Comparative safety evaluation of riot control agents of synthetic and natural origin.

Riot control agents (RCA) are lachrymatory, irritating compounds which temporarily incapacitate the uncontainable crowd. Ortho-Chlorobenzylidene-malononitrile (CS), 2-chloroacetophenone (CN), dibenz[b,f]1:4-oxazepine (CR), and nonivamide (PAVA) are synthetic RCAs, while oleoresin extract of chili known as oleoresin capsicum (OC) a natural irritant has been in use by various law enforcement agencies. Though efficacy of these agents is beyond doubt, they suffer from certain drawbacks including toxicity, production cost, and ecological compatibility. Presently, we have evaluated the safety of CR, OC, and PAVA on inhalation variables along with oral lethality. Additionally, the liver function test (LFT) in serum and lungs function was evaluated in broncho-alveolar-lavage fluid (BALF), both collected on the 14th day after RCA exposure. Animals then sacrificed and histopathology of liver and lungs was carried out. Results showed OC and PAVA to be more toxic than CR with an oral LD50 of 150 and 200 mg/kg body weight, respectively, while CR was safe at >3 g/kg body weight. All three agents caused severe impairment of respiratory variables bringing down normal respiration by >80% with rise in sensory irritation. Recovery from the irritating effect of CR was more rapid than OC and PAVA. LFT and BALF variables were not significantly different from that of control. There were no remarkable histopathological changes in liver and lungs. Hence, as per results, CR is safest among all synthetic and natural origin RCAs and can be safely used for effective dispersion of disobedient mob.

[On the stability of the irritant dibenz-[b,f]-[1,4]-oxazepine (substance CR)].

The objective of the present study was to determine the duration and conditions of persistence of the irritant dibenz-[b,f]-[1,4]-oxazepine (substance CR) on the environmental objects. The quantitative analysis of the substance on cotton fabric specimens was carried out using the approved method of high performance liquid chromatography (HPLC) with UV detection at different time periods and envirobmental conditions. It was shown that the main factor determining the lifetime of dibenz-[b,f]-[1,4]-oxazepine on the cotton fabric is the ambient conditions. By way of example, the "open" and "closed", storage of such specimens during 300 days resulted in the decrease of the amount of substance CR to 22.5 and 79% of the initial level respectively. By the end experiment (day 600) these values lowered to 3 and 52.5% respectively. Taken together, the results of the study indicate that dibenz-[b,f]-[1,4]-oxazepine can be described as a substance resistant to environmental impacts. It is comparable in terms of stability with such known irritants as capsicum oleoresin and pelargonic acid morpholide.

[The biological activity of the irritant dibenz-[b,f] - [1,4]-oxazepine (substance CR) persisting during a long period at the environmental objects].

The objective of the present study was to determine the biological activity of the irritant dibenz-[B,F]-[1,4]-oxazepine (substance CR) contained in the environmental samples in case of their "closed" storage during different periods of time. The experiments were carried out using male and female rabbits of the Chinchilla strain with the initial body mass of 3000-4000 g. The animals were administered an aqueous alcoholic extract from the tissue samples of the rabbit eye coat as described in the "Methodological guidelines on the medico-biological assessment of the safety of personal protection devices". The results of experiments indicate that extracts from tissue samples elicit irritation in the eyes of the laboratory animals even after their storage as long as 600 days. This observation suggests that substance CR retains the ability to cause irritation during a prolonged period.

Tear gasses CN, CR, and CS are potent activators of the human TRPA1 receptor.

The TRPA1 channel is activated by a number of pungent chemicals, such as allylisothiocyanate, present in mustard oil and thiosulfinates present in garlic. Most of the known activating compounds contain reactive, electrophilic chemical groups, reacting with cysteine residues in the active site of the TRPA1 channel. This covalent modification results in activation of the channel and has been shown to be reversible for several ligands. Commonly used tear gasses CN, CR and CS are also pungent chemicals, and in this study we show that they are extremely potent and selective activators of the human TRPA1 receptor. To our knowledge, these are the most potent TRPA1 agonists known to date. The identification of the molecular target for these tear gasses may open up possibilities to alleviate the effects of tear gasses via treatment with TRPA1 antagonists. In addition these results may contribute to the basic knowledge of the TRPA1 channel that is gaining importance as a pharmacological target.

Tear gases and irritant incapacitants. 1-chloroacetophenone, 2-chlorobenzylidene malononitrile and dibenz[b,f]-1,4-oxazepine.

Irritant incapacitants, also called riot control agents, lacrimators and tear gases, are aerosol-dispersed chemicals that produce eye, nose, mouth, skin and respiratory tract irritation. Tear gas is the common name for substances that, in low concentrations, cause pain in the eyes, flow of tears and difficulty in keeping the eyes open. Only three agents are likely to be deployed: (i) 1-chloroacetophenone (CN); (ii) 2-chlorobenzylidene malononitrile (CS); or (iii) dibenz[b,f]-1,4-oxazepine (CR). CN is the most toxic lacrimator and at high concentrations has caused corneal epithelial damage and chemosis. It has accounted for at least five deaths, which have resulted from pulmonary injury and/or asphyxia. CS is a 10-times more potent lacrimator than CN but is less systemically toxic. CR is the most potent lacrimator with the least systemic toxicity and is highly stable. CN, CS and CR cause almost instant pain in the eyes, excessive flow of tears and closure of the eyelids, and incapacitation of exposed individuals. Apart from the effects on the eyes, these agents also cause irritation in the nose and mouth, throat and airways and sometimes to the skin, particularly in moist and warm areas. In situations of massive exposure, tear gas, which is swallowed, may cause vomiting. Serious systemic toxicity is rare and occurs most frequently with CN; it is most likely to occur when these agents are used in very high concentrations within confined non-ventilated spaces. Based on the available toxicological and medical evidence, CS and CR have a large safety margin for life-threatening or irreversible toxic effects. There is no evidence that a healthy individual will experience long-term health effects from open-air exposures to CS or CR, although contamination with CR is less easy to remove.

Effect of inhaled aerosol of 1-chloroacetophenone (CN) and Dibenz (b,f)-1,4 oxazepine (CR) on lung mechanics and pulmonary surfactants in rats.

Inhalation toxicity following exposure to 1-Chloroacetophenone (CN) and Dibenz(b,f)-1,4 oxazepine (CR) aerosols for 60 min at sublethal concentrations were studied in rats. The dynamic surface tension (gamma max and stability ratio) of lung homogenate increased significantly on CN exposure. The lung mechanics studies revealed a significant increase in compliance in CN exposed rats. CR, on the other hand did not influence any of the above variables except for a decrease in compliance. Total lung phospholipids and sphingomyelin contents decreased significantly following exposure to CN, while CR exposure produced an increase in sphingomyelin, reduction in phosphatidylcholine and ethanolamine, with no change in total phospholipid contents. Histomorphological observations indicated cellular degeneration in the epithelium of the bronchiole and alveolar septal-wall thickening due to the presence of an increased number of mononuclear cells in CN exposed rats. However, CR induced inflammatory reaction and enlargement of respiratory air spaces. It is concluded that of the two sensory irritants (tear gases) examined, CN is potentially more toxic compared to CR in rats.

Toxicological evaluation of 1-chloroacetophenone and dibenz[b,f]-1,4-oxazepine after repeated inhalation exposure in mice.

Toxicological evaluation was made on the effects of two peripheral sensory irritants (tear gases): 1-chloroacetophenone (CN) and dibenz[b,f]-1,4-oxazepine (CR). Animals had a 15-min daily inhalation exposure to average vapour concentrations of 87.6 mg CN m-3 or 1008 mg CR m-3 (both equal to 0.05 LC50) for 5 or 10 days and were sacrificed 24 h after the last exposure, when biochemical and histopathological observations were made. Both chemicals caused a significant decrease in body weight gain. Histological changes in lung, liver and kidneys were more severe after 10 than after 5 days of exposure and were more severe in CN-exposed than in CR-exposed mice. Organ weight to body weight ratios remained normal except for the spleen to body weight ratio, which decreased in CN-exposed mice after both 5 and 10 days of exposure. Biochemical indicators showed a toxic response only in CN-exposed mice, but the only consistent change was an increase in blood glucose. Hepatic alkaline phosphatase was not influenced, malondialdehyde concentration and acid phosphatase activity were increased only after 5 days of exposure and liver GSH concentration decreased after 10 days of exposure. Results indicate that CN is not only more toxic than CR in absolute terms but is also more toxic at the 5% level of their LC50.

Time dependent histomorphological assessment of lung damage induced by inhaled dibenz(b,f)-1-4-oxazepine (CR) and 1-chloroacetophenone (CN) in rats.

Lung damage caused by inhalation (single exposure for 60 min) of sublethal concentration of pure aerosols of dibenz(b,f)-1,4-oxazepine (CR) and 1-chloroacetophenone (CN) have been examined at different time intervals in rats. The damage was not severe with CR (2830 mg.m-3) but in the case of CN (60.26 mg.m-3) it was evident up to 30th day post exposure. Necrobiosis, attenuation of bronchiolar epithelium, edema in the air ways and also in the lumen of alveoli leading to substantial changes in the histoarchitecture of the lung were observed during CN exposure. On the other hand CR caused degenerative changes which disappeared on 30th day.

Effect of nitroxazepine on bone marrow cells of mice.

The antidepressant drug, nitroxazepine, was tested in mice for mutagenicity in bone marrow cells by a micronucleus test. The drug was administered orally at a dose of 4.5, 9, and 13.5 mg per mouse. The results show that the drug is found to be mutagenic in mouse bone marrow cells.

Biochemical evidence that high concentrations of the antidepressant amoxapine may cause inhibition of mitochondrial electron transport.

Overdosage with the antidepressant amoxapine causes metabolic acidosis and may lead to brain damage and death. To better understand the metabolic disturbances caused by amoxapine overdose, its effects on three simple systems were studied: growth of Saccharomyces cerevisiae, mitochondrial energy metabolism, and an electron transport system in microsomal membranes. Growth of yeast on all substrates except lactate was inhibited by amoxapine at 50-100 micrograms ml-1. Growth on lactate was observed at 200 micrograms ml-1 of amoxapine. In beef heart mitochondria, amoxapine at 100 micrograms ml-1 inhibited reactions involving large sections of the electron transport chain. Energy-linked reactions in submitochondrial particles were also inhibited. Electron microscopy showed some disruption of the mitochondrial internal structure by amoxapine and a change from orthodox to condensed conformation. Microsomal NADH-cytochrome b5 reductase was inhibited by amoxapine, but at higher amoxapine concentrations than mitochondrial reactions. The results suggest amoxapine disrupts reactions of membrane-associated enzyme complexes, and mitochondrial energy conservation may be one of the first systems affected. We speculate that lactic acid accumulation in patients with amoxapine overdose may be caused by loss of electron acceptor activity in tissues.

Chemical irritant algesia assessed using the human blister base.

The chemical irritants o-chlorobenzylidene malononitrile (CS), n-nonanoylvanillylamine (VAN) and dibenzoxazepine (CR) and several of its derivatives have been assayed using the human blister base. The relative potencies found by this method, CR greater than VAN greater than CS, conflicted with those found in non-human test systems but the rank order of potency of CS and CR reflected that reported in tests on the human eye and tongue. Data derived from humans thus appear to be of importance when assessing irritant potency. Interactions between CS, CR, VAN, capsaicin and bradykinin were investigated to discover any common pathways of irritant activity. Self-desensitization developed on repeated application of all agents to the blister base and selective cross-desensitization also occurred.

A repeated dose study of the toxicity of technical grade dibenz-(b.f.)-1,4 oxazepine in mice and hamsters.

The repeated dose inhalation toxicology of technical grade dibenz-(b.f.)-1,4 oxazepine (CR) was studied in mice and hamsters. The animals were exposed 5 days/week for 18 weeks and retained until 1 year after the start of exposure. CR, at high doses, affected survival of both species, nevertheless the material produced little specific organ-directed toxicity.

A repeated dose study of the toxicity of CR applied to the skin of mice.

Dibenz (b,f)-1-4 oxazepine (CR) was applied to the skin of C3H and Porton-strain mice, daily for 12 weeks. After a further 80 weeks the animals were sacrificed and examined grossly and histologically. The results were compared with appropriate solvent and untreated controls. No abnormalities were found that could be ascribed to CR, but a high incidence of fatty infiltration of the liver in 1 strain of mice might have been due to the solvent in which CR was dissolved, namely acetone. There were marked differences in the incidence of several lesions in the 2 strains of mice, alveologenic carcinoma being much more common in the Porton mice. CR appeared to have little effect on the skin but both the test and solvent groups of male Porton mice shared an increased incidence of acanthosis.

Ultrastructure of rat lungs following exposure to aerosols of dibenzoxazepine (CR).

Three groups of 18 animals were exposed respectively to the following large doses of dibenz (b.f)-1:4 oxazepine (CR) aerosols, 78,200,140,900 and 161,300 mg/min/m3. Animals were killed at intervals from 15 min to 2 days, and the lungs examined macroscopically, by electron microscopy and conventional histology. There were no deaths during or after exposure. Macroscopically the lungs from all rats appeared normal. Microscopically there were a few areas of mild congestion, haemorrhage and emphysema, but there was little variation between the different groups. Electron micrographs revealed some morphological alteration of the epithelium and endothelium but only occasional changes in the interstitium. The alterations took the form of "ballooning" of the endothelium with isolated foci of swelling and thickening of the epithelium. Interstitial oedema was observed in one animal only which was exposed to the highest concentration. The effects appeared similar in all groups, and are thought to be transient. The results of this investigation suggest that even high doses of CR aerosols cause minimal damage to the lung, and the structural alterations which do occur are believed to be due to the stress to which the animals were subjected during the exposure period.

The acute mammalian toxicology of dibenz(b,f)-1,4-oxazepine.

Dibenz(b,f)-1,4-oxazepine (CR), a potent peripheral sensory irritant material, has been shown to have a very low acute lethal and sub-lethal toxicity by intravenous, intraperitoneal, oral, percutaneous and inhalation routes to several species of laboratory mammal. There was no organ-specific pathology. Comparison of the acute toxicity of CR with that of two other peripheral sensory irritants, 1-chloroacetophenone (CN) and 2-chlorobenzyl-lidene malononitrile (CS), shows CR to be significantly less toxic than either of them. Pyrotechnically generated CR smoke was more toxic than pure (thermally generated) aerosols of CR; this was due to the presence of pyrotechnic decomposition products in the atmosphere from the burning of the smoke generating composition. However, the median lethal toxicity of pyrotechnically generated CR smoke was very significantly less than that of either pyrotechnically generated CN or CS smokes. Short-term cumulative toxicity did not occur following multiple oral dosing with CR. The acute toxicology of three ether intermediates encountered in the synthesis of CR from 1-chloro-2-nitrobenzene and sodium phenoxide (2-nitrodiphenyl ether, 2-aminodiphenyl ether and 2-formamidodiphenyl ether) was investigated; all three ethers were found to be less acutely toxic than CR itself.

The comparative ophthalmic toxicology of 1-chloroacetophenone (CN) and dibenz(b.f)-1:4-oxazepine(CR).

Rabbit eyes were contaminated with 1-chloroacetophenone (CN) and dibenz(b.f)-1:4-oxazepine (CR) in solution (1 to 10% in polyethylene glycol 300), as a solid (0.1 to 5 mg), and as aerosols (15 min exposure to 360 to 719 mg/m3). In solution, CN caused marked and persistent inflammatory effects, the severity and duration being related to the concentration used. Corneal damage was marked and persistent with 5 and 10% solutions; the lowest concentration causing just detectable keratitis in a small proportion of animals was 2%. Solid CN was even more damaging to the eye than similar amounts in solution. In marked contrast, CR in solution caused mild to moderate inflammatory effects, usually of only a few days duration, even at the higher concentrations. With 1 and 2%, these effects were just detectable to mild, lasting for 24 hrs or less. 10% CR caused just detectable keratitis of usually only a few days duration; the lowest concentration causing just detectable keratitis in a small proportion of treated animals was 5%. Solid CR merely caused minor irritation of the conjunctivae and eyelids for about 1 hr. Aerosols of CN and CR did not damage the eye, but irritation of the lids and conjunctivae was more marked and persistent with CN. Solutions of CN and CR caused concentration-related increases in corneal thickness and intraocular tension, being more marked and sustained with CN.