Towards Development of a Dermal Pain Model: In Vitro Activation of Rat and Human Transient Receptor Potential Ankyrin Repeat 1 and Safe Dermal Injection of o-Chlorobenzylidene Malononitrile to Rat.

During clinical development of analgesics, it is important to have access to pharmacologically specific human pain models. o-Chlorobenzylidene malononitrile (CS) is a selective and potent agonist of the transient receptor potential ankyrin repeat 1 (TRPA1), which is a transducer molecule in nociceptors sensing reactive chemical species. While CS has been subject to extensive toxicological investigations in animals and human beings, its effects on intradermal or subcutaneous injection have not previously been reported. We have investigated the potential of CS to be used as an agonist on TRPA1 in human experimental pain studies. A calcium influx assay was used to confirm the capacity of CS to activate TRPA1 with >100,000 times the selectivity over the transient receptor potential vanilloid receptor 1. CS dose-dependently (EC50 0.9 µM) released calcitonin gene-related peptide in rat dorsal root ganglion cultures, supporting involvement in pain signalling. In a local tolerance study, injection of a single intradermal dose of 20 mM CS to rats resulted in superficial, circular crusts at the injection sites after approximately 4 days. The histopathology evaluation revealed a mild, acute inflammatory reaction in the epidermis and dermis at the intradermal CS injection site 1 day after administration. After 14 days, the epidermal epithelium was fully restored. The symptoms were not considered to be adverse, and it is suggested that doses up to 20 µL of 20 mM CS can be safely administered to human beings. In conclusion, our data support development of a CS human dermal pain model.

The development of an analytical method for urinary metabolites of the riot control agent 2-chlorobenzylidene malononitrile (CS).

The analysis of biomedical samples such as urine and blood can provide evidence of exposure to chemicals for a range of applications including occupational exposure monitoring, detection of drugs of abuse, performance enhancement in sport and investigations of poisoning and incapacitation. This paper reports the development of an analytical method for two suspected urinary metabolites of the riot control agent 2-chlorobenzylidene malononitrile (CS): 2-chlorohippuric acid and 2-chlorobenzyl-N-acetylcysteine. 2-Chlorohippuric acid was identified in all 2h post-exposure samples from a set of urine samples taken from army recruits exposed to low levels of thermally dispersed CS during training. 2-Chlorobenzyl-N-acetylcysteine, a metabolite known to be formed in the rat, was not identified in any of the samples. The lower limit of detection (LLOD) for 2-chlorohippuric acid and 2-chlorobenzyl-N-acetylcysteine was 1ng/ml and 0.5ng/ml in pooled urine from the pre-exposed subjects. 2-Chlorohippuric acid was rapidly excreted but was detectable in the urine of 17 of the 19 subjects tested 20h after exposure.

Unintended cutaneous reactions to CS spray.

CS spray (2-chlorobenzylidene malononitrile 5% w/v in methyl isobutyl ketone) has been used by the police force in the UK as an incapacitant for nearly a decade. It causes a number of well-recognized cutaneous reactions, which are generally regarded as short-lived. These include skin burning, erythema and blistering. However, a range of unpredictable cutaneous reactions to CS spray may also occur. We have found contact allergy, leukoderma, initiation or exacerbation of seborrhoeic dermatitis, and aggravation of rosacea following CS spray exposure in 6 police officers and 1 doorman. These skin reactions have required long-term changes in working practice for the affected individuals. Police officers may have repeated exposure to CS spray during their training and in their work, and designated police officers carry CS spray canisters daily in the line of duty. They may therefore be at greater risk of exposure to CS spray and its unintended effects than many assailants.

Short-term and long-term physical effects of exposure to CS spray.

CS gas (2-chlorobenzylidene malonitrile) is widely used in an incapacitant spray that causes intense lacrimation, blepharospasm and burning sensations in the throat and nose. Questions have been raised about its safety. We obtained information on short-term and long-term symptoms, and performed ear, nose and throat examinations and respirometry at 8-10 months, in 34 young adults who had been exposed to CS spray in a confined space during a confrontation with police. The group was subdivided into those who had been sprayed directly on the face (n=10) and those exposed indirectly. At one hour, all but 2 individuals still had symptoms; respiratory and oral symptoms were significantly more prevalent in the directly exposed group. At one month, only oral symptoms were significantly more prevalent. At 8-10 months, symptoms were still reported but there were no differences between the groups and clinical examinations revealed no specific abnormalities. There was no convincing evidence of long-term physical sequelae from exposure to CS spray.

Comparative acute toxicity of o-chlorobenzylidene malononitrile (CS) and oleoresin capsicum (OC) in awake rats.

Tear gases are largely used to control civil unrest. Their incapaciting effects involve eyes, skin and respiratory tract. This study was performed to compare acute respiratory effects of o-chlorobenzylidene malononitrile (CS), oleoresin capsicum (OC) and their respective solvents in awake rats, using an integrated system of nose-only exposure and multiple monitoring of breathing. Aerosols were generated by a Collison Nebulizer from the solutions held in tear gas sprays. The reduction of minute ventilation, observed during a 5 min exposure, was significantly more important with CS than with OC: minute ventilation represented 29+/-8 and 50+/-6% of pre-exposure minute ventilation respectively (P<0.05). The reduction of minute ventilation observed with CS and OC solvents alone was not significantly different from that observed with the tear gases themselves. The decrease in minute ventilation observed, between the second and the fifth minute of exposure, was of the same level for repeated exposure separated by 24 h. Time necessary to recover to 80% of pre-exposure minute ventilation was not significantly different between the two tear gases: 722+/-272 and 691+/-262 s for CS and OC respectively (NS). Histological analysis of the trachea, performed at the end of exposures, revealed an increase in mucus secretion after exposure to OC and cytoplasmic vacuoles in epithelial cells after exposure to CS. In the lungs, interstitial oedema was observed after exposure to OC and emphysema after exposure to CS.

The comparative acute mammalian toxicity of 1-chloroacetophenone (CN) and 2-chlorobenzylidene malononitrile (CS).

The comparative acute toxicity of two peripheral sensory irritant materials, 1-chloroacetophenone (CN) and 2-chlorobenzylidene malononitrile (CS), has been investigated in several species of small mammal using solutions in polyethylene glycol 300 for intravenous, intraperitoneal and oral administration, and as pure aerosols for inhalation exposure. Additionally, the comparative potency for inducing primary contact dermatitis was studied. CN and CS were found to be about equitoxic by intravenous and intraperitoneal injection, but CS was significantly less toxic by the oral and inhalation routes and less likely to cause non-lethal tissue damage than CN.