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Journal of Medical Chemical, Biological and Radiological Defense
J Med CBR Def  |  Volume 5, 2007
Received 9 March 2007 |  Accepted 8 June 2007  |  Published 18 June 2007

Evaluation Of The Antidotal Efficacy Of Atropine Sulfate And Pralidoxime Chloride Given By Autoinjectors Against Nerve Agent (Sarin) Toxicity

R. Vijayaraghavan*, Neeti Jain, Anshoo Gautam, Manoj Sharma, Seema Singh, Deo Kumar, Ram Singh, Pravin Kumar, A.S.B. Bhaskar, A.K. Gupta and Suman Jain

Defence Research and Development Establishment,

Gwalior – 474002



* Corresponding Author:

R. Vijayaraghavan

Defence Research and Development Establishment,

Gwalior – 474002


Tel.: 91-751-2233494 | Fax: 91-751-2341148 | E-mail:



The antidotal effectiveness of atropine sulfate and pralidoxime chloride delivered by autoinjectors against sarin toxicity was evaluated in rabbits.  Sarin, diluted in saline was administered at a dose of 5, 10 or 20 µg/kg, topically to one eye of the rabbits.  Anticholinesterase activity was assessed by measurements of ocular pupillary diameter and plasma cholinesterase activity.  Atropine sulfate (1.8 ± 0.1 mg) and/ or pralidoxime chloride (36 ± 2 mg) were administered by autoinjectors to evaluate their antidotal efficacy individually and as combined treatment. The pupillary diameter was decreased to less than 50% of pretreatment values in the sarin-treated eyes within 5 minutes, without any change in the contralateral eye. Sarin given by topical application to the eye caused a significant inhibition of plasma cholinesterase activity. Tremor and paralysis of the limbs were observed at 20 µg/kg dose. When the antidotes were given alone, the effects of sarin on pupillary diameter and plasma cholinesterase activity were only marginally protective. More significant protection was obtained when atropine sulfate and pralidoxime chloride were given as a combined antidote.  These findings confirm that the administration of atropine sulfate and pralidoxime chloride by the autoinjectors is an effective way of protecting nerve agent toxicity.



The nerve agents are chemical warfare agents and are also potentially a threat from the terrorist organisations  (Tokuda et al, 2006).  During the World War II huge quantities of the nerve agents were stockpiled, and, there were allegations of their use during the Iraq-Iran war (LeJeune et al, 1998; Newmark, 2004).  In 1995 the Aum Shinrikyo group of Japan used an extremely toxic nerve agent, sarin in a Tokyo subway killing 12 people and injuring about 5000 people (Vale, 2005).  Though the Chemical Weapons Convention (CWC) has been signed and ratified by more than 180 countries, the threat persists, and several countries have developed preparedness for such an eventuality.

The nerve agents (also known as nerve gases) are organophosphorus compounds (OP). All OP compounds do not qualify as war gases and are not a threat, due to their differential toxicity.  Some of the OP compounds that are less toxic to humans are used as insecticides. Agents that fall in the nerve agent category are tabun, sarin, soman and Vx (Maynard and Beswick, 1992; Marrs et al, 1996).  The nerve agents are classified under Schedule I of the CWC (Organisation for the Prohibition of Chemical Weapons, OPCW,1993).  The absorption of these agents into the system is through inhalation, dermal absorption, and through mucous membranes.If the skin is also exposed, these agents can be absorbed appreciably.  Nerve agents irreversibly inhibit the enzyme acetylcholinesterase (AChE), which results in accumulation of acetylcholine leading to cholinergic crisis  (Koelle, 1992).  

As soon as the nerve agents enter the system, symptoms of poisoning appear. The nerve agents affect the muscarinic and nicotinic receptors and on the receptors within the central nervous system. These effects  include constriction of pupil (miosis), increased production of saliva, a running nose, increased perspiration, urination, defecation, bronchosecretion, bronchoconstriction, decreased heart rate and blood pressure, muscular twitches and cramps, cardiac arrhythmias, tremors and convulsions. The most critical effects are paralysis of respiratory muscles and inhibition of respiratory centre.  Ultimately death results due to respiratory arrest (Worek et al., 1995).  If the concentration of the nerve agent is high, death is immediate.

The treatment of nerve agent casualty requires artificial respiration and drug treatment. The recommended drugs are atropine sulfate and pralidoxime chloride (Holstege et al, 1997). Atropine sulfate is a competitive inhibitor of muscarinic receptors and the recommended human dose in the field is 2 mg intramuscularly and subsequently increased to 4 or 6 mg, and sometimes higher.  Pralidoxime chloride is used as a cholinesterase reactivator and the recommended dose is 500 to 1000 mg intramuscularly or intravenously (Johnson et al., 1996).  Death following nerve agent exposure is very rapid and in the field it is not possible for medical personnel to administer the drugs when large numbers of persons are affected. For this reason, autoinjectors are being used by several countries for the immediate field administration of the drugs by the affected individuals or by their buddies (Schier et al., 2004).  Autoinjectors commonly use atropine sulfate and pralidoxime chloride together but historically they were used one after the other.  

The present study is aimed to evaluate the antidotal efficacy of atropine sulfate and pralidoxime chloride given by autoinjectors against sarin toxicity in rabbits.



Animals : Randomly bred New Zealand white rabbits (male and female) weighing between 2.5 to 3.0 kg from the Institute’s animal facility were used for the study. They were housed in stainless steel cages individually under controlled environmental conditions with free access to pellet diet (Ashirwad Brand, India), sprouted gram, seasonal vegetables and water. The care and maintenance of animals were as per the approved guidelines of the ‘Committee for the Purpose of Control and Supervision of Experiments on Animals” (CPCSEA, India).  Food and water were withheld three hours prior to the experiment. This study had the approval of the Institute’s Animal Ethical Committee.

A total of 18 rabbits were used for the study.  For the various treatments the rabbits were reused after a period of 15 days.  They were randomised for all the individual experiments. Since the sarin exposure was a single exposure through the eye, the cholinesterase was monitored, and the rabbits were used after 15 days, when the cholinesterase level was normal.  This reduced the use of large number of animals needed.

Chemicals :  Sarin (isopropyl methylphosphonofluoridate) was synthesised in the synthetic chemistry division and was found to be more than 95 % pure by gas chromatographic analysis.  Extreme care was taken during the synthesis and storage of sarin, as per the approved guidelines of the Institute.

Autoinjectors : The technology of production of the autoinjectors and drug cartridges has been transferred to the industries by Defence R & D Organisation, and these same autoinjectors and drug cartridges have been used in this evaluation [Fig. 1].  These autoinjectors are reusable, i.e., once the drugs shelf life expires, the drug cartridge can be replaced with fresh cartridges.  The atropine sulfate cartridge consisted of 1 mg/mL of atropine sulfate (2.2 mL) and after the actuation of the autoinjector, 1.8 ± 0.1 mg per rabbit was injected intramuscularly.  The dose was calculated based on the injection capability test of the autoinjector, i.e., actuating the autoinjector on a piece of 6 mm closed rubber foam of 30 shore hardness, above which was placed a piece of the NBC suit (nuclear biological and chemical protection suit).  The quantity delivered was measured (1.8 ± 0.1 mL) to arrive at the dose. The quantity delivered was calculated using an in vitro drug assay. The pralidoxime chloride cartridge consists of 300 mg/mL of pralidoxime chloride in 2.2 mL.  Since this dose of pralidoxime is toxic to the rabbits, a diluted solution of 20 mg/mL (2.2 mL) was used in the cartridge.  The dose of pralidoxime chloride was 36 ± 2 mg per rabbit.  The autoinjectors were administered in the gluteal muscle of the rabbits.  Atropine sulfate autoinjector was administered first, two minutes after sarin eye instillation and 30 seconds later the pralidoxime chloride autoinjector was administered.

Drug assay:  The concentration of atropine sulfate was determined using a high performance liquid chromatographic, (HPLC), method.  The HPLC system consisted of a multisolvent delivery system, equipped with rheodyne manual injector, 10 mL sample loop, spherisorb column ODS2 5 µm (4.6 x 150 mm) and dual l absorbance detector (Waters Corporation, USA). The mobile phase consisted of solvent A (50 mM KH2PO4 in 1 % triethylamine in water) and solvent B (100 % acetonitrile) in the ratio of 85:15.  The pH of the mobile phase was adjusted to 3.5 by adding ortho phosphoric acid.  The atropine sulfate was monitored at 211 nm. Quantitation of atropine sulfate was done by calculating the area under the curve, and compared with a standard atropine sulfate (Sigma, USA).

The concentration of pralidoxime chloride was determined spectrophotometrically as per the I.P. method (Pharmacopoea of India, 1985).  Briefly, to the diluted solution of pralidoxime chloride, 1 N sodium hydroxide was added and the OD was measured at 336 nm in a spectronic 1201 spectrophotometer (Spectronic Corporation, USA) and compared with a standard.

Efficacy of atropine sulfate and pralidoxime chloride autoinjectors :  The initial pupillary diameter was measured and blood was withdrawn from the ear vein of the rabbits before the administration of sarin.  Three doses of sarin were used in this study, viz., 5, 10 and 20 µg/kg.  (A dose of 40 µg/kg instilled through the eye caused convulsions and death.  Hence the maximum dose was limited to 20 µg/kg. The LD50 for rabbit by  percutaneous exposure is 925 ug/kg [Marrs, T.C., Maynard, R.L., Sidell, F.R.; Chemical Warfare Agents. Toxicology and Treatment. John Wiley & Sons, New York, NY. (1996),  p. 89. Thus the exposure through the eyes is more toxic.). Sarin was dissolved in normal saline and used within 15 minutes.  The concentration of sarin was adjusted in such a manner that 20 ± 2 µL was instilled in the lower sac of the left eye of the rabbit.  In the right eye normal saline was instilled.  The treatment for the animals was grouped as follows:

Group 1 : Sarin only, 5, 10 and 20 µg/kg.
Group 2 : Atropine sulfate autoinjector only
Group 3 : Pralidoxime chloride autoinjector only
Group 4 : Sarin (5, 10 and 20 µg/kg) + atropine sulfate autoinjector
Group 5 : Sarin (5, 10 and 20 µg/kg) + pralidoxime chloride autoinjector.
Group 6 : Sarin (5, 10 and 20 µg/kg) + atropine sulfate and pralidoxime chloride autoinjector.

For each group and dose five rabbits were used (The rabbits were reused after a period of 15 days and every time they were randomly assigned to groups).

Measurement of pupillary diameter:  The experiment was carried out in a well lit room (200  lux).  The pupillary diameter was measured using a digital vernier calliper.  The mean of a minimum of three measurements of the left eye (sarin-instilled) was used. 

Estimation of plasma cholinesterase:  The blood was withdrawn from the ear vein of the rabbits.  The ear was cleaned with absolute alcohol.  A 22 gauge needle was inserted in the vein for the collection of blood (about 500 µl) in heparinised tubes.  The tubes were then centrifuged to separate the plasma.  Plasma cholinesterase was estimated by the method of Ellman et al., using acetylthiocholine as substrate (Ellman et al., 1961). 

Statistical analysis :  All the variables were analysed by one-way ANOVA followed by Student-Newman-Keuls multiple comparisons test.  A probability of less than 0.05 is taken as statistically significant.  SigmaStat (SPSS Inc., USA) was used for the statistical analysis.



The normal pupillary diameter of the rabbits was 8.0 ± 1.7 mm.  Sarin instillation caused an immediate constriction of the pupil.  The pupillary constriction was observed within two to three minutes of sarin instillation following 5, 10 or 20 µg/kg [Fig. 2].  As sarin was instilled in the left eye, constriction was observed only in the left eye and not in the right eye.  A dose dependent papillary constriction was observed in the groups with sarin doses of 5, 10 and 20 µg/kg.  The pupillary constriction remained even 24 hours after sarin instillation. Administration of either atropine sulfate or pralidoxime chloride alone through the autoinjectors marginally protected the animals, as noted by pupillary constriction, 30 minutes after the drug administration, but significantly protected affected pupillary constriction 120 minutes after the drug administration. The exception was for the 20 µg/kg dose group at 120 minutes, however by 24 hours, pupillary constriction was comparable with the lower dose groups.  When atropine sulfate and pralidoxime chloride were administered together, they significantly protected pupillary constriction by 30 minutes after the combined drug administration [Table 1].  Somewhat surprisingly, there was no significant change in the right eye, in which normal saline was instilled, throughout the experiment.  Administration of atropine alone or pralidoxime chloride alone also did not show any significant change in the pupillary diameter (116.4 and 116.6 % respectively) in the left right eye.

Normal plasma cholinesterase was found to be 0.90 ± 0.07 nmoles/10 µL of plasma.  Administration of 5, 10 or 20 µg/kg of sarin produced a dose dependent decrease in the plasma cholinesterase level.  Though the level of cholinesterase returned to normal in 24 hr after 5 µg/kg sarin dose, in other two groups (10 and 20 µg/kg) it was still less than the control. Tremor and paralysis of the limbs were observed at 20 µg/kg dose.  Compared to the administration of atropine sulfate alone, pralidoxime chloride administration improved the cholinesterase level. Administration of atropine sulfate and pralidoxime chloride together significantly improved the level of plasma cholinesterase even after 30 minutes.  The cholinesterase level returned to normal 72 hours after sarin administration in all the groups.  The cholinesterase level was monitored up to 7 days after sarin instillation and there was no significant change compared to the control levels. The return of the cholinesterase activity was higher than that before sarin instillation, but we have not found a reason for this.



Among the various chemical warfare agents, specific antidotes are available for the nerve agents.  An antagonist of acetylcholine, such as atropine sulfate and a reactivator of the inhibited acetylcholinesterase such as pralidoxime chloride are the recommended drugs for the treatment of organophosphorus intoxications, including the nerve agents (Brimblecombe et al., 1970; Johnson and Stewart, 1970; Boskovic et al., 1984).  The role of atropine is as a competitive antagonitsm of acetylcholine at the muscarinic receptors.  The inhibition of cholinesterase by the nerve agents stimulates not only the muscarinic receptors but also the nicotinic receptors (Koelle, 1992).  Hence for treating the nerve agent poisoning along with atropine sulfate, a cholinesterase reactivator is also required, such as the pralidoxime chloride, to counter the nicotinic effects. In the present study, in terms of constriction of pupil and reactivation of plasma cholinesterase, it is observed that the administration of atropine sulfate and pralidoxime chloride together resulted in better protection compared to the individual protection from each drug.  It has also been observed in previous work that the protection of nerve agent poisoning by the combined administration of cholinolytic and cholinesterase reactivator is better than the individual drugs (Crook et al., 1962; Das Gupta et al., 1991). 

Atropine sulfate is the drug of choice for the treatment of all the nerve agents like, tabun, sarin, soman and Vx (Davies et al., 1959; Crook et al., 1962; Johnson and Stewart, 1970).  A number of cholinesterase reactivators are known and their ability to provide protection for nerve agent poisonings vary.  Obidoxime is a better cholinesterase reactivator and some countries are using this in their autoinjectors.  Since it is more potent the dose required is also less.  Obidoxime is also more toxic than pralidoxime chloride (Marrs, 1991).  Both obidoxime and pralidoxime are unsuitable for soman poisoning, and another cholinesterase reactivator, viz., HI-6 has been found to be more effective (Clement and Lockwood, 1982; Shih et al., 1991).

There is an advantage to the reusable autoinjectors over the single time autoinjectors, since the drug cartridges can be changed after the expiry of the drugs.  The autoinjectors are very easy to use by the soldiers in an emergency.  They are preferred over manual injection of the drugs, since the drug is delivered in the various layers of the muscle unlike the conventional (manual) injection where the whole drug is released at one place.  The absorption of the drug is expected to be faster in the autoinjector delivery (Friedl et al., 1989). 

After the sarin attack on the Tokyo subway, various threat analyses have cautioned that terrorists may try to use chemical warfare agents again.  In such a situation the autoinjectors will be very useful for the medical team. The autoinjectors are also useful in the case of OP insecticide poisoning, as they can be stored in the health centres.  However, their effectiveness for different OP pesticides varies.

Dermal absorption and inhalation are the major routes of entry of the nerve agents.  As weapons they are dispersed as aerosols and produce vapours under normal atmospheric conditions.  For the experimental work on antidote evaluation, the nerve agents are either administered subcutaneously or by inhalation.  In the case of nerve agent vapours, such as sarin, nerve agents penetrate the mucosal membranes of the eye. The present study was planned with nerve agent administration through the eye. In this experimental set-up systemic poisoning was observed as the animals showed paralysis of the muscles and tremor.  However, the right eye was not affected demonstrating that the systemic toxicity need not necessarily show pupillary constriction (Holstege et al., 1997).  

The present study showed that administration of atropine sulfate or pralidoxime chloride individually did not give significant protection as measured by the pupillary constriction and plasma cholinesterase level, while both the autoinjectors together gave significant protection.  Our study also showed that administration of atropine sulfate and pralidoxime chloride using the autoinjectors, one after the other but close in time, is an effective way of protecting against nerve agent toxicity.  Atropine sulfate antagonises the actions of excess acetylcholine and PAM chloride reactivates the inhibited AChE. Both actions together offer better protection against nerve agents.



The authors are grateful to Mr. K. Sekhar, Defence R & D Establishment, Gwalior for his encouragement and keen interest in the present study.


Table 1. Effect of sarin instillation (left eye) on the pupillary diameter (% normal diameter) and its protection by atropine sulfate and pralidoxime chloride. [go to text reference]

Atropine sulfate alone 116.4 + 19.3 (n = 3)
Pralidoxime chloride alone 116.6 + 3.8 (n = 3)
GROUPS 5 µg/kg 10 µg/kg 20 µg/kg
5 min after sarin instillation
Sarin alone 47.7 + 0.4 40.9 + 1.4 37.0 + 0.3
30 min after sarin instillation
Sarin alone 43.9 + 1.3 41.7 + 2.0 40.9 + 2.8
Sarin + atropine sulfate 69.8 + 4.6* 55.0 + 14.2 54.8 + 2.8*
Sarin + pralidoxime chloride 45.9 + 4.1 51.7 + 9.1 52.4 + 4.8*
Sarin + atropine sulfate and pralidoxime chloride 75.6 + 6.3* 93.3 + 9.1* 88.9 + 2.5*
120 min after sarin instillation
Sarin alone 42.6 + 3.2 41.8 + 0.5 45.2 + 9.5
Sarin + atropine sulfate 83.2 + 4.2* 65.1 + 9.3* 56.5 + 8.7
Sarin + pralidoxime chloride 63.3 + 4.0* 67.7 + 8.0* 59.0 + 3.8
Sarin + atropine sulfate and pralidoxime chloride 75.6 + 4.5* 99.8 + 6.3* 105.3 + 1.9*
24 hours after sarin instillation
Sarin alone 78.0 + 2.2 68.3 + 3.4 70.0 + 7.0
Sarin + atropine sulfate 79.2 + 1.7 73.6 + 12.0 55.7 + 6.0
Sarin + pralidoxime chloride 79.3 + 4.3 80.5 + 2.7 83.2 + 2.4
Sarin + atropine sulfate and pralidoxime chloride 85.1 + 2.7 111.2 + 8.8* 107.7 + 1.5*


Mean + SE (n = 5)

*Statistically significant from sarin group

Normal pupillary diameter = 8.0 + 1.7 mm (The normal pupillary diameter is the pooled value of all the animals before any treatment has been given).

There was no change in the pupillary diameter of the right eye (data not shown). 


Table 2. Effect of sarin instillation on the plasma cholinesterase (% activity) and its protection by atropine sulfate and pralidoxime chloride.

Atropine sulfate alone 115.0 + 18.1 (n = 3)
Pralidoxime chloride alone 99.9 + 4.4 (n = 3)
GROUPS 5 µg/kg 10 µg/kg 20 µg/kg
30 min after sarin instillation
Sarin alone 45.7 + 1.9 45.6 + 2.0 36.3 + 5.6
Sarin + atropine sulfate 77.4 + 3.8 57.9 + 12.4 70.0 + 8.7*
Sarin + pralidoxime chloride 80.6 + 7.8 89.4 + 5.8* 84.6 + 2.9*
Sarin + atropine sulfate and pralidoxime chloride 128.1 + 21.8* 102.9 + 12.9* 103.1 + 3.1*
120 min after sarin instillation
Sarin alone 57.5 + 18.0 50.3 + 8.2 44.5 + 8.8
Sarin + atropine sulfate 55.4 + 16.1 63.1 + 16.2 61.2 + 27.0
Sarin + pralidoxime chloride 113.8 + 6.2* 120.9 + 27.8* 63.2 + 21.5
Sarin + atropine sulfate and pralidoxime chloride 153.3 + 4.6* 158.4 + 17.8* 123.4 + 6.5*
24 hours after sarin instillation
Sarin alone 98.6 + 21.0 69.8 + 2.4 66.0 + 10.1
Sarin + atropine sulfate 80.6 + 11.0 99.1 + 6.1 97.3 + 10.9
Sarin + pralidoxime chloride 84.3 + 31.0 136.7 + 31.5 86.3 + 13.6
Sarin + atropine sulfate and pralidoxime chloride 152.9 + 15.4 139.6 + 18.7 98.4 + 11.7
48 hours after sarin instillation
Sarin alone 93.4 + 11.7 86.2 + 15.7 70.9 + 17.6
Sarin + atropine sulfate 88.3 + 10.0 131.6 + 17.6 115.2 + 10.0
Sarin + pralidoxime chloride 110.6 + 23.9 131.1 + 19.0 80.2 + 13.7
Sarin + atropine sulfate and pralidoxime chloride 158.2 + 20.0 107.6 + 3.0 97.9 + 9.5



Mean + SE (n = 5)

*Statistically significant from sarin group

Normal activity = 0.90 + 0.07 nmoles/10 µl of plasma (The normal activity is the pooled value of all the animals before any treatment has been given)



Picture of reusable autoinjectors

Figure 1. Atropine sulfate and pralidoxime chloride reusable autoinjectors. [go to text reference]


Picture of normal eye Picture of pupillary constriction

Figure 2. Effect of sarin instillation on the left eye of the rabbit. (a) normal eye (b) pupillary constriction, 5 min after 5 µg/kg of sarin instillation. [go to text reference]




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