#232 
 
STUDIES TO EVALUATE THE SAFETY OF 
RESIDUES OF VETERINARY 
DRUGS IN HUMAN FOOD: 
GENERAL APPROACH TO ESTABLISH AN 
ACUTE REFERENCE DOSE (ARFD) 
 
Guidance for Industry 
 
VICH GL54 
 
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, Room 1061, Rockville, MD 
20852.  Identify all comments with the Docket No. FDA-2015-D-1804. 
 
For further information regarding this document, contact AskCVM@fda.hhs.gov. 
 
Additional copies of this guidance document may be requested from the Policy and 
Communications 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 
August 2017 
    Contains Non-Binding Recommendations 
 
VICH GL54 (SAFETY)  ARfD 
 
For Implementation at Step 7 
 
 
 
 
 
S E S  
TUDIES TO  VALUATE THE  AFETY OF
R V D  
ESIDUES OF  ETERINARY  RUGS IN
H F : 
UMAN  OOD
G A E  
ENERAL  PPROACH TO  STABLISH
A R D (AR D) 
AN  CUTE  EFERENCE  OSE  F
                 
 
 
 
 
Adopted at Step 7 of the VICH Process by the VICH Steering Committee in November 2016 
for implementation by November 2017. 
 
 
 
 
 
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. 
 
   
2  Contains Non-Binding Recommendations 
 
Table of Contents 
 
 
1.  Introduction......................................................................................................................... 4 
1.1  Objective .................................................................................................................................................. 4 
1.2  Background ............................................................................................................................................. 4 
1.3  Scope of the current guidance ........................................................................................................... 5 
2.  Guidance for an ARfD ........................................................................................................ 6 
2.1  Stepwise procedure .............................................................................................................................. 6 
2.2  Information and studies to support an ARfD ................................................................................ 7 
2.2.1  Use of traditional repeat-dose toxicology studies .................................................. 7 
2.2.2  Acute studies ........................................................................................................... 8 
2.3  How to derive an ARfD ....................................................................................................................... 8 
3.  Glossary .............................................................................................................................. 9 
4.  References ......................................................................................................................... 11 
5.  Annex 1 - Procedure for the Derivation of an Appropriate ARfD .................................... 12 
   
3  Contains Non-Binding Recommendations 
 
STUDIES TO EVALUATE THE SAFETY OF RESIDUES OF VETERINARY DRUGS IN HUMAN FOOD: 
GENERAL APPROACH TO ESTABLISH AN ACUTE REFERENCE DOSE (ARFD) 
 
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 it 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. 
 
  Introduction 
 
1.1  Objective 
 
The current guidance addresses the nature and types of data that can be useful in determining a 
toxicological acute reference dose (ARfD) for residues of veterinary drugs, the studies that may 
generate such data, and how the ARfD may be calculated based on these data. 
 
In general, FDAs guidance documents do not establish legally enforceable responsibilities.  
Instead, guidances describe the Agencys 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 Agency guidances means that something is suggested or recommended, but 
not required. 
 
1.2  Background 
 
The safety of residues of veterinary drugs in human food is most commonly addressed through 
the conduct of toxicology studies in test animal species that provide for the determination of a 
no-observed-adverse-effect level (NOAEL)1 and an acceptable daily intake (ADI) by application 
of appropriate safety/uncertainty factors (UF(s)).2  The ADI, generally expressed as microgram 
(g) or milligram (mg)/kg body weight per day, is defined as the daily intake which, for up to an 
entire lifetime, appears to be without adverse effects or harm to the health of the consumer (see 
Glossary). 
 
It has been recognized that there is the potential for some veterinary drug residues to cause 
adverse effects in the human consumer following a single meal.  The ADI may not be the 
appropriate value in such cases for quantifying the level above which exposure after a single 
                                                 
1 Both the terms NOEL (no-observed-effect level) and NOAEL (no-observed-adverse-effect level) have historically 
been used to establish an ADI.  In practice, NOEL and NOAEL have had similar meanings when used for this 
purpose. 
2 While some regulatory authorities use the term safety factor and others use the term uncertainty factor, there is 
a general agreement in the application of these terms to address variability between  groups (e.g., from animal 
models to humans) and within groups (e.g., animal to animal or human to human variability).  For the purpose of 
this document, UFs will be used to represent the use of either safety or uncertainty factors. 
4  Contains Non-Binding Recommendations 
 
meal or over one day can produce acute adverse effects.  Determining the ARfD is an 
appropriate approach to address this concern. 
 
The ARfD approach has been developed to provide a human health guidance value for pesticides 
and other chemicals, including veterinary drugs, when their use can result in residues high 
enough to cause adverse effects following acute or short-term exposures in people consuming 
large portions of food containing the residue.  This contrasts with the use of ADIs, which are 
established to address potential adverse effects following chronic or long-term exposures to 
residues in foods. 
 
Various publications which describe the ARfD approach are available.  In 2005, some members 
of the United Nations Joint Food and Agriculture Organization (FAO)/World Health 
Organization (WHO) Meeting on Pesticide Residues (JMPR) published a paper describing the 
development of the ARfD for acute health risk assessment of agricultural pesticides (Solecki et 
al., 2005).  The Organization for Economic Co-Operation and Development (OECD) has 
finalized Guidance No. 124, Guidance for the Derivation of an Acute Reference Dose, which 
is primarily intended for pesticides, biocides, and veterinary drugs (IOMC, 2010).  The OECD 
Guidance No. 124 describes a tiered approach that is intended to maximize the use of available 
data and minimize the need for studies specifically designed to derive an ARfD.  This approach 
is consistent with the 3-Rs (Replacement, Refinement and Reduction) minimizing the use of 
animals in the development of veterinary drugs.  In addition, the Joint FAO/WHO Expert 
Committee on Food Additives (JECFA) has noted that certain substances ..... e.g., some metals, 
mycotoxins, or veterinary drug residues, could present an acute risk, i.e., could raise concern 
regarding acute health effects in relation to short periods of intake at levels greater than the ADI 
or TDI3.  JECFA agreed that, building on the experience of and the guidance developed by 
JMPR ..... the need to establish an ARfD should be considered on a case-by-case basis, and only 
if the substance, on the basis of its toxicological profile and considering the pattern of its 
occurrence and intake, is likely to present an acute health risk resulting from exposure in a period 
of 24 h or less (JECFA, 2005).  JECFA and JMPR have contributed to the International 
Program on Chemical Safety (IPCS) Environmental Health Criteria (EHC) 240 describing the 
derivation of an ARfD in the application of a maximum residue limit (MRL), a tolerance, or 
other national or regional tools used to establish an acceptable concentration of residues of the 
veterinary drug in the edible tissues of treated animals (IPCS, 2009).  In 2016, JECFA published 
a draft guidance for monographers on the use and interpretation of the ARfD.  This document 
continues to be under development. 
 
1.3  Scope of the current guidance 
 
This guidance can be used to address the nature and types of data that should be useful in 
determining an ARfD, the studies that may generate such data, and how the ARfD can be 
calculated based on these data.  The current guidance is limited to the application of 
toxicological and pharmacological endpoints and offers special consideration for residues of 
veterinary drugs in contrast to the available guidances and guidances that address the derivation 
and use of the ARfD for human exposure to pesticides, contaminants, and chemicals other than 
veterinary drugs.  The guidance provides internationally harmonized technical recommendations 
                                                 
3 TDI  tolerable daily intake. 
5  Contains Non-Binding Recommendations 
 
for an ARfD used in support of veterinary product registration/approval.  Detailed guidance on 
the derivation of an ARfD may be found in OECD Guidance 124 (IOMC, 2010). 
 
This guidance does not, except in very broad terms, address  
 
  When an ARfD would or would not be appropriate to address the concerns of a national 
or regional regulatory authority. 
  Evaluation of specific pharmacological or toxicological adverse effects that may lead to 
the determination of an ARfD. 
  Human dietary exposure data that may be appropriate for use with an ARfD in the 
derivation of an MRL, a tolerance or other national or regional tools used to refine an 
acceptable concentration of the veterinary drug residue in food. 
  Refinement of the exposure calculation for the acute health risk assessment. 
  Routes of human exposure to veterinary drugs other than the oral route. 
 
Recognizing international efforts to address possible acute effects of residues of an antimicrobial 
veterinary drug on the human intestinal microbiota following acute human exposure, the current 
guidance only provides a harmonized approach to a toxicological ARfD at this time. 
 
Finally, this guidance does not seek to limit the studies that can be performed to establish the 
safety of residues in human food with respect to acute toxicity.  Neither does it preclude the 
possibility of alternative approaches that can offer an equivalent assurance of safety, including 
scientifically-based reasons as to why such data are not warranted. 
 
  Guidance for an ARfD 
 
2.1  Stepwise procedure 
 
Before examining the endpoints of acute pharmacological effects and toxicity, and before 
designing studies, careful consideration should be given to the 3-Rs principles.  Therefore, the 
following stepwise approach is recommended before conducting an acute toxicity study: 
 
  Step 1.  Evaluate available pharmacological and toxicological data and information, 
including data from repeated-dose toxicity studies, in order to establish whether or not 
acute endpoints (attributable to the first 24 hours of dosing) have been adequately 
addressed. 
 
  Step 2.  If additional acute toxicity information is needed, consideration to the 3-Rs 
principle should be given, for example, by integrating observations/examinations related 
to acute endpoints in planned standard toxicity studies. 
 
  Step 3.  If the two options in Steps 1 and 2 are insufficient to provide adequate 
information on acute endpoints, then a new, specifically designed toxicity study(ies) can 
be considered. 
 
  See also the decision tree in Annex 1.   
6  Contains Non-Binding Recommendations 
 
2.2  Information and studies to support an ARfD 
 
The first consideration should be to examine available data and information that describe the 
physical, chemical, pharmacological, and toxicological characteristics of the veterinary drug.  
This information can be available from data provided to support human food safety as per VICH 
GL33 or through published peer reviewed literature.  In addition the studies provided under 
VICH GL33 to support safety may provide useful information for the evaluation of acute toxicity 
endpoints that support the assessment of an ARfD.  It is recommended that all information on a 
specific veterinary drug be considered in the derivation of a chemical specific ARfD. 
 
2.2.1  Use of traditional repeat-dose toxicology studies 
 
The following are key points for consideration when evaluating information regarding the 
potential for acute toxicity: 
 
  In the absence of data to the contrary, all relevant indications of acute adverse 
pharmacological and toxicological effects observed in repeated-dose studies can be 
considered as potentially relevant to setting an ARfD. 
  Particular emphasis should be given to observations and investigations at the beginning 
of repeated dose studies. 
 
Examples of potential endpoints of acute toxicity in standard toxicity studies include those 
described in OECD Guidance No. 124 (see paragraphs 36 through 59) and in EHC 240 (see 
section 5.2.9.5).  Endpoints could include, but are not limited to, haematoxicity, immunotoxicity, 
neurotoxicity, hepatotoxicity, nephrotoxicity, developmental effects, reproductive effects, 
pharmacological effects, and direct effects on the gastrointestinal tract as well as clinical 
findings.  In keeping with the goal of reducing the number of animals for testing, in some cases, 
it may be possible to modify the standard toxicology study protocols to provide more relevant 
information for the assessment of the ARfD without compromising the original objective of the 
study.  For example, a veterinary drug might be anticipated to cause acute haematological 
changes; the protocol for a repeat-dose oral toxicity study in rats could be modified to include 
satellite groups where blood is sampled from control and treated animals beginning on the first 
day through the first two weeks of dosing to evaluate whether this endpoint occurs after one or 
just a few doses.  If no effects are observed in the high dose group then no further evaluation of 
the collected samples would be warranted.  Further, in this example a lower bound for potential 
acute toxicity may be established based on the high dose group in the study.  In addition to the 
endpoints mentioned in EHC 240, adverse effects observed at the beginning of the study should 
be taken into consideration. 
 
Prior to modification of an existing protocol, consideration should be given to available data and 
information that describe the physical, chemical, pharmacological, and toxicological 
characteristics of the veterinary drug, including its possible mode of action (MOA).  While the 
relevant dosing for assessment of an ARfD is anticipated to be an acute dose (a single dose or up 
to a single days dosing), the timing for measurement of effects should be based on an 
understanding of available pharmacokinetics and pharmacodynamics of the veterinary drug.  
Particular emphasis should be given to observations and investigations at the beginning of the 
7  Contains Non-Binding Recommendations 
 
repeat-dose study in the determination of potential acute toxicity.  The inclusion of selected 
endpoints for the evaluation of acute toxicity beyond those described in the guidance documents 
should be considered on a case-by-case basis. 
 
Consideration should be given to dose selection, numbers of animals, and the use of satellite 
groups.  A high dose group within the repeat-dose toxicity study protocol that is relevant to 
concerns related to acute exposure to the human consumer could inform an ARfD evaluation.  
Elements of study design described in OECD Guidance No. 124, Annex 2, can be incorporated 
into modifications of an existing repeat-dose toxicity study.  Dose selection is also critical when 
developing a point of departure (POD) for the derivation of the ARfD.  The POD from the most 
sensitive endpoint relevant to human food safety in the most appropriate species should be used. 
 
2.2.2  Acute studies 
 
In some cases, an appropriate POD to determine an ARfD is not available from existing 
information.  Studies intended to address chronic toxicity may not provide sufficient information 
to allow a robust estimate of the ARfD.  In such cases, a single exposure study specifically 
designed to support an ARfD for a given veterinary drug may be warranted.  In all cases, it is 
recommended that the design of an acute effect study specifically to derive an ARfD include 
consideration of all available relevant physical, chemical, pharmacological, and toxicological 
information, and also consider the MOA (particularly of the pharmacologically active substance) 
where relevant. 
 
Specific guidance on the conduct of a single exposure toxicity study can be found in Annex 2 of 
OECD Guidance No. 124. 
 
2.3  How to derive an ARfD 
 
The basic approach for the derivation of an ARfD is based on the identification of an appropriate 
POD, or threshold, for the pharmacological or toxicological endpoint of concern.  This is 
typically identified as a NOAEL dose or benchmark dose lower confidence limit (BMDL).  The 
ARfD is determined by dividing this POD by an appropriate UF(s).  The ARfD can be reported 
as an amount of the substance expressed on a per person or body weight basis (e.g., mg/person or 
mg/kg body weight) 
 
POD
ARfD=  
UF
 
Where: 
 
POD is the point of departure or threshold for pharmacological or toxicological effects of 
concern (see Glossary). 
 
UF is an uncertainty or safety factor, or series of factors that typically account for considerations 
such as animal to animal variability, interspecies extrapolation, quality of data, severity of 
response, etc. (see Glossary).  Additional recommendations on the selection of an appropriate 
8  Contains Non-Binding Recommendations 
 
uncertainty factor (described as a chemical specific assessment factor) are provided in Step One 
of the Tiered-Approach for the Derivation of an appropriate ARfD in OECD Guidance No 124 
(IOMC, 2010). 
 
Consideration should be given to the discussion of uncertainty factors in OECD Guidance No. 
124 (see page 21) and EHC 240 (see section 5.2.3).  The selection of appropriate UFs for inter-
species and human inter-individual variabilities should be considered based on available data.  
To provide for the quantitative incorporation of differences in the toxicokinetic/toxicodynamics 
for a chemical, the default 10-fold factor for inter-species variability and the default 10-fold 
factor for human inter-individual variability can be used.  When available, chemical-specific UFs 
on one or more specific sources of variability could replace the default values to adjust sub-
factors for inter-species and human inter-individual variabilities.  If chemical specific 
toxicokinetic and toxicodynamic data are inadequate to justify data based UFs, consider any 
information (e.g., quantitative structure-activity relationship (QSAR) or MOA, of closely related 
compounds) that would indicate reduced or increased uncertainty. 
 
When an ARfD could be determined based on toxicological and/or pharmacological endpoints, 
the ARfD should be based on the endpoint that is most relevant for protecting public health. 
 
  Glossary 
 
The following definitions apply for purposes of this guidance: 
 
3-Rs  Replacement, Refinement, Reduction.  VICH is committed to approaches that 
reduce, refine or replace the use of laboratory animals (the 3Rs) while maintaining appropriate 
scientific standards. The 3Rs principles were first introduced in Russell and Burch's 1959 book, 
'The principles of humane experimental technique'. 
 
ADI  Acceptable Daily Intake is the daily intake which, during up to an entire life of a 
human, appears to be without adverse effects or harm to the health of the consumer.  The ADI 
most often will be set on the basis of the drugs toxicological, microbiological, or 
pharmacological properties.  It is usually expressed in micrograms or milligrams of the chemical 
per kilogram of body weight per day. 
 
ARfD  Acute Reference Dose.  An estimate of the amount of residues expressed on a 
body weight basis that can be ingested in a period of 24 h or less without adverse effects or harm 
to the health of the human consumer. 
 
BMD  Benchmark Dose.  A dose of a substance associated with a specified low 
incidence of response, generally in the range of 1 to 10%, of a health effect, or a dose associated 
with a specified measure or change of a biological effect.  See Benchmark Dose Software 
(BMDS) (US Environmental Protection Agency, 2015), and PROAST (National Institute for 
Public Health and the Environment (RIVM), 2014). 
 
BMDL  Benchmark Dose Lower Confidence Limit.  A dose producing an appropriate, 
low, and measurable response at a defined lower bound response level based on the lower one-
9  Contains Non-Binding Recommendations 
 
sided confidence limit of a 95% confidence interval extrapolated from a line fitted to available 
data for an appropriate endpoint. 
 
EHC  Environmental Health Criteria. International Program on Chemical Safety (IPCS) 
documents that provide international critical reviews on the effects on human health and the 
environment of chemicals or combinations of chemicals, including veterinary drugs, as well as 
physical and biological agents. 
 
IPCS  International Program on Chemical Safety.  A joint program of the World Health 
Organization, International Labor Organization and the United Nations Environment 
Programme. 
 
MOA  Mode of Action.  A biologically plausible sequence of key events leading to an 
observed effect supported by robust experimental observations and mechanistic data.  A mode of 
action describes key cytological and biochemical events, that is, those that are both measurable 
and necessary to the observed effect in a logical framework. 
 
NOEL  No Observed Effect Level.  The highest administered dose that was observed not 
to cause an effect in a particular study. 
 
NOAEL  No Observed Adverse Effect Level.  The highest administered dose that was 
observed not to cause an adverse effect in a particular study. 
 
OECD  Organization for Economic Co-operation and Development brings together the 
governments of various countries to support sustainable economic growth, boost employment, 
raise living standards, maintain financial stability, assist other countries economic development 
and contribute to world trade. 
 
POD  Point of Departure.  A reference point for hazard characterization; typically a 
point on a dose-response curve at which the response first becomes apparent, and represents 
toxicological or pharmacological effects of concern; often classified as a NOEL, NOAEL, or 
BMDL. 
 
QSAR  Quantitative Structure Activity Relationship.  A quantitative relationship between 
a biological activity (e.g., toxicity) and one or more molecular descriptions that are used to 
predict activity. 
 
Satellite Groups  Additional groups of animals typically treated following all or some of the 
study treatment protocol and then examined for endpoints that differ from the main study group 
or are in other ways treated differently.  For example, a satellite group of rats receiving all 
treatments but limited to a few animals per treatment group can be used for 
pharmacokinetic/toxicokinetic measurements, or a satellite group containing all treatment groups 
but only receiving a single dose can be used to examine acute effects in a subchronic repeat dose 
study. 
 
10  Contains Non-Binding Recommendations 
 
UF  Uncertainty Factors.  Typically UFs are intended to account for uncertainty in 
extrapolating animal data to humans (inter-species variability), the variation in sensitivity among 
humans (inter-individual variability), quality of data, severity of response, or other concerns, 
where available sources of variability can be replaced with chemical specific information to 
refine the UF, such as toxicokinetics, toxicodynamics, QSAR, MOA, and information on closely 
related compounds. 
 
  References 
 
US Environmental Protection Agency (EPA).  Benchmark dose software (BMDS) and Users 
Manual.  Available from:  https://www.epa.gov/bmds. 
 
European Food Safety Agency (EFSA).  Guidance of the Scientific Committee on a request from 
EFSA on the use of the benchmark dose approach in risk assessment.  The EFSA Journal.  2009. 
1150:1-72. 
 
Inter-Organization Programme for the Sound Management of Chemicals (IOMC).  2010.  OECD 
Environment Directorate Joint Meeting of the Chemicals Committee and the Working Party on 
Chemicals, Pesticides and Biotechnology. Series on Testing and Assessment. No. 124. Guidance 
for the Derivation of an Acute Reference Dose. Env/JM/MONO(2010)15. 
 
Joint FAO/WHO Expert Committee on Food Additives.  Sixty-fourth meeting.  Rome, 8-17 
February 2005.  Summary and Conclusions.  Available from: 
ftp://ftp.fao.org/es/esn/jecfa/jecfa64_summary.pdf. 
 
International Programme on Chemical Safety.  2009.  Environmental Health Criteria 240.  
Principles and Methods for the Risk Assessment of Chemicals in Food.  Food and Agriculture 
Organization of the United Nations and World Health Organization. 
 
Solecki, R, Davies L, Dellarco V, Dewhurst I, van Raaij M, and Tritscher A. 2005.  Guidance on 
setting of acute reference dose (ARfD) for pesticides.  Food and Chemical Toxicology 43:1569-
1593. 
 
VICH.  2009. VICH Guidance 33, Safety Studies for Veterinary Drug Residues in Human Food: 
General Approach to Testing. 
 
   
11  Contains Non-Binding Recommendations 
 
  Annex 1 - Procedure for the Derivation of an Appropriate ARfD 
 
 
When ARfD is appropriate 
Evaluate Existing Database 
Select the Appropriate Endpoints 
Incorporate Appropriate Endpoints 
into Repeat-Dose Study, or 
Conduct Acute Toxicity Study 
No 
Is the 
Available 
Need more information 
Information 
Adequate? 
Yes 
Select the Appropriate Point of Departure 
Select an Uncertainty Factor 
Calculate ARfD 
 
12