**3. Conclusions**

338 Toxicity and Drug Testing

a. The choice of animal model should be appropriate for the product and clinical indication. Often rabbits are used for parenteral vaccine toxicity because their muscle mass may receive a volume equivalent to a full human clinical dose (e.g., 0.5 mL). b. High dose should be at least 1 – 10 times the actual highest planned clinical dose;

c. To determine if the observed effects are dose-related (and to potentially identify an equivalent to a No-Observed-Adverse-Effect Level), 2 or 3 concentrations, to cover the range of proposed clinical doses, in addition to a vehicle and/or adjuvant control, should be used. At a minimum, the highest proposed human dose should be tested.

e. The period of study varies, depending on the frequency of dose administration (episodic, not daily), which may be abbreviated compared to the proposed clinical dosing schedule. The duration of the GLP safety studies is dependent on the study design. Tissue samples

g. A minimum of 5 animals per gender per dose should be included for each time point of

h. Same route of administration as the proposed clinical route (with same delivery device,







should be processed and data analyzed after intermediate and terminal sacrifice. f. Timepoints for sacrifice: 1-3 days post-last inoculation; 2-4 weeks post-last inoculation

sacrifice – this number may vary depending on animal model chosen.


studies if repeated doses are planned in the clinical evaluation.


only a select list is examined histopathologically)

sometimes preferably not scaled on weight or body area.

d. Number of proposed clinical inoculations plus one.

i. Minimal endpoints examined should include:


urinalysis measurements


(recovery).

whenever possible).

histopathology

inoculations)

studies

DNA vaccination is a continuously evolving and exciting field with many challenges to face. Methodological and regulatory frames are also developing every day. One important issue

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for a promising future in this vaccination strategy is to demonstrate efficacy in humans in a safe context. In this scenario, the pre-clinical protocols are very relevant and should be based on the design of the proposed clinical assessment. General regulatory frames rule the administration of exogenous substances, but given the nature and proposed mechanism of action for DNA vaccines additional specific considerations should be taken into account. Safety evaluation of DNA vaccines against infectious diseases should be carried out considering the particular characteristics of the disease and the causal pathogen, including the expected use, preventive or therapeutic, and target population, in a case by case approach. Fortunately, no major adverse events have been observed so far after DNA immunization in humans on clinical trials. The establishment of strong tests for releasing the product is very relevant in lots consistency and reproducibility of results at both pre-clinical and clinical level. New frontiers are opening and attention should be given to novel preparations including original adjuvants/immunostimulatory molecules or employing modern delivery vehicles for DNA vaccines, as well as the long-term pharmaco-vigilance.
