Title 40—Protection of Environment--Volume 1
PART 799—IDENTIFICATION OF SPECIFIC CHEMICAL SUBSTANCE AND MIXTURE TESTING REQUIREMENTS
§ 799.9748   TSCA metabolism and pharmacokinetics
(a) Scope. (1) This section is intended to meet the testing requirements under section 4 of the Toxic Substances Control Act (TSCA). (1) Testing of the disposition of a test substance is designed to obtain adequate information on its absorption, distribution, biotransformation, and excretion and to aid in understanding the mechanism of toxicity. Basic pharmacokinetic parameters determined from these studies will also provide information on the potential for accumulation of the test substance in tissues and/or organs and the potential for induction of biotransformation as a result of exposure to the test substance. These data can be used to assess the adequacy and relevance of the extrapolation of animal toxicity data (particularly chronic toxicity and/or carcinogenicity data) to human risk assessment.
(2) Metabolism data can also be used to assist in determining whether animal toxicity studies have adequately addressed any toxicity concerns arising from exposure to plant metabolites, and in the setting of tolerances, if any, for those metabolites in raw agricultural commodities.
(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 870.7485 (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.
Metabolism (biotransformation) is the sum of the processes by which a foreign chemical is subjected to chemical change by living organisms.
LOEL is the lowest observable effects level.
NOEL is the no observable effects level.
Pharmacokinetics is the quantitation and determination of the time course and dose dependency of the absorption, distribution, biotransformation, and excretion of chemicals.
(d) Good laboratory practice standards. The pharmacokinetics and metabolism tests outlined in this guideline must conform to the laboratory practices stipulated in 40 CFR Part 792—Good Laboratory Practice Standards.
(e) Test Procedures. Test procedures presented below utilize a tier system to minimize the use of resources and to allow flexibility in the conduct of metabolism studies. The proposed tier system consists of a basic data set (Tier 1) and additional studies (Tier 2). These additional studies may be requested based upon the existing toxicology data base and/or the results of Tier 1 testing which are found to impact upon the risk assessment process. For Tier 1 testing, the oral route will typically be required; however, if the use pattern results in other types of exposure, other routes (dermal and/or inhalation) may be required for initial testing of the disposition of a chemical substance. The registrant should justify the route of exposure to the Agency. Complete descriptions of the test procedures for these other routes of exposure can be found in paragraph (i) of this section. Except in unusual circumstances, the tiered approach to metabolism testing should apply to all listed routes of exposure.
(1) Pilot studies. The use of pilot studies is recommended and encouraged for the selection of experimental conditions for the pharmacokinetics and metabolism studies (mass balance, analytical procedures, dose-finding, excretion of CO 2 , etc.).
(2) Animal selection —(i) Species. The rat must normally be used for testing because it has been used extensively for metabolic and toxicological studies. The use of other or additional species may be required if critical toxicology studies demonstrate evidence of significant toxicity in these species or if metabolism is shown to be more relevant to humans in the test species.
(ii) Strain. Adult animals of the strain used or proposed to be used for the determination of adverse health effects associated with the test substance.
(3) Material to be tested —(i) Test substance. (A) A radiolabeled test substance using 14 C should be used for all material balance and metabolite identification aspects of the study. Other radioactive and stable isotopes may be used, particularly if the element is responsible for or is a part of the toxic portion of the compound. If it can be demonstrated that the material balance and metabolite identification requirements can be met using unlabeled test substance, then radiolabeled compound need not be used. If possible, the radiolabel should be located in a core portion of the molecule which is metabolically stable (it is not exchangeable, is not removed metabolically as CO 2 , and does not become part of the one-carbon pool of the organism). Labeling of multiple sites of the molecule may be necessary to follow the metabolic fate of the compound.
(B) The label should follow the test compound and/or its major metabolites until excreted. The radiopurity of the radioactive test substance shall be the highest attainable for a particular test substance (ideally it should be greater than 95%) and reasonable effort should be made to identify impurities present at or above 2%. The purity, along with the identity of major impurities which have been identified, shall be reported. For other segments of the study, nonradioactive test substance may be used if it can be demonstrated that the analytical specificity and sensitivity of the method used with nonradioactive test substance is equal to or greater than that which could be obtained with the radiolabeled test substance. The radioactive and nonradioactive test substances shall be analyzed using an appropriate method to establish purity and identity. Additional guidance will be provided in chemical specific test rules to assist in the definition and specifications of test substances composed of mixtures and methods for determination of purity.
(ii) Administration of test substance. Test substance should be dissolved or suspended homogeneously in a vehicle usually employed for acute administration. A rationale for the choice of vehicle should be provided. The customary method of administration will be by oral gavage; however, administration by gelatin capsule or as a dietary mixture may be advantageous in specific situations. Verification of the actual dose administered to each animal should be provided.
(4) Tier testing. (i) The multiplicity of metabolic parameters that impact the outcome of toxicological evaluations preclude the use of a universal study design for routine toxicological evaluation of a test substance. The usefulness of a particular study design depends upon the biological activity of a compound and circumstances of exposure. For these reasons, a tiered system is proposed for evaluation of the metabolism/kinetic properties of a test substance.
(ii) The first tier data set is a definitive study by the appropriate route of exposure conducted in male rats to determine the routes and rate of excretion and to identify excreted metabolites. First tier data will also provide basic information for additional testing (Tier 2) if such testing is considered necessary. In the majority of cases, Tier 1 data are expected to satisfy regulatory requirements for biotransformation and pharmacokinetic data on test chemicals.
(iii) Second tier testing describes a variety of metabolism/kinetic experiments which address specific questions based on the existing toxicology data base and/or those results of Tier 1 testing impacting significantly on the risk assessment process. For conduct of these studies, individualized protocols may be necessary. Protocols for these studies, if required, can be developed as a cooperative effort between Agency and industry scientists.
(f) Tier 1 data requirements (minimum data set). At this initial level of testing, biotransformation and pharmacokinetic data from a single low dose group will be required. This study will determine the rate and routes of excretion and the type of metabolites generated.
(1) Number and sex of animals. A minimum of four male young adult animals must be used for Tier 1 testing. The use of both sexes may be required in cases where there is evidence to support significant sex-related differences in toxicity.
(2) Dose selection. (i) A single dose is required for each route of exposure. The dose should be nontoxic, but high enough to allow for metabolite identification in excreta. If no other toxicity data are available for selection of the low dose, a dose identified as a fraction of the LD 50 (as determined from acute toxicity studies) may be used. The magnitude of the dose used in Tier 1 studies should be justified in the final report.
(ii) For test substances of low toxicity a maximum dose of 1,000 mg/kg should be used; chemical-specific considerations may necessitate a higher maximum dose and will be addressed in specific test rules.
(3) Measurements —(i) Excretion. (A) Data obtained from this section (percent recovery of administered dose from urine, feces, and expired air) will be used to determine the rate and extent of excretion of test chemical, to assist in establishing mass balance, and will be used in conjunction with pharmacokinetic parameters to determine the extent of absorption. The quantities of radioactivity eliminated in the urine, feces, and expired air shall be determined separately at appropriate time intervals.
(B) If a pilot study has shown that no significant amount of radioactivity is excreted in expired air, then expired air need not be collected in the definitive study.
(C) Each animal must be placed in a separate metabolic unit for collection of excreta (urine, feces and expired air). At the end of each collection period, the metabolic units must be rinsed with appropriate solvent to ensure maximum recovery of radiolabel. Excreta collection must be terminated at 7 days, or after at least 90% of the administered dose has been recovered, whichever occurs first. The total quantities of radioactivity in urine must be determined at 6, 12, and 24 hours on day 1 of collection, and daily thereafter until study termination, unless pilot studies suggest alternate or additional time points for collection. The total quantities of radioactivity in feces should be determined on a daily basis beginning at 24 hours post-dose, and daily thereafter until study termination. The collection of CO 2 and other volatile materials may be discontinued when less than 1% of the administered dose is found in the exhaled air during a 24-hour collection period.
(ii) Tissue distribution. At the termination of the Tier 1 study, the following tissues should be collected and stored frozen: Liver, fat, gastrointestinal tract, kidney, spleen, whole blood, and residual carcass. If it is determined that a significant amount of the administered dose is unaccounted for in the excreta, then data on the percent of the total (free and bound) radioactive dose in these tissues as well as residual carcass will be requested. Additional tissues must be included if there is evidence of target organ toxicity from subchronic or chronic toxicity studies. For other routes of exposure, specific tissues may also be required, such as lungs in inhalation studies and skin in dermal studies. Certain techniques currently at various stages of development, e.g., quantitative whole-body autoradiography, may prove useful in determining if a test substance concentrates in certain organs or in determining a specific pattern of distribution within a given tissue. The use of such techniques is encouraged, but not required, and may be employed to limit the number of tissues collected to those shown to contain a measurable amount of radioactivity.
(iii) Metabolism. Excreta must be collected for identification and quantitation of unchanged test substance and metabolites as described in paragraph (f)(3)(i) of this section. Pooling of excreta to facilitate metabolite identification within a given dose group is acceptable. Profiling of metabolites from each time period is recommended. However, if lack of sample and/or radioactivity precludes this, pooling of urine as well as pooling of feces across several time points is acceptable. Appropriate qualitative and quantitative methods must be used to assay urine, feces, and expired air from treated animals. Reasonable efforts should be made to identify all metabolites present at 5% or greater of the administered dose and to provide a metabolic scheme for the test chemical. Compounds which have been characterized in excreta as comprising 5% or greater of the administered dose should be identified. If identification at this level is not possible, a justification/explanation should be provided in the final report. Identification of metabolites representing less than 5% of the administered dose might be requested if such data are needed for risk assessment of the test chemical. Structural confirmation should be provided whenever possible. Validation of the methods used in metabolite identification should be included.
(g) Tier 2 data requirements. Studies at the Tier 2 level are designed to answer questions about the disposition of test chemicals based on the existing toxicology data base and/or results of Tier 1 testing which may have a significant impact on the risk assessment for the test chemical. Such studies may address questions regarding absorption, persistence, or distribution of the test chemical, or a definitive alteration in the metabolic profile occurring with dose which may be of toxicological concern. At the Tier 2 level, only those studies which address a specific concern are required, and if required must be conducted according to mutual agreement between the registrant and the Agency. Flexibility will be allowed in the design of specific experiments as warranted by technological advances in this field.
(1) Absorption. (i) If the extent of absorption cannot be established from Tier 1 studies, or where greater than 20% of the administered dose is present in feces, a study to determine the extent of absorption will be required. This can be accomplished either through intravenous administration of test material and measurement of radioactivity in excreta or after oral administration of test material and measurement of radioactivity in bile.
(ii) For the intravenous study, a single dose (not to exceed the oral dose used in Tier 1) of test chemical using an appropriate vehicle should be administered in a suitable volume (e.g., 1 mL/kg) at a suitable site to at least three male rats (both sexes might be used if warranted). The disposition of the test chemical should be monitored for oral dosing as outlined in paragraph (f)(3)(i) of this section. Metabolite identification will not be required for this study.
(iii) If a biliary excretion study is chosen the oral route of administration may be requested. In this study, the bile ducts of at least three male rats (or of both sexes, if warranted) should be appropriately cannulated and a single dose of the test chemical should be administered to these rats. Following administration of the test chemical, excretion of radioactivity in bile should be monitored as long as necessary to determine if a significant percentage of the administered dose is excreted via this route.
(2) Tissue distribution time course. (i) A time course of tissue distribution in selected tissues may be required to aid in the determination of a possible mode of toxic action. This concern may arise from evidence of extended half-life or possible accumulation of radioactivity in specific tissues. The selection of tissues for this type of study will be based upon available evidence of target organ toxicity and/or carcinogenicity, and the number of time points required will be based upon pharmacokinetic information obtained from Tier 1 data. Flexibility will be allowed in the selection of time points to be studied.
(ii) For this type of study, three rats per time point will be administered an appropriate oral dose of test chemical, and the time course of distribution monitored in selected tissues. Only one sex may be required, unless target organ toxicity is observed in sex-specific organs. Assessment of tissue distribution will be made using appropriate techniques for assessment of total amount distributed to tissue and for assessment of metabolite distribution.
(3) Plasma kinetics. The purpose of this experiment is to obtain estimates of basic pharmacokinetic parameters (half-life, volume of distribution, absorption rate constant, area under the curve) for the test substance. Kinetic data may be required if the data can be used to resolve issues about bioavailability and to clarify whether clearance is saturated in a dose-dependent fashion. For this experiment a minimum of three rats per group is required. At least two doses will be required, usually the NOEL and LOEL from the critical toxicology study. Following administration of test substance, samples should be obtained from each animal at suitable time points appropriate sampling methodology. Total radioactivity present (or total amount of chemical, for nonradioactive materials) should be analyzed in whole blood and plasma using appropriate methods, and the blood/plasma ratio should be calculated.
(4) Induction. (i) Studies addressing possible induction of biotransformation may be requested under one or more of the following conditions:
(A) Available evidence indicates a relationship between induced metabolism and enhanced toxicity.
(B) The available toxicity data indicate a nonlinear relationship between dose and metabolism.
(C) The results of Tier 1 metabolite identification studies show identification of a potentially toxic metabolite.
(D) Induction can plausibly be invoked as a factor in such effects where status may depend on the level of inducible enzymes present. Several in vivo and in vitro methods are available for assessment of enzyme induction, and the experiments which best address the issue at hand can be determined between Agency and industry scientists. If induction is demonstrated, the relationship of this phenomenon to toxicity observed from subchronic and/or chronic toxicity studies will need to be addressed.
(ii) [Reserved]
(iii) If toxicologically significant alterations in the metabolic profile of the test chemical are observed through either in vitro or in vivo experiments, characterization of the enzyme(s) involved (for example, Phase I enzymes such as isozymes of the Cytochrome P450-dependent mono-oxygenase system, Phase II enzymes such as isozymes of sulfotransferase or uridine diphosphate glucuronosyl transferase, or any other relevant enzymes) may be requested. This information will help establish the relevance of the involved enzyme(s) to human risk, as it is known that certain isozymes are present in animal species which are not present in humans, and vice versa.
(5) Physiologically-based modeling. Traditional methods of modeling have been used to determine kinetic parameters associated with drug and xenobiotic disposition, but have assumed a purely mathematical construct of mammalian organisms in their operation. On the other hand, more recent models which take into account the physiological processes of the animal have been used with success in defining biological determinants of chemical disposition as well as the relationship between tissue dose and tissue response. These so-called physiologically-based models, also allow for cross-species extrapolation which is often necessary in the risk-assessment process. The use of physiologically-based modeling as an experimental tool for addressing specific issues related to biotransformation and pharmacokinetics of a test substance is encouraged. Information as derived from physiologically-based modeling experiments may aid in the comparison of biotransformation and pharmacokinetics of a test substance between animal species and humans, and in the assessment of risk under specific exposure conditions. At the discretion of the Agency, or by mutual agreement, results of physiologically based pharmacokinetic (PBPK) studies with parent compound may be submitted in lieu of other studies, if it is determined that such data would provide adequate information to satisfy this guideline.
(h) Reporting of study results. In addition to the reporting requirements specified under EPA Good Laboratory Practice Standards at 40 CFR part 792, subpart J, the completed study (Tier 1 or Tier 2) should be presented in the following format:
(1) Title/cover page. Title page and additional requirements (requirements for data submission, good laboratory practice, statements of data confidentiality claims and quality assurance) if relevant to the study report, should precede the content of the study formatted below. These requirements are to be found in 40 CFR parts 790, 792, and 799.
(2) Table of contents. A concise listing must precede the body of the report, containing all essential elements of the study and the page and table number where the element is located in the final report of the study. Essential elements of the table of contents should include a summary, an introduction, the materials and methods section, results, discussion/conclusions, references, tables, figures, appendices, and key subsections as deemed appropriate. The table of contents should include the page number of each of these elements.
(3) Body of the report. The body of the report must include information required under this section, organized into sections and paragraphs as follows:
(i) Summary. This section of the study report must contain a summary and analysis of the test results and a statement of the conclusions drawn from the analysis. This section should highlight the nature and magnitude of metabolites, tissue residue, rate of clearance, bioaccumulation potential, sex differences, etc. The summary should be presented in sufficient detail to permit independent evaluation of the findings.
(ii) Introduction. This section of the report should include the objectives of the study, guideline references, regulatory history, if any, and a rationale.
(iii) Materials and methods. This section of the report must include detailed descriptions of all elements including:
(A) Test substance. ( 1 ) This section should include identification of the test substance—chemical name, molecular structure, qualitative and quantitative determination of its chemical composition, and type and quantities of any impurities whenever possible.
( 2 ) This section should also include information on physical properties including physical state, color, gross solubility and/or partition coefficient, and stability.
( 3 ) The type or description of any vehicle, diluents, suspending agents, and emulsifiers or other materials used in administering the test substance should be stated.
( 4 ) If the test substance is radiolabeled, information on the following should be included in this subsection: The type of radionuclide, position of label, specific activity, and radiopurity.
(B) Test animals. This section should include information on the test animals, including: Species, strain, age at study initiation, sex, body weight, health status, and animal husbandry.
(C) Methods. This subsection should include details of the study design and methodology used. It should include a description of:
( 1 ) How the dosing solution was prepared and the type of solvent, if any, used.
( 2 ) Number of treatment groups and number of animals per group.
( 3 ) Dosage levels and volume.
( 4 ) Route of administration.
( 5 ) Frequency of dosing.
( 6 ) Fasting period (if used).
( 7 ) Total radioactivity per animal.
( 8 ) Animal handling.
( 9 ) Sample collection.
( 10 ) Sample handling.
( 11 ) Analytical methods used for separation.
( 12 ) Quantitation and identification of metabolites.
( 13 ) Other experimental measurements and procedures employed (including validation of test methods for metabolite analysis).
(D) Statistical analysis. If statistical analysis is used to analyze the study findings, then sufficient information on the method of analysis and the computer program employed should be included so that an independent reviewer/statistician can reevaluate and reconstruct the analysis. Presentation of models should include a full description of the model to allow independent reconstruction and validation of the model.
(iv) Results. All data should be summarized and tabulated with appropriate statistical evaluation and placed in the text of this section. Radioactivity counting data should be summarized and presented as appropriate for the study, typically as disintegrations per minute and microgram or milligram equivalents, although other units may be used. Graphic illustrations of the findings, reproduction of representative chromatographic and spectrometric data, and proposed metabolic pathways and molecular structure of metabolites should be included in this section. In addition the following information is to be included in this section if applicable:
(A) Justification for modification of exposure conditions, if applicable.
(B) Justification for selection of dose levels for pharmacokinetic and metabolism studies.
(C) Description of pilot studies used in the experimental design of the pharmacokinetic and metabolism studies, if applicable.
(D) Quantity and percent recovery of radioactivity in urine, feces, and expired air, as appropriate. For dermal studies, include recovery data for treated skin, skin washes, and residual radioactivity in the covering apparatus and metabolic unit as well as results of the dermal washing study.
(E) Tissue distribution reported as percent of administered dose and microgram equivalents per gram of tissue.
(F) Material balance developed from each study involving the assay of body tissues and excreta.
(G) Plasma levels and pharmacokinetic parameters after administration by the relevant routes of exposure.
(H) Rate and extent of absorption of the test substance after administration by the relevant routes of exposure.
(I) Quantities of the test substance and metabolites (reported as percent of the administered dose) collected in excreta.
(J) Individual animal data.
(v) Discussion and conclusions. (A) In this section the author(s) should:
( 1 ) Provide a plausible explanation of the metabolic pathway for the test chemical.
( 2 ) Emphasize species and sex differences whenever possible.
( 3 ) Discuss the nature and magnitude of metabolites, rates of clearance, bioaccumulation potential, and level of tissue residues as appropriate.
(B) The author(s) should be able to derive a concise conclusion that can be supported by the findings of the study.
(vi) Optional sections. The authors may include additional sections such as appendices, bibliography, tables, etc.
(i) Alternate routes of exposure for Tier 1 testing —(1) Dermal —(i) Dermal treatment. One (or more if needed) dose levels of the test substance must be used in the dermal portion of the study. The low dose level should be selected in accordance with paragraph (f)(2) of this section. The dermal doses must be dissolved, if necessary, in a suitable vehicle and applied in a volume adequate to deliver the doses. Shortly before testing, fur is to be clipped from the dorsal area of the trunk of the test animals. Shaving may be employed, but it should be carried out approximately 24 hour before the test. When clipping or shaving the fur, care should be taken to avoid abrading the skin, which could alter its permeability. Approximately 10% of the body surface should be cleared for application of the test substance. With highly toxic substances, the surface area covered may be less than approximately 10%, but as much of the area as possible is to be covered with a thin and uniform film. The same nominal treatment surface area must be used for all dermal test groups. The dosed areas are to be protected with a suitable covering which is secured in place. The animals must be housed separately.
(ii) Dermal washing study. (A) A washing experiment must be conducted to assess the removal of the applied dose of the test substance by washing the treated skin area with a mild soap and water. A single dose must be applied to two animals in accordance with paragraph (f)(2) of this section. After application (2 to 5 minutes) the treated areas of the animals must be washed with a mild soap and water. The amounts of test substance recovered in the washes must be determined to assess the effectiveness of removal by washing.
(B) Unless precluded by corrosiveness, the test substance must be applied and kept on the skin for a minimum of 6 hours. At the time of removal of the covering, the treated area must be washed following the procedure as outlined in the dermal washing study. Both the covering and the washes must be analyzed for residual test substance. At the termination of the studies, each animal must be sacrificed and the treated skin removed. An appropriate section of treated skin must be analyzed to determine residual radioactivity.
(2) Inhalation. A single (or more if needed) concentration of test substance must be used in this portion of the study. The concentration should be selected in accordance with paragraph (f)(2) of this section. Inhalation treatments are to be conducted using a “nose-cone” or “head-only” apparatus to prevent absorption by alternate routes of exposure. If other inhalation exposure conditions are proposed for use in a chemical-specific test rule, justification for the modification must be documented. A single exposure over a defined period must be used for each group—a typical exposure is 4-6 hours.
§ 799.9780   TSCA immunotoxicity.
(a) Scope. This section is intended to meet the testing requirements under section 4 of TSCA. This section is intended to provide information on suppression of the immune system which might occur as a result of repeated exposure to a test chemical. While some information on potential immunotoxic effects may be obtained from hematology, lymphoid organ weights and histopathology (usually done as part of routine toxicity testing), there are data which demonstrate that these endpoints alone are not sufficient to predict immunotoxicity (Luster et al., 1992, 1993 see paragraphs (j)(8) and (j)(9) of this section). Therefore, the tests described in this section are intended to be used along with data from routine toxicity testing, to provide more accurate information on risk to the immune system. The tests in this section do not represent a comprehensive assessment of immune function.
(b) Source. The source material used in developing this TSCA test guideline is the OPPTS harmonized test guideline 870.7800 (June 1996 Public Draft). This source is available at the address in paragraph (j) of this section.
(c) Definitions. The following definitions apply to this section.
Antibodies or immunoglobulins (Ig) are part of a large family of glycoprotein molecules. They are produced by B cells in response to antigens, and bind specifically to the eliciting antigen. The different classes of immunoglobulins involved in immunity are IgG, IgA, IgM, IgD, and IgE. Antibodies are found in extracellular fluids, such as serum, saliva, milk, and lymph. Most antibody responses are T cell-dependent, that is, functional T and B lymphocytes, as well as antigen-presenting cells (usually macrophages), are required for the production of antibodies.
Cluster of differentiation (CD) refers to molecules expressed on the cell surface. These molecules are useful as distinct CD molecules are found on different populations of cells of the immune system. Antibodies against these cell surface markers (e.g., CD4, CD8) are used to identify and quantitate different cell populations.
Immunotoxicity refers to the ability of a test substance to suppress immune responses that could enhance the risk of infectious or neoplastic disease, or to induce inappropriate stimulation of the immune system, thus contributing to allergic or autoimmune disease. This section only addresses potential immune suppression.
Natural Killer (NK) cells are large granular lymphocytes which nonspecifically lyse cells bearing tumor or viral antigens. NK cells are up-regulated soon after infection by certain microorganisms, and are thought to represent the first line of defense against viruses and tumors.
T and B cells are lymphocytes which are activated in response to specific antigens (foreign substances, usually proteins). B cells produce antigen-specific antibodies (see the definition for “antibodies or immunoglobulins”), and subpopulations of T cells are frequently needed to provide help for the antibody response. Other types of T cell participate in the direct destruction of cells expressing specific foreign (tumor or infectious agent) antigens on the cell surface.
(d) Principles of the test methods. (1) In order to obtain data on the functional responsiveness of major components of the immune system to a T cell dependent antigen, sheep red blood cells (SRBC), rats and/or mice 1 shall be exposed to the test and control substances for at least 28 days. 2 The animals shall be immunized by intravenous or intraperitoneal injection of SRBCs approximately 4 days (depending on the strain of animal) prior to the end of the exposure. At the end of the exposure period, either the plaque forming cell (PFC) assay or an enzyme linked immunosorbent assay (ELISA) shall be performed to determine the effects of the test substance on the splenic anti-SRBC (IgM) response or serum anti-SRBC IgM levels, respectively.
1 If absorption/distribution/metabolism/excretion (ADME) data are similar between species, then either rats or mice may be used for the test compound in question. If such data are lacking, both species should be used.
2 Because there is a fairly rapid turnover of many of the cells in the immune system, 28 days is considered sufficient for the purposes of the anti-SRBC tests.
(2) In the event the test substance produces significant suppression of the anti-SRBC response, expression of phenotypic markers for major lymphocyte populations (total T and total B), and T cell subpopulations (T helpers (CD 4 ) and T cytotoxic/suppressors (CD 8 )), as assessed by flow cytometry, may be performed to determine the effects of the test substance on either splenic or peripheral-blood lymphocyte populations and T cell subpopulations. When this study is performed, the appropriate monoclonal antibodies for the species being tested should be used. If the test substance has no significant effect on the anti-SRBC assay, a functional test for NK cells may be performed to test for a chemical's effect on non-specific immunity. 3 For tests performed using cells or sera from blood (ELISA or flow cytometry), it is not necessary to destroy the animals, since immunization with SRBCs at 28 days is not expected to markedly affect the results of other assays included in subchronic or longer-term studies (these tests are discussed in the reference under paragraph (j)(7) of this section). The necessity to perform either a quantitative analysis of the effects of a chemical on the numbers of cells in major lymphocyte populations and T Cell subpopulations by flow cytometry, or a splenic NK cell activity assay to assess the effects of the test compound on non-specific immunity shall be determined on a case-by-case basis, depending upon the outcome of the anti-SRBC assay.
3 When these optional tests are included, the phenotypic or NK cell analyses may be performed at 28 days of exposure, or at a later timepoint if ADME data suggest that a longer exposure is more appropriate.
(e) Limit test. If a test at one dose level of at least 1,000 mg/kg body weight (or 2 mg/L for inhalation route of exposure) 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. Expected human exposure may indicate the need for a higher dose level.
(f) Test procedures —(1) Animal selection —(i) Species and strain. These tests are intended for use in rats and/or mice. Commonly used laboratory strains shall be employed. 4 All test animals shall be free of pathogens, internal and external parasites. Females shall be nulliparous and nonpregnant. The species, strain, and source of the animals shall be identified.
4 The study director shall be aware of strain differences in response to SRBC. For example, if the B 6 C 3 F 1 hybrid mouse is used in the PFC assay, a response of 800-1,000 PFC/10 6 spleen cells in control mice should be the minimally acceptable PFC response.
(ii) Age/weight. (A) Young, healthy animals shall be employed. At the commencement of the study, the weight variation of the animals used shall not exceed ±20% of the mean weight for each sex.
(B) Dosing shall begin when the test animals are between 6 and 8 weeks old.
(iii) Sex. Either sex may be used in the study; if one sex is known or believed to be more sensitive to the test compound, then that sex shall be used.
(iv) Numbers. (A) At least eight animals shall be included in each dose and control group. The number of animals tested shall yield sufficient statistical power to detect a 20% change based upon the interanimal variation which may be encountered in these assays.
(B) To avoid bias, the use of adequate randomization procedures for the proper allocation of animals to test and control groups is required.
(C) 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 shall 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 shall be at 22 ±3 °C.
(C) The relative humidity of the experimental animal rooms shall be between 30 and 70%.
(D) Where lighting is artificial, the sequence shall be 12 hrs light, 12 hrs dark.
(E) Control and test animals shall be maintained on the same type of bedding and receive feed from the same lot. The feed shall be analyzed to assure adequacy of nutritional requirements of the species tested and for impurities that might influence the outcome of the test. Rodents shall be fed and watered ad libitum with food replaced at least weekly.
(F) The study shall not be initiated until the animals have been allowed an adequate period of acclimatization or quarantine to environmental conditions. The period of acclimatization shall be at least 1 week in duration.
(2) Control and test substances. (i) The test substance shall be dissolved or suspended in a suitable vehicle. Ideally, if a vehicle or diluent is needed, it shall not elicit toxic effects or substantially alter the chemical or toxicological properties of the test substance. It is recommended that an aqueous solution should be used. If solubility is a problem a solution in oil may be used. Other vehicles may be considered, but only as a last resort.
(ii) One lot of the test substance shall be used, if possible, throughout the duration of the study, and the research sample shall be stored under conditions that maintain its purity and stability. Prior to the initiation of the study, there shall be a characterization of the test substance, including the purity of the test compound and if technically feasible, the name and quantities of any known contaminants and impurities.
(iii) If the test or positive control substance is to be incorporated into feed or another vehicle, the period during which the test substance is stable in such a mixture shall be determined prior to the initiation of the study. Its homogeneity and concentration shall also be determined prior to the initiation of the study and periodically during the study. Statistically randomized samples of the mixture shall 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. (i) A concurrent, vehicle-treated control group is required.
(ii) A separate untreated control group is required if the toxicity of the vehicle is unknown.
(iii) A positive control group with a known immunosuppressant (e.g., cyclophosphamide) shall be included in the study. A group of at least eight animals shall be given the immunosuppressive chemical.
(4) Dose levels. (i) In repeated-dose toxicity tests, it is desirable to have a dose-response relationship and a no observed immunotoxic effect level. Therefore, at least three dose levels and a negative control shall be used, unless a limit test is performed as specified under paragraph (e) of this section.
(ii) The highest dose level shall not produce significant stress, malnutrition, or fatalities, but ideally should produce some measurable sign of general toxicity (e.g., a 10% loss of body weight).
(iii) The lowest dose level ideally shall not produce any evidence of immunotoxicity.
(5) Administration of the test substance. (i) The test substance, vehicle, or positive control substance shall be administered for at least 28 days for the anti-SRBC assay. The route of administration of the test material will usually be oral; however, this shall be determined by the likely route of occupational or indoor exposure. Therefore, under certain conditions, the dermal or inhalation route of exposure may be more relevant for the study. All animals shall be dosed by the same method during the entire experimental period.
(ii) If the test substance is administered by gavage, the animals are dosed with the test substance ideally on a 7-days-per-week basis. However, based primarily on practical considerations, dosing by gavage on a 5-days-per-week basis shall be acceptable. If the test substance is administered in the drinking water, or mixed directly into the diet, then exposure shall be on a 7-days-per-week basis.
(A) 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 in parts per million (ppm) or a constant dose level in terms of the animal's body weight shall be used; the alternative used should be specified.
(B) For a substance administered by gavage, the dose shall be given at approximately the same time each day, and adjusted at intervals (weekly for mice, twice per week for rats) to maintain a constant dose level in terms of the animal's body weight.
(iii) If the test substance is administered dermally, use paragraphs (f)(5)(iii)(A) through (f)(5)(iii)(D) of this section.
(A) Dose levels and dose selection. ( 1 ) In this test, 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 should 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.
( 2 ) 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.
( 3 ) The intermediate dose levels should be spaced to produce a gradation of toxic effects.
( 4 ) The lowest dose level should not produce any evidence of toxic effects.
(B) Preparation of animal skin. Shortly before testing, fur should 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 hrs 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.
(C) Preparation of test substance. ( 1 ) Liquid test substances are generally used undiluted, except as indicated in paragraph (f)(5)(iii)(A)( 2 ) of this section.
( 2 ) 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.
( 3 ) The volume of application should be kept constant, e.g. less than 300 <greek-m≤L for the rat; different concentrations of test solution should be prepared for different dose levels.
(D) Administration of test substance. ( 1 ) The duration of exposure should be at least for 90 days.
( 2 ) The animals should be treated with test substance for at least 6 hrs/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.
( 3 ) The test substance should be applied uniformly over the treatment site.
( 4 ) 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.
( 5 ) During the exposure period, the test substance should be held in contact with the skin with a porous gauze dressing. The test site should 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.
(iv) If the test substance is administered by the inhalation route, use the procedures under paragraphs (e)(2), (e)(3), (e)(6), (e)(8), (e)(9), and (e)(10) of 40 CFR 799.9346. The exposure time for the anti-SRBC test shall be at least 28 days.
(6) Observation period. Duration of the observation period shall be at least 28 days.
(7) Observation of animals. (i) Observations shall be made at least once each day for morbidity and mortality. Appropriate actions shall be taken to minimize loss of animals to the study (e.g., necropsy of those animals found dead and isolation or euthanasia of weak or moribund animals).
(ii) A careful clinical examination shall be made at least once a week. Observations shall be detailed and carefully recorded, preferably using explicitly defined scales. Observations shall 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 motor activity, gait and posture, reactivity to handling or sensory stimuli, grip strength, and stereotypes or bizarre behavior (e.g., self-mutilation, walking backwards).
(iii) Signs of toxicity shall be recorded as they are observed, including the time of onset, degree and duration.
(iv) Food and water consumption shall be determined weekly.
(v) Animals shall be weighed immediately prior to dosing, weekly (twice per week for rats) thereafter, and just prior to euthanasia.
(vi) Any moribund animals shall be removed and euthanized when first noticed. Necropsies shall be conducted on all moribund animals, and on all animals that die during the study.
(vii) The spleen and thymus shall be weighed in all animals at the end of the study.
(g) Immunotoxicity tests —(1) Functional tests. Either a splenic PFC assay or an ELISA shall be used to determine the response to antigen administration.
(i) Antibody plaque-forming cell (PFC) assay. If the antibody PFC assay is performed, the criteria listed under paragraphs (g)(1)(i)(A) through (g)(1)(i)(F) of this section shall be adhered to. Assays described in the references under paragraphs (j)(2) and (j)(4) of this section may be used.
(A) The T cell-dependent antigen, SRBC, shall be injected intravenously or intraperitoneally, usually at 24 days after the first dosing with the test substance. 5 Although the optimum response time is usually 4 days after immunization, some strains of test animal may deviate from this time point. The strain to be used shall be evaluated for the optimum day for PFC formation after immunization.
5 If the SRBCs are administered by the intraperitoneal route, the study director should be aware that a low percentage of animals may not respond because the antigen was accidentally injected into the intestinal tract.
(B) The activity of each new batch of complement shall be determined. For any given study, the SRBCs shall be from a single sheep, or pool of sheep, for which the shelf life and dose for optimum response has been determined.
(C) Modifications of the PFC assay described in paragraph (g)(1)(i) of this section exist and may prove useful; however, the complete citation shall be made for the method used, any modifications to the method shall be reported, and the source and, where appropriate, the activity or purity of important reagents shall be given. Justification or rationale shall be provided for each protocol modification. Discussions of modifications of the PFC assay are available in the references under paragraphs (j)(5),(j)(6), and (j)(10) of this section
(D) Samples shall be randomized and shall be coded for PFC analysis, so that the analyst is unaware of the treatment group of each sample examined.
(E) Spleen cell viability shall be determined.
(F) The numbers of IgM PFC per spleen, and the number of IgM PFC per 10 6 spleen cells shall be reported.
(ii) Immunoglobulin quantification. As an alternative to a PFC assay, the effects of the test substance on the antibody response to antigen may be determined by an Enzyme-Linked Immunosorbent Assay (ELISA). Comparison between the PFC and ELISA assays for immunotoxicity assessment are discussed in the references under paragraphs (j)(5), (j)(6), and (j)(10) of this section. Test animals shall be immunized with SRBCs as for the PFC assay. IgM titers in the serum of each test animal shall be determined (usually 4 days after immunization). As with the PFC assay, the optimum dose of SRBCs and optimum time for collection of the sera shall be determined for the species and strain of animal to be tested. Several methods are described in the reference under paragraph (j)(11) of this section).
(iii) Natural killer (NK) cell activity. The methods described in the reference under paragraph (j)(3) of this section may be used to demonstrate the effects of at least 28 days of exposure to a test substance on spontaneous cytotoxic activity. In this assay, splenocytes from treated and untreated test animals are incubated with 51 Cr-labeled YAC-1 lymphoma cells. The amount of radiolabel released from the target cells after incubation with the effector cells for four hrs is used as a measure of NK cytolysis. The following points shall be adhered to when using the NK cell assay:
(A) Assay controls shall be included to account for spontaneous release of radiolabel from target cells in the absence of effector cells, and also for the determination of total release of radiolabel.
(B) Target cells other than YAC-1 lymphoma cells may be appropriate for use in the assay. In all cases, target cell viability shall be determined.
(C) Modifications of the protocol exist that may prove useful. However, complete citation shall be made to the method used. Modifications shall be reported, and where appropriate, the source, activity, and/or purity of the reagents should be given. Justification or rationale shall be provided for each protocol modification.
(2) Enumeration of splenic or peripheral blood total B cells, total T cells, and T cell subpopulations. The phenotypic analysis of total B cell, total T cell, and T cell subpopulations from the spleen or peripheral blood by flow cytometry should be performed after at least 28 days of dosing; this may be performed at a later timepoint, if ADME data suggest that a longer exposure is more appropriate. If an exposure period longer than 28 days is used, then these tests may be performed in conjunction with subchronic (ninety day oral, dermal, or inhalation) toxicity studies, when these studies are required. Methods described in the references under paragraphs (j)(1) and (j)(5) of this section may be used.
(h) 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 effects, the types of effects and the percentage of animals displaying each type of effect.
(ii) All observed results, quantitative and incidental, shall be evaluated by an appropriate statistical method. Any generally accepted statistical methods may be used; the statistical methods including significance criteria shall be selected during the design of the study.
(2) Evaluation of study results. The findings of an immunotoxicity study shall be evaluated in conjunction with the findings of preceding studies and considered in terms of other toxic effects. The evaluation shall include the relationship between the dose of the test substance and the presence or absence, and 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 test shall provide a satisfactory estimation of a no-observed-effect level. It may indicate the need for an additional study and provide information on the selection of dose levels.
(3) Test report. In addition to the reporting requirements as specified under 40 CFR part 792, subpart J, the following specific information shall be reported. Both individual and summary data should be presented.
(i) The 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) The test system shall contain data on:
(A) Species, strain, and rationale for selection of animal species, if other than that recommended.
(B) Age, body weight data, and sex.
(C) Test environment including cage conditions, ambient temperature, humidity, and light/dark periods.
(D) When inhalation is the route of exposure, a description of the exposure equipment and data shall be included as follows:
( 1 ) Description of test conditions; the following exposure conditions shall be reported:
( i ) 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.
( ii ) The equipment for measuring temperature, humidity, and particulate aerosol concentrations and size should be described.
( 2 ) Exposure data shall be tabulated and presented with mean values and a measure of variability (e.g., standard deviation) and include:
( i ) Airflow rates through the inhalation equipment.
( ii ) Temperature and humidity of air.
( iii ) Actual (analytical or gravimetric) concentration in the breathing zone.
( iv ) Nominal concentration (total amount of test substance fed into the inhalation equipment divided by volume of air).
( v ) Particle size distribution, calculated mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD).
( vi ) 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.
(E) Identification of animal diet.
(iii) The test procedure shall 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.
(iv) Test results should include the following data:
(A) Group animal toxic response data shall be tabulated 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 shall be presented, as well as summary (group mean data).
(C) Date of death during the study or whether animals survived to termination.
(D) Date of observation of each abnormal sign and its subsequent course.
(E) Absolute and relative spleen and thymus weight data.
(F) Feed and water consumption data, when collected.
(G) Results of immunotoxicity tests.
(H) Necropsy findings of animals that were found moribund and euthanized or died during the study.
(I) Statistical treatment of results, where appropriate.
(i) 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 the 40 CFR Part 792—Good Laboratory Practice.
(j) 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) Cornacoff, J.B., Graham, C.S., and LaBrie, T.K. Eds. Burleson, G.R., Dean, J.H., and Munson, A.E. Phenotypic identification of peripheral blood mononuclear leukocytes by flow cytometry as an adjunct to immunotoxicity evaluation. Vol. 1. Methods in Immunotoxicology (Wiley-Liss, Inc., New York, 1995) pp. 211-226.
(2) Cunningham, A.J. A method of increased sensitivity for detecting single antibody-forming cells. Nature. 207:1106-1107 (1965).
(3) Djeu, Julie Y. Eds. Burleson, G.R., Dean, J.H., and Munson, A.E. Natural Killer Activity. Methods in Immunotoxicology. pp. 437-449 (1995).
(4) Holsapple, M.P. Eds. Burleson, G.R., Dean, J.H., and Munson, A.E. The plaque-forming cell (PFC) response in Immunotoxicology: An approach to monitoring the primary effector function of B lymphocytes. Vol. 1. Methods in Immunotoxicology (Wiley-Liss, Inc., New York, 1995) pp. 71-108.
(5) Ladics, G.S. and Loveless, S.E. Cell surface marker analysis of splenic lymphocyte populations of the CD rat for use in immunotoxicological studies. Toxicology Methods. 4: 77-91 (1994).
(6) Ladics, G.S., Smith, C., Heaps, K., and Loveless, S.E. Evaluation of the humoral immune response of CD rats following a 2-week exposure to the pesticide carbaryl by the oral, dermal, or inhalation routes. Journal of Toxicology Environmental Health. 42:143-156 (1994).
(7) Ladics., G.S., Smith, C., Heaps, K., Elliot, G.S., Slone, T.W., and Loveless, S.E. Possible incorporation of an immunotoxicological functional assay for assessing humoral immunity for hazard identification purposes in rats on standard toxicology study. Toxicology. 96:225-238 (1995).
(8) Luster, M.I., Portier, C., Pait, D.G., White, K.L., Jr., Gennings, C., Munson, A.E., and Rosenthal, G.J. Risk assessment in immunotoxicology I. Sensitivity and predictability of immune tests. Fundamental Applied Toxicology. 18:200-210 (1992).
(9) Luster, M.I., Portier, C., Pait, D.G., Rosenthal, G.J. Germolec. D.R., Corsini, E., Blaylock, B.L., Pollock, P., Kouchi, Y., Craig, W., White, D.L., Munson, A.E., and Comment, C.E. Risk Assessment in Immunotoxicology II. Relationships Between Immune and Host Resistance Tests. Fundamental Applied Toxicology. 21:71-82 (1993).
(10) Temple, L., Kawabata, T. T., Munson, A. E., and White, Jr., K. L. Comparison of ELISA and plaque-forming cell assays for measuring the humoral immune response to SRBC in rats and mice treated with benzo[a]pyrene or cyclophosphamide. Fundamental Applied Toxicology. 21:412-419 (1993).
(11) Temple, L., Butterworth, L., Kawabata, T.T., Munson, A.E., and White, Jr., K.L. Eds. Burleson, G.R., Dean, J.H., and Munson, A.E. ELISA to Measure SRBC Specific Serum IgM: Method and Data Evaluation. Vol. 1. Methods in Immunotoxicology (Wiley-Liss, Inc., New York, 1995) pp. 137-157.