§ 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.

(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 ) and T cytotoxic/suppressors (CD )), 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.

(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.

(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.