**5. Acute toxicity test**

Characteristically, in the presence of an unknown substance, the first step in the search for pharmacological activity begins with a toxicological study and in particular, the evaluation of the lethal dose 50 (LD 50), that is. the dose, which causes the mortality of 50% of the animals. In virtue of which, increasing doses of extracts are administered to rats and mice until mortality is achieved. Although this technique is highly controversial from an ethical point of view, it nevertheless provides highquality information:

1.Firstly, it determines the toxicity of the substance and the therapeutic margin; in other words, the ratio between the active dose and the toxic dose for the animal

*Methods for Evaluating the* In Vivo *Analgesic and Anti-Inflammatory Activity of Essential Oils DOI: http://dx.doi.org/10.5772/intechopen.113832*

species tested; this is an essential step in the use of any substance for therapeutic purposes.

2.Observation of the first symptoms of toxicity reveals the target organs; in other words, those which are preferentially affected by the toxicant; toxicity stands alike for an excellent criterion for orienting research into pharmacological activity [41].

More to the point, essential oils are not products that can be used without risk. Similarly as all natural products, 'just because it is natural does not mean it is safe for the body' [23]. The acute toxicity test for essential oil extracts can quickly be carried out using the method described by the organisation for economic cooperation and development (OECD) [43]. This test consists of administering experimental doses to the animals and monitor them for signs of poisoning, including drooling, convulsions, unusual activity, loss of consciousness, coma or even death. These observations are performed regularly for up to 48 hours [44].
