CVM GFI #243 (VICH GL56) Studies to Evaluate the Metabolism and Residue Kinetics of Veterinary Drugs in Food-Producing Species: Study Design Recommendations for Residue Studies in Honey for Establishing Maximum Residue Limits and Withdrawal Periods

Administrative guidance, not enacted legislation. This captured publisher edition preserves the listed document and attachments; later developments are not independently consolidated.

#243
Studies to Evaluate the Metabolism and Residue
Kinetics of Veterinary Drugs in Food-Producing Species:
Study Design Recommendations for Residue Studies in
Honey for Establishing MRLs and Withdrawal Periods
Guidance for Industry
VICH GL56
Submit comments on this guidance at any time. Submit electronic comments to
https://www.regulations.gov/. Submit written comments to the Dockets Management
Staff (HFA-305), Food and Drug Administration, 5630 Fishers Lane, Rm. 1061,
Rockville, MD 20852. All comments should be identified with the docket number FDA-
2016-D-4461.
For further information regarding this document, contact AskCVM@fda.hhs.gov.
Additional copies of this guidance document may be requested from the Policy and
Regulations Staff (HFV-6), Center for Veterinary Medicine, Food and Drug
Administration, 7500 Standish Place, Rockville, MD 20855, and may be viewed on the
Internet at either https://www.fda.gov/AnimalVeterinary/default.htm or
https://www.regulations.gov/.
U.S. Department of Health and Human Services
Food and Drug Administration
Center for Veterinary Medicine
November 2018

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VICH GL 56 (MRK) – RESIDUES IN H ONEY
For Implementation at Step 7
Studies to Evaluate the Metabolism and
Residue Kinetics of Veterinary Drugs
in Food-Producing Species: Study
Design Recommendations for Residue
Studies in Honey for Establishing
MRLs and Withdrawal Periods
Adopted at Step 7 of the VICH Process by the VICH Steering Committee
in June 2018
for implementation by June 2019
This Guidance has been developed by the appropriate VICH Expert Working Group and has
been subject to consultation by the parties, in accordance with the VICH Process. At Step 7 of
the Process the final draft is recommended for adoption to the regulatory bodies of the
European Union, Japan and USA.

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Table of Contents
1. 	INTRODUCTION.......................................................................................................... 4
1.1. 	Objective of guidance ................................................................................................. 4
1.2 	Background .................................................................................................................. 4
2. 	GUIDANCE ................................................................................................................... 4
2.1. 	Purpose........................................................................................................................ 4
2.2. 	Scope ........................................................................................................................... 5
2.3. 	Residue Studies ........................................................................................................... 5
2.3.1. 	General considerations ............................................................................................ 5
2.3.2. 	Test Article .............................................................................................................. 5
2.3.3. 	Residues to monitor ................................................................................................. 6
2.3.4. 	Bees and beekeeping conditions .............................................................................. 6
2.3.5. 	Dosing and method of Administration .................................................................... 6
2.3.6. 	Study design ............................................................................................................ 7
2.3.7. 	Sampling .................................................................................................................. 8
2.4. 	Analytical Method for Assay of Residues .................................................................. 8
3. 	GLOSSARY ................................................................................................................... 9

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Studies to Evaluate the Metabolism and Residue Kinetics of
Veterinary Drugs in Food-Producing Species: Study Design
Recommendations for Residue Studies in Honey for
Establishings MRLs and Withdrawal Periods
Guidance for Industry
This guidance represents the current thinking of the Food and Drug Administration (FDA
or Agency) on this topic. It does not establish any rights for any person and is not binding
on FDA or the public. You can use an alternative approach if the approach satisfies the
requirements of the applicable statutes and regulations. To discuss an alternative
approach, contact the FDA office responsible for this guidance as listed on the title page.
1. 	INTRODUCTION
1.1. 	Objective of guidance
The objective of this guidance is to provide study design recommendations which will
facilitate the universal acceptance of the generated residue depletion data to fulfill the
national/regional requirements in order to establish appropriate Maximum Residue Limits
(MRLs) or other safe limits in honey following the treatment of honeybees with veterinary
drug products, or to justify withdrawal periods in honey for registration or approval
purposes, as applicable, when an MRL already exists.
Use of veterinary drug products in honeybee production is considered as a minor use in
minor species in most jurisdictions.
In general, FDA’s guidance documents do not establish legally enforceable
responsibilities. Instead, guidances describe our current thinking on a topic and should be
viewed only as recommendations, unless specific regulatory or statutory requirements are
cited. The use of the word should in FDA guidances means that something is suggested or
recommended, but not required.
1.2 	Background
This guidance is one of a series developed to facilitate the mutual acceptance of residue
chemistry data for veterinary drug products used in food-producing animals. This
guidance was prepared after consideration of the current national/regional requirements
and recommendations for evaluating veterinary drug residues in the VICH regions.
2. 	GUIDANCE
2.1. 	Purpose
Residue studies in honey are recommended for registration or approval, as applicable, of a
veterinary drug product for use in honeybees.

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These studies can be used to:
• 	measure the residues in honey
• 	generate data suitable for establishment of appropriate maximum residue limits.
• 	justify the withdrawal period for a veterinary drug product in accordance with an
existing MRL and/or generate data suitable for the establishment of risk-
management measures (e.g., use restrictions) in order to address consumer safety
concerns. It is generally accepted that the most practical withdrawal period in
honey is a zero-day withdrawal, meaning that all harvested honey should be safe
for human consumption. (Honey is harvested when at least 75% of the honeycells
within a frame are filled and capped.) However, additional measures (e.g., the
time interval between end of treatment and start of honey flow) may be
recommended to be followed.
Design elements for residue studies in honey differ in many respects from those in
commodities from other food producing species because honey is a unique food of animal
origin. There is minimal pharmacokinetic depletion of residues in honeybees following
treatment. When present in honey, residue concentrations are reduced mainly by dilution
as more honey is produced during the honey flow. Residue concentrations might also be
influenced by dissipation (if volatile), thermal degradation (as temperature inside the hive
reaches 32-36 ºC), acidic hydrolysis (honey pH ranges 3.4 – 6.1), or other chemical
reactions with honey matrix components. Honey production rate depends on factors such
as temperature, rain, season of the year, climatic zone, food source/type, and honeybee
species/subspecies.
2.2. 	Scope
The intention is that one set of residue studies, conducted in multiple locations within one
or more regions, would satisfy the data recommendations for establishment of appropriate
safe limits for a veterinary drug (i.e., the specific active substance)/veterinary drug product
in honey.
Studies should be conducted in conformity with the applicable principles of Good
Laboratory Practice (GLP).
2.3. 	Residue Studies
2.3.1. 	General considerations
When conducting residue studies for honey, treatments are typically applied to honeybee
colonies in accordance with Good Beekeeping Practice. Treatments are generally applied
once per year after honey harvesting and should be completed before honey flow
commences.
2.3.2. 	Test Article
The test article (the veterinary drug product) used for the study should be representative of
the commercial formulation. Final Good Manufacturing Practices (GMP) manufactured

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material (pilot scale or commercial scale) is the preferred source of test article; however,
laboratory scale preparations characterized with respect to GLP could also be appropriate.
2.3.3. 	Residues to monitor
Metabolic or total residue studies using radiolabelled drugs are not requested for MRL
assessment/approval of veterinary drug products used in honeybees. It is anticipated that
in most cases the residue to monitor would be the parent drug. However, data on
physicochemical properties of the active drug substance and other scientific information
might be useful to reveal the identity of putative transformation and/or degradation
products. If data indicate transformation or degradation of parent drug, an alternative
residue or combination of residues may be recommended to be monitored. For substances
that are prone to transformation/degradation, prior to conducting residue studies,
additional (in-vitro) studies can be used to determine their stability in honey (during its
production and up to its harvest). Variables to be tested include pH, moisture content,
temperature, time and exposure to (UV)-light. The selected conditions should be justified.
2.3.4. 	Bees and beekeeping conditions
Healthy and strong colonies should be used (See Glossary for ‘colony strength’). The
honeybee species/subspecies should be recorded. The colonies per site (See 2.3.6.) should
be uniform in adult honeybee population. The hives per site should be uniform in the
number of frames and box size and should be uniquely identified. Hive construction
should be described. The hives should consist of one brood box only. A super box with
frames should be added at the start of honey flow. The number of frames in each box
should be recorded.
Neither the colonies, boxes, nor frames should have a history of exposure to the veterinary
drug.
The study should be conducted at locations that mimic the conditions found at the time of
the year when apiarists would normally treat colonies with the particular veterinary drug
product.
2.3.5. 	Dosing and method of Administration
The design should cover the maximum treatment regimen. The method of applying the
veterinary drug product in the study should be representative of the intended commercial
use. The route of administration should be described in detail. All colonies per site
should receive the same treatment on the same day.
If the veterinary drug product is intended to be applied by more than one method, a
separate residue study for each method of administration is recommended. Alternatively,
a single study representing the worst-case scenario can be conducted with the resulting
safety parameters (i.e., proposed MRLs, use restrictions) being applicable to all methods.
Full justification of the worst-case scenario should be provided.

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2.3.6. 	Study design
Residue studies should be conducted in four sites of differing agro-ecological areas within
one or more regions. If the residue studies are intended to support an application for a
national license, then depending on the country of application (size, variety of landforms,
and climatic conditions), two to three sites (of differing agro-ecological areas) may be
considered appropriate. In such cases the national authorities should be consulted. For the
duration of the studies, information on climate (temperature, rainfall, and any other
parameter considered relevant for the performance of the veterinary drug product) and
beekeeping management practices should be provided. In addition, data on the plants in
the area in which honeybees forage during the study should be reported. If supplemental
feeding is needed to prevent starvation of a colony, it should be justified that this does not
influence honey production, and information (e.g., type, amount, duration) on the
supplemental feeding should be provided. Otherwise, the colony should be removed from
the study.
The time of treatment, the approximate time of the start of honey flow and the time of
sample collection/colony should be provided.
2.3.6.1 	Colonies
Six colonies per site should be treated, resulting in 24 colonies per residue study.
Depending on the type of application fewer treatment sites may be considered appropriate
(See 2.3.6 referring to application for a national license). A single sampling timepoint per
colony is considered appropriate. This is the first timepoint when honey from each colony
is ready to be harvested for human consumption (super honey from one or more frames).
Honey harvest refers to the collection of honey from the honeycombs once they are filled
with capped honey; at least 75% of the honeycells of the selected honeycombs should be
filled and capped. Alternative criteria to determine when to harvest honey should be
justified by the sponsor.
Figure 1 outlines a theoretical scheme (example) of sample collection per site.
2.3.6.2. Residues in Comb Honey for Lipophilic Drug Substances Only
Residue studies should be conducted as described in 2.3.6.1. In addition, for the last
colony harvested per site, a pooled wax sample (all available from the single colony)
should be collected and analyzed following honey extraction (Figure 1). To determine
residues in comb honey from the separate concentrations in honey and wax, consider that
1 kg of comb honey consists of 22/23 kg honey and 1/23 kg wax.1
2.3.6.3. Treatment during Honey Flow
In cases of veterinary drug products that could be used during the honey flow, the basic
study design should be followed. The sponsor should provide justification for
modifications to the basic study design. Points to consider are transfer of the veterinary
drug to existing and newly produced honey.
1 Beeswax Production, Harvesting, Processing and Products, Coggshall and Morse.Wicwas Press. 1984-06-01. p. 41.
ISBN 1878075063.

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2.3.7. 	Sampling
2.3.7.1. Sample Preparation
Super honey
The honey from all honeycombs in the collected frames from each colony should be
harvested. The honey should be extracted, filtered, and thoroughly mixed to produce a
pooled sample to represent that particular colony. Extraction of honey from a honeycomb
may be facilitated by centrifugation. Sample processing (all activities after sampling and
up to analysis) should take into account the stability properties of the residues. The
amount of the pooled honey produced should be provided. The pH and moisture content
of all pooled honey samples should be measured and reported.
Wax
For wax samples, the combs should be homogenized after honey extraction. Sample
processing (all activities after sampling and up to analysis) should take into account the
stability properties of the residues. The amount of the bulk wax sampled should be
provided.
2.3.7.2. Sample storage
If the chemical analysis cannot be completed immediately following sample collection, the
samples should be stored appropriately. If samples are stored after collection, the Sponsor
bears the responsibility for demonstrating residue stability through to the time of assay.
The parameters and recommendations to assess sample storage are discussed in CVM GFI
#208/VICH GL 49(R), “Studies to Evaluate the Metabolism and Residue Kinetics of
Veterinary Drugs in Food Producing Animals: Validation of Analytical Methods Used in
Residue Depletion Studies” (March 2015).2
2.4. 	Analytical Method for Assay of Residues
The Sponsor should submit a validated analytical method for the determination of the
residues in the samples generated. The method(s) should be capable of reliably
determining concentrations of the residues that encompass the appropriate proposed
reference point for honey (i.e., MRL).
The parameters to be included in the method validation are fully discussed in CVM GFI
#208/VICH GL 49(R), “Studies to Evaluate the Metabolism and Residue Kinetics of
Veterinary Drugs in Food Producing Animals: Validation of Analytical Methods Used in
Residue Depletion Studies” (March 2015) (See Footnote 2).
2 CVM GFI #208/VICH GL 49(R): Studies to Evaluate the Metabolism and Residue Kinetics of Veterinary Drugs in
Food Producing Animals: Validation of Analytical Methods Used in Residue Depletion Studies,
EMA/CVMP/VICH/463202/2009.
https://www.fda.gov/downloads/AnimalVeterinary/GuidanceComplianceEnforcement/GuidanceforIndustry/UCM20794
2.pdf

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3. 	GLOSSARY
The following definitions are applied for purposes of this document.
Beehive is a place used for housing a colony of bees, commonly stackable wooden boxes
consisting of a bottom board, brood box/es and super box/es containing movable frames.
Bee colony is the aggregate of worker bees, drones, queen, and developing brood living
together as a family unit in a hive or other dwelling.
Brood box (brood chamber) is a box in which the queen is confined and brood is reared.
Brood refers to eggs, embryo’s larval and pupal stages of bees.
Colony strength is evaluated by estimating the adult honey bee population in the hive and
depends on the time of the year and colony management practices.
Comb honey is honey stored by bees in the cells of freshly built broodless combs or thin
comb foundation sheets made solely of beeswax and sold in sealed whole combs or
sections of such combs (Revised Codex Standard for honey, 2001).
Frame is a rectangular wooden support designed to hold combs, usually 10 to each (brood
or super) box. In frames, a wax foundation is usually installed.
Good Beekeeping Practice refers to best practice recommendations found in numerous
references, for example: Hygiene in the apiary (A manual for hygienic beekeeping) Ed.
Dalibor Titera, BRI Dol 2009; National best management practice for beekeeping in the
Australian environment, The Australian honey bee industry council, 2007.
Honey is ‘the natural sweet substance produced by honeybees from the nectar of plants or
from secretions of living parts of plants or excretions of plant sucking insects on the living
parts of plants, which the bees collect, transform by combining with specific substances of
their own, deposit, dehydrate, store and leave in the honey comb to ripen and mature’
according to the Codex definition (Revised Codex Standard for honey, 2001).3
Honey flow is a period of time when one or more nectar or honeydew sources are in
abundance such that honeybees can store a surplus of honey.
Honey harvest refers to the collection of honey from the honeycombs once they are filled
with capped honey; at least 75% of the honeycells in a frame should be filled and capped.
Honeybees are a subset of bees in the genus Apis, primarily distinguished by the
production and storage of honey and the construction of perennial, colonial nests out of
wax. Although there are seven species of honeybees (A. andreniformis, A. cerana, A.
dorsata, A. florea, A. koschevnikovi, A. mellifera, A. nigrocincta) only two of them A.
mellifera (Western or European honeybee) and A. cerana are maintained by beekeepers,
with the former being the most commonly domesticated species. A. mellifera is native to
Europe, Asia and Africa and was introduced to North America in the early 1600s. Since
3 Revised Codex Standard for honey (2001). CODEX STAN 12-1981, Rev.1 (1987), Rev.2 (2001).
http://www.fao.org/fao-who-codexalimentarius/sh-
proxy/en/?lnk=1&url=https%253A%252F%252Fworkspace.fao.org%252Fsites%252Fcodex%252FStandards%252
FCODEX%2BSTAN%2B12-1981%252Fcxs_012e.pdf

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then it has spread worldwide. There are many subspecies that have adapted to the local
geographic and climatic environments, and in addition hybrid strains have been bred [e.g.,
the Africanized bee (A. mellifera lingustica X A. m. scutellata)].
Honeycomb is a mass of hexagonal wax cells built by honeybees to contain their stores of
honey and pollen (honeycomb in the super box) or for raising brood (broodcomb in the
brood box).
Lipophilic substance refers to a chemical substance having high (log Kow≥3)
octanol/water partition coefficient.
Maximum residue limit (MRL) is the maximum concentration of a veterinary drug
residue that is legally permitted or recognized as acceptable in or on a food as set by a
national or regional regulatory authority. The term ‘tolerance,’ used in some countries,
can be, in many instances, synonymous with MRL.
Residue means the veterinary drug (parent) and/or its metabolites. In the case of honey
this may include transformation and degradation products.
Super box is a box in which the honeybees store honey (super honey) and that is placed
above a queen excluder and the brood chamber.
Wax (or comb) foundation is a plate made of wax with the base of the honeycomb on
which honeybees will construct a complete comb.
Withdrawal period is the period necessary between the last administration of a veterinary
drug product to animals and the production of foodstuffs from such animals, in order to
protect public health by ensuring that such foodstuffs do not contain residues in quantities
in excess of the maximum residue limits established.
Zero-day withdrawal refers to a label indication that allows entry of edible tissues/animal
products into the food chain without regard to the time of last drug administration.

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Figure 1. A theoretical scheme of sample collection per site.
The figure outlines a theoretical example of sample collection per site. A single sampling
timepoint per colony is considered to be appropriate. This is the first timepoint when
honey from each colony can be harvested for human consumption (only super honey from
one or more frames). Honey harvest refers to the collection of honey from the
honeycombs which are filled with capped honey. The figure illustrates that the time points
when honey is mature (at least 75% of the honeycells in one or more frames are filled and
capped) and the number of mature honeycombs per colony usually varies from hive to
hive. The first honey harvest is considered the worst case scenario in terms of residues.