Commission Regulation (EC) No 429/2008Show full title

3.1. Studies concerning the safety of use of the additive for the target animals

The studies included in this section are intended to assess:

• the safety of use of the additive in the target species per se; and

• any risk associated with the selection and/or transfer of resistance to antimicrobials and increased persistence and shedding of enteropathogens.

3.1.1. Tolerance studies for the target species

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.

3.1.1.1. The design of a tolerance test includes a minimum of three groups:

• 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.

3.1.1.2. Duration of tolerance trials

Table 1

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.

Table 2

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.

Table 3

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.

Table 4

Duration of tolerance trials: Sheep

Target animals	Duration of the studies	Characteristic of the target animals
Lambs for rearing and for fattening	28 days

Table 5

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.

Table 6

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

Table 7

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).

3.1.1.3. Experimental conditions

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:

(1)

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;

(2)

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;

(3)

date and exact duration of testing: date and nature of the examinations performed;

(4)

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;

(5)

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;

(6)

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;

(7)

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

(8)

therapeutic/preventive treatments, if necessary, shall not interact with the proposed mode of action of the additive and shall be recorded individually.

3.1.2. Microbial studies

Studies shall be provided to determine the ability of the additive to induce cross-resistance to antibiotics used in human or veterinary medicine, to select resistant bacterial strains under field conditions in target species, to give rise to effects on opportunistic pathogens present in the digestive tract, to cause shedding or to excrete zoonotic micro-organisms.

If the active substance(s) possesses antimicrobial activity at the feed concentration level, the minimum inhibitory concentration (MIC) for relevant bacterial species shall be determined, according to standardised procedures. Where relevant antimicrobial activity is demonstrated, the ability of the additive to select resistant bacterial strains in vitro and in the target species, and to induce cross-resistance to relevant antibiotics shall be established(1).

Tests at the recommended use level shall be provided for all microbial additives, and for those other additives in which an effect on the gut micro-flora can be anticipated. These studies shall demonstrate that use of the additive does not create conditions conducive to an overgrowth and shedding of potentially pathogenic micro-organisms.

The choice of micro-organisms to be monitored will depend on the target species, but shall include relevant zoonotic species, regardless of whether or not they produce symptoms in target animals.

(1)

A non-exhaustive list is available in: www.efsa.europa.eu/en/science/feedap/feedap_opinion/993.html