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Toxicity testing in the 21st century refining the army's toolbox.

In 2007, the National Academy of Sciences (NAS) published a comprehensive report, "Toxicity Testing in the 21st Century: A Vision and a Strategy" (1) in response to a US Environmental Protection Agency request to the National Research Council to develop a plan using innovative methods to advance toxicity testing. Toxicity determination in the previous half-century required animal testing of all new chemicals (medicine, food additives, industrial, consumer, and agricultural chemicals) for potential to cause cancer, birth defects, and other adverse health effects. Animal testing is slow, expensive, uses many animals, and often requires assumptions and controversial extrapolations. (1)

The NAS vision discussed harnessing technologies developed in emerging fields such as systems biology (eg, use of computational models fused with in vitro laboratory data) and bioinformatics (computational models to analyze massive data sets), with high throughput screening assays of effects of chemicals on human cells, cellular components, and tissues, at low levels, which are typically more relevant. (1) The report recommended identification not only of changes at the molecular level, but of signal transduction and other pathways that, when perturbed, lead to adverse effects. Study of these influences may lead to improved predictability of health effects in human populations. Mapping such "toxicity pathways" and then discerning actual or predicted perturbations by means of computational models and high throughput screening of chemicals:

   could reduce the backlog of the large number of industrial
   chemicals that have not yet been evaluated under the current
   testing system. (1(p30))

The new approach would most likely reduce animal use as well. However, many of these methods are new; their value remains to be validated in predicting effects or determining safe levels of exposure for humans.

In 1985, the Office of The Surgeon General (OTSG) designated the Army Institute of Public Health (AIPH), now part of the Army Public Health Command, as lead agent of the Health Hazard Assessment Program. (2) In fulfillment of that mission, the AIPH Toxicology Portfolio is charged with evaluating the toxicity of specific military-unique chemicals in materials entering the Army supply system. (3) The primary goals have been (1) to identify health hazards associated with exposure to new substances used in military applications, (2) to provide a technical foundation for approvals (or disapprovals) to eliminate or control hazards associated with manufacturing-related exposures and use and disposal of weapons, equipment, clothing, training devices and other materials. (3) The Toxicity Clearance (TC) is the instrument used for this evaluation. It provides a technical basis to help the acquisition program manager make important life cycle decisions. Solvents, fire extinguishing agents, repellents, fabric finishes, refrigerants, explosives, energetics, propellants, pyrotechnics, hydraulic fluids, metals/alloys, and pest control agents are examples of substances used in systems that have been evaluated. Toxicity Clearances are provided for specific applications and are generally not applicable to systems with different use conditions. (2)

In accordance with the NAS recommendations and Department of Defense mandates to improve efficiency in research, development, and acquisition, the Toxicology Portfolio has implemented a phased approach to toxicity testing and has expanded its toolbox of in silico, in vitro, as well as in vivo methods to better evaluate potential health and environmental threats, ideally, before a new substance is approved for entry into the Army supply chain. Used in a relative manner, these emerging methods can be used in side-by-side comparison to determine which substance is likely to cause health effects from exposure and use and which would present a lower hazard risk.

The phased approach to toxicity testing is intended to identify and characterize occupational and environmental human toxicity concerns as early as possible in the science and technology (S&T) phase of research, prior to transition to an advanced developer. Even before a new material has been synthesized, its properties and performance can be estimated and evaluated using computer modeling techniques that allow toxicity and physical properties to be assessed. (4) Modification, reformulation, or even substitution at the S&T stage of development, generally in budget activity levels (BA) 1 to 3 would most likely be significantly less time-consuming and less costly than comparable changes at BA stages 4 to 8. An example of the phased approach to toxicity testing is underway with the upgrade for the 2.75-inch Hydra rocket:

   The Hydra is one of the most extensively used munitions in the
   Army, but environmental concerns are associated with it. The
   training warhead for the rocket contains perchlorate. The
   propellant contains lead ... The phased approach to environmental
   safety and occupational health (ESOH) was used to replace
   components of the Hydra with safer formulations. These replacement
   compounds are now entering the final stages of approval and
   implementation. (4)

Another success story involved the reformulation of the propellant for the M-115/116/177 (whistle, bang, flash) simulators. The original formulation used ammonium perchlorate as the propellant that resulted in significant contamination of training ranges where used. The new formulation contained a more traditional black powder mix that was just as effective on the ranges, without resulting in toxic environmental residues, loss of performance, or significant addition to cost. Both formulations were evaluated side-by-side and recommendations made using this approach.

Rather than waiting for an upgrade, phased toxicity testing should be a prerequisite to reach a given Technology Readiness Level (TRL) for new products or systems in initial development. The type of toxicity assessment would be selected to be compatible with the stage of development. For example, at the risk of repetitiveness, even before a new material has been synthesized, properties and performance of the substance can be initially evaluated using computer modeling techniques to identify potential toxicity. More sophisticated (and more extensive) tools would be used later in development, but still prior to transition to a weapon system or platform. For example, in silico assays that provided reasonable estimates of confidence regarding toxicity would be followed by in vitro assays, including measures of mutagenesis, genotoxicity, and cytotoxicity assays. These measures provide data that help address targets of toxicity and potential mechanisms for extrapolation to soldiers, civilians, and the environment. Focused animal testing would be reserved for those candidates selected as most likely to be efficacious and safe, in preparation for transition to a program executive officer or advanced developer like the US Army Medical Materiel Development Activity.

Currently, a Programmatic Environmental, Safety, and Occupational Health Evaluation (PESHE) is not required until Milestone B, at which the product must be at TRL 6. New S&T products are often considered for transition about the BA-3 level at roughly TRL 3-4. It is generally accepted that a primary cause of failure to transition includes "lack of technical maturity." The National Environmental Policy Act of 1969 (Pub L No. 91-90, 83 Stat 852 (1969)) mandates full disclosure of possible impacts, alternatives and environmental mitigation measures. Moreover, if not evaluated alongside S&T, the program manager runs the risk of development of a system that may cause injury to the Warfighter or worker, or not lend itself for sustainable use at testing and training ranges. Therefore, it behooves the S&T community to examine the potential for toxicity as part of technology maturity determination, prior to or at least as part of the transition process into an acquisition program of record. Codifying the need for appropriate toxicity assessment in the technology transfer agreement is therefore important. Additionally, since the PESHE is currently the first requirement addressing toxicity and is not required prior to the product advancement to TRL 6 and Milestone B, some products may require reformulation or replacement due to toxicity. This would be fairly late in the acquisition pipeline, by which time the Army might have already committed hundreds of thousands of dollars to a product. This practice is inconsistent with Executive Order 13514 ("Federal Leadership in Environmental, Energy and Economic Performance, 2009) which required:

   ... minimizing the generation of waste and pollutants. [and]
   reducing and minimizing the quantity of toxic and hazardous
   chemicals and materials acquired ... (5(p3))

There is no guidance on what data are needed to help make environmental safety and occupational health (ESOH) decisions for the PESHE or elsewhere regarding toxicity testing, therefore, program managers accept risks based solely on available ESOH data. (6) The proposed phased approach:

   ... seeks to make an ESOH evaluation compatible with each stage of
   the development process by applying appropriate assessment tools.
   [and] adds a data requirement to each stage for which managers can
   plan and program, ... (6(p53))

A proposed requirement for appropriate toxicity data at each BA level is illustrated in the Figure.

In addition to a phased approach to early toxicity testing, the Toxicology Portfolio has implemented a number of targeted assays to help research, development, test and evaluation (RDT&E) scientists and managers make funding decisions based upon ESOH risks. Moreover, individuals in the Toxicology Portfolio have been working with the Technical Cooperative Program, Key Technical Area 4-42 to develop internationally harmonized methods for use in the development of new weapon systems or platforms to ascertain ESOH hazards. (7)

Although currently a "hot topic" in discussions of toxicity testing and foreseen in the NAS vision as a valuable in silico method, most high throughput methods are still in the research stage. Much work remains to validate their ability to determine maximum safe exposure levels. Use of high throughput methods will require refined dose metrics for calculation of a safe level of exposure using in vitro results. However, these methods can currently be used for notional new substances in RDT&E where only small amounts are available. High throughput methods can determine if substances have characteristics with a high probability of causing illness in soldiers and workers, or of adversely affecting range sustainment. Computer projections can be made by comparing the molecular structure of a new compound with databases of compounds in which structure has been correlated with toxicity. Such projections typically provide relative measures of confidence in estimates, and can also help identify pathways of toxicity. The latter inform future study design. Affirmation of functionality of a new substance also helps identify potential toxic endpoints. This, in turn, helps industrial hygienists and occupational health physicians conduct meaningful surveillance in the workforce.

The AIPH Toxicology Portfolio attempts to assess high priority military-related substances using the above philosophy wherever possible. In Fiscal Year 14 alone, it completed 39 Toxicity Clearances, 21 technical reports (of which 10 are Toxicity Assessments), and 17 peer-reviewed manuscripts. Doctoral level subject matter experts (SMEs) in endocrine disruption, ecotoxicity, developmental toxicity, genotoxicity, immunotoxicity, and quantitative structure activity relationships make up the 2 branches of the Portfolio (Toxicity Evaluation Program; Health Effects Research Program). (6) Technical experts in inhalational toxicity testing, dermal sensitization, mutagenicity and novel methods team with the SMEs to develop and execute good laboratory practices-compliant protocols using rodents and select sentinel species.

The Toxicology Portfolio is funded by a combination of core Defense Health Program funds and investments by other Department of Defense organizations in collaborative arrangements. As important as toxicity testing is, the contribution of the AIPH Toxicology Portfolio is not widely known. Years ago, new products and materials were fielded based on efficacy and the ability to meet military operational requirements. Only relatively recently have regulatory guidelines mandated that new military products be not only effective (can it function as designed?), but also safe for Soldiers. Some reports state that human male fertility in certain developed nations declined in the 20th century, (8) although others dispute the claim. Regardless, regulatory guidelines for safety assessment of pharmaceuticals and chemicals currently include screens for effects on reproduction and fertility, including stage-aware histopathological examination of the testis, as a sensitive method for detecting disturbances in spermatogenesis. (9-11) These are services which AIPH Toxicology Portfolio personnel routinely perform as part of their mission.

As the nation grows increasingly motivated to spend taxpayer dollars efficiently and to protect the environment to preserve our future, the need for phased and targeted toxicity assessment will most likely become a prerequisite for all R&D investments, and it is reasonable to expect that the AIPH Toxicology Portfolio will remain a leader in this effort.


(1.) National Research Council of the National Academies. Toxicity Testing in the 21st Century: A Vision and a Strategy. Washington, DC: National Academies Press; 2007. Available at: /openbook.php?record_id=11970. Accessed May 14, 2015.

(2.) Mughal MR, Houpt J, Kluchinsky TA. Health hazard assessment and the toxicity clearance process. US Army Med Dep J. July-September 2014:59-60.

(3.) McCain WC, Salice CJ, Johnson MS, et al. USA-CHPPM toxicology: maintaining readiness and protecting the environment. US Army Med Dep J. July-September 2004:10-14.

(4.) Johnson MS. Phased approach to ESOH assessment streamlines search for replacement substances. One Health. Spring, 2012:19-20.

(5.) Executive Order 13514: Federal Leadership in Environmental, Energy, and Economic Performance. 74 Federal Register 194 (2009). Available at: ments/2009fedleader_eo_rel.pdf. Accessed May 14, 2015.

(6.) Eck WS, Watts K, Lieb NJ, Johnson MS. Avoiding environmental risk. Army AL&T. April-June 2013:50-56. Available at: wp-content/uploads/2013/04/April-June2013_army_al.pdf. Accessed May 14, 2015.

(7.) Brochu S, Hawari J, Monteil-Rivera F, et al. Assessing the Potential Environmental and Human Health Consequences of Energetic Materials: A Phased Approach. The Technical Cooperation Program; April 2014. TTCP Technical Report CP 4-42 [TR-WPN-TP04-15-2014].

(8.) Creasy DM. Evaluation of testicular toxicity in safety evaluation studies: the appropriate use of spermatogenic staging. Toxicol Pathol. 1997;25(2):119-131.

(9.) International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for human Use. Guideline for Industry: Detection of Toxicity to Reproduction for Medicinal Products. Geneva, SUI: ICH Secretariat; 1994. ICH-S5A. Available at: /Drugs/GuidanceComplianceRegulatoryInformation /Guidances/ucm074950.pdf. Accessed May 14, 2015.

(10.) Organisation for Economic Cooperation and Development. OECD Guideline for Testing of Chemicals: Reproduction/Developmental Toxicity Screening Test. Paris, France: OECD; 1995. OECD Test Guideline 421.

(11.) Organisation for Economic Cooperation and Development. OECD Guideline for Testing of Chemicals: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screen Test. Paris, France: OECD; 1996. OECD Test Guideline 422.

LTC Erica Eggers Carroll, VC, USA

Mark S. Johnson, PhD, DABT


LTC Carroll is Chief, Division of Toxicologic Pathology, Toxicology Portfolio, Army Institute of Public Health, US Army Public Health Command, Aberdeen Proving Ground, Maryland.

Dr Johnson is Director, Toxicology Portfolio, Army Institute of Public Health, US Army Public Health Command, Aberdeen Proving Ground, Maryland.
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Author:Carroll, Erica Eggers; Johnson, Mark S.
Publication:U.S. Army Medical Department Journal
Date:Jul 1, 2015
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