Riot control agents: pharmacology, toxicology, biochemistry and chemistry.

The desired effect of all riot control agents is the temporary disablement of individuals by way of intense irritation of the mucous membranes and skin. Generally, riot control agents can produce acute site-specific toxicity where sensory irritation occurs. Early riot control agents, namely, chloroacetophenone (CN) and chlorodihydrophenarsazine (DM), have been replaced with 'safer' agents such as o-chlorobenzylidene malononitrile (CS) and oleoresin of capsicum (OC). Riot control agents are safe when used as intended: however, the widespread use of riot control agents raises questions and concerns regarding their health effects and safety. A large margin exists between dosages that produce harassment and dosages likely to cause adverse health effects for modern riot control agents such as CS and dibenz[b,f]1 : 4-oxazepine (CR). Yet, despite the low toxicity of modern riot control agents, these compounds are not entirely without risk. The risk of toxicity increases with higher exposure levels and prolonged exposure durations. Ocular, pulmonary and dermal injury may occur on exposure to high levels of these substances, and exposure to riot control agents in enclosed spaces may produce significant toxic effects. Reported deaths are few involving riot control agents, and then only under conditions of prolonged exposure and high concentrations. Recently, concern has focused on the deaths resulting from law enforcement use of OC, a riot control agent generally regarded as safe because it is a natural product. As with other xenobiotics, not enough is known concerning the long-term/chronic effects of riot control agents. Clearly, there is considerable need for additional research to define and delineate the biological and toxicological actions of riot control agents and to illuminate the full health consequences of these compounds as riot control agents.

Use of methyl salicylate as a simulant to predict the percutaneous absorption of sulfur mustard.

Exposure to chemical vesicants such as sulfur mustard (HD) continues to be a threat to military forces requiring protectant strategies to exposure to be evaluated. Methyl salicylate (MS) has historically been the simulant of choice to assess HD exposure. The purpose of this study was to compare the percutaneous absorption and skin deposition of MS to HD in the isolated perfused porcine skin flap (IPPSF). The HD data were obtained from a previously published study in this model wherein 400 microg cm(-2) of ](14)C[-MS or ](14)C[-HD in ethanol were topically applied to 16 IPPSFs and experiments were terminated at 2, 4 or 8 h. Perfusate was collected at increasing time intervals throughout perfusion. Radioactivity was determined in perfusate and skin samples. Perfusate flux profiles were fitted to a bi-exponential model Y(t) = A(e(-bt) - e(-dt)) and the area under the curve (AUC), peak flux and time to peak flux were determined. Sulfur mustard had more pronounced and rapid initial flux parameters (P < 0.05). The AUCs determined from observed and model-predicted parameters were not statistically different, although the mean HD AUC was 40--50% greater than MS. The HD skin and fat levels were up to twice those seen with MS, but had lower stratum corneum and residual skin surface concentrations (P < 0.05). Compared with other chemicals studied in this model, HD and MS cutaneous disposition were very similar, supporting the use of MS as a dermal simulant for HD exposure.

Evaluation of neutralized chemical agent identification sets (CAIS) for skin injury with an overview of the vesicant potential of agent degradation products.

Vesication and skin irritation studies were conducted in hairless guinea-pigs to determine the vesicant and skin irritation potential of chemically-neutralized Chemical Agent Identification Sets (CAIS). The CAIS are training items that contain chemical warfare-related material--sulfur mustard (HD), nitrogen mustard (HN) or lewisite (L)--and were declared obsolete in 1971. Animals were dosed topically with 'test article'--neat HD, 10% agent/chloroform solutions or product solutions (waste-streams) from neutralized CAIS--and evaluated for skin-damaging effects (gross and microscopic). Product solutions from the chemical neutralization of neat sulfur mustard resulted in microvesicle formation. All agent-dosed (HD or agent/chloroform solutions) sites manifested microblisters as well as other histopathological lesions of the skin. Waste-streams from the neutralization of agent (agent/chloroform or agent/charcoal) were devoid of vesicant activity. Cutaneous effects (erythema and edema) were consistent with the skin-injurious activity associated with the neutralizing reagent 1,3-dichloro-5,5-dimethylhydantoin (DCDMH). Chemical neutralization of CAIS was effective in eliminating/reducing the vesicant property of CAIS containing agent in chloroform or agent on charcoal but was inefficient in reducing the vesicant potential of CAIS containing neat sulfur mustard.

Acute inhalation toxicity of neutralized chemical agent identification sets (CAIS) containing agent in chloroform.

An acute head-only inhalation study was conducted in rats exposed for 1 h to product solution (wastestream) resultant from the chemical neutralization of Chemical Agent Identification Sets (CAIS) containing agent (sulfur mustard (HD), nitrogen mustard (HN-1) or lewisite (L)) in chloroform. Groups of Sprague-Dawley rats were exposed to varying concentrations (24000, 18000, 12000 or 6000 ppm) of CAIS wastestream. An additional group was exposed to the vehicle (chloroform/t-butanol) only, at a concentration equivalent to the concentration of vehicle at the highest exposure level. Animals were evaluated for toxic effects, including assessment of toxicant-induced alterations to the ocular and respiratory systems. Mortality on exposure to 24000 ppm of test article or to vehicle alone was high. Mortality in the other exposure groups was roughly proportional to the concentration of test article (wastestream). Toxic signs were consistent with exposure to solvent system components (chloroform/t-butanol) and to agent decomposition products/by-products. Incidence and severity of ocular effects were similar in vehicle control and treatment groups. The salient respiratory effect observed was a decreased minute volume, which was also noted in vehicle and treatment groups.

Change in hen sciatic nerve calcium after a single oral dose of tri-o-tolyl phosphate.

Six trace elements were monitored in neural tissue homogenates from White Leghorn hens orally dosed with tri-o-tolyl phosphate (TOTP) or tri-m-tolyl phosphate (TMTP) (200 mg/kg). Treated birds were monitored daily for development of delayed neurotoxicity, and concentrations of calcium, copper, iron, magnesium, manganese, and zinc were measured with atomic absorption spectroscopy at the time of maximal locomotor impairment (27-35 days postdosing). TOTP-treated birds manifested motor deficit by 15 days postdosing, while hens administered TMTP exhibited no signs of delayed neurotoxicity. Total calcium content in the sciatic nerve homogenates from TOTP-dosed hens was significantly less (P < 0.05) at the time of maximal locomotor impairment, while no shifts in the other trace elements were found. Therefore, the ortho isomer of tritolylphosphate elicited symptoms of delayed neurotoxicity in the hen (i.e., organophosphorus ester-induced delayed neurotoxicity or OPIDN) and caused a decrease in total calcium content in the sciatic nerve homogenates, in contrast to effects of the meta isomer. Analysis of neural homogenates at time of maximal locomotor impairment reflected secondary events in the degradative processes, since the initial assault of TOTP happens early after administration. Therefore, at fully developed OPIDN alteration of calcium balance in sciatic nerves is an indicator of axonopathy in a degenerated nerve following chemical injury.

Histochemical demonstration of neurotoxic esterase.

We developed a histochemical method for localizing neurotoxic esterase (NTE), defined as the phenylvalerate (PV)-hydrolyzing esterase that is resistant to 40 microM paraoxon (A) but inactivated by paraoxon plus 50 microM mipafox (B). NTE is considered to be the target enzyme in the production of organophosphorus ester-induced delayed neurotoxicity (OPIDN). Cryostat sections were incubated in a medium containing alpha-naphthyl valerate and 6-benzamido-4-methoxy-m-toluidine diazonium chloride (fast violet B) after treatment with the above-mentioned inhibitors, leading to formation of an aqueous insoluble precipitate at sites of enzymatic activity. NTE activity was estimated as staining detectable in A but not in B. In the central nervous system (CNS) of chicken, NTE appeared to be present primarily in the somata of most neurons, but at sites indistinguishable from those of the other inhibitor-resistant and -sensitive alpha-naphthyl valerate-hydrolyzing esterases. It could not be distinguished in the CNS of cat, probably because it constitutes less than 3% of the total PV-hydrolyzing activity in the CNS of that species.

Effect of acute tri-o-tolyl phosphate exposure on 2', 3'-cyclic nucleotide 3'-phosphohydrolase activity in hen neural tissues.

The activity of 2', 3'-cyclic nucleotide 3'-phosphohydrolase (CNP, EC 3.1.4.37), a myelin-associated enzyme, was monitored in brain, spinal cord, and sciatic nerve homogenates from tri-o-tolyl phosphate (TOTP) and tri-m-tolyl phosphate (TMTP) treated hens. Atropinized adult White Leghorn hens were orally dosed with TOTP (200 mg/kg) or with TMTP (200 mg/kg). The treated birds were monitored daily for development of delayed neurotoxicity, and CNP activity was measured via spectrophotometry at the time of maximal locomotor impairment (27-35 days post dosing). The TOTP-treated birds manifested locomotor deficit by 15 days postdosing and exhibited T2-T4 ataxia at maximal locomotor impairment. The hens administered TMTP exhibited no signs of delayed neurotoxicity. CNP activity of sciatic nerve preparations from TOTP-dosed hens was significantly inhibited (p less than 0.05) at the time of maximal locomotor impairment. There was also a significant correlation between decreased CNP activity and the degree of ataxia at the time of maximal locomotor impairment. This decrease in sciatic nerve CNP activity was most likely associated with nerve fiber degeneration. The level of CNP activity in spinal cord and brain homogenates from TOTP-dosed birds was not markedly altered. TMTP-treated birds exhibited no change in neural tissue CNP activity. The results suggest that the criterion of decreased CNP activity may serve as a useful biochemical adjunct to established clinical, biochemical, and morphological methods in the assessment of chemically-induced neuropathies.

Neurotoxicity assessment of O-ethyl-O'-(2-diisopropylaminoethyl) methylphosphonite (QL) in hens.

O-ethyl-O'-(2-diisopropylaminoethyl) methylphosphonite (QL), an intermediate in the formation of ethyl S-2-diisopropylaminoethyl methylphosphonothiolate, was evaluated for neurotoxicity in the adult hen. Birds were given a single oral dose of QL ranging from 635 to 6080 mg kg-1. The QL-treated hens were observed for up to 24 h after dosing for acute toxicologic effects and over a 24 d post-dose period for evaluation of delayed neurotoxicity. The oral LD50 in hens is 1186 mg kg-1. Neurologic dysfunction, as evidenced by motor incapacitation, was observed at 6 days and thereafter after treatment with QL. Neurotoxic esterase (NTE) activity was not inhibited at 24 h after exposure to compound. Neural damage, multifocal in nature, was noted in the peripheral nervous system of QL-treated hens at dose levels greater than or equal to 635 mg kg-1, oral dose. These findings indicate neural damage tendencies following QL exposure.

Effect of acute organophosphorus exposure on 2', 3',-cyclic nucleotide 3'-phosphohydrolase activity in hen neural tissue.

The effect of various organophosphorus (OP) compounds on 2', 3'-cyclic nucleotide 3'-phosphohydrolase (CNP) activity and the relationship to neurotoxic response was studied. Adult White Leghorn hens were dosed with either diisopropyl fluorophosphate (DFP), tri-o-cresyl phosphate (TOCP), leptophos, or paraoxon. CNP activity was determined utilizing crude homogenates or purified myelin preparations from whole brain, spinal cord, and sciatic nerves at maximal neurologic dysfunction, evaluated as locomotor impairment. Enzyme assays of CNP revealed a decrease in brain CNP activity at the time of maximal locomotor impairment after a single, oral dose of leptophos. Decreased CNP activity was also noted in spinal cord preparations from DFP-treated hens at the time of maximal locomotor impairment. Sciatic nerve CNP activity was not altered following treatment with OP-compounds. Paraoxon, a non-neurotoxic OP-compound, had little effect on neural tissue CNP activity. Neurotoxic OP-compounds, that cause secondary degeneration of myelin (Wallerian), may be associated with decreased brain and spinal cord CNP activity at the time of maximal locomotor impairment.

Membrane-bound and soluble esterase activities in various hen brain regions after diisopropyl phosphorofluoridate and trichlorfon treatment.

Acetylcholinesterase, neurotoxic esterase, and nonspecific esterase activities were studied in subcellular fractions obtained from homogenates of forebrain, cerebellum, and brainstem areas of hen brain. The hens were given a single, oral dose (1.0 mg/kg of Dyflos (diisopropyl phosphorofluoridate) or 100 mg/kg of trichlorfon (dimethyl -2,2,2-trichloro-1-hydroxyethylphosphonate). The animals were killed 24 hr after dosing. The level of neurotoxic esterase and soluble nonspecific esterase activities were consistent from region to region; however, acetylcholinesterase activity among the brain regions varied significantly. Both organophosphorus compounds produced definite inhibition of acetylcholinesterase. The cerebellar acetylcholinesterase activity was the most susceptible to organophosphorus ester-induced inhibition. A marked inhibition of neurotoxic esterase (70-80%) was produced by diisopropyl phosphorofluoridate in all areas. Trichlorfon produced little or no inhibition of neurotoxic esterase (0-10%). Little variation in soluble nonspecific esterase activity (alpha -naphthyl acetate) was noted between the three brain regions studied. Both diisopropyl phosphorofluoridate and trichlorfon failed to significantly alter soluble nonspecific esterase activity among the brain regions.

Mutagenicity of alkyl-(omega-hydroxyalkyl) nitrosamines related to dibutylnitrosamine.

Various alkyl-(omega-hydroxyalkyl) derivatives related to dibutylnitrosamine (DBN) were investigated for mutagenicity in the absence of liver-activation system. Butyl-(4-hydroxybutyl)-, butyl-(3-hydroxypropyl)-, and butyl-(2-hydroxyethyl)-nitrosamines were so tested and found to be mutagenic for TA 1535 strain of Salmonella typhimurium. In all cases, a simple dose-response relationship was observed. Furthermore, no significant (p less than 0.05) differences in the mutagenicity of the various test compounds were observed as the alkyl sidechain possessing the OH group increased in length. From these results it is siggested that mutagenesis in S. typhimurium by the higher dialkylnitrosamines is partially due to the formation of omega-hydroxylated derivatives in addition to the major mutagenic metabolite derived from alpha-carbon dealkylation.