3.1.1.1.The design of a tolerance test includes a minimum of three groups:U.K.
an unsupplemented group;
a group with the highest recommended dose; and
an experimental group with the multi-fold level of the highest recommended dose.
In the experimental group the additive shall generally be given at ten times the highest recommended dose. Test animals shall be routinely monitored for visual evidence of clinical effects, performance characteristics, product quality where relevant, haematology and routine blood chemistry and for other parameters likely to be related to the biological properties of the additive. Critical end-points known from the toxicological studies in laboratory animals shall be considered. Any adverse effect detected during efficacy trials shall also be reported in this section. Unexplained deaths in the tolerance test shall be investigated by necropsy and, if appropriate, histology.
If a 100 times the maximum recommended dose can be shown to be tolerated, no haematology or routine blood chemistry would be required. If the product is tolerated only at lower level than ten times of the highest recommended dose, the study shall be designed in such a way that a margin of safety for the additive can be calculated and additional end-points (by necropsy, histology if relevant, and other appropriate criteria) shall be provided.
For some additives depending on their toxicology and metabolism or use, it may not be necessary to carry out tolerance tests.
The experimental design used must include consideration of adequate statistical power.