VETERINARY SERVICES MEMORANDUM NO. 800.206 

TO: Veterinary Services Leadership Team

 Directors, Center for Veterinary Biologics

 Biologics Licensees, Permittees, and Applicants

FROM: Jack A. Shere

 Deputy Administrator

SUBJECT: General Licensing Considerations: Preparing Outlines of 

Production for Vaccines, Bacterins, Antigens, Toxoids, and 

Diagnostic Test Kits

I. PURPOSE

This memorandum provides guidance on preparing Outlines of Production for

vaccines, bacterins, antigens, toxoids, and diagnostic test kits in compliance with title

9, Code of Federal Regulations (9 CFR), part 114.9. 

II. CANCELLATION

This memorandum replaces Veterinary Services (VS) Memorandum No. 800.206 

dated March 26, 2014.

III. BACKGROUND

The Animal and Plant Health Inspection Service (APHIS) regulations in 9 CFR 114.9

provide broad subject headings for Outlines of Production. These standardized 

headings ensure consistent placement of key information used by the Center for 

Veterinary Biologics (CVB) in regulating veterinary biologics. The headings are

broad to accommodate the diversity of today’s biological products, but this also 

results in considerable variation in content provided by individual licensees and 

permittees. This memorandum provides guidance regarding writing an effective 

Outline of Production.

Animal and Plant

Health Inspection

Service

Veterinary Services

1400 Independence 

Ave, SW

Washington, DC

20250 for
VETERINARY SERVICES MEMORANDUM NO. 800.206

Page 2

IV. GUIDANCE ON PREPARING OUTLINES OF PRODUCTION

A. General Considerations

1. Use accurate terms

Write Outlines in a manner that ensures consistent production and testing 

methods. Pay particular attention to terms that may allow for variability in 

production or testing. Ensure the language used is appropriate for the purpose.

a. “Should” means the procedure or value is recommended, but not 

mandatory. Deviations from the described procedure are not necessarily 

out of compliance with the filed Outline of Production. Likewise, “may” 

permits a procedure, but does not require it.

b. “Shall,” “must,” or “will” means that the procedure or action is 

mandatory. Deviations are out of compliance. 

c. “And” versus “or.” “And” means that both conditions must be met. “Or” 

means that either condition (but not necessarily both) must be met.

Avoid language that allows variability in production or testing. 

Manufacturing and testing procedures should not be subject to undue 

variability. Alternatives may be acceptable to document minor variations 

between domestic and exported versions of a product, or to allow 

equivalent alternatives in domestic production where such alternatives are 

necessary.

If the language allows variability, clearly describe the decision criteria to 

determine the option to use for an individual serial. CVB may request data 

to support the equivalence of alternative methods.

2. Avoid duplicated text

The codified section headings for Outlines may appear to promote some 

overlap in content among various sections. To the practical extent, avoid 

duplicating text in sections. This reduces the potential for introducing errors 

when revising repetitive sections.

a. Include text in the most applicable section and cite this section (e.g., “See 

section III.A”) in other sections where overlap occurs.

b. Direct the reader to the source of the full text. Avoid “citation chains” that 

direct the reader to a location that, in turn, directs them to a third location.
VETERINARY SERVICES MEMORANDUM NO. 800.206

Page 3

c. If citations refer to another Outline, ensure that the Outline is for an 

actively licensed product. Do not cite Outlines for terminated or prelicense 

products. Cite which sections of the Outline apply.

d. If citations refer to a document CVB published (e.g., supplemental assay 

methods), refer to the “current version” of the document rather than a 

specific version number. This prevents referring to obsolete documents.

3. Subsection identification

Adequate numbering and subsection identification facilitate review of 

Outlines and references to specific text. When text for a single section or 

subsection spans more than one page of a paper document, identify the 

section and subsection number at the top of each page. Alternatively, include 

the complete identifier in the format for each subheading level (i.e., write 

“V.C.3” instead of “3”). This avoids returning to a previous page to find the 

section number.

For electronic Outlines, follow the portal-specific guidance for creating 

navigable headings as an acceptable means of subsection identification.

a. Add subsections beyond those specified in 9 CFR 114.9 as needed, but 

add them at the end of the existing subsections. Do not insert them in a 

location that requires renumbering the existing subsections.

b. If a section or subsection exceeds more than a brief paragraph or two,

consider dividing the text into additional subsections. This helps reviewers 

refer to specific text in their comments.

4. Describe variable parameters

Describe parameters with inherent variability as an acceptable range rather 

than an absolute value. Examples include time and temperature (e.g., 20-

25 °C instead of 23 °C) and ingredients added to a target concentration 

instead of by direct measurement.

a. Avoid vague, subjective descriptors such as “overnight” or “room 

temperature.”

b. Use mechanical ranges within the accuracy of the equipment (i.e., 

reasonable for the normal negligible variability that one might expect to 

encounter in adequately controlled daily production).

c. Limit other ranges to concentrations reasonably expected in the course of 

routine production. Ranges must reflect consistent manufacture.
VETERINARY SERVICES MEMORANDUM NO. 800.206

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d. Define upper and lower limits; avoid open-ended ranges.

5. Note equivalent materials

In general, Outlines of Production should have enough detail to ensure 

consistent manufacture of all serials. This may necessitate including 

manufacturing sources and product identification numbers to describe 

component materials adequately. In certain cases, the licensee may use 

equivalent materials from a number of sources. When using equivalent 

materials, specify the preferred product source in the Outline, followed by 

the phrase “or equivalent.” Specifying the preferred material provides a basis 

for determining equivalency of any alternative material that may be used.

6. Document exemptions

If CVB grants an exemption to a codified requirement, document the 

exemption in the applicable section of the Outline. Include the CVB approval 

date and the CVB mail log number under which the approval was granted, if 

known. If the product was initially licensed under a different establishment 

number, indicate the establishment number under which the exemption was 

granted.

7. Document pivotal study approvals

When documenting pivotal regulatory study approvals in an Outline or 

special outline, include the study identifier (if applicable), the CVB approval 

date, and the CVB mail log number (if known). If the product was initially 

licensed under a different Establishment number, indicate the Establishment 

number under which the study was granted.

8. Use of Special Outlines

Use Special Outlines to document processes or tests that pertain to more than 

one product. Cite the single Special Outline at the appropriate location in 

each applicable Outline of Production. In addition, use Special Outlines when 

a lengthy detailed description is required for a product or test. Moving these 

descriptions to a separate document can improve the overall readability and 

flow of the Outline.

Structure Special Outlines in the same way as Outlines of Production. 

Include a cover page, appropriate section and subsection identification, and 

document any changes. Submit Special Outlines in the same manner as 

Outlines of Production.
VETERINARY SERVICES MEMORANDUM NO. 800.206

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9. Include summary of changes

a. For each change include the specific reason for the change. If the change 

is justified by data on file with CVB, cite the study number or other data 

identifier and CVB mail log number/date of CVB acceptance. If a change 

was made in response to prior CVB correspondence, indicate the CVB 

mail log number and date of the correspondence. CVB may return 

Outlines unprocessed at the discretion of the CVB reviewer, if adequate 

justifications are not provided.

b. National Center for Animal Health (NCAH) portal submitters: Use the 

“track changes” functionality of the word processing software to show 

where insertions and deletions are made. Use the “comment” feature to 

insert the reason and justification for the change next to the applicable 

edited text. No separate “summary” of changes is required, but may be 

prepared in addition to tracked changes and contextual comments, if 

desired.

c. Paper submitters: List every change made to the Outline in the summary 

of changes (see 9 CFR 114.9(a)(6)). Cite the page and paragraph in which 

each change occurs.

B. Section-specific considerations

See appendix I for a section-by-section discussion of Outline content for 

vaccines, bacterins, antigens, toxoids, and immunomodulators. See appendix II 

for additional information regarding products involved in split manufacture 

agreements. See appendix III for guidance regarding Outlines for diagnostic test 

kits.

V. GUIDANCE ON SUBMITTING OUTLINES OF PRODUCTION

A. Format

Configure all Outlines according to the headings described in 9 CFR 114.9. Do 

not change headings or insert new headings, except as discussed in section 

IV.A.3.a of this memorandum. 

Submit electronic Outlines via the NCAH Portal only on the template provided by 

CVB or another CVB-approved template created by your firm. See portal-related 

guidance for details on necessary electronic template requirements.

B. Number of Copies to Submit 
VETERINARY SERVICES MEMORANDUM NO. 800.206

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1. Submission by the NCAH portal: Follow portal-specific guidance on the CVB 

web site.

2. Paper submissions: Submit an original and at least one copy. The original and 

first copy must each bear an original signature, preferably in a different ink 

color (e.g., blue) than the text.

C. Transmittal Forms

1. NCAH portal submitters. Submit Outlines with APHIS Form 2049. Do not 

use APHIS Form 2015. Submit only complete revisions.

2. Paper submitters

a. Submit Outlines with APHIS Form 2015, “Transmittal of Labels and 

Circulars or Outlines.”

b. Ensure the date submitted (block 4) corresponds to the preparation date 

of the Outline (or date of submitted pages). If the mailing date of the 

submission differs from the preparation date of the Outline, indicate the 

mailing date of the submission in block 13, “Comments.”

c. Enter the preparation date of the last complete revision in block 12, “Date 

of Previous Outline.” Do not use the date of individual pages amended 

after the last complete revision.

3. Do not use an APHIS Form 2015 to submit Outlines of Production for 

products exported under the Food and Drug Administration’s Export Reform 

and Enhancement Act of 1996 (FDA-EREA). Submit FDA-EREA Outlines 

as correspondence with a cover letter regardless of whether submitting by 

portal or paper.

D. Complete Revisions versus Page Amendments

1. NCAH portal submitters. Submit only Complete Revisions. Because of the 

electronic nature of the document, CVB does not accept individual page 

amendments that need to be collated with older pages.

2. Paper submitters. Update Outlines by submitting complete revisions or 

individual page amendments. Either approach is acceptable in many cases. 

However, CVB recommends a complete revision in lieu of individual page 

amendments when at least half the pages in the document have been 

amended since the last complete revision.

E. Date of Superseded Version. 
VETERINARY SERVICES MEMORANDUM NO. 800.206

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If an Outline submission replaces a previously-filed version, state the date of the 

superseded version (“supersedes date;” see 9 CFR 114.9(a)(4)).

1. If the Outline is new, state, “New” on the cover page and all subsequent page 

headers. 

When an Outline is part of a merger or acquisition, it may be “new” only to 

the establishment acquiring the product. In such cases, specify the document 

on which the new Outline is based. Place this information on the “Supersedes” 

line of the cover page. Example: Establishment Z submits a “new” Outline for 

Product B. This product was formerly licensed to Establishment X as Product 

A. State on the cover page of the “new Outline” for Est. Z, “Supersedes: 

Identical to Est X, Product A Outline of Production” or equivalent. Including 

this cross reference on the cover page does not automatically inactivate the 

cited document.

2. Update the supersedes date on a paper cover page only when submitting a 

complete revision of the Outline. Use the preparation date of the last complete 

revision, rather than a later date when individual pages were amended.

3. For the supersedes date on an individual paper page header, show the 

preparation date of the previous version of that page if updated after the last 

complete revision. For a complete paper revision, state “Complete Revision” 

instead of a supersedes date on the individual page headers.

4. When resubmitting an Outline that CVB returned unprocessed, use the 

preparation date of the last filed Outline for the supersedes date, rather than 

the date of the unprocessed submission. However, on the APHIS Form 2015

for the current submission, note the preparation dates of unprocessed outlines 

in block 13, “Comments.” 

F. Version number (NCAH Portal Submissions only)

Follow the portal-specific guidance for electronic Outlines found on the CVB 

website. 

VI. IMPLEMENTATION/APPLICABILITY

This memorandum applies to all products, licensed and unlicensed, as of the date of this 

memorandum. For licensed products, Outlines of Production should comply within a year 

from the date of this memorandum.

Appendices
VETERINARY SERVICES MEMORANDUM NO. 800.206

Appendix I, Page 1 

Appendix I

Section-Specific Guidelines for Preparing Outlines of Production: Vaccines, 

Bacterins, Antigens, and Toxoids

Cover Page

Each Outline and special outline must have a cover page, containing the information 

specified in title 9 of the Code of Federal Regulations (9 CFR), part 114.9(a)(2). 

Paper submitters only: In general, licensees and permittees submitting paper copy should 

replace the cover page only when submitting a complete revision, not for individual page 

changes. When there is a necessary cover page change, (e.g., a name change for the 

existing establishment) but no other revisions warranting a complete revision, submit an 

updated cover page without submitting a complete revision. Continue to list the date of 

the last complete revision on the cover page. Underneath this date, add a statement “cover 

page updated [date].”

The following guidance applies to vaccines, bacterins, antigens, and toxoids in general. 

See VS Memorandums 800.213 and 800.214 for additional guidance specific to platform 

and prescription platform product Outlines.

Section I. Composition of the product

Complete this section in each Outline. Do not cite another related Outline.

A. Microorganisms used. For each microorganism used to produce the product, 

provide the following information:

• Organism name.

• Complete lot number (or other unique identifier), exactly as it appears on 

the stored Master Seed container and licensee’s records.

• Isolation and known preparation history, including the number of passages 

since isolation. Specify the type of medium, cell culture, or animal used 

for each passage.

• The date CVB approved the Master Seed for use in production. Include 

the CVB Mail Log number of the approval letter, if known.

• For viruses and other obligate intracellular organisms, approvals are 

specific for one or more animal species, depending on the extraneous 

agent testing performed (9 CFR 113.47). List each of the animal species 

for which the approval applies.
VETERINARY SERVICES MEMORANDUM NO. 800.206

Appendix I, Page 2

• Name and license number of the firm at the time CVB approved the 

Master Seed for use in production. Do not list the date that CVB licensed 

the product containing the Seed.

If inventories of the original Master Seed are depleted and CVB has authorized 

the use of a passage of the original Seed as the “new” Master Seed, list the lot 

identification and approval date for the current Master Seed lot, not the depleted 

original lot.

B. Source and date of accession of each microorganism. For each microorganism 

used to produce the product, indicate the date and from whom the current licensee 

obtained the Master Seed. If the Master Seed was obtained through a merger or 

sublicensing agreement with a current or former U.S. Department of Agriculturelicensed firm, provide the name and license number of the firm and the effective 

date of the relevant company merger or agreement.

C. Strains. Provide the strain or isolate designation for each microorganism used to 

produce the product. If the microorganism is a generally recognized strain, 

specify the strain. Otherwise, provide the isolate designation as it appears on 

containers used to store the Master Seed.

D. Proportions of each strain. 

• If the product contains only one strain or isolate for each fraction, mark this 

section “Not Applicable.”

• If a fraction of the product contains more than one strain or isolate, but the 

strains or isolates can be distinguished in the serial release potency test, mark 

this section “Not Applicable.”

• If a fraction contains more than one strain or isolate that cannot all be 

distinguished in the serial release potency test, indicate the proportion of each 

strain or isolate in the fraction. Base the proportions of the strains in that 

fraction on antigenic mass, not on volume of harvest fluids or other criteria. 

For each fraction, the sum of the proportions must total 100 percent. For other 

fractions in the product that meet either of the first two criteria, indicate “Not 

Applicable.”

Section II. Cultures

Antigen production methods are often common to multiple products. If desired, compile 

the information in this section in a Special Outline and cite the Special Outline in each 

applicable Outline of Production. This is preferable in lieu of maintaining product 

licenses for monovalent products solely for the purpose of citing the Outline for the 

production of a particular antigen.
VETERINARY SERVICES MEMORANDUM NO. 800.206

Appendix I, Page 3

A. Methods of identifying each microorganism. Specify the stages when identity 

testing is performed (e.g., working seeds or production cultures). Specify the tests 

that will be performed. Include the frequency with which methods are applied 

(e.g., when each working seed is established).

B. Virulence and purity of cultures and the determination and maintenance thereof. 

Range of subcultures and passages to be used in production. Specify the number 

of passages beyond Master Seed allowed in production. This is determined by the 

number of passages used to produce the serial with which product efficacy was 

demonstrated. Typically, it is limited to five passages beyond Master Seed.

The portion of the heading pertaining to the virulence and purity of the culture

and maintenance thereof is largely obsolete since the implementation of the 

Master Seed system. Historically, the virulence of some cultures was maintained 

by periodic passage through animals. This is no longer an acceptable practice. 

Seeds recovered after animal passage are now considered to be “new” Master 

Seeds and are subject to full testing before use.

C. Composition and reaction of media used for seed and production cultures. 

Provide information on Master Cell Stocks and eggs, tissues, and primary cells in 

each Outline. Do not cite related Outlines.

This section must include a statement regarding the source of any ingredients of 

animal origin. The preferred wording can be found in VS Memorandum No. 

800.51, section IV.A.2.

1. Master Cell Stocks. For each cell line used to produce the product, provide the 

following information:

• Name of cell line.

• Complete lot number (or other unique identifier), exactly as it appears 

on the stored Master Cell Stock container and the licensee’s records.

• Source and history.

• Maximum approved passage level.

• Date when CVB approved the Master Cell Stock for use in production 

and the CVB mail log number of the approval letter, if known.

• Master Cell Stock approvals are specific for one or more animal 

species, depending on the extraneous agent testing performed 

(9 CFR 113.47). Include each of the animal species to which the 

approval applies.

• Name and license number of the firm at the time CVB approved the 

Master Cell Stock for use in production.
VETERINARY SERVICES MEMORANDUM NO. 800.206

Appendix I, Page 4

• Antigens in this product that are propagated with this cell.

2. Eggs, tissue, or primary cells. For each type used, specify:

• Type of cellular substrate. Indicate the age of embryos, as applicable.

• VS Memorandum No. 800.65 provides guidance on eggs used in 

production. If eggs are used in production, include a statement in the 

Outline that ensures compliance with this memorandum.

• Tests to determine that eggs, tissues, and cells are free of 

contamination. Include the stage at which the tests are performed and 

criteria for a satisfactory test.

• Antigens propagated with each substrate.

3. Composition of media. For each microbiological medium used in 

manufacture, include the following:

• Name of medium.

• Composition (ingredients and amounts): The composition of Seed 

media used during scale-up may be subject to some lot-to-lot variation, 

depending on the immediate needs of individual cultures. If certain 

ingredients are expressed as a range to allow for customization, ensure 

that the upper and lower limits reflect concentrations reasonably used 

in routine production. However, prepare final production media in a 

consistent manner.

• Storage conditions, temperature, and dating period.

• How medium is used (for example, as production culture for 

Mannheimia haemolytica fraction).

• Exemptions, if any, to 9 CFR 113.53 (testing ingredients of animal 

origin).

D. Character, size, and shape of containers used for growing cultures. Specify the 

following:

• Culture system (e.g., stationary flasks, roller bottles, or bioreactors).

• Range of vessel sizes that reasonably may be used.

• Vessel composition (e.g., glass or plastic).

• Vessel configuration (e.g., height-to-diameter ratio and baffled or 

unbaffled tank).

• Type of mixing system.
VETERINARY SERVICES MEMORANDUM NO. 800.206

Appendix I, Page 5

• Type and number of agitation impellers, if applicable.

• How aeration or other gas is provided to the culture, if applicable.

• Type of sealing used with agitation system to ensure integrity of the 

vessel.

• Closure systems used on containers and vessels.

E. Storage conditions of Seed cultures. Indicate how the Master Seed is stored. If 

Working Seeds are prepared for long-term storage, indicate how they are stored.

F. Methods of preparing suspensions for seeding or inoculation. Indicate how the 

Seed is recovered from scale-up cultures for inoculation into subsequent cultures.

G. Technique of inoculation. Describe the following:

• Acceptable range of inoculum concentration (or multiplicity of infection).

• Cell density, if cellular cultures are involved.

• Volume of medium for each size and type of culture container.

If these parameters vary depending on the type of passage (scale-up versus final 

production passage), clarify the differences in subsections II.G.1 and II.G.2.

H. Conditions for incubation used for each microorganism or group of

microorganisms. For each antigen, describe:

• Incubation conditions (time, temperature, atmospheric conditions). If the 

conditions vary depending on the type of passage, clarify the differences.

• Handling that may occur during the incubation period (e.g., adding 

nutrients, candling eggs).

• List critical parameters under process control. Briefly describe how the 

parameters are measured.

I. Character and amount of growth; observation as to contamination of growth. For 

each antigen, describe:

• Parameters (e.g., optical density, percent of cells with cytopathic effect) 

that determine when incubation is complete.

• Tests performed at this stage in production to evaluate purity of the 

culture.

J. Method of attenuation before use in production. APHIS wrote this section 

heading before implementing the Master Seed concept. Procedures performed to 

attenuate a Seed organism before establishment of the Master Seed lot should be 

described in section I.A. APHIS no longer accepts attenuation of post-Master 
VETERINARY SERVICES MEMORANDUM NO. 800.206

Appendix I, Page 6

Seed cultures on a serial-by-serial basis. Thus, in most cases, section II.J should 

be marked “Not Applicable.”

Section III. Harvest

A. Handling and preparation of cultures and media before removal of 

microorganisms or tissues for production purposes. Describe any procedures or 

testing performed on a completed culture while it is still in the culture vessel or 

system. If egg culture is involved, indicate whether dead eggs are discarded or 

handled separately. Describe conditions (e.g., refrigeration) to maintain eggs 

before harvest.

B. Minimum and maximum period of time elapsing from time of inoculation until 

harvest.

• Specify the incubation time for each individual culture, not the total time 

elapsed from inoculation of the first scale-up culture to the harvest of the 

final production culture.

• Clarify any differences in incubation time that may exist between scale-up 

cultures and the final production culture.

A. Technique of harvesting microorganisms or tissues for production purposes. 

Describe how the microorganisms are removed from the culture vessel and any 

processing that occurs at this point in manufacture:

• If the organism is propagated in eggs, indicate which parts (e.g., 

chorioallantoic membrane, embryo, allantoic fluid) are retained.

• Describe any grinding or coarse filtration.

B. Specification for acceptable harvest material. Include all critical parameters, 

including minimum antigen concentration and purity. Include the evaluation 

methods used to determine acceptability of the culture. If a spectrophotometer is 

used to measure antigen concentration, specify the wavelength and type of 

spectrophotometer used.

C. Handling of discarded material not used in production. VS Memorandum 

No. 800.56 describes acceptable methods to discard material. Cite this 

memorandum if one of the described methods is used. Fully describe any 

exceptions.

D. Additional pertinent information. Use this section to describe any critical 

information regarding manufacture not covered under another heading.
VETERINARY SERVICES MEMORANDUM NO. 800.206

Appendix I, Page 7

Section IV. Preparation of the product

A. Method of inactivation, attenuation, or detoxification. Include:

• Procedure for inactivation or detoxification.

• Test method to confirm completeness of inactivation or detoxification.

• If applicable, additional procedures to be performed if the initial test 

demonstrates incomplete inactivation.

• Documentation of CVB acceptance of inactivation kinetics data to support 

the inactivation time and procedure. For products licensed before 2013,

and for which there is no explicit date on which inactivation data were 

accepted, the acceptance date is the date of the Outline in effect at the time 

of product licensure.

B. Composition of preservative, adjuvant, or stabilizer.

1. Adjuvant. Include:

• Chemical composition of adjuvant (and trade name, if applicable). Include 

any antibiotics or preservatives added to the adjuvant.

• Proportion of adjuvant used, stated so that the concentration can be 

calculated.

2. Preservative. 

• If a preservative (antibiotics and/or other) is added at batching/serial 

assembly, specify the identity and the actual concentration added. Do not 

merely cite 9 CFR 114.10.

• If the final product may contain residual amounts of antibiotics or other 

preservatives because of upstream processes (e.g., growing organisms in 

media containing antibiotics or thimerosal), indicate which substances 

may be present in residual amounts and identify the stages at which they 

were added. You do not need to estimate the concentration of residual 

antibiotic or preservative in the final product if there is no material risk 

that this level exceeds codified maximum amounts (e.g., 9 CFR 114.10).

C. Method and degree of concentration. If the microbial harvests may be 

concentrated before batching/serial assembly, specify:

• Criteria that determine whether concentration is necessary.

• Method of concentration, including specific parameters (such as g-force or 

filter molecular weight cutoff).
VETERINARY SERVICES MEMORANDUM NO. 800.206

Appendix I, Page 8

• Maximum permissible concentration factor.

D. Standardization of product. If the product is standardized for antigen 

concentration, describe the procedures and calculations.

E. Assembly of units to make a serial.

1. Provide an example of a typical serial, including all components and 

proportions added. If the product may be administered at different dose 

volumes, depending on indications for use, base calculations on the most 

common dose volume.

2. Volume of average serial.

3. Volume of maximum serial.

4. Other pertinent information.

F. Volume of fill. Include an acceptable range for the fill volume for each size of 

container.

G. Method and technique of filling and sealing final containers. Include exemptions

to 9 CFR 114.6, if applicable.

H. Desiccation, including moisture control. If the product is lyophilized, include:

• How live product is handled between harvest and desiccation to maintain 

viability.

• Procedure for desiccation.

• Permissible temperature ranges during desiccation.

• Permissible time period for desiccation, including the longest time the 

product may be held at maximum temperature.

• Test method for residual moisture (9 CFR 113.29).

• Allowable percent residual moisture in finished product.

I. Amount of antigenic material per dose in final container. Describe, in the most 

objective terms possible (e.g., preinactivation cell count, spectrophotometry or 

nephelometer readings) the minimum antigen content in the product at the time of

serial assembly. Avoid characterizing the amount of antigen as the amount 

sufficient to pass serial release potency tests, or as a volume of non-standardized 

antigen. 
VETERINARY SERVICES MEMORANDUM NO. 800.206

Appendix I, Page 9

Section V. Testing

Include stepwise procedures in sufficient detail so a laboratory technician experienced in 

general laboratory techniques could perform the assay. Many standard requirements 

specify only general assay architecture (e.g., perform a serum neutralization test), so 

additional detail beyond the 9 CFR citation is often required. If testing is performed by a 

third party under contract, include the information specified in VS Memorandum No. 

800.115.

Mark section V as “not applicable” in Outlines of Production for combination packages 

(i.e., two or more individually licensed component products that may be marketed with a 

recommendation to combine the components before administration) because the 

combination package product is not tested. Do not refer the reader to Outlines for the 

component products.

Stepwise procedures for section V tests may be described in Special Outlines. If Special 

Outlines are used, retain key information regarding the assay and interpretation of results

in each Outline of Production so the information can be retrieved easily during the serial 

release process. Information to remain in section V when a Special Outline is used 

includes:

• Criteria for a valid test

• Criteria for a satisfactory test

• Criteria for an inconclusive test

• Lot number of the current potency reference preparation, if applicable, and the 

original approval date and current expiration date.

• Preparation specific to a particular lot of potency reference (such as working 

dilution).

Where the Special Outline refers to the above information, direct the reader to “see the 

Outline of Production for the product being tested.” Do not duplicate this information in 

the Special Outline.

A. Purity.

• Specify the media volumes to be used when testing according to

9 CFR 113.26 or 9 CFR 113.27. Determine these volumes according to the 

dilution of preservative procedure in 9 CFR 113.25(d) and Supplemental 

Assay Method 903. Document CVB acceptance of the dilution of preservative 

study supporting the specified media volume.

• Other purity tests (e.g., Mycoplasma, Salmonella, lymphoid leukosis, chicken 

anemia virus) may be required depending on product ingredients and 

manufacturing method. List any exemptions to these requirements.
VETERINARY SERVICES MEMORANDUM NO. 800.206

Appendix I, Page 10

B. Safety.

• If an animal safety test is required, specify a single consistent vaccination 

route (e.g., subcutaneous).

• If an exemption to animal safety testing has been granted, per VS 

Memorandum No. 800.116, document the exemption but retain a 

description of the safety tests that have been exempted. This is necessary 

so that if the exemption is rescinded for individual high-risk serials 

according to the provisions in VS Memorandum No. 800.116, the testing 

to be completed is documented.

• Test poultry and aquaculture products in the species for which the product 

is recommended or in a suitable surrogate species.

C. Potency

• Include a potency test for every antigenic fraction associated with a label 

claim.

• Document CVB acceptance of the pivotal immunogenicity and efficacy 

study for each antigenic fraction (live or killed). Include the age and 

species of the animals and the route of administration used to establish 

product efficacy. If the fraction is a live organism, specify the titer of the 

efficacy serial. 

• If multiple immunogenicity and efficacy studies were performed to 

support claims for a specific antigenic fraction (such as short-term efficacy 

versus duration of immunity claims or claims for different disease 

syndromes caused by a single agent), include the above information on 

each approved study.

• If software is required to calculate relative potency, document the 

procedure for using the software in the Outline (or Special Outline) or in a 

separate standard operating procedure submitted for CVB review. Specify 

the software version(s) allowed, and if a specific template/protocol file is 

also used, specify its version-controlled file name. Document the 

procedure in sufficient detail that a user in an independent laboratory, 

possibly using different software, would be able to reproduce the 

calculation. Such details include pertinent software options and settings, 

the data model, validity criteria, and (if necessary) the region of the 

response curves that will be utilized to estimate relative potency.

D. Moisture (if desiccated). Describe the assay procedure for moisture testing, as 

well as criteria for acceptable results, in section IV.H. In section V, direct the user 

to see section IV.H.
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Appendix I, Page 11

Section VI. Post-preparatory steps

A. Form and size of final containers. Specify the composition and nominal fill 

volumes of the final containers.

This section also provides a uniform location for miscellaneous information.

1. Diluent: If the product is packaged with a diluent, indicate the name of the 

diluent. If the diluent is purchased, specify the supplier. If it is made in-house, 

specify the Special Outline containing the production method. This is not 

applicable to combination packages.

2. International testing: In general, do not include testing conducted solely for 

the purpose of international distribution in the Outline of Production filed with 

APHIS. If, however, the inclusion of this testing facilitates export, place 

international testing requirements in section VI.A. Filed Outlines with such 

testing in section V should be revised at the time of the next annual review. 

Include the following disclaimer for any international testing that appears in 

section VI.A: “The testing described in this section is conducted solely for 

international purposes. It is not used by APHIS in serial release 

determinations.”

B. Collection, storage, and submission of representative samples.

• Indicate the production steps where samples are taken.

• For samples taken during the fill process in accordance with 9 CFR 113.3, 

include any applicable information regarding specific authorizations to 

submit partial-fill samples or quantities other than those described in 

9 CFR.

• If samples are shipped to designated recipients before APHIS release, list 

the approved recipients and maximum number/volume of samples to be 

shipped. Describe any approvals required before shipping samples.

C. Expiration date.

• According to 9 CFR 114.12 the expiration date for each serial is computed 

from the date of the initiation of the first potency test. The start date is 

considered to be the date the serial is introduced into the test system (the 

first day the serial is injected into the animal or the first day the serial is 

added to an in vitro test plate). Format this section to read: “The expiration 

date is (or will not exceed) XX months after the date of initiation of the 

first potency test.”
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Appendix I, Page 12

• 9 CFR 114.13 requires real-time stability data to confirm the acceptability 

of product dating. Document the CVB acceptance of the supporting study. 

If the dating has not yet been confirmed, indicate that appropriate data are 

being collected. Update the Outline when the CVB accepts the stability 

data.

• If a product is a combination package, format section VI.C to read:

“The expiration date of the combination package is the earliest 

expiration date of the individual product components in accordance 

with 9 CFR 114.13. This combination package is composed of 

released product of Codes ____________.”

Do not list the dating period for the component products in the 

combination package Outline.

D. Use, dosage, and route of administration. 

Include the following:

• All APHIS-approved prophylactic and therapeutic efficacy claims, even if

not currently stated on product labeling. Format these claims in 

accordance with the provisions in VS Memorandum No. 800.54.

• All APHIS-approved safety claims (such as whether the product is safe for 

use in pregnant animals).

• Dose volume.

• Animal species in which the product is used.

• Routes of administration.

• Number of doses in primary vaccination series and dosing interval.

• Minimum age for administration.

• Timing of administration, if applicable (for example, vaccination 4 to 6 

weeks before breeding).

• Revaccination recommendations (beyond primary vaccination series).

• Duration of immunity.

• Slaughter withdrawal period (for livestock, equine, poultry, and 

aquaculture products).

• All antibiotics or other preservatives (e.g., thimerosal) in the product, 

including those that may be present in trace amounts. If antibiotics added 

to preliminary manufacturing components are reduced by downstream 
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Appendix I, Page 13

processing to undetectable levels in the final product and CVB has 

accepted supporting data, indicate the date CVB accepted the data.

• APHIS-mandated warnings or cautionary statements.

• Any other product-specific instructions for use.

Many establishments reproduce the complete product label text in the Outline. 

This is not required, but it helps ensure all required elements on the label have 

been included in the Outline.

E. Site of Manufacture. If the licensee has licensed premises in multiple 

geographically separated locations (i.e., cities), specify the location at which each 

step of production occurs. Specify one location for each step unless the step 

routinely occurs in more than one location. This does not preclude moving 

production to other licensed premises, with APHIS approval, as production needs 

dictate. Follow the guidance in VS Memorandum No. 800.87 when transferring 

locations.
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Appendix II, Page 1 

Appendix II

Guidance for Outlines for Products Involved in Split Manufacture

I. If a product is part of a split manufacturing agreement (i.e., a For Further Manufacture 

(FFM) product or a Final-Use Product (FUP) that contains FFM components), detail in 

each Outline in the manufacturing chain only those processes done by that 

manufacturer at that point in production.

A. In each section of an Outline utilizing manufacturing precursors provided by a 

FFM manufacturer, cite the Establishment Number and Product Code of the FFM 

product for details of upstream processes conducted by the FFM establishment. Do 

not reproduce the information found in the FFM Outline.

B. In FFM Outlines, do not describe how the product is processed after leaving the 

FFM manufacturer. Mark sections of the FFM Outline covering downstream 

processes “Not Applicable.”

II. Indicate in section III.F (Other Pertinent Information) of Outlines for products that 

use FFM components:

A. Minimum specifications for acceptance of each FFM component.

B. Tests performed to ensure the incoming product meets these specifications.

III. Include in section VI.A of Outlines for FFM products:

A. Name (and U.S. Veterinary Establishment Number, if applicable) of each 

recipient of the FFM product.

B. Type and size of containers in which the FFM product is shipped.

C. Shipping conditions, including the required shipping temperature range. Include 

any monitoring procedures that are in place during shipment.

D. Responsible party (e.g., commercial carrier, transfer by establishment employees) 

for shipping between establishments

IV. Frequently, the FFM manufacturer is involved in serial release testing of splitmanufactured products. In such situations:

A. Describe in section IV.J of the FFM Outline any serial release testing performed 

by the FFM manufacturer
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Appendix II, Page 2

B. When the FFM manufacturer performs a serial release test, indicate in section V 

of the FUP Outline that the FFM provider conducts the test and cite section IV.J 

of the FFM Outline for the details of the test procedure. Section V of the FUP 

should include all items expected to be in section V of a product completely 

manufactured by a single firm. The items listed in V.C. of appendix I that are 

routinely kept in the main Outline of Production (i.e., reference information, 

criteria for valid test, criteria for satisfactory test, retest criteria) apply to a FUP 

outline, as well as the outline of production of a product completely 

manufactured by a single firm.

V. If the FFM product is inactivated, the results of testing to confirm complete 

inactivation must be reported on APHIS Form 2008 for each FFM serial. In section 

V of the FFM Outline, describe the inactivation confirmation test procedure, the test 

validity criteria, the criteria for satisfactory results, the date of CVB acceptance, and 

CVB mail log number, if known. For products licensed before 2013 and for which 

there is no explicit date on which inactivation data were accepted, list the date of the 

Outline in effect at the time of licensure.
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Appendix III, Page 1 

Appendix III

Guidance for Preparing Outlines of Production for Diagnostic Test Kits

Introduction

Briefly specify the type of assay and describe how it works. Specify the analyte 

detected (e.g., antibody, antigen), the sample types (e.g., serum) used, the list of kit 

components, any equipment included (e.g., droppers), any specialized equipment 

required, and items that are licensed under a split manufacturing agreement. Include

recommendations regarding use, result interpretations, and test result limitations. 

The kit description should not specify or imply the test kit is a quantitative assay. 

Do not use statements such as “proportional to the OD value,” unless the kit is a 

poultry antibody test kit. Such statements are allowed only for poultry antibody test 

kits.

Section I: Antibody Components

Every kit in which ingredients of animal origin are used in manufacture must 

contain a statement regarding the origin of these ingredients. The recommended 

statement is in VS Memorandum No. 800.51, section IV.A.2. Place the statement 

at the beginning of section I for consistency, even if the kit does not contain 

antibody components. If there are no ingredients of animal origin, state that there 

are no such ingredients.

Antibody may be purchased or prepared on licensed premises. If antibody is purchased, 

specify the identity, vendor and catalog number, country of origin, acceptance criteria, and 

any acceptance testing performed on each lot. The statement, “accepted under a

Certificate of Analysis” is not acceptable. If the antibody is prepared on licensed premises, 

describe production steps and specify acceptance criteria. Details of production should 

include species, age, weight, conditions, and general health of any animals used. See 

9 CFR 114.9(f) regarding required pre-injection, immunization, and harvest information.

1. Purchased Monoclonal Antibody

Purchased monoclonal antibodies must be full characterized. In addition to 

the information listed above, specify the clone designation or reacting

epitope.

2. Hybridomas

Hybridomas used in the preparation of monoclonal antibodies prepared on 

licensed premises must meet the applicable Master Cell requirements in 9 CFR 

113.52. Include the information specified in appendix I, section II.C.1, in the 

Outline.
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Appendix III, Page 2

Section II: Antigen Preparation, including PCR Primers, Master Seed and Master 

Cell Stock 

Antigen may be purchased or prepared on licensed premises.

1. Purchased Antigen

If antigen is purchased, specify the antigen identity, vendor and catalog number, 

country of origin, and lot acceptance criteria for each lot. Include testing 

performed by the vendor and testing performed on licensed premises. The 

statement “accepted under a Certificate of Analysis” is not acceptable.

2. Antigen Prepared on Licensed Premises

Antigen prepared on licensed premises must be prepared from Master Seed. 

Include in the Outline all information specified in appendix I, sections I.A 

through I.C. Also specify suppliers, catalog numbers, acceptance criteria, and 

shelf life for media and media ingredients. If media are prepared on licensed 

premises, describe the formulation and production steps.

3. Synthetic Antigen or Sequences

When synthetic antigens or oligonucleotides are used, specify the amino acid

sequence, nucleotide sequence, or carbohydrate composition, as applicable, 

along with any other critical structural specifications and criteria necessary to

ensure quality. Synthetic antigens or oligonucleotides may be purchased. If so, 

specify the product identity, vendor and catalog number, country of origin, lot 

acceptance criteria, and any testing done on licensed premises.

Section III: Preparation of Standard Reagents

Test kit components are subject to the requirements and restrictions indicated in the 

following chart. 
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Appendix III, Page 3

Component Source Same 

Lot for 

Entire 

Serial

Source 

Identified 

and/or 

Formulae in 

Outline

Submit Data 

Before Changing

Production 

Method

Component 

Dating 

Cannot 

Exceed 

Dating of 

Kit Serial

Anti-species 

Antibody or 

Conjugate

Licensed 

Premises 

or 

Vendor

Required Required Required Required

Agent Antigen or 

Antibody

Licensed 

Premises 

or 

Vendor

Required Required Required Required

PCR Master Mix Licensed 

Premises

Required Required Required Required

Sample Diluent Licensed 

Premises 

or 

Vendor

Not 

Required

Required Required Required

Controls Licensed 

Premises 

or 

Vendor

Required Required Required Required

Stop Solution Licensed 

Premises 

or 

Vendor

Not 

Required

Required Required Not 

Required

Coating of Solid 

Phase

Licensed 

Premises

Required Required Required Required

1. Controls

Describe the manufacture of all controls in the kit. The controls may be purchased. If 

so, specify the identity, vendor and catalog number, country of origin, lot acceptance 

criteria, and testing performed on licensed premises for each lot. The statement

“accepted under a Certificate of Analysis” is not acceptable. Specify control 

acceptance criteria in this section. 

2. Conjugate 

The conjugate is any reagent used to amplify or report an antigen-antibody reaction. 

This category includes anti-species antibody, immunoglobulin binding proteins (such 
VETERINARY SERVICES MEMORANDUM NO. 800.206

Appendix III, Page 4

as A, G, or L; colloidal gold; biotin), or enzyme-labeled versions of any of these.

Conjugate does not have to be prepared on licensed premises, but acceptance criteria 

for each lot must be specified. If conjugate is prepared on licensed premises, describe

the production process.

3. Substrate

The substrate is the substance that undergoes a color change or other detectable 

reaction when catalyzed by an enzyme-labeled kit component. It may be purchased. If 

so, specify the identity, vendor and catalog number, acceptance criteria, and testing

performed on each lot. If substrate is prepared on licensed premises, describe the 

production process.

4. Buffers, Diluents, or Other Reagents Included in the Kit

Specify all buffers, diluents, and other reagents included in the kit. These may be 

purchased or manufactured on licensed premises. If manufactured on licensed 

premises, describe the preparation and acceptance criteria. Describe the source 

and formula of each buffer, diluent, or other reagent in the kit. If purchased, 

specify the vendor, catalog number, and acceptance criteria for each lot. Specify 

methods used to stabilize these liquids against bacterial contamination, 

including the maximum time between manufacture and stabilization.

Section IV: Preparation of the Product

1. Preservatives

List the chemical name (e.g., sodium azide) or the full Trade Name (e.g., ProclinTM

950) of each preservative used in kit manufacture. If purchased, specify the vendor, 

catalog number, and acceptance criteria for each lot. If preservatives are formulated on 

licensed premises, describe the formulation.

2. Solid Phase

When coated solid-phase components (e.g., immunoassay plates, beads, or 

membranes) are prepared, identify the solid-phase component type and the coating 

process. Each lot of coated solid-phase component should be prepared with a single lot

of coating reagent and a single lot of solid-phase substrate. Include the formula for

reagents used to prepare the solid-phase components.

3. Fill volume

List the minimum and maximum fill volumes for each final container to ensure there 

is enough of the component to adequately perform the test(s).
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Appendix III, Page 5

4. Disposal 

Describe the method(s) used to dispose of unsatisfactory material. Alternatively, the

Outline may refer to VS Memorandum No. 800.56.

Section V: Testing for Serial Release

1. Test kits are exempt from the sterility and purity tests described in 9 CFR 113.26, 

113.27, and 113.28, and from animal safety tests. Specify the exemptions in the 

Outline. Include the statement, “Test kits are exempt from purity tests,” to section 

V.A. Include the statement, “Test kits are exempt from animal safety tests,” to 

section V.B.

2. See VS Memorandum No. 800.73 for guidance on the reports and data needed for 

CVB approval of potency tests. Potency is typically evaluated using a panel of 

well-characterized samples approved by CVB. Include the identifier for each panel 

member, along with criteria for valid and satisfactory tests. Include storage 

conditions. Include plate diagrams where applicable. Include the assay protocol.

As a minimum, include panel members that serve as examples of:

• Samples from negative/uninfected animals.

• Samples from strongly positive animals.

• Samples from weakly positive animals.

Section VI: Post-preparatory Steps

1. List the number of final component containers in each kit box. Multiple 

presentations, with various quantities of kit components, are acceptable.

2. Describe the collection of samples from each serial to be retained and submitted 

to CVB. See 9 CFR 113.3(b)(7), 113.3(e)(1) and VS Memorandum No. 800.59. 

Specify conditions for storage and shipment of the kits, in accordance with 9

CFR 114.11.

3. Each lot of each component in the kit shall be assigned an expiration date based 

on the stability of the individual component. The expiration date may be indicated 

on the component label. The expiration date of the serial shall be calculated from 

the date of initiation of the first potency test, but shall not exceed the expiration 

date of any of the components. The dating for test kits shall not exceed 12 months 

unless real-time stability data to justify a longer interval have been approved by 

CVB. Indicate the date CVB approved the real-time data confirming the dating 

period.

4. Information in section VI.D should be consistent with the user instructions 
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Appendix III, Page 6

provided in regulated labeling. Include all recommendations, qualifications,

limitations, and interpretations. Describe the acceptable qualities and the potential 

impact for a test sample to be suitable for analysis in the test kit. All potentially 

infective material must be appropriately labeled. Include chemical safety and 

disposal instructions. 

5. Section VI.E: Site of Manufacture. See appendix 1, section VI.E for additional 

information.