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Epidemiologic Notes and Reports An Evaluation of the Immunotoxic Potential of Isobutyl Nitrite

Initial epidemiologic studies indicated that the use of inhalant drugs, such as amyl nitrite, isobutyl nitrite (IBN), and butyl nitrite, may be a risk factor for acquired immunodeficiency syndrome (AIDS) (1,2). Because the immunotoxic potential of these drugs was unknown, CDC undertook an immunotoxicologic evaluation of one of the most commercially available inhalants--IBN.

Balb/c mice were exposed to IBN at vapor concentrations of 20, 50, and 300 parts per million (ppm) for 6.5 hours a day, 5 days a week, for 3, 7, 13, or 18 weeks. At selected intervals, mice exposed to either 50 or 300 ppm of IBN were removed from the exposure chambers and tested for immunocompetency by the following assays: 1) antibody-producing cells were counted by localized hemolysis in gel assay (Jerne Plaque Assay) (3) 4 days after the mice had been immunized intraperitoneally with sheep erythrocytes; 2) radiometric skin testing with PPD (purified protein derivative) was performed 21 days after immunization with Freund's complete adjuvant (4); 3) the lymphocyte blast transformation (LBT) assay was performed by using splenic lymphocytes stimulated at several concentrations of the following mitogens: phytohemagglutinin, concanavalin A, pokeweed mitogen, or lipopolysaccharide. Each assay was performed on at least 10 (five male, five female) exposed animals and 10 control animals each time, and, except for the skin testing, assays for each animal were done in replicates of three (plaque assay) or four (LBT).

In addition to the immunocompetency testing, all animals were weighed weekly; their spleens, thymuses, and livers were weighed at necropsy, when hematologic measurements, including white-cell counts, red-cell counts, differential white-cell counts, and methemaglobin levels, were also determined. Fifteen major organs were removed and processed for histologic and pathologic analysis.

None of the animals exposed to IBN showed any evidence of immunotoxic reactions. Methemaglobinemia was noted in animals exposed to 300 ppm of IBN, and some evidence of thymic atrophy, possibly stress-related, was found in this group. All detailed histologic examinations have not been completed. Reported by Immunology Section, Laboratory Investigations Br, Div of Respiratory Disease Studies; Chronic Toxicology Section, Experimental Toxicology Br, Div of Biomedical and Behavioral Sciences, National Institute of Occupational Safety and Health; AIDS Activity and Div of Host Factors, Center for Infectious Diseases; Office of the Director, Center for Environmental Health, CDC.

Editorial Note

Editorial Note: Aliphatic nitrites, such as IBN, are commercially available as room odorizers but are commonly used as inhalant "street" drugs. The results of the present study, as well as the occurrence of AIDS among populations not commonly using inhalant nitrites, suggests that these drugs are not responsible for the basic immune defects characteristic of AIDS.

Although the data obtained in this study indicate that IBN was not immunotoxic for mice, these drugs do have toxic effects. They have been shown to be mutagenic in vitro (5) and are highly flammable. Reported side effects include: dizziness, headache, tachycardia, syncope, hypotension, and increased intraocular pressure; nitrites have also been associated with methemoglobinemia and, rarely, sudden death (6). Nitrite inhalants do not appear to be implicated as a cause of the immunosuppression seen in AIDS, but their role as a cofactor in some of the illnesses found in this syndrome has not been ruled out.


  1. Goedert JJ, Neuland CY, Wallen WC, et al. Amyl nitrite may alter T lymphocytes in homosexual men. Lancet 1982;1:412-6.

  2. Marmor M, Friedman-Kien AE, Laubenstein L, et al. Risk factors for Kaposi's sarcoma in homosexual men. Lancet 1982;1:1083-7.

  3. Dresser DW. Assays of immunoglobulin-secreting cells. In: Weir DM, ed. Handbook of experimental immunology, 3rd edition. Oxford: Blackwell Scientific Publications, 1978. pp 28.1-28.25.

  4. Luster MI, Dean JH, Moore JA. Evaluation of immune functions in toxicology. In: Hayes W, ed. Methods in toxicology. New York: Raven Press, 1982, pp 516-86.

  5. Quinto I. Mutagenicity of alkylnitrites in the Salmonella test. Boll Soc Ital Biol Sper. 1980;56:816-20.

  6. Bruckner JV, Peterson RG. Toxicology of aliphatic and aromatic hydrocarbons. In: National Institute on Drug Abuse (research monograph no. 15). In: Sharp CW, Brehm ML, eds. Review of inhalants: euphoria to dysfunction. Washington, D.C.: Government Printing Office, 1977;124-63.

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