#171 
 
Demonstrating Bioequivalence for Soluble 
Powder Oral Dosage Form Products and 
Type A Medicated Articles Containing Active 
Pharmaceutical Ingredients Considered to Be 
Soluble in Aqueous Media 
 
Guidance for Industry 
 
This guidance document makes revisions to the guidance published in May 2021.  This revision updates 
the recommendations for using the dose adjusted approach for solubility determinations in poultry, as 
well as updates the appropriate buffers for use in solubility determinations involving cattle.  
 
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-2019-D-3764. 
 
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 https://www.fda.gov/animal-
veterinary, https://www.fda.gov/regulatory-information/search-fda-guidance-documents, or 
http://www.regulations.gov. 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Veterinary Medicine (CVM) 
June 2023
 
  Contains Nonbinding Recommendations 
 
Table of Contents 
 
I.  Introduction .......................................................................................................................... 1 
II.  Background .......................................................................................................................... 2 
III.  Human Food Safety Considerations .................................................................................. 3 
IV.  Biowaivers for Soluble Powder Oral Dosage Form Products .......................................... 3 
A.  Qualifying for a waiver from the requirements for performing an in vivo 
bioequivalence study (biowaiver) .............................................................................. 3 
B.  Data to support a waiver from the requirement to perform an in vivo 
bioequivalence study (biowaiver) request .................................................................... 3 
1.  Information about the API(s): ................................................................................ 4 
2.  Information about the formulation: ....................................................................... 4 
V.  Biowaivers for Type A Medicated Articles Manufactured from Water-Soluble Active 
Pharmaceutical Ingredients ................................................................................................ 4 
A.  Qualifying for a waiver from the requirements for conducting an in vivo 
bioequivalence study (biowaiver) ................................................................................. 4 
B.  Data to support a waiver from the requirement to perform an in vivo 
bioequivalence study (biowaiver) request .................................................................... 5 
1.  Information about the API(s): ................................... Error! Bookmark not defined. 
2.  Information about the formulation: ....................................................................... 6 
3.  Solubility Data:......................................................................................................... 6 
C.  Manufacturing process .................................................................................................. 6 
D.  CVM evaluation of solubility data................................................................................ 6 
1.  Calculation based on the Maximum Daily Dose: .................................................. 7 
2.  Calculations based on the Dosage Adjusted Approach: ....................................... 7 
E.  Solubility determination ................................................................................................ 8 
F.  Technical parameters to consider include: .................................................................. 9 
1.  Use of the Shaker flask: ........................................................................................... 9 
2.  Media composition and pH considerations: .......................................................... 9 
3.  Other considerations: ............................................................................................ 10 
VI.  GLOSSARY........................................................................................................................ 10 
i  Contains Nonbinding Recommendations 
 
Demonstrating Bioequivalence for Soluble Powder Oral Dosage 
Form Products and Type A Medicated Articles Containing Active 
Pharmaceutical Ingredients Considered to Be Soluble in Aqueous 
Media 
 
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 staff 
responsible for this guidance as listed on the title page. 
 
I.  Introduction 
This document describes how the Center for Veterinary Medicine (CVM) intends to evaluate 
requests for waiving the requirement for performing in vivo bioequivalence studies (biowaivers) 
for animal drugs administered orally as soluble powders or as Type A medicated articles 
manufactured from active pharmaceutical ingredients (APIs) considered to be soluble in aqueous 
media (water-soluble APIs).  This document expands upon CVMs Guidance for Industry (GFI) 
#35, Bioequivalence Guidance,1 to include biowaivers for soluble powder oral dosage form 
products as well as Type A medicated articles manufactured from APIs considered to be soluble 
in aqueous media, and it offers particular focus on criteria for the waiver of the requirements for 
submitting in vivo bioequivalence study data.  This guidance does not address Type A medicated 
articles manufactured from APIs considered to be insoluble in aqueous media. 
 
This guidance is applicable to generic investigational new animal drug (JINAD) files and to 
abbreviated new animal drug applications (ANADAs).  Although the recommendations in this 
guidance refer to ANADAs, the general principles described may also be applicable to new 
animal drug applications (NADAs), supplemental NADAs, and investigational new animal drug 
(INAD) files. 
 
The recommendations in this guidance are premised on the assumption that a sponsor will be 
bridging between identical dosage forms (e.g., their Type A medicated article for use in complete 
feed to the approved reference Type A medicated article for use in complete feed).  Therefore, 
you should not use the recommendations in this guidance to compare the solubility of two drug 
products where the API will be administered in differing manners (e.g., drinking water versus 
complete feed; complete feed for administration throughout the day versus top dress).  CVM 
encourages sponsors to contact the Center to discuss that type of comparison. 
 
The granting of a waiver from the requirement to perform an in vivo bioequivalence study does 
not imply that a drug product is approvable.  For drug product approval, all applicable legal 
 
1 https://www.fda.gov/media/70115/download 
1  Contains Nonbinding Recommendations 
 
requirements must be met (see, e.g., sections 512(c)(2) and 512(n) of the Federal Food, Drug, 
and Cosmetic Act (FD&C Act)). 
 
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 guidance means that something is suggested or recommended, but 
not required. 
 
II.  Background 
An ANADA must include information to show that the proposed generic new animal drug and 
reference listed new animal drug (RLNAD) are bioequivalent.2  This requirement is patterned 
very closely on the approval requirements for human generic drugs.3 
 
The Center for Drug Evaluation and Researchs (CDER) regulations implementing the 
bioequivalence requirement for human generic drugs can be found in 21 CFR part 320.  In most 
cases, there must be an in vivo demonstration of no significant differences in the rate and extent 
of drug availability associated with the proposed generic and reference drug products when 
administered at the same molar dose under similar conditions.4  In certain circumstances, 
however, the demonstration of bioequivalence does not need to be established on the basis of in 
vivo studies.5  For several categories of human drug products, including oral solutions, 
bioequivalence is considered self-evident under specified conditions.6  In other circumstances, a 
large body of research on human intestinal physiology has been used to support a determination 
of product bioequivalence of solid oral dosage forms based on the use of in vitro approaches.  In 
this regard, the human Biopharmaceutics Classification System (BCS) criteria have been applied 
to support the use of an in vitro approach to document product bioequivalence for highly soluble, 
rapidly dissolving, and orally administered drug products.7  However, because of the 
physiological differences between the gastrointestinal (GI) tract of humans and that of veterinary 
species, and because of the additional complexities associated with drugs that are administered as 
medicated feeds, the human BCS criteria cannot be applied to support the use of an in vitro 
approach for demonstrating product bioequivalence for orally administered veterinary drug 
products. 
 
CVM has issued guidance on in vivo bioequivalence studies, which includes a list of some of the 
product categories, including oral solutions and other solubilized forms, that may be eligible for 
a waiver from the requirement to perform in vivo bioequivalence studies.8  This guidance 
 
2 Section 512(n)(1)(E) of the FD&C Act 
3 Section 505(j)(2)(A)(iv) of the FD&C Act 
4 21 CFR 320.1(e) and 320.21(b)  
5 21 CFR 320.21(b) and (f), and 320.22 
6 21 CFR 320.22(b)(3) 
7 See CDER Guidance for Industry, Waiver of In Vivo Bioavailability and Bioequivalence Studies for Immediate-
Release Solid Oral Dosage Forms Based on a Biopharmaceutics Classification System, December 2017, pages 2 
and 11-12.  (https://www.fda.gov/media/70963/download) 
8 See footnote 2. 
2    Contains Nonbinding Recommendations 
 
provides additional information and recommendations regarding bioequivalence waivers for 
soluble powder oral dosage form products intended for use in animal drinking water, and Type A 
medicated articles manufactured from APIs considered to be soluble in aqueous media and 
intended for use in animal feed. 
 
III.  Human Food Safety Considerations 
The granting of a waiver from the requirement to perform an in vivo bioequivalence study 
(biowaiver) does not imply that a drug product is approvable.  For drug product approval, all 
applicable legal requirements must be met, which includes addressing the tissue residue portion 
of the Human Food Safety (HFS) technical section of the application (sections 
512(c)(2)(A)(viii), 512(c)(2)(B), and 512(n)(1)(A) of the FD&C Act).  For products that receive 
a biowaiver using the concepts of this guidance, the sponsor should contact the Division of 
Human Food Safety (HFV-150) directly to discuss what, if any, additional information may be 
needed to satisfy the requirements for approval. 
 
IV.  Biowaivers for Soluble Powder Oral Dosage Form Products 
A.  Qualifying for a waiver from the requirements for performing an in vivo 
bioequivalence study (biowaiver) 
CVM believes it is appropriate to grant biowaivers for oral dosage forms known as 
soluble powders that meet the solubility requirements discussed in this guidance.  Such 
products are intended for administration to animals via the drinking water that is provided 
on an ad libitum basis under most husbandry systems. 
 
The conceptual basis for granting biowaivers for soluble powders is that if an API is in 
solution before administration, the drug products formulation will usually not influence 
the bioavailability of the active ingredient.  If the test and reference formulations contain 
essentially the same inactive ingredients, then from a mechanistic perspective, the rate-
limiting step in systemic API absorption will be either: (a) the rate of gastric transit; or (b) 
the permeability of the API across the gastrointestinal (GI) mucosal membranes.  
Typically, both these variables are formulation independent, relying solely on the API and 
the characteristics of the GI tract of that animal species.  Similarly, because the rate-
limiting step is the API movement down the GI tract and its lateral diffusion across the 
viscous intestinal contents, if an API acts locally within the GI tract (i.e., not systemically 
absorbed), the local exposure to the dissolved API in the proposed and reference drug 
product formulations will be equivalent if the API is already in solution.  The only 
exceptions of which CVM is aware are when the formulation of the drug product contains 
substances other than the API that could cause a direct pharmacologic effect (e.g., altered 
GI transit time, membrane permeability, or drug metabolism) or when there is inactivation 
of the API. 
 
B.  Data to support a waiver from the requirement to perform an in vivo 
bioequivalence study (biowaiver) request 
For soluble powder oral dosage form products, CVM recommends that justification of 
requests for biowaivers be made by a demonstration of solubility.  CVM recommends that 
3    Contains Nonbinding Recommendations 
 
sponsors make these biowaiver requests before submitting an ANADA.  The following 
should be provided in the biowaiver request: 
 
1.  Information about the API(s) 
CVM recommends that the API(s) used to support a biowaiver request for soluble 
powder oral dosage forms be provided from the same supplier of the API(s) that will be 
used to formulate the proposed drug product during production.  CVM recommends 
that the applicant identify the source of the API(s) and provide a certificate of analysis 
(COA) for the API(s).  If the API has a United States Pharmacopeia (USP) monograph, 
the API should at a minimum comply with the monograph specifications.  The 
acceptability of the API(s) will be determined upon review of the Chemistry, 
Manufacturing, and Controls (CMC) Technical Section. 
 
2.  Information about the formulation 
The formulation of the proposed drug product should be submitted.  The formulation 
should be such that there are no differences between the RLNAD and the proposed 
drug product that would have an effect on the bioavailability of the API.  There should 
be no inactive ingredients in the proposed drug products formulation likely to cause 
adverse pharmacologic effects.  The API should be qualitatively (Q1) and 
quantitatively (Q2) the same as the API used in the RLNAD.9  The solubility of the 
drug product should be determined under the range of physical conditions that a user of 
the product would typically encounter when reconstituting the soluble powder with 
animal drinking water (i.e., well or municipal water) in the field.  This study is the same 
as the reconstitution study outlined in GFI #5, Drug Stability Guidelines, section 
IV.F. Soluble Powders and Drinking Water.10  The reconstitution data for soluble 
powder oral dosage form products should be submitted in the Chemistry, 
Manufacturing, and Controls (CMC) Technical Section. 
 
V.  Biowaivers for Type A Medicated Articles Manufactured from Water-Soluble Active 
Pharmaceutical Ingredients 
A.  Qualifying for a waiver from the requirements for conducting an in vivo 
bioequivalence study (biowaiver) 
Type A medicated articles that contain APIs that are not classified as water soluble are not 
the subject of this guidance.  The determination of the water solubility of APIs is discussed 
in section V.E. Solubility Determination. 
 
With respect to eligibility for a biowaiver, CVM believes there is no reasonable basis for 
drawing a distinction between APIs intended for administration to animals via drinking 
water and APIs intended to be administered via feed, provided these APIs have similar 
solubility characteristics.  A water-soluble API present in a Type A medicated article and 
 
9 Current Standards & Emerging Issues of Pharmaceutical Equivalence. AAPS/EUFEPS/FIP Workshop. Mei-Ling 
Chen, Ph.D.  November 1314, 2010. 
10 https://www.fda.gov/media/69957/download 
4    Contains Nonbinding Recommendations 
 
mixed into a feed matrix rapidly dissolves when exposed to the fluids of the GI tract.  If 
such an API readily goes into solution across the range of physiological pH values, it will 
also readily go into solution when exposed to the fluids in the GI tract.  Accordingly, such 
medicated feeds will effectively behave as oral solutions immediately after consumption by 
the animal.  Therefore, CVM intends to review biowaiver requests for Type A medicated 
articles containing water soluble APIs based on a demonstration of solubility of the API, as 
well as an evaluation of the drug product formulation, to ensure that there are no 
ingredients in the proposed formulation likely to cause pharmacologic or 
pharmacodynamic differences from the RLNAD. 
 
The criteria detailed in this guidance assume that the in vivo dissolution of the test and 
reference products represents the only factor influencing the relative bioavailability of both 
products.  However, for compounds that are fully soluble and systemically absorbed, there 
is the potential for certain excipients to alter drug absorption and or pre-systemic 
metabolism (liver or intestinal), and hence affect the relative bioavailability of the products.  
Therefore, for Type A medicated articles containing water-soluble APIs that are 
systemically absorbed, the formulation of the proposed product should be such that there 
are no differences in the formulations of the proposed drug product and the RLNAD that 
would have an effect on the bioavailability of the API.  Further, there should be no inactive 
ingredients in the proposed drug products formulation likely to cause adverse 
pharmacologic effects. 
 
Type A medicated articles may contain biomass drug substances, which are APIs produced 
through fermentation that are not subjected to extensive post-fermentation purification. 
Biomass drug substances may contain the active molecule(s), microorganisms used for 
production, other metabolites produced by the microorganisms used for production, and 
media components.  Biomass drug substances are only used as the drug substance in Type 
A medicated article formulations.  Because certain biomass drug substances are well-
accepted and routinely used components of Type A medicated articles, CVM will consider 
the potential for the biomass drug substance component of a Type A medicated article to 
cause adverse pharmacologic effects or effects on API bioavailability in the same manner 
that it considers these effects with respect to other excipients. 
 
B.  Data to support a waiver from the requirement to perform an in vivo 
bioequivalence study (biowaiver) request 
For Type A medicated articles containing water-soluble APIs, CVM recommends that 
requests for biowaivers be made by a demonstration of solubility.  CVM recommends that 
sponsors make these biowaiver requests before submitting an ANADA.  The following 
should be provided in the biowaiver request: 
 
1.  Information about the API(s) 
CVM recommends that the API(s) used to support a biowaiver request be provided 
from the same supplier of the API(s) that will be used to formulate the proposed drug 
product during production.  CVM recommends that the applicant identify the source of 
the API(s), indicate if the API(s) is a biomass drug substance (if applicable), and 
provide a COA for the API(s).  If the API has a USP monograph, the API should at a 
5    Contains Nonbinding Recommendations 
 
minimum comply with the monograph specifications.  The acceptability of the API(s) 
will be determined upon review of the Chemistry, Manufacturing, and Controls (CMC) 
Technical Section. 
 
2.  Information about the formulation 
The formulation of the proposed drug product should be submitted.  The formulation 
should be such that there are no differences between the RLNAD and the proposed 
drug product that would have an effect on the bioavailability of the API.  There should 
be no inactive ingredients in the proposed drug products formulation likely to cause 
adverse pharmacologic effects.  The API should be qualitatively (Q1) and 
quantitatively (Q2) the same as the API used in the RLNAD.  Sponsors should address 
the inactive ingredients in their formulation and any potential impact they may have on 
the bioavailability of the API(s) or the likelihood to cause adverse pharmacologic 
effects.  If supporting data or information regarding any potential impact of the inactive 
ingredients on the bioavailability of the API(s) or their likelihood to cause adverse 
pharmacologic effects is available, it should be submitted. 
 
3.  Solubility Data 
Solubility data including method validations, and chromatograms as appropriate, should 
be submitted with each biowaiver request for Type A medicated articles.  The solubility 
of the API should be determined in aqueous media.  The inherent ability of the API to 
dissolve in aqueous media is critical to using the solubility data in support of any 
request for granting a biowaiver.  Solubility data should represent a pH range that is 
inclusive of all the pH values expected in the gastric/ruminal environment for all the 
major species on the RLNAD label.  
 
CVM recommends that the Shaker flask method be used for solubility determinations, 
employing an appropriate dissolution time and temperature, relevant to the labeled 
species.  CVM-recommended times and temperatures for major species are documented 
in Table 1 of section V.E. Solubility Determination. 
 
C.  Manufacturing process 
If the process used to manufacture the Type A medicated article has the potential to alter 
the solubility of the API, thereby potentially impacting bioequivalence of the generic 
product to the RLNAD, then CVM may request additional data regarding solubility of the 
API in the final formulation. 
 
D.  CVM evaluation of solubility data 
Sponsors should submit solubility data, analytical method validations, and additional 
documentation such as chromatograms in support of their biowaiver request.  CVM will 
evaluate the acceptability of the solubility data for the purposes of a biowaiver based on the 
criteria as documented below.  If a sponsor wishes to propose an alternative to meeting the 
bioequivalence requirements, the sponsor may request a meeting with CVM to discuss their 
approach prior to requesting a biowaiver.  In general, CVM will evaluate data submitted 
with a biowaiver request using the following criteria: 
6    Contains Nonbinding Recommendations 
 
 
1.  Calculation based on the Maximum Daily Dose 
In this scenario, the entire daily dose is administered in a single feeding event.  
Therefore, a demonstration that the maximum administered dose, as indicated on the 
RLNAD label, is soluble in the minimum fluid volume, in the specified time, for the 
indicated species (Table 1) will suffice as evidence for granting a biowaiver. 
 
2.  Calculations based on the Dosage Adjusted Approach 
In this scenario, the dose is administered as multiple feeding events through the day.  
The number of feeding events by which the dose is divided is species dependent (Table 
1).  Therefore, the aqueous solubility is to be evaluated based on the highest expected 
mg of API per mL of gastric fluid at any point in time.  This assessment is determined 
based on the API concentration in the feed and characteristics of the gastric physiology 
of the target animal species. 
 
a.  API concentration 
The API concentration should be calculated by using the amount of API in the Type 
A medicated article likely to be consumed per feeding event, e.g., dividing the daily 
dose into the number of feeding events that the target animal species typically takes 
to consume their daily ration (Table 1).  This scenario is typically applicable to 
situations where the animals do not consume their rations on a continual basis 
through the day.  An exception to this approach is seen in poultry, where 
differences in feeding practices can influence the amounts of Type A Medicated 
article consumed per bird over time.  Therefore, in poultry, a demonstration of 
product bioequivalence using the dosage adjusted approach will be evaluated on a 
case-by-case basis to determine the appropriate quantity of the API to use in the 
associated solubility studies.  Accordingly, for Type A medicated articles intended 
for administration to poultry, sponsors should contact CVM to discuss the 
conditions most suitable for demonstrating product bioequivalence. 
 
b.  Gastric physiology 
The animal physiology is critical as it determines the gastric residence time (how 
long it takes for the consumed medicated feed to exit the stomach or rumen), the 
gastric fluid volume of the target animal species (Table 1), the pH range over which 
solubility measurements must be made, and the temperature at which solubility 
should be determined. 
 
The solubility of the API, when tested based on the API concentration, is defined by 
the highest expected daily dose (mg of API) that will go into solution when tested in 
a manner consistent with the most conservative conditions associated with the 
approved product label (e.g., the largest dose to fluid volume ratio, minimum time 
of dissolution, and the broadest pH range over which solubility is demonstrated).  
When this level of solubility of the API is demonstrated, CVM will consider the 
API to be eligible for a biowaiver. 
 
7    Contains Nonbinding Recommendations 
 
If there are multiple species on the labeling, solubility should be based on the most 
conservative mg/mL scenario (i.e., use the species that produces the highest dose to 
gastric fluid volume ratio). 
 
This method of defining API solubility is similar to that described for categorizing 
compounds when using the BCS and to the BCS-based approach described in 
CDER guidance.11  In this case, the appropriate fluid volume for testing API 
solubility depends on the target animal species/class for which the medicated feed is 
intended.  For example, a conservative estimate of ruminal fluid volume (fluid 
volume available for drug solubilization) for steers is 47 L.  The sponsor should 
provide the estimated daily drug intake (mg/kg body weight) based on the labeled 
drug concentration (grams of drug per ton) in the feed administered to the animal 
(e.g., the Type C medicated feed) and the highest amount of medicated feed 
(kg/day) expected to be consumed by an individual animal.  When using this 
approach, CVM recommends using the species-specific animal data estimates 
summarized in Table 1. 
 
E.  Solubility determination 
For the purposes of granting a biowaiver under this guidance, CVM is interested in the 
physiologically relevant solubility of the API in aqueous solutions.  As such, the addition 
of compounds, such as surfactants used to increase the solubility of the API in aqueous 
media, is not considered acceptable.  If the use of surfactants is required to achieve 
acceptable solubility data, then the API is not considered to be fully soluble in water, and 
therefore is no longer covered under the scope of this guidance.  If the use of a buffer 
results in any effect that may increase the solubility of the API or drug product, that waiver 
request will also be denied. 
 
The preferred method for determination of solubility is by use of the Shaker flask method12 
containing an aqueous media for the indicated time and temperature relevant to the labeled 
species (Table 1).  Solubility should be confirmed using a validated assay procedure that 
can distinguish the API from its degradation products.  The media used for testing should 
represent a pH range that is inclusive of all the pH values expected in the gastric 
environment for all the major species on the RLNAD label and as described in section 
V.F.2. Media composition and pH considerations below.  When the conditions within the 
GI tract are markedly different across labeled species, appropriate media composition for 
testing API solubility should be discussed with CVM. 
 
 
 
 
 
 
 
 
11 See CDER Guidance for Industry, Waiver of In Vivo Bioavailability and Bioequivalence Studies for Immediate-Release 
Solid Oral Dosage Forms Based on a Biopharmaceutics Classification System, December 2017.   
12 USP 1236 
8    Contains Nonbinding Recommendations 
 
Table 1. Standard Conditions for Use in Determining Experimental Conditions 
Gastric Fluid  Gastric resident  Temperature  Feeding events per 
Species * 
Volume in Liters  times (hours)  (0C)  day 
Cattle 1, 3  47 #  8  36.7  39.3  2 
Swine 3  0.5  1  38.7  39.8  2 
Horse 2, 3  1.5  0.25  37.2  38.2  2 
Chicken 3  0.01   2  40.6  43.0  Variable 5 
Turkey 4  0.04   2  40.6  41.5  Variable 5 
* CVM acknowledges that the estimates for the indicated species are very conservative.  If alternate data points are 
used, that use must be adequately justified.  CVM notes that in certain instances the appropriate variable may be less 
than that indicated in the table, as is the case when dealing with younger animals. 
# Fluid volume of the rumen. 
 Includes the fluid volumes of both the proventriculus and the ventriculus. 
1 Martinez, M. N., Apley, M. D. Drug solubility classification in the bovine. J. vet. Pharmacol. Therap. 35 (Suppl. 1), 
9397, 2012. 
2 The Equine Hospital Manual first edition, ed. Kevin Corley and Jennifer Stephen, Chapter 5, page 282. Wiley 
Blackwell, 2008. 
3 The Merck Veterinary Manual: http://www.merckvetmanual.com/appendixes/reference-guides/normal-rectal-
temperature-ranges (accessed June 8, 2023). 
4 Some Factors Affecting Body Temperature of Turkeys: Poultry Science, Volume 34, Issue 2, 1 March 1955, Pages 
369371, https://doi.org/10.3382/ps.0340369 (accessed June 8, 2023.) 
5 Feeding events per day will be determined on a case-by-case basis due to the variability in feeding practices for 
poultry.  
F.  Technical parameters to consider include: 
1.  Use of the Shaker flask 
  The temperature of the solution within the flask should be within the 
physiological range of the healthy animal for the major indicated species on the 
label.  Temperatures should be maintained within  0.5C of the indicated 
temperature throughout the study to ensure solubility is not affected by variation 
in temperature. 
  After the appropriate period of shaking (based on Gastric residence time as 
described in Table 1), prior to sampling, there should be an excess of API 
apparent on the bottom of the flask. 
  The supernatant (liquid above the solid) should be filtered to remove 
undissolved particles not apparent to the naked eye. 
  Samples for analysis should be taken from the filtered supernatant.  The 
supernatant solution may need to be diluted before analysis to be within the 
linear range of the analytical method and to avoid possible precipitation. 
2.  Media composition and pH considerations 
  For monogastric animals:  pH 1.2, 4.6 (acetate buffer), and 7.5 phosphate 
buffer. 
9    Contains Nonbinding Recommendations 
 
  For ruminants:  For the purposes of generating data for a biowaiver request, the 
media should be buffered at a pH of 4.5 (acetate buffer) and 7.5 (phosphate 
buffer). 
  For poultry:  The sponsor should justify the sets of conditions under which 
solubility will be tested.  Without agreed upon justification, the default 
conditions would be the same conditions as those described for monogastric 
animals. 
  Solution pH should be verified after addition of the API to the buffer solution.  
If the pH changes significantly after addition of the API, we would conclude 
that the buffers selected are inadequate to control fluctuations in pH. 
  The pH of the solution should be verified to be consistent throughout the 
assessment of the API solubility.  At a minimum the pH should be verified at 
the time the sample is taken for analysis. 
Note that the buffers used may need to be modified if there are concerns that the buffer 
itself may alter the inherent solubility of the API or drug product (e.g., common ion 
effect).  However, the inclusion of organic compound-containing buffer systems is not 
considered to be acceptable. 
 
3.  Other considerations 
  CVM recommends a minimum of three replicate determinations of solubility 
under each pH condition.  Depending on study variability, additional replication 
may be necessary to provide a reliable estimate of solubility. 
  A visual determination of solubility alone is insufficient.  For the determination 
of solubility, the concentration of the API in the selected media must be 
determined using a validated quantitative assay method.13 
VI.  GLOSSARY 
For purposes of this guidance document, the following definitions apply: 
 
Active Pharmaceutical Ingredient (API):  A substance used in a finished pharmaceutical 
product, intended to furnish pharmacological activity or to otherwise have direct effects in the 
diagnosis, cure, mitigation, treatment, or prevention of disease or to have direct effect in 
restoring, correcting, or modifying physiological functions of the body. 
 
Bioavailability:  The rate and extent to which the active ingredient or active moiety is absorbed 
from a drug product and becomes available at the site of action.  For drug products that are not 
intended to be absorbed into the bloodstream, bioavailability may be assessed by measurements 
intended to reflect the rate and extent to which the active ingredient or active moiety becomes 
available at the site of action. 
 
 
13 21 CFR 514.1(b)(5)(vii)(a) 
10    Contains Nonbinding Recommendations 
 
Bioequivalence:  The absence of a significant difference in the rate and extent to which the 
active ingredient or active moiety in pharmaceutical equivalents or pharmaceutical alternatives 
becomes available at the site of drug action when administered at the same molar dose under 
similar conditions in an appropriately designed study. 
 
Biomass Drug Substance:  An active ingredient, which is an unpurified fermentation product 
derived from the cultivation of microorganisms, that is intended to furnish pharmacological 
activity or other direct effects in the diagnosis, cure, mitigation, treatment, or prevention of 
disease or to affect the structure or any function of the animal. 
 
Biowaiver:  A waiver from the requirement to perform an in vivo bioequivalence study (21 USC 
360b(n)(1)(E)). 
 
Qualitative (Q1), Quantitative (Q2) and Structural Similarity (Q3):  Q1 means qualitative 
similarity between generic and reference listed products, while Q2 represents quantitative 
similarity of composition.  Q3 describes structural similarity and refers to the arrangement of 
matter and state of aggregation of the product. 
11