Chemical warfare agent simulants for human volunteer trials of emergency decontamination: A systematic review

Abstract Incidents involving the release of chemical agents can pose significant risks to public health. In such an event, emergency decontamination of affected casualties may need to be undertaken to reduce injury and possible loss of life. To ensure these methods are effective, human volunteer trials (HVTs) of decontamination protocols, using simulant contaminants, have been conducted. Simulants must be used to mimic the physicochemical properties of more harmful chemicals, while remaining non‐toxic at the dose applied. This review focuses on studies that employed chemical warfare agent simulants in decontamination contexts, to identify those simulants most suitable for use in HVTs of emergency decontamination. Twenty‐two simulants were identified, of which 17 were determined unsuitable for use in HVTs. The remaining simulants (n = 5) were further scrutinized for potential suitability according to toxicity, physicochemical properties and similarities to their equivalent toxic counterparts. Three suitable simulants, for use in HVTs were identified; methyl salicylate (simulant for sulphur mustard), diethyl malonate (simulant for soman) and malathion (simulant for VX or toxic industrial chemicals). All have been safely used in previous HVTs, and have a range of physicochemical properties that would allow useful inference to more toxic chemicals when employed in future studies of emergency decontamination systems.


| INTRODUCTION
A chemical incident is traditionally defined as an unexpected or uncontrolled release of a chemical from its containment. While rare, incidents involving the exposure of large numbers of people to chemical contaminants have taken place (WHO, 1999(WHO, , 2002. Chemical incidents may involve the accidental or deliberate release of a chemical contaminant, and the majority of incidents involve an acute release, accompanied by a rapidly rising exposure risk (WHO, 2002). Chemical incidents may be small or large in scale, and can give rise to multiple primary or secondary chemical casualties and fatalities (Baker, 2005;Duarte-Davidson, Orford, Wyke, et al., 2014).
In a chemical incident, emergency decontamination of affected casualties needs to be undertaken to reduce injury and possible loss of life. In a real incident, decontamination must protect against potential highly toxic and hazardous chemicals, such as toxic industrial chemicals (TICs) and chemical warfare agents (CWAs) (Balali-Mood & Balali-Mood, 2008;Brennan, Waeckerle, Sharp, & Lillibridge, 1999;Duarte-Davidson et al., 2014). TICs are defined as any substances (gas, liquid or solid) that are produced, stored, transported and widely used by industry and can cause harm to human health or the environment when not properly contained. They possess chemical hazards (e.g., as carcinogens or corrosives) and/or physical hazards (e.g., flammable or explosive properties). TICs are normally produced in large quantities, which differentiate them from highly toxic speciality chemicals that are produced in only limited volumes ('Toxic CWAs are highly toxic synthetic chemicals that can be dispersed as a gas, liquid (including aerosols) or adsorbed on to particles to become a powder. CWAs have either lethal or incapacitating effects on humans, and differ from explosive chemicals where the destructive effects are localized and caused by shear force. There are thousands of toxic substances, but only a few are considered CWAs based on their characteristics, such as high toxicity, rapid action and persistency (Ganesan, Raza, & Vijayaraghavan, 2010; Technical Secretariat of Organization for Prohibition of Chemical Weapons, 1997). CWAs are generally classified according to the physiological effects on humans and include nerve agents, vesicants (blistering agents), blood agents (cyanogenic agents), choking agents (pulmonary agents), riotcontrol agents (tear gases), psychomimetic agents and toxins (Occupational Safety and Health Administration).
Studies to optimize the effectiveness of emergency decontamination processes using human volunteers must, for ethical reasons, use simulants to mimic the physicochemical properties of more harmful chemicals, while remaining non-toxic at the dose applied. A simulant is a compound that can mimic the behaviour of the chemical of interest (e.g., has similar physicochemical properties) or is a functional analogue of a more harmful chemical (Jenkins, Buchanan, Merriweather, et al., 1994), and can be used in human volunteer trials (HVTs) with minimal risk (Amlôt, Larner, Matar, et al., 2010;Josse, Comas, Bui-Tho, et al., 2011;Larner, Matar, Riddle, et al., 2007;Ribordy, Rocksén, Dellgar, et al., 2012;Torngren, Persson, Ljungquist, et al., 1998).
While there are a range of simulants reported in the literature (in vitro, in vivo and HVTs of decontamination studies), it is often difficult to identify simulants that adequately represent the diverse physicochemical properties and physiological effects of TICs and CWAs (Lavoie, Srinivasan, & Nagarajan, 2011)

| Inclusion criteria and literature screening
Inclusion criteria to focus on relevant papers included: • Language: All articles must be in English due to the lack of time and resources that would be required for the translation of papers.
• Publication status: All types of publication, including grey literature (letters, articles, PhD theses, internal reports, evaluations and working papers) were included in the review.
• Range of search: The fields of the publication that were included in the search strategy were the title, abstract and keywords. If the use of simulants was not initially obvious from the title or the abstract, the paper was omitted.
• Simulant specificity: All papers had to contain references to simulant quantity and specific use. These could include papers referring to qualitative or quantitative studies as long as the simulant's unit of measurement (including those described by the qualitative term 'concentrated') were included and that the simulant was a suitable simulant (based on physicochemical properties) for a TIC and/or CWA.
Initially, search terms were applied for all fields (title, abstract, keywords), and then the search result was narrowed down through stages of manual screening that included assessing relevancy based on the title, the abstract and then the full text ( Figure 1).

| Persistence
Simulants should be relatively stable and persistent, i.e., they should be stable at a range of temperatures and light conditions consistent with conducting a decontamination study in human volunteers. If bioavailable following dermal application then the half-life should be appropriate to assess uptake through 24 hour urine collection (half-life should not be too long and urinary excretion should be the dominant route). Simulants should also have a low vapour pressure, i.e., should remain in/on the body long enough to be sampled and detected by relatively routine analytical methods. To assess persistence, the relative vapour pressure of each simulant was determined from reliable scientific literature. The lower the vapour pressure, the more persistent the simulant and the less likely it would be to evaporate prematurely. Indications of volatility (likely/unlikely to volatilise) were obtained from the UK Recovery Handbook for Chemical Incidents (Wyke, Brooke, Dobney, Baker, & Murray, 2012).

| Evaluation criteria
Potentially suitable simulants were shortlisted and evaluated according to their physicochemical properties, including the relative toxicity of the compound, stability within the human body (biological half-life), vapour pressure and water solubility.

| Simulant suitability
Simulants were objectively evaluated according to a suitability matrix (Table 2) with criterion for suitability colour coded; red (indicating limited suitability), yellow (indicating moderate suitability) and green (indicating good suitability). The suitability colours are qualitative indications of how suitable the identified simulants may be for use in HVT studies. The most suitable simulant identified in this review may not be the best to use in every study, as every study is contextually dependent.

| Bias
Bias was minimized by using keywords to search multiple databases and specific search criteria to refine the identified literature. All identi-

| RESULTS
Peer reviewed papers, scientific reports and literature (n = 1055) were retrieved from all searched databases and reduced to 940 following removal of duplicates. After an initial assessment (review of title and abstract), 865 papers were discounted according to the inclusion criteria ( Figure 1). Twenty-two of these excluded papers referred to 'simulations'of in vitro decontamination procedures using CWAs and as such did not use simulants.   Riviere, Smith, Budsaba, et al., 2001 In vitro study on the efficacy of combined decontamination methods on decontamination efficacy of hair exposed to MeS vapours. Spiandore, Piram, Lacoste, et al., 2016 An in vitro study (using skin mounted on diffusion cells) into the effects of hydrodynamics, detergents and delays on the effectiveness of the ladder pipe decontamination system. Matar, Atkinson, Kansagra, et al., 2014 Human trial Human trial testing the efficacy and functionality of the environment within a mass decontamination unit when contaminating humans with CWA simulants.

Ribordy et al., 2012
Evaluation of the efficacy of a decontamination station following exposure of volunteers to ethyl lactate and MeS, simulants of sarin and mustard respectively. Torngren et al., 1998 Human volunteer trial assessing an optimised decontamination protocol as part of the ORCHIDS project. Larner et al., 2007 In silico Use of cheminformatics to determine suitable CWA simulant choice. Lavoie et al., 2011 Diethyl malonate (DM) -(soman) In vitro Qualitative determination of the effect of wet decontamination on skin hydration, and subsequent issues with decontamination of CWA simulant diethylmalonate. Loke et al., 1999 Simulation of soman decontamination using diethyl malonate and a showering method. Reifenrath, Mershon, Brinkley, et al., 1984 In silico Use of cheminformatics to determine suitable CWA simulant choice. Lavoie et al., 2011 Ethyl lactate (EL) -(chlorine/ Sarin)

Human trial
Evaluating the impact of environmental factors on decontamination within a mass decontamination unit when contaminating humans with CWA simulants.

Ribordy et al., 2012
Evaluation of the efficacy of a decontamination station following exposure of volunteers to EL and MeS, simulants of sarin and sulphur mustard respectively. Torngren et al., 1998 Malathion ( In vitro Comparative study of breakthrough times of protective clothing, with sulphur mustard and a simulant, DCP. Singh et al., 2000Singh et al., et al., 2013Boulware, Fields, McIvor, et al., 2012;Powell, Boulware, Thames, Vasquez, & MacLeod, 2010).
Some compounds were not initially regarded as 'too toxic' for human use. For example, parathion and its metabolite paraoxon were identified as potential simulants. However, exposure to parathion (and its subsequent metabolite paraoxon) can result in headaches, nausea, respiratory depression, seizures and significant and irreversible effects arising from the inhibition of acetylcholinesterase (Edwards, Yedjou, & Tchounwou, 2013;Reigart, 2013;Satar, Tap, & Ay, 2015).
Therefore, parathion and paraoxon were eliminated as potential simulants for HVTs. However, malathion, the other organophosphate considered as a simulant, has a much lower order of toxicity after dermal exposure and so was not eliminated from consideration.
Tetrahydrothiophene was another potential candidate due to its relatively low toxicity; however, it also has an intensely unpleasant

| DISCUSSION
The aim of this study was to undertake a systematic literature review to identify chemical simulants that have previously been used in in vivo and in vitro assessments of decontamination processes, with the goal of identifying which simulant(s) would be most suitable for use in HVTs of emergency decontamination processes. The suitability of the shortlisted simulants was evaluated using a matrix that considered relative toxicity, biological half-life, persistence (vapour pressure), water solubility, partition coefficient (K ow ) and physicochemical similarity to their corresponding TIC or CWA.
Ultimately, the suitability of a simulant for use in HVTs will be dependent on the toxicity of the chemical. However, there is an inherent prob- 4.1 | 1,3-dichloropropane as a simulant for sulphur mustard 1,3-Dichloropropane (DCP), although used in one study (Table 1) is extremely volatile. DCP vapours are known to cause respiratory distress.
With LC 50 inhalation levels as low as 2000 ppm h −1 (in rats and mice) (Smyth, Carpenter, Weil, et al., 1969), the inhalational risk to human volunteers would be too great, and for these reasons DCP was excluded.

| Ethyl lactate as a simulant for chlorine or sarin
Ethyl lactate (EL) has been reported as an effective simulant for chlorine and sarin; however, EL is highly volatile and has low persistence, which makes sampling (and subsequent analysis) during a HVT challenging and potentially inaccuratethere is a risk that during a HVT a large proportion of the applied simulant could evaporate off the subject before and during any decontamination procedures. This could result in a false positive, indicating that the decontamination methods being tested are more effective than they actually are. If high concentrations of EL are present in the air there is also a potential respiratory risk to consider (the reported LC 50 (inhalation) in rats is approximately 5400 mg m −3 ) (Bingham, Cohrssen, & Powell, 2001), which would significantly reduce the suitability of EL as a simulant for use in HVTs. The suitability for using EL will, however, depend on the research question being addressed and the study design; for example, EL may be more suitable for testing the inhalational risk associated with decontamination processes for a sarin-like contaminant.

| Diethyl malonate as a simulant for soman
Diethyl malonate has been identified as a potentially suitable simulant to mimic the behaviour of soman, as it has similar water solubility but slightly different persistence and volatility. However, there is also a risk that dermal absorption of diethyl malonate may be significantly increased during decontamination procedures. Loke, U, Lau, et al. (1999) suggested that the enhancement effect was attributed to either the spreading of the chemical over the skin during washing, or the transient skin hydration during washing, leading to a decrease in skin barrier properties. However, as diethyl malonate is metabolized into a relatively non-hazardous compound (malonic acid) (Opdyke, 1975), the simulant itself has a relatively low dermal toxicity. Diethyl malonate was also identified as the lowest toxicity (based on rat oral data) simulant for soman in terms of potential exposure after volatilization (Bartelt-Hunt et al., 2008). However, gastrointestinal irritation can occur through ingestion and could pose an issue when applying the simulant to human volunteers, meaning greater consideration should be given to application methodology to avoid accidental ingestion.

| Malathion as a simulant for organophosphate toxic industrial chemicals and VX
One of the more suitable simulants identified from the literature for HVTs of emergency decontamination was malathion. Malathion is an organophosphate insecticide best known as a chemical used for the treatment of lice (pediculosis). It is present in some head lice treatment shampoos commonly used with children, and left on the hair and scalp for between 8 and 12 hours, indicating a suitably low projected human dermal toxicity value. The dermal LD 50 of malathion in rats is >4000 mg kg −1 (Gallo & Lawryk, 1991), therefore, if malathion was applied to a 70 kg human, the potential total dermal LD 50 would be a dose of about 280 g (a difficult dermal dose to achieve). Malathion is persistent, has low vapour pressure and volatility (i.e., it does not off-gas), which makes malathion a good candidate as a simulant for TICs and VX. Malathion is also one of the least toxic organophosphate insecticides available and therefore can be used to mimic exposure to more toxic organophosphate pesticides, i.e., TICs. None the less, even with a low relative toxicity, obtaining ethical approval to use an organophosphate for HVTs may be a limiting factor. In recent years, there has been considerable controversy about the scientific value and ethical acceptability of studies involving experimental exposure of human volunteers to low doses of pesticides, in the context of regulatory risk assessment (London, Coggon, Moretto, et al., 2010). is of similar persistence, volatility and water solubility to sulphur mustard. While MeS is likely to volatilise it is recoverable and has been used for several human volunteer decontamination studies (Larner et al., 2007;Ribordy et al., 2012;Torngren et al., 1998). Furthermore, previous literature has determined that MeS is the least toxic simulant when mimicking the adsorption/desorption of sulphur mustard in the environment (Bartelt-Hunt et al., 2008). The oral LD 50 of MeS in humans is 500 mg kg −1 whereas the dermal LD 50 in rabbits is >5000 mg kg −1 indicating poor dermal uptake. The abundance of literature that reports using MeS as a simulant also leads to greater availability of optimized and validated methods of simulant application, recovery and analysis, and a clearer indication of the suitable dose for HVTs of emergency decontamination. MeS is suitably persistent to be measureable as part of HVTs, with a high enough volatility to simulate sulphur mustard in vapour exposure studies in decontamination units (Torngren et al., 1998).
MeS as a potential simulant for sulphur mustard has also been modelled to assess the similarity (Tanimoto coefficient) and dissimilarity (Euclidean Distance) according to molecular weight, solubility, vapour pressure and partition coefficients while also taking into account molecular parameters such as bond connectivity, stereochemistry, conformational variability and substructural fingerprints. When compared to distilled mustard, MeS has a Tanimoto coefficient of 0 (1 = identical, 0 = no similarity) and a Euclidean distance of 0.587 (0 = identical), which suggest that when mathematically modelled, MeS has very little similarity to sulphur mustard (Lavoie et al., 2011), which contrasts with other opinions in the literature (Bartelt-Hunt et al., 2008).
Comparisons can be made between this review and a previous study (Bartelt-Hunt et al., 2008), which identified MeS as one of the most suitable simulants for a range of parameters, while also being low enough in toxicity to be used in HVTs.