The following abstract presents results of a study conducted by a contract laboratory for the National Toxicology Program. The findings have not been peer reviewed and were not evaluated in accordance with the levels of evidence criteria established by NTP in March 2009. The findings and conclusions for this study should not be construed to represent the views of the NTP or the U.S. Government.
2,4-Diaminotoluene (DAT) represents an amino substituted aromatic which has been proposed for immunotoxicity testing by the National Toxicology Program (NTP) because of the results of the carcinogenicity tests. Testing is also proposed because of its use in intermediate production of toluene diisocyanate, in the production of dyes, and in the preparation of impact-resistance resins, polyimides, hydraulic fluids, urethane foams, fungicide stabilizers and sensitizers for explosives. Production volume averages about 200 million pounds per year.
The doses selected for evaluation of the immune system as a target for toxicity were 25, 50 and 100 mg/kg. From probe studies, doses of 150 mg/kg/day for 14 days had lethal actions in the B6C3F1 mouse; thus, the high dose used in the immunotoxicity evaluation is considered to be a maximum tolerated dose for 14 days of oral exposure.
DAT, at doses between 25 and 100 mg/kg per day for 14 days by gavage, targeted the hepatic and immune system. The toxicology studies are summarized in Table ES-1. Mice exposed to DAT in doses up to 100 mg/kg for 14 days by gastric gavage showed no change in body weight and only a slight decrease in body weight gain. There was a 41.8% increase in liver weight and a slight decrease in spleen weight. The only gross pathology observed was an increase in liver size. Histopathology evaluation of selected organs showed the liver to be the major target with morphological changes. The changes were dose-dependent with the high dose (100 mg/kg) being associated with moderate centrilobular necrosis. No abnormal structure was noted in the spleen, lungs, thymus, kidney or mesenteric lymph nodes. The liver toxicity was associated with elevation in SGPT levels The only change noted in selected hematological parameters was a 63.8% increase in peripheral blood leukocytes which may be a response to the liver damage.
The immunology studies are summarized in Table ES-2. DAT, administered by gastric gavage in doses between 25 and 100 mg/kg, was associated with a number of immunological changes. The IgM and IgG response to the T-dependent sRBC represents a comprehensive assay requiring appropriate participation of T and B cells and appropriate production of the various interleukins. Mice exposed to DAT showed a decreased IgM and IgG response to sRBC. The deficient immunoglobulin response was associated with a decrease in spleen cell number. Thus, IgM & IgG responses were not significantly changed when expressed on a per spleen cell basis but the response was decreased on a whole spleen basis. The decrease was not a function of a decreased number of B cells since the number of B cells increased dose-dependently. Proliferative capacity of T lymphocytes was not affected by exposure to DAT as measured by response to the mitogens (Concanavalin A, phytohemagglutinin) and allogeneic cells (MLR). The increased number of B cells was associated with an increase in B cell responsiveness to the B cell mitogen bacterial lipopolysaccharide. The delayed hypersensitivity response to KLH represents a holistic assay measuring most functions associated with cell-mediated immunity. The DHR response to KLH in mice exposed to DAT was increased. This may be a compensatory action in response to the decrease in antibody production and NK cell activity. Natural Killer Cell activity was decreased dose-dependently and may be a spleen cell pool shift since the number of B cells increased in the presence of a decreasing spleen size. One of the serum complement components, C3, was suppressed dose-dependently. Phagocytosis of peritoneal macrophages was unaffected by DAT exposure, but the macrophages of the spleen were suppressed. Phagocytic ability of the Kupffer cells was unchanged although the data indicates an increased number of Kupffer cells.
The host resistance studies are summarized in Table ES-3. Two bacterial and two tumor host resistance models were employed in these studies. DAT exposure for 14 days decreased host resistance to the bacterial challenge, Streptococcus pneumoniae and Listeria monocytogenes. The decrease may be related to the decrease in serum complement (C3) and spleen phagocytosis, respectively. Host resistance to the pulmonary tumor model, B16F10, and the PYB6 Fibrosarcoma were unaffected by DAT exposure. The lowest dose that produced an immunological effect was 50 mg/kg which was seen in the number of B cells and Natural Killer Cell activity. Thus, the no effect level is 25 mg/kg for a 14 day exposure.
The studies were conducted at the Medical College of Virginia Immunotoxicology Laboratory under NTP Contract No. ES 55094. The in-life phase of the studies was conducted between 30 June 1987 and 13 November 1987. The animals were housed in the animal facility of the Strauss Building.