2.2.1. Irwin test study

Behavior, physiological and neurological response of Autonomic Nervous System of mice inoculated with m-TiO₂NPs were studied.

The modified Irwin test and the Irwin test were performed during this research [36]. The modified Irwin test consisted on the observation of the mice pre- and post-applications by injecting the m-TiO₂NPs, at times 0, 15, and 30 min; the Irwin test consisted of follow-up observations at 1, 4 and 24 h [11, 37]. During this time, observation tests were performed with and without human presence (Figure 3).

Irwin test results by comparison with control mice: (A) strength test on a grid, (B) color of skin, eyes, and ears, as well as eye shape in a normal test mouse, and (C) normal group behavior.

We evaluated Group D and E with the modified Irwin test and the Irwin test, observing the following: if the animal was awake or asleep; if it was restless, showed fear, aggressiveness or indifference with regards to the experimenter; and if it was silent or made noises. The position of the body, locomotor activity (movements of grooming), abnormal behavior (tremors, shaking or convulsions); changes in the skin, fur, mucous membrane, eyes, respiration, behavior patterns were also observed [9]. Each parameter was scored on a 0 to 3 scale, with 0 representing the response in a normal group animal and 3 representing a maximally impaired animals [11, 38] (Table 2).

Irwin test scoring method.