The aim of the tolerance test is to provide a limited evaluation of short-term toxicity of the additive to the target animals. It is also used to establish a margin of safety, if the additive is consumed at higher doses than recommended. Such tolerance tests must be conducted to provide evidence for safety for each of the target species/animal categories for which a claim is made. In some cases it is acceptable to include some elements of the tolerance test in one of the efficacy trials provided that the requirements given below for these tests are met. All studies reported in this section must be based on the additive described in Section II.
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.
Duration of tolerance trials: Pigs
| Target animals | Duration of the studies | Characteristic of the target animals |
|---|---|---|
| Suckling piglets | 14 days | Preferably from 14 days to weaning |
| Weaned piglets | 42 days | For 42 days after weaning |
| Pigs for fattening | 42 days | Body weight at start of the study ≤ 35 kg |
| Sows for reproduction | 1 cycle | From insemination to the end of the weaning period |
If suckling and weaned piglets are applied for, a combined study (14 days suckling piglets and 28 days weaned piglets) would be considered sufficient. If the tolerance for weaned piglets has been shown, no separate study for pigs for fattening is required.
Duration of tolerance trials: Poultry
| Target animals | Duration of the studies | Characteristic of the target animals |
|---|---|---|
| Chickens for fattening/reared for laying | 35 days | From hatching |
| Laying hens | 56 days | Preferably during the first third of the laying period |
| Turkeys for fattening | 42 days | From hatching |
Tolerance data from chickens for fattening or turkeys for fattening can be used to demonstrate tolerance for chickens or turkeys reared for laying/breeding respectively.
Duration of tolerance trials: Bovines
| Target animals | Duration of the studies | Characteristic of the target animals |
|---|---|---|
| Calves for fattening | 28 days | Initial bodyweight ≤ 70kg |
| Calves for rearing; cattle for fattening or reproduction | 42 days | |
| Dairy cows | 56 days |
If calves for rearing and cattle for fattening were applied for, a combined study (28 days for each period) would be considered sufficient.
Duration of tolerance trials: Sheep
| Target animals | Duration of the studies | Characteristic of the target animals |
|---|---|---|
| Lambs for rearing and for fattening | 28 days |
Duration of tolerance trials: Salmonidae and other fish
| Target animals | Duration of the studies | Characteristic of the target animals |
|---|---|---|
| Salmon and trout | 90 days |
As an alternative to a 90-day duration, a study could be performed where the fish increase their initial body weight at the start of the trial by least a factor of two.
If the additive is intended to be used for brood stock only, the tolerance tests shall be carried out as close to the spawning period as possible. The tolerance tests shall last for 90 days and attention shall be paid to the egg quality and survival of the eggs.
Duration of tolerance trials: Pets and other non food-producing animals
| Target animals | Duration of the studies | Characteristic of the target animals |
|---|---|---|
| Dogs and cats | 28 days |
Duration of tolerance trials: Rabbits
| Target animals | Duration of the studies | Characteristic of the target animals |
|---|---|---|
| Rabbits for fattening | 28 days | |
| Breeding does | 1 cycle | From insemination to the end of the weaning period |
If rabbits suckling and weaned are applied for, a period of 49 days (beginning one week after birth) would be considered sufficient and must include the does until weaning.
If an additive is applied for a specific and shorter period than given by the animal category definition, it shall be administered according to the proposed conditions of use. However, the observation period shall not be shorter than 28 days and shall involve the relevant end-point (e.g. for sows for reproduction the number of piglets born alive when considering the gestation period, or the number and weight of weaned piglets when considering the lactation period).
The studies shall be reported individually, giving details of all experimental groups. The trial protocol shall be carefully drawn up with regard to general descriptive data. In particular, the following shall be recorded:
herd or flock: location and size; feeding and rearing conditions, method of feeding; for aquatic species, size and number of tanks or pens at the farm, light conditions and water quality including water temperature and salinity;
animals: species (for aquatic species intended for human consumption identification shall be made by their colloquial name followed in parenthesis by the Latin binomial), breed, age (size for aquatic species), sex, identification procedure, physiological stage and general health;
date and exact duration of testing: date and nature of the examinations performed;
diets: description of manufacture and quantitative composition of the diet(s) in terms of ingredients used, relevant nutrients (analysed values) and energy. Feed intake records;
concentration of the active substance(s) or agent(s) (and, where that is the case, substances used for comparative purposes) in the feedingstuffs shall be established by a control analysis, using the appropriate recognised methods: reference number(s) of the batches;
number of test and control groups, number of animals in each group: the number of animals involved in the trials must permit statistical analysis. The methods of statistical evaluation used should be stated. The report shall include all animals and/or experimental units involved in the trials. Cases which cannot be assessed due to a lack or loss of data shall be reported, and their distribution within the groups of animals classified;
the timing and prevalence of any undesirable consequences of treatment in individuals or groups must be reported (give details of the observation programme used in the study); and
therapeutic/preventive treatments, if necessary, shall not interact with the proposed mode of action of the additive and shall be recorded individually.