Table of Contents
This guideline is adapted from the European Medicines Agency’s Committee for Medicinal Products for Veterinary Use (CVMP) Guideline on demonstration of target animal safety and efficacy of veterinary medicinal products intended for use in farmed finfish.
This is a guideline about the sorts of information an applicant may submit to address the safety and efficacy criteria for veterinary chemical products intended for use in farmed finfish. It also provides guidance about how the information might be presented and analysed. For further information on the safety and efficacy criteria, refer to satisfying the statutory criteria.
If you are seeking to register such a product, you should submit efficacy and safety data to support all label claims. We encourage you to apply to us for technical advice before submitting an application.
In addition to efficacy and safety, the Australian Pesticides and Veterinary Medicines Authority (APVMA) also considers aspects such as chemistry and manufacture, public health, environmental fate, and occupational health and safety for veterinary chemical products. We also encourage you to review the Veterinary Labelling Code, the Standard for the Uniform Scheduling of Medicines and Poisons (SUSMP), and the FAISD Handbook before applying.
You should follow the APVMA guidelines where possible, although they may not be applicable for all veterinary chemical products for use in finfish. If you modify or omit particular aspects, you should provide justification for doing so.
In principle, you may use the results of all trials as documentation, irrespective of where the trials have been carried out. However, you should take into account the various conditions of the trials (for example, climatic conditions, the disease situation, water temperature and salinity), as they may influence the outcome and hence the applicability of the studies.
The following terms are used in this guideline:
- Finfish: this term distinguishes true fish from shellfish, crayfish, jellyfish, etc. All the species of fish mentioned in this guideline are true finfish.
- Degree-day: this is a measure of cooling or heating. It is determined by multiplying the water temperature each day by the number of days. For example, 10 days at 5 °C equals 50 degree-days.
- Positive control: a positive control group is treated with an authorised reference product, approved for the same indication as the test product, according to the label instructions in order to compare the test and reference products.
- Negative control: a negative control group is treated with a placebo, either saline or vehicle (the finished product deprived of the active substance) in order to compare treatment with the test product with no treatment.
3. General considerations
We encourage you to standardise study protocols and study reports as far as possible to facilitate the comparison of study results and the possible extrapolation between different species of fish.
If the product is intended for in-feed administration, you should consider and, if appropriate, investigate the possible impact of the feed composition. The feed composition and manufacturing process may influence the medicinal product because of physico-chemical compatibility. Fish have very marked senses of taste and smell. To ensure that they will accept the final product, we recommend that the palatability of the active substance and, if relevant, the excipients be investigated prior to clinical trials.
Conditioning and pelleting are the main factors affecting stability during the manufacture of medicated feedstuffs. Those processes can subject the feed to high temperatures and pressures, which can cause the degradation of active constituents, excipients or other feed constituents. Other quality points for consideration are homogeneity and segregation of these products. To account for fish being poikilothermic animals, the term ‘degree-days’ should be used wherever relevant.
Several farmed fish species are kept in areas where the water temperatures can vary considerably during the year. Laboratory studies should therefore be carried out to cover the relevant temperature range. The optimal temperature for the disease should also be taken into consideration when planning the studies. Normally, studies should be carried out at 2 water temperatures. You should justify your choice of water temperature(s) in relation to the fish species, product and indication. If you do not carry out studies at 2 different temperatures, you should justify that choice.
More limited investigations may be acceptable for a compound previously authorised for use in another relevant species. This is elaborated under the appropriate topic (see 'Safety in the target species' and ‘Clinical studies’).
When justified, data from non-aquatic species can be used as supportive information.
The origin and genetics of the experimental fish are important to obtain valid and reproducible results, and any variation should be addressed. All finfish species should be identified by their colloquial name, followed in parentheses by the Latin or Linnean description.
All experimental techniques should be described in such detail as to allow them to be reproduced. You should also establish their validity and describe in detail each experimental trial or field trial and the conditions under which it was performed. You should provide separate reports on all trials, whether the results are favourable or not. Adequate summaries of groups of trials based on the same protocols may be provided.
Refer to Brattelid and Smith (2000) for detailed guidance on the contents of the study report.
You should report adverse events in sufficient detail to enable a proper assessment of safety in the target animal. You should explain non-specific mortalities and comment on any physical or behavioural abnormalities.
For clinical studies, you should clearly state the onset and the duration of relevant disease outbreaks. This information will allow the evaluation of coincidental mortality data and its potential threat to the statistical power of the study. You should explain how the data continues to be valid and fit for purpose.
General study design
As water quality has been identified as an important element for maintaining healthy fish and ensuring valid experimental results, water quality parameters such as temperature and salinity should be addressed in detail.
You should state the efficacy of the veterinary chemical product as a function of dose, frequency and duration of treatment. The criteria used for the evaluation of efficacy in the trials should be pre-determined. In confirmatory clinical trials, you should usually identify one primary efficacy endpoint and you may report one or more secondary endpoints. The primary endpoints should accurately reflect the intended benefit of the product. You should present the results in a way that is suited for adequate statistical evaluation. The clinical trials should cover all claimed indications, and each indication should be discussed and reported separately. You should perform statistical analysis of the results whenever relevant.
You should justify the observation unit (such as individual fish or cage) and the number of samples collected on each sampling occasion. The sample sizes should be sufficiently large, statistically justified and based on clinically relevant endpoints.
In studies of products intended for use against aquatic one-host parasites (such as sea lice on salmon), we recommend sampling a limited number of fish from many cages instead of many fish from a small number of cages in order to take into account clustering, which occurs naturally with such parasites.
4. Preclinical studies
The objective of preclinical studies is to characterise the active component or formulation, either to collect information of relevance when designing clinical trials or to document that clinical data obtained previously could be used for a new formulation, a new route of administration or administration in new temperature or salinity conditions.
When deciding which preclinical studies are relevant for efficacy and safety, you should take into account the mode of action (if known) and route of administration. For example, for waterborne products acting directly on ectoparasites, neither pharmacodynamic nor pharmacokinetic parameters in the target species are relevant from an efficacy point of view. However, it is important to know the mode of action and the effective concentration for the parasite.
On the other hand, for ectoparasiticides administered orally, pharmacokinetic parameters of the target species are relevant, as the active substance needs to reach the site where it is presented to the parasites (for example, blood, tissue fluids or mucus layer) in sufficient amounts.
Great care should be taken to ensure that the fish receive the intended dose. For single-dose studies of orally administered products, we recommended that you administer the test substance orally by gavage. For repeated dose studies of pre-mixes intended for medicated pellets, examples of applicable control methods are as follows:
- Small number of test fish: count the pellets before they are given to the fish. After dosing, count the uneaten pellets and then calculate the average dose received.
- Large number of test fish: small X-ray-dense glass beads (ballotini) can be incorporated when manufacturing medicated feed pellets for the trial, at a known concentration of beads per pellet (this can be determined by X-raying the pellets). When the number of beads per pellet is known, a representative number of fish can be X-rayed to reveal the average number of pellets ingested by the fish. It is also possible, by using small and large pellets alternate days, to reveal how many pellets were ingested on 2 different days in a row.
You should describe the pharmacodynamic (PD) effects, including the mode of action of the active ingredient(s), as the basis for the recommended use of the product. In some cases, this information will be available in the safety part of the dossier.
You should present all available relevant data (such as published references), including data from other animal species, where appropriate.
For antimicrobials, microbiological studies in vitro should be carried out according to the Guideline for the demonstration of efficacy for veterinary medicinal products containing antimicrobial substances. State the study parameters in detail. Notably, temperature and possibly salinity in the medium may affect minimum inhibitory concentration (MIC) values and should be stated. In vivo studies to investigate the mode of action are generally not recommended.
For antiparasiticides, the mode of action could be investigated in vitro according to standard protocols. For in vivo studies, the principles of VICH GL7 (Efficacy requirements for anthelmintics: General requirements) apply.
Because fish are poikilothermic and significant temperature-related effects can be expected, you should justify your choice of the temperatures at which in vivo studies are conducted. In addition, in the case of topically administered products, other water quality parameters such as water salinity, pH and hardness should be taken into account.
Note that southern bluefin tuna can maintain selected tissues at temperatures above the environmental temperature and are considered to be heterotherms (Crockett and Londraville 2006).
We recommend that pharmacokinetic studies in finfish be carried out in accordance with the principles in the CVMP guideline for the conduct of pharmacokinetic studies in target animal species, as far as they may be applied to finfish. That guideline describes all relevant steps for pharmacokinetic studies, regardless of species. The main difference in fish is that repeated samples from the same fish may not be possible. Thus, samples from different fish at different time-points may be necessary. When modelling the results, all fish sampled are regarded as one ‘mega-fish’. Statistical methods are available to determine confidence intervals for pharmacokinetic parameters, such as bootstrapping (see, for example, Nordgreen et al 2009).
You should submit pharmacokinetic studies where there is a need to document the residue depletion profile. However, we also strongly recommend that you consider pharmacokinetics as a tool to help reduce the total amount of clinical data – and hence the number of test fish needed.
In experimental studies in which systemic pharmacodynamics or tolerance is documented, pharmacokinetic data recorded in the study will increase the total amount of information, as effects could then be linked to exposure rather than to dose only. Also, in studies in which the individual dose is not known (as in the case of group oral medication), this will allow the estimation of an exposure-effect relationship at the individual level.
Pharmacokinetic data can also be used for bridging purposes. For instance, if clinical data are recorded in a study in which a certain formulation is used, those data could also be relevant to support the therapeutic performance of another formulation, provided there are comparative pharmacokinetic data to show that the 2 formulations are similar in terms of exposure.
Comparative pharmacokinetic studies could also be considered to bridge data between different environments, such as different salinity or different temperatures (the high and low ends of natural variation).
Performance of tests
You should perform pharmacokinetic studies under relevant conditions (such as salinity and temperature) because kinetic parameters, such as bioavailability, may be significantly affected by such factors.
The choice of study design will depend on the objectives of the study. Because it is difficult to collect a comprehensive set of data from an individual fish, analysis on a group level is normally used. Standard pharmacokinetic parameters could be calculated based on data from groups of animals sacrificed at different time points.
When using studies of distribution to support efficacy (for example, for substances that require distribution to skin or mucus for effect), studies should be planned with the combined use in mind. You could use either standard chemical analyses or methods using radiolabelled substances (such as whole body autoradiography and scintillation counting).
Sampling fish out of a small group may cause stress and produce effects such as decreased food uptake among the remaining fish, which may affect the outcome of the study, so you should take measures to reduce the animals’ stress. We recommended that the group be large enough to avoid stressing the remaining fish during all sampling procedures throughout the study. You should justify the group size chosen for each study.
You should discuss the mechanism for resistance and the frequency of resistance and include information on possible transmission. Possible cross-resistance to other active substances used in farmed fish should be stated.
For ectoparasiticides, experience of the development of antiparasitic resistance should be included, if relevant. Experience related to the development of resistance from other areas of use (for example, as pesticides) should also be included when available.
Safety in the target species
You should determine target animal safety in all of the target species, as you have defined them, unless you can justify not doing so. Studies performed in one species of fish may be considered relevant for the evaluation of safety in a second species of fish of the same genus or taxonomic family, provided that they are kept under the same environmental conditions. In such a case, there should be supportive safety data from clinical trials in the second species. For example, it may be considered unnecessary to carry out formal target animal safety studies in trout if such studies have been carried out on other species of salmonid, and if supportive safety data from clinical studies in trout are available.
Excipients normally used in pharmaceutical products for terrestrial animals might not be well tolerated by aquatic species. You should determine the safety of excipients and justify any lack of appropriate data.
It is important to take into account possible adverse effects on development (malformations) if the medication is applied to young fish (embryos, larvae and juveniles) and the product can easily interfere with growth. Indicate the range of sizes and weights of fish recruited for the trial, as the same treatment might not have the same effect in fish of different sizes.
Studies of repeated dose tolerance are relevant only for products intended for repeated dose administration.
This and the following points apply to all target animal safety studies.
We recommend that you use the final formulation of the medicinal product. If the formulation used in studies differs from the final commercial formulation, you should demonstrate that the bioavailability of the formulations is the same. Substances administered by gavage should have a suitable formulation (for example, solution, suspension, capsule or in feed). All formulations used in the tests should be assayed for the concentrations of the active substances before the start of the trials.
Negative control groups
Studies with in-feed medication should be carried out with the medicated group (preferably using the final formulation of the test product) and an untreated (feed-alone) group.
In all tests, the test product and placebo should be administered in the same manner as intended for the finished product. Handle untreated controls identically to treated fish.
For studies other than in-feed medication, the control substance should be either saline or vehicle (the finished product deprived of the active substance). You should justify your choice of control substance, taking into account that the excipients may have some effects of their own.
The fish to be tested should be in a normal physiological condition and be feeding well during 2 weeks of acclimatisation. You should allocate fish into groups randomly the day before the administration of the test product, using an appropriate method. To reduce stress caused by handling the fish, and for practical reasons, it could be acceptable to allocate and/or mark fish groups immediately before or during the administration of the test product, if justified. At sea, it may be difficult to allocate fish into cages. In this situation, allocation may be earlier than is mentioned above.
Acclimatisation is not applicable for embryonal stages.
The following conditions of exposure are recommended.
- Waterborne administration: a maximum of one gram of fish per litre of water
- Oral administration: a maximum of 5 grams of fish per litre of water
- Parenteral administration: a maximum of 5 grams of fish per litre of water
These stocking densities are low and are suitable for tank-based operations. They may be adjusted for field studies.
Higher loading is acceptable – that is, higher stocking density than quoted above may be used.
Group size and number
You should justify the numbers of fish per group, which should not be less than 10 with a minimum of 2 tanks per dose and 2 control tanks. If you choose to depart from this recommendation, you should consider applying to us for technical advice before beginning the trial. Trials with southern bluefin tuna should be conducted in a field situation, as it may not be feasible to hold this species in tanks.
We recommend that you use fish of the most sensitive category (size or age and physiological status) for which the product is intended.
Necropsy histopathology examinations and blood analyses
As a minimum, tissues from all fish in the highest dose group and control group should be examined macroscopically and microscopically. Where the toxicity of the test product is expected to be relatively high, you should consider different necropsy schemes to include gross and microscopic examinations for all fish or for randomly preselected fish. If lesions are found in any tissue from the highest dose group, you should examine samples from fish in the second highest dose group macroscopically and microscopically, until a no-observed-adverse-effect level is determined. In addition, tissues from all fish showing systemic clinical signs should be examined macroscopically and microscopically.
Haematology and blood chemistry should be performed in laboratory studies on target animal safety. If blood chemistry and haematology parameters are unremarkable in the highest dose group, you may choose not to test in the lower dose groups.
You should justify the parameters you have chosen for testing. For substances already approved for other animal species, the decision on whether blood chemistry and haematology are performed should be based on the previous findings in those other species. You should include in the documentation a discussion or justification of the decision on whether blood chemistry was done, and, if so, of which parameters were chosen for testing.
Dose justification and duration of dosage
You should justify your choice of dose levels and the duration of exposure.
The chosen levels should be adequate to demonstrate a sufficient margin of safety for the product when it is used under field conditions. This means that the dosage levels should be high enough to account for the fact that varying degrees of unintended overdosing will commonly occur in practice with such types of medicinal products intended for waterborne or in-feed treatment.
For single-dose studies, at least 3 dose levels should be tested. The selection of dose levels should be based on the proposed therapeutic dosing regimen.
For repeated-dose studies, the selection of dose level(s) and the duration of the treatment period should be based on the proposed therapeutic dosing regimen and on results from single-dose studies.
You should provide detailed records of feed uptake and concomitant daily dose.
For solutions and suspensions given by gavage, adjust the concentration of the active ingredient so that, if possible, no more than 0.5 gram or millilitre test product per 100 grams of fish is used to achieve the required dose. These maximum dosage recommendations are based on the practical dosage limitations in fish.
Dipping and bathing are considered to be methods of waterborne administration.
Waterborne treatment should usually have a very broad margin of safety due to the difficulty of accurate dosing and estimating water volume in raceways or sea cages.
The duration of treatment should be equal to, or longer than, the proposed length of treatment. The dosage of a veterinary medicinal product (as in mammals) is principally a function of treatment concentration and exposure period. For sedatives and anaesthetics for use in finfish, the length of exposure is the main parameter available for adjustment during treatment.
Both the test and the control product should be administered by injection. The same volume of test solution should preferably be administered to the fish in both the test and the control groups. You should also provide the maximal volume of the product administered in one injection site and an assessment of the reaction in the injection site.
In some cases, investigating fewer than 3 dose levels could be justified because there are practical limitations to the volume that can be injected into fish. In addition, the test solution may have limited solubility, which restricts the maximum concentration in a restricted volume. This can make it difficult to obtain more than 2 dose levels with a significant difference.
5. Clinical studies
The main purpose of the documentation of efficacy is to prove the therapeutic value of a new veterinary medicinal product for farmed finfish and to define an optimal dose and dosage regimen.
Clinical trials should be conducted for each proposed indication and for all target species in which efficacy is claimed. For some products, such as waterborne treatments that act directly on ectoparasites and that are independent of the pharmacokinetics in the fish, you may choose not to submit clinical trials in a second species if the clinical data obtained for the main fish species can be shown to be relevant to the second species. In such cases, you should provide sufficient justification for the omission of clinical studies, together with documentation of target animal tolerance.
All studies should be performed under appropriate conditions according to the proposed method of use of the product. For example, the study should be carried out in water temperature(s) in which the test product is likely to be used, considering the different climatic conditions within Australia. The studies should be blinded, unless otherwise justified.
Normally, you should submit data from both laboratory and full-scale field trials. Where appropriate, you should justify the lack of relevant data.
The omission of field studies and the submission of challenge studies only may be accepted if you can adequately justify it. For example, in the case of a second species closely related to a first species for which the efficacy of the product is fully documented, challenge studies may be sufficient to document efficacy in the second species.
In all studies, the final formulation or an essentially similar formulation should be used and administered by the proposed route. If a similar formulation is used, bioequivalence should be confirmed.
The clinical trials should include control groups, and you should justify the choice of control group (positive or negative). If a positive control is used, you should explain how the study design has sufficient sensitivity to detect effects above placebo level. You should consider and discuss all variables likely to confound results and the methods used to reduce or avoid them.
You should remove fish from the trial when they show definitive signs of disease and/or when there has been pathological confirmation of disease in the holding unit, rather than wait for death to occur. Only where the removal of fish showing clinical signs would significantly diminish the value of the data should animals showing such signs be left in the enclosures. In all circumstances, humane endpoints should be applied.
You should monitor and record the nature and frequency of adverse drug reactions.
For oral medication, you should record the daily uptake of medicated feed together with the daily dose of the active substance, if possible. Pre-mixes should be administered as medicated feed prepared by the procedure recommended by the manufacturer, preferably using a standardised feed.
The test conditions can be controlled and standardised in land- or sea-based test facilities. Experimental trials should be performed for the main target species.
The fish to be included in the trials should be of similar age and size, be susceptible to the disease in question and be of known origin and health status. The allocation of fish into groups should be done randomly using an appropriate method.
Every study should be designed to allow for appropriate statistical evaluation. You should present a sample size analysis. Significant differences might be found between different groups of fish kept under identical conditions owing to the fact that they are kept in separate tanks. Therefore, you should always use at least 2 groups kept under identical conditions but in separate tanks.
You should justify the choice of parameters recorded for evaluation and the statistical evaluation methods.
If negative controls are used, studies with in-feed medication should be carried out with the medicated group (using the test product) and an untreated (feed-alone) group. For products intended for other routes of administration, one negative control group is usually sufficient.
You should discuss challenge models (cohabitant, waterborne, injection) and their relevance to natural conditions (time of challenge, time of treatment, infection pressure, etc.).
The test animals should not previously have been exposed to the challenge organism, as such exposure has the potential to alter study results. You should provide specific justification if you use such animals.
The challenge organism should be of a strain relevant for the current disease situation. It should be isolated and characterised by the most appropriate method (preferably a standard method used by the national reference laboratory), which should be described in detail. The timing and performance of the challenge and the design of the study should be justified. You should report the results of the introduction of the challenge organism based on parasite counting, microbiological analyses or other pertinent investigations. If appropriate, a statistical analysis should be provided.
Dose determination trials
Dose determination studies are normally laboratory trials with or without challenge. Their purpose is to determine the optimal dose, dosage interval and total period of treatment for the claimed indications. By integrating pharmacokinetic (PK) and pharmacodynamic (PD) data as a basis for the pre-selection of a treatment dose, the need for more extensive studies, such as traditional dose determination studies, might be reduced. The estimated dose should be confirmed by dose confirmation studies.
You should establish a dose-response relationship for therapeutic effect and, if possible, for adverse effects. Dose determination trials may be performed as field trials. Data from well-controlled experimental studies are preferred where relevant models are available, and field studies could then be used to confirm the findings from the controlled trials. The final dosage recommendations should be supported by documentation showing that satisfactory efficacy is obtained within the relevant temperature range.
Tests should be carried out in seawater and freshwater, if relevant to the proposed use, unless it is documented that the pharmacokinetics of the active substance are not affected by salinity.
If there are validated endpoints or models available, dose determination could be performed only as PK/PD modelling. In such cases, it is important that a sufficiently large exposure range is investigated, which implies that more than one dose level should normally be investigated.
For antimicrobials, MIC data could be used together with pharmacokinetic parameters to estimate the appropriate level of exposure. Such data could be used for dose determination, provided the PK/PD surrogate marker used is adequately validated. Note that these parameters differ between antimicrobials and bacteria, so you should justify the choice of parameter in each case. The pharmacokinetic endpoints should be derived from plasma, and the free (non-protein bound) fraction of active substance concentration should be used for calculations.
Dose confirmation trials
Separate dose confirmation trials can be replaced by field trials performed with the final formulation of the product administered in the recommended dosage regimen.
The aim of field trials is to ensure that the product is efficacious and safe in the diversified conditions found in Australian aquaculture. The field studies should preferably be performed in accordance with good clinical practice.
If the trials use positive controls, a product that the APVMA has approved should preferably be used in the control group(s).
For products intended against diseases representing a potential threat to animal welfare, negative controls should only be used if no product is authorised for the claimed indication. The control group can be treated once an adequate estimation of difference in effect can be established.
Selection of farms
You should justify the number and suitability of the sites selected for field trials. The sites should be geographically well distributed to maximise the possibility of diversified environmental conditions, disease situations and management practices. Each site should have several pens or tanks with fish of the relevant size or age and physiological condition (smoltification, sexual maturation, etc.) for the proposed use of the product. At least 2 pens or tanks of fish, and preferably 12 to 14 (that is, several pairs), should be used in the trial. The farmer should be experienced in keeping detailed records on all important factors concerning the farm and its fish. Records of the source of fish and the disease history in different pens or tanks should be kept. Previous medication and the use of chemicals and vaccines should be known. Daily records of outbreaks of disease, mortality and medication are required, as well as known and stable management practices concerning, for example, hygiene, feeding, handling and the use of feed additives or biocides. Daily records of water temperature should be kept.
Selection of groups
All fish in one tank or pen are considered as one group. You should use a minimum of 2 groups in each trial, one of which should be a control group, which in most cases will be a positive control group. The groups should be allocated randomly, using an appropriate method. The prevalence of disease, daily mortality, clinical symptoms and other relevant parameters should be comparable in the treated and control groups at the start of the study.
Field trials in commercial fish farms should preferably be performed in spontaneous outbreaks of the diseases for which efficacy is claimed. Trials should thus be conducted at the time of year and under conditions in which a successful natural challenge can be defined by the investigator. Your report should include the method of identification of the causal agent. Information from trials performed with unsuccessful natural challenges may be provided, along with an explanation of the failures. All trials should be performed with adequate controls. Field trials with anaesthetics or other non-therapeutics should be performed with healthy fish. All trials should be planned so that suitable data are available for statistical analysis. You should choose clinical endpoints relevant for the proposed indication and specify primary and secondary endpoints.
You should confirm the presence of the investigated diseases in all groups included in the trial, and report the criteria for establishing an accurate diagnosis. Standard diagnostic methods should preferably be used. The same criteria are to be used in all trials and should include post mortem examination of a sufficient number of fish (at least 6 fish from each group). You should report the precise disease condition and the identification of any pathogenic organism.
Diseases caused by microorganisms should be diagnosed by isolating and characterising the pathogen by the most appropriate microbiological method (preferably a standard method), which should be described in detail. Samples from at least 6 fish per group are recommended.
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