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Acute systemic toxicity data are used by a number of U.S. federal agencies, most commonly for hazard classification and labeling and/or risk assessment for acute chemical exposures. To identify opportunities for the implementation of non-animal approaches to produce these data, the regulatory needs and uses for acute systemic toxicity information must first be clarified. Thus, we reviewed acute systemic toxicity testing requirements for six U.S. agencies (Consumer Product Safety Commission, Department of Defense, Department of Transportation, Environmental Protection Agency, Food and Drug Administration, Occupational Safety and Health Administration) and noted whether there is flexibility in satisfying data needs with methods that replace or reduce animal use. Understanding the current regulatory use and acceptance of non-animal data is a necessary starting point for future method development, optimization, and validation efforts. The current review will inform the development of a national strategy and roadmap for implementing non-animal approaches to assess potential hazards associated with acute exposures to industrial chemicals and medical products. The Acute Toxicity Workgroup of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), U.S. agencies, non-governmental organizations, and other stakeholders will work to execute this strategy.

Perchlorate is an important oxidizer used in propellants, pyrotechnics, and as a gas generator in commercial airbags, fireworks, and roadside flares. It is highly water soluble, interferes with thyroidal iodide uptake and is an environmental contaminant. By changing the reaction chemistry, 5-aminotetrazole (5-AT) and nitrates replace perchlorate in some propellants. The short term toxicity of 5-AT was evaluated. Using a modified Ames assay, 5-AT was not mutagenic with or without S9 metabolic activation. 5-AT was considered “slightly toxic” with an EC50 of 28.8 mg 5-AT/L for a 15 min exposure in Aliivibrio fischeri. In the in vitro sodium iodide symporter test, 5-AT did not inhibit the uptake of iodine. In the acute rat oral test, no adverse effects and no mortalities were observed at the limit dose of 2000 mg 5-AT/kg. In the 14-day sub-acute study, there were no clinical signs of toxicity or morbidity up to 623 mg 5-AT/kg-day; the highest dose tested. No differences were observed in hematology, clinical chemistry, organ weight, body weight, food consumption, histopathology, or DNA damage (peripheral blood micronucleus assay) of treatments compared with controls. The No Observed Adverse Effect Level (NOAEL) was 623 mg 5-AT/kg-day, the highest dose in the subacute oral bioassay.

United States regulatory and research agencies may rely upon skin sensitization test data to assess the sensitization hazards associated with dermal exposure to chemicals and products. These data are evaluated to ensure that such substances will not cause unreasonable adverse effects to human health when used appropriately. The U.S. Consumer Product Safety Commission, the U.S. Environmental Protection Agency, the U.S. Food and Drug Administration, the Occupational Safety and Health Administration, the National Institute for Occupational Safety and Health, and the U.S. Department of Defense are member agencies of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM). ICCVAM seeks to identify opportunities for the use of non-animal replacements to satisfy these testing needs and requirements. This review identifies the standards, test guidelines, or guidance documents that are applicable to satisfy each of these agency’s needs; current use of animal testing and flexibility for using alternative methodologies; information needed from alternative tests to fulfill the needs for skin sensitization data; and whether data from non-animal alternative approaches are accepted by these U.S. federal agencies.

New approaches are needed to assess the effects of inhaled substances on human health. These approaches will be based on mechanisms of toxicity, an understanding of dosimetry, and the use of in silico modeling and in vitro test methods. In order to accelerate wider implementation of such approaches, development of adverse outcome pathways (AOPs) can help identify and address gaps in our understanding of relevant parameters for model input and mechanisms, and optimize non-animal approaches that can be used to investigate key events of toxicity. This paper describes the AOPs and the toolbox of in vitro and in silico models that can be used to assess the key events leading to toxicity following inhalation exposure. Because the optimal testing strategy will vary depending on the substance of interest, here we present a decision tree approach to identify an appropriate non-animal integrated testing strategy that incorporates consideration of a substance's physicochemical properties, relevant mechanisms of toxicity, and available in silico models and in vitro test methods. This decision tree can facilitate standardization of the testing approaches. Case study examples are presented to provide a basis for proof-of-concept testing to illustrate the utility of non-animal approaches to inform hazard identification and risk assessment of humans exposed to inhaled substances.

This chapter presents the wildlife toxicity assessment for nitroguanidine (NQ), a stable explosive compound used as a propellant in cartridges. This wildlife toxicity assessment addresses only toxicity to mammals, as data for amphibians, reptiles, and birds were lacking. Acute, chronic, and reproductive studies represented three mammalian species (rats, mice, and rabbits) in two orders, and showed limited toxicity of NQ to mammals. Water consumption was the most consistent effect. At high doses (1,000 mg/kg/day) other affects included death, reduced body weight gain, and reduced food consumption, which were attributed to decreased palatability and interference with digestion at 1,000 mg/kg/day. In developmental/reproduction studies, reduced fetal body weight and increased incidence of minor developmental alterations were noted. Thus, in mammals, the no and low observed adverse effect levels (NOAEL and LOAEL) for NQ were estimated to be 316 mg/kg/day and 1,000 mg/kg/day, respectively.

Biomedical, toxicological, and ecological databases were searched for reports and studies about the toxicology of the pesticide aldrin on wildlife (mammals, birds, reptiles, and amphibians). Citations were evaluated for biologically relevant endpoints such as reproduction, development, behavior, growth, and survival. Valid citations were used to develop toxicity reference values. Ingestion toxicity reference values were developed for mammals, birds, and amphibians. A paucity of available studies precluded the creation of ingestion toxicity reference values for reptiles. Inhalation and dermal contact toxicity reference values could not be developed for birds, reptiles, and amphibians due to a paucity of available studies.

This chapter presents the wildlife toxicity assessment (WTA) for chlordane, an organochlorine pesticide that was widely used in the U.S. from 1947 to 1978. Its use was curtailed following an increased concern about its cancer-causing effects, and it was entirely banned for use in 1988. It is highly persistent in the environment, causing a concern for wildlife toxicity. This WTA addresses chlordane’s toxicity to mammals, birds, and reptiles as there were no sufficient data on amphibian toxicity. The studies utilized indicate a moderate toxicity of chlordane, with effects on reproduction in mammals, birds, and reptiles. Based upon the available data in the mammalian studies, a chronic no observed adverse effect level (NOAEL) and low observed adverse effect level (LOAEL) were determined to be 2.1 and 21 milligrams per kilogram per day (mg/kg/day), respectively.

The treatment of chronic myelogenous leukemia (CML) with specific tyrosine kinase inhibitors typically results in clinical success, although therapeutic failure frequently occurs. In order to investigate the biological consequences of treating CML cells with such drugs, we previously reported that the antioxidant selenoprotein glutathione peroxidase-1 (GPx-1) was induced by imatinib in both patient samples and cultured cells. Here, we extend these findings to demonstrate that the treatment of CML cell lines, but not non-CML cells, results in an approximately four-fold increase in the levels of another important antioxidant protein, manganese superoxide dismutase (MnSOD), without altering the steady state levels of the corresponding transcript.

Glutathione peroxidase activity was previously determined to be elevated in lymphocytes obtained from patients treated with the Bcr-Abl kinase inhibitor imatinib mesylate. In order to expand upon this observation, the established chronic myelogenous leukemia cell lines KU812 and MEG-01 were treated with imatinib and the effect on several anti-oxidant proteins was determined. The levels of GPx-1 were significantly increased following treatment with imatinib. This increase was not due to altered steady-state mRNA levels, and appeared to be dependent on the expression of Bcr-Abl, as no increases were observed following imatinib treatment of cells that did not express the fusion protein. The nutrient-sensing signaling protein, mammalian target of rapamycin (mTOR), can be activated by Bcr-Abl and its activity regulates the translation of many different proteins. Treatment of those same cells used in the imatinib studies with rapamycin, an inhibitor of mTOR, resulted in elevated GPx-1 and GPx-4 protein levels independent of Bcr-Abl expression. These proteins all belong to the selenoprotein family of peptides that contain the UGA-encoded amino acid selenocysteine. Collectively, these data provide evidence of a novel means of regulating anti-oxidants of the selenoprotein family via the mTOR pathway.