Fifteen Saw-scaled vipers, Echis carinatus were captured from different regions of Central Punjab of Pakistan. The snakes were kept in captivity for two weeks before their milking was performed. All snakes selected for milking were adult. Milking was performed without anesthetics. A specialized team was responsible for scientific classification, milking of specimens and storage of the venom. Venoms of all snake specimens were pooled and dissolved in saline (final concentration: 10mg/ml). All the venom samples were stored at 4 oC until used to avoid any disruption of their natural toxic properties.

Fifty adult albino white mice belonging to both sexes were purchased from Manawa Research Institute (MRI) Lahore, Punjab. The pooled venom was injected intraperitoneally; separately into groups of 10 mice with doses ranging from 1 to 10-mg/kg-body wt. As all the snakes used in the experiment belonged to the same species, pooled venom was suggested and preferred to the individual venom in order to have a cumulative effect of the toxins present in a particular snake species. Survival times (time between injection and death) of each animal for 24 hours were recorded. The LD₅₀ of each snake species was determined according to the mathematical scheme adopted by Meier and Theakston (1986) some modification was however made in their method where by the maximum three-hour survival time was increased up to 24 hours for bringing convenience and authenticity to results.

No variables related to age, geographical origin, sex, and diet of snakes were controlled although variation in venom composition, even in natural conditions, may be associated with these variables. The effects of these variables on the toxicity of the venom in mice, although certainly present, were therefore not observed.

Survival times observed after intraperitoneal (i.p.) injection of different doses of Echis carinatus venom in mice are given in table 1. The scheme for the calculation of Approximate LD₅₀ is given table 2.

Figure 1

Table 1: Survival times observed after i.p. Injection of different doses of venom in mice.

Figure 2

Table 2: Calculation Scheme Of Approximate LD of

The pharmacological effects of the crude venom were characterized on a number of mice, however some significant effects observed in various models with visible symptoms are described.

When a mouse was injected intraperitoneally with a non – lethal dose 1 mg/kg of viper venom, it grew surprisingly energetic and hypersensitive. It ate more feed than the normal and, during one of the countless attempts of escape from the finely wired jar; it sometimes excreted the fecal matter that was larger, almost double the normal fecal matter size. The mouse remained alive and active and did not by any means seem to suffer badly from the effects of the non-lethal dose. It showed no symptoms of pain or agitation.

When a sub lethal dose (2 mg/kg) was injected to a mouse, there was a little sloughing of the dead tissue around the injection site after a day or so. The being grew weaker, ate poorly, and appeared generally miserable for about two days before it recovered its health.

As the venom of the viper Echis carinatus literally dissolves and liquefies some tissues that it contacts, one mouse with a sub lethal dose bled to death not due to the lethal effect of the venom but more because of the large wound at the site of the injection. The wound grew so black and large that the entire viscera of the mouse literally began to pop out of the wound and the mouse died almost 12 hours after the injection. The survival time of this mouse was not included in the data

When a partially lethal (4.5 mg/kg) dose was injected to a mouse, the weakness and difficulty in breathing developed and lasted for a few hours. The tissues at the site of the injection were dissolved. The animal would be obviously uncomfortable from the start; it scratched, rubbed at the injection site and was generally irritable and hyperactive and hypersensitive. Next came a quiet period during which the animal would sit huddled up but alert. The injection site was dark. There would be a short period of almost normal activity followed by another quiet stage that eventually terminated in collapse and death. The skin around the injection site grew gangrenous. The mouse became flat, immobile with its hind legs paralyzed. The survival time of the mouse was about 18 hours (table 1).

When a mouse was injected intraperitoneally with a lethal, but not overwhelming, dose of viper venom (5 mg/kg), it showed little immediate evidence of pain or discomfort. For a short time it behaved normally but soon it started showing signs of weakness. Its breathing got labored, and its flanks became peculiar, punched in appearance. Soon it could no longer get to its feet with the breathing becoming ever more difficult. As respiration ceased, the heart continued to beat for a few minutes. The survival time of the mouse was noted to be about 16 hours and 20 minutes (table 1).

At the next lethal doses (6 to 12 mg/kg) the sequence of events was not altogether different. The duration of the each event was brief and it started quicker in time and closer to each other. The mice would sometimes lick the place where the venom was inoculated. Within a few minutes the breathing became irregular and abdominal respiration began which was also spasmodic. Soon the mice became prone, motionless with their hind legs paralyzed. In some mice powerful convulsive spasms rarely passed from the tail through the whole body. The bodies of the mice became stiff near death. The place of the skin where the venom was inoculated became darker in color and black spot appeared at the site of injection within half an hour or so. The area of the skin around the site of injection became hairless and acute tissue digestion began at the site. This showed the obvious necrotic effect of the venom. The survival time of the mouse having the highest dose (12 mg/kg) of venom was recorded to be about 20 minutes only.

Pharmacological studies clarify the clinical and pathological features of human fertility. Furthermore these help to understand specific resistance and susceptibility of various warm blooded and cold-blooded species (Perez et al. 1978, 1979). This could lead to gauging the extent of physiological tolerance to natural snakebites and thereby enable preparation of better antisera to neutralize the venom activity.

Two type of toxic effects were most easily observable in the all the mice, i.e. the change in color and dissolution of the skin at the site of the injection and the mice becoming prone, motionless with their hind legs paralyzed. The dissolution of the skin is an evident consequence of the myotoxic effect of envenomation. Paralysis of hind legs making the mouse flat and immobile reflects the neurotoxic effect of the venom.

In the Approximate LD₅₀ the number of the animals sacrificed is significantly reduced i.e. up to 10 mice for one type of venom. This small number becomes negligible when compared with the use of more than 200 mice for one type of venom through the classical LD₅₀. Moreover the use of 8-10 animals for one test gives LD₅₀ values within the same statistical range as that obtained using the classical LD₅₀ assay which employs much more experimental animals.

The intraperitoneal Toxicity of Echis carinatus determined through Approximate LD₅₀ in our study was 6.23 (Table 2). This value is authenticated by the intravenous toxicity of E. carinatus determined through Approximate LD₅₀, which was 2.98. The difference between the two figures is mainly due to two factors. Firstly the two experiments were performed under different conditions and circumstances Secondly there is always substantial difference between an intraperitoneal and an intravenous dose of the same drug (in this case the venom).

As classical LD₅₀ experiments, owing to their high level of variability, are far from satisfactory, the approximately method of LD₅₀ suggested for scientific, economic and ethical reasons was tried and found relatively unproblematic, suitable and effective. Approximate method has thus been found a satisfactory alternative to classical LD₅₀ determinations for animal venoms, owing to its exactitude and reproducibility for most purposes of lethality testing in animal models.