and 60%, but in certain instances (e.g., use of water vehicle) this may not be practicable.
(9) Physical measurements. Measurements or monitoring shall be made of the following:
(i) The rate of airflow shall be monitored continuously but recorded at least three times during the exposure.
(ii) The actual concentrations of the test substance shall be measured in the animal's breathing zone. During the exposure period, the actual concentrations of the test substance shall be held as constant as practicable and monitored continuously or intermittently depending on the method of analysis. Chamber concentration may be measured using gravimetric or analytical methods as appropriate. If trial run measurements are reasonably consistent ±10% for liquid, aerosol, gas, or vapor; ±20% for dry aerosol), then two measurements should be sufficient. If measurements are not consistent, three to four measurements should be taken. Whenever the test substance is a formulation, or it is necessary to formulate the test substance with a vehicle for aerosol generation, the analytical concentration must be reported for the total formulation, and not just for the active ingredient (AI). If, for example, a formulation contains 10% AI and 90% inerts, a chamber analytical limit concentration of 2 mg/L would consist of 0.2 mg/L of the AI. It is not necessary to analyze inert ingredients provided the mixture at the animal's breathing zone is analogous to the formulation; the grounds for this conclusion must be provided in the study report. If there is some difficulty in measuring chamber analytical concentration due to precipitation, nonhomogeneous mixtures, volatile components, or other factors, additional analyses of inert components may be necessary.
(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. The MMAD particle size range should be between 1-3 µm. The particle size of hygroscopic materials should be small enough when dry to assure that the size of the swollen particle will still be within the 1-3 µm range. Measurements of aerodynamic particle size in the animal's breathing zone should be measured during a trial run. If MMAD valves for each exposure level are within 10% of each other, then two measurements during the exposures should be sufficient. If pretest measurements are not within 10% of each other, three to four measurements should be taken.
(iv) Temperature and humidity shall be monitored continuously and recorded at least three times during an exposure.
(10) Feed and water during exposure period. Feed shall be withheld during exposure. Water may also be withheld during exposure.
(11) Observation of animals. (i) During and following exposure, observations are made and recorded systematically; individual records should be maintained for each animal. It is not always possible to observe animals during exposure in a whole-body chamber.
(ii) Observations shall be made at least once each day for morbidity and mortality. Appropriate actions should be 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) A careful clinical examination shall be made at least once weekly. Observations should be detailed and carefully recorded, preferably using explicitly defined scales. Observations should include, but not be limited to, evaluation of skin and fur, eyes and mucous membranes, respiratory and circulatory effects, autonomic effects such as salivation, central nervous system effects, including tremors and convulsions, changes in the level of activity, gait and posture, reactivity to handling or sensory stimuli, altered strength, and stereotypes or bizarre behavior (e.g., self-mutilation, walking backwards).
(iv) Signs of toxicity should be recorded as they are observed including the time of onset, degree and duration.
(v) Individual weights of animals shall be determined shortly before the test substance is administered, and weekly thereafter.
(vi) Food consumption shall also be determined weekly if abnormal body weight changes are observed.
(vii) Moribund animals should be removed and sacrificed when noticed and the time of death should be recorded as precisely as possible.
(viii) At termination, all survivors in the treatment groups shall be sacrificed.
(12) Clinical pathology. Hematology and clinical chemistry examinations shall be made on all animals, including controls, of each sex in each group. The hematology and clinical chemistry parameters should be examined at terminal sacrifice at the end of the study. Overnight fasting of the animals prior to blood sampling is recommended. Overall, there is a need for a flexible approach in the measures examined, depending on the observed or expected effects from a chemical, and in the frequency of measures, depending on the duration of potential chemical exposures.
(i) Hematology. The recommended parameters are red blood cell count, hemoglobin concentration, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration, white blood cell count, differential leukocyte count, platelet count, and a measure of clotting potential, such as prothrombin time or activated partial thromboplastin time.
(ii) Clinical chemistry. (A) Parameters which are considered appropriate to all studies are electrolyte balance, carbohydrate metabolism, and liver and kidney function. The selection of specific tests will be influenced by observations on the mode of action of the substance and signs of clinical toxicity.
(B) The recommended clinical chemistry determinations are potassium, sodium, glucose, total cholesterol, urea nitrogen, creatinine, total protein and albumin. More than 2 hepatic enzymes, (such as alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, sorbitol dehydrogenase, or gamma glutamyl transpeptidase) should also be measured. Measurements of addtional enzymes (of hepatic or other origin) and bile acids, may also be useful.
(C) If a test chemical has an effect on the hematopoietic system, reticulocyte counts and bone marrow cytology may be indicated.
(D) Other determinations that should be carried out if the test chemical is known or suspected of affecting related measures include calcium, phosphorus, fasting triglycerides, hormones, methemoglobin, and cholinesterases.
(iii) Optionally, the following urinalysis determinations could be performed during the last week of the study using timed urine volume collection: appearance, volume, osmolality or specific gravity, pH, protein, glucose, and blood/blood cells.
(13) Ophthalmological examination. Ophthalmological examinations shall be made on all animals prior to the administration of the test substance and on all high concentration and control groups at termination. If changes in the eyes are detected, all animals in the other concentration groups shall be examined.
(14) 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, brain, and gonads shall be trimmed and weighed wet, as soon as possible after dissection to avoid drying.
(iii) The following organs and tissues, or representative samples thereof, shall be preserved in a suitable medium for possible future histopathological examination:
(A) Digestive system.
( 1 ) Salivary glands.
( 2 ) Esophagus.
( 3 ) Stomach.
( 4 ) Duodenum.
( 5 ) Jejunum.
( 6 ) Ileum.
( 7 ) Cecum.
( 8 ) Colon.
( 9 ) Rectum.
( 10 ) Liver.
( 11 ) Pancreas.
( 12 ) Gallbladder (dogs).
(B) Nervous system.
( 1 ) Brain (multiple sections).
( 2 ) Pituitary.
( 3 ) Peripheral nerve(s).
( 4 ) Spinal cord (three levels).
( 5 ) Eyes (retina, optic nerve).
(C) Glandular system.
( 1 ) Adrenals.
( 2 ) Parathyroids.
( 3 ) Thyroids.
(D) Respiratory system.
( 1 ) Trachea.
( 2 ) Lung.
( 3 ) Pharynx.
( 4 ) Larynx.
( 5 ) Nose.
(E) Cardiovascular/hematopoietic system.
( 1 ) Aorta (thoracic).
( 2 ) Heart.
( 3 ) Bone marrow.
( 4 ) Lymph nodes.
( 5 ) Spleen.
( 6 ) Thymus.
(F) Urogenital system.
( 1 ) Kidneys.
( 2 ) Urinary bladder.
( 3 ) Prostate.
( 4 ) Testes.
( 5 ) Epididymides.
( 6 ) Seminal vesicle(s).
( 7 ) Uterus.
( 8 ) Ovaries.
(G) Other.
( 1 ) Lacrimal gland.
( 2 ) Mammary gland.
( 3 ) Skin.
( 4 ) Skeletal muscle.
( 5 ) All gross lesions and masses.
( 6 ) Sternum and/or femur.
(15) Histopathology. (i) The following histopathology shall be performed:
(A) Full histopathology on the respiratory tract and other organs and tissues, listed under paragraph (e)(15)(iii) of this section, of all animals in the control and high exposure 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.
(D) Lungs of all animals. Special attention to examination of the respiratory tract should be made for evidence of infection as this provides a convenient assessment of the state of health of the animals.
(E) When a satellite group is used, histopathology shall be performed on tissues and organs identified as showing effects in the treated groups.
(ii) If excessive early deaths or other problems occur in the high exposure group compromising the significance of the data, the next concentration should be examined for complete histopathology.
(iii) An attempt should be made to correlate gross observations with microscopic findings.
(iv) Tissues and organs designated for microscopic examination should be fixed in 10% buffered formalin or a recognized suitable fixative as soon as necropsy is performed and no less than 48 hrs prior to trimming. Tissues should be trimmed to a maximum thickness of 0.4 cm for processing.
(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 qualitative) should be evaluated by an appropriate statistical method. Any generally accepted statistical method may be used; the statistical methods including significance criteria should be selected during the design of the study.
(2) Evaluation of study 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. It also can indicate the need for an additional longer-term study and provide information on the selection of concentrations.
(3) Test report. In addition to reporting requirements specified under 40 CFR part 792, subpart J, the following specific information shall be reported. Both individual and summary data should be presented.
(i) Test substance characterization shall include:
(A) Chemical identification.
(B) Lot or batch number.
(C) Physical properties.
(D) Purity/impurities.
(E) Identification and composition of any vehicle used.
(ii) Test system information shall include:
(A) Species and strain of animals used and rationale for selection if other than that recommended.
(B) Age, sex, and body weight.
(C) Test environment including cage conditions, ambient temperature, humidity, and light/dark periods.
(D) Identification of animal diet.
(E) Acclimation period.
(iii) Test procedure information shall include:
(A) Method of randomization used.
(B) Full description of experimental design and procedure.
(C) Exposure regimen including concentration levels, methods, and volume.
(D) Description of test conditions; the following exposure conditions shall be reported:
( 1 ) Description of exposure apparatus including design, type, volume, source of air, system for generating 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.
(E) Exposure data shall be tabulated and presented with mean values and a measure of variability (e.g., standard deviation) and include:
( 1 ) Airflow rates through the inhalation equipment.
( 2 ) Temperature and humidity of air.
( 3 ) Actual (analytical or gravimetric) concentration in the breathing zone.
( 4 ) Nominal concentration (total amount of test substance fed into the inhalation equipment divided by volume of air).
( 5 ) Particle size distribution, calculated mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD).
( 6 ) Explanation as to why the desired chamber concentration and/or particle size could not be achieved (if applicable) and the efforts taken to comply with this aspect of the section.
(iv) Test results information shall include:
(A) Group animal data. Tabulation of toxic response data by species, strain, sex and exposure level for:
( 1 ) Number of animals exposed.
( 2 ) Number of animals showing signs of toxicity.
( 3 ) Number of animals dying.
(B) Individual animal data. Data should be presented as summary (group mean) as well as for individual animals.
( 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 consumption data, when collected.
( 5 ) Results of ophthalmological examination, when performed.
( 6 ) Results of hematological tests performed. .
( 7 ) Results of clinical chemistry tests performed.
( 8 ) Results of urinalysis tests performed.
( 9 ) Necropsy findings, including absolute and relative organ weight data.
( 10 ) Detailed description of all histopathological findings.
( 11 ) Statistical treatment of results, where appropriate.
(g) Quality control. A system shall be developed and maintained to assure and document adequate performance of laboratory staff and equipment. The study shall be conducted in compliance with 40 CFR part 792—Good Laboratory Practice Standards.
(h) References. For additional background information on this test guideline, the following references should be consulted. These references are available at the addresses in § 700.17(b)(1) and (2) of this chapter.
(1) Cage, J.C. Ed. Paget, G.E. Experimental Inhalation Toxicology, Methods in Toxicology. (F.A. Davis Co., Philadelphia, PA, 1970) pp. 258-277.
(2) Casarett, L.J. and Doull. Chapter 9. Toxicology: The Basic Science of Poisons (New York: Macmillan Publishing Co., Inc., 1975).
(3) U.S. Environmental Protection Agency, Office of Pesticide Programs, Health Effects Division. Interim policy for particle size and limit concentration issues in inhalation toxicity studies (February 1, 1994).
(4) MacFarland, H.N. Ed. Hayes, W.J. Vol. 7. Respiratory Toxicology, Essays in Toxicology. (Academic Press, New York, NY, 1976) pp. 121-154.
(5) Organisation for Economic Co-operation and Development. Guidelines for testing of chemicals, section 4-health effects, part 413. Subchronic Inhalation Toxicity Studies (Paris, 1981).