Series 810 Guidelines FAQ
The following Frequently Asked Questions (FAQs) refer to the revised Product Performance Test
Guidelines OCSPP 810.2000, 810.2100, and 810.2200 dated February 2018. The test guidelines may be
accessed at the following location: https://www.epa.gov/test-guidelines-pesticides-and-toxic-
substances/series-810-product-performance-test-guidelines. Documents pertaining to the revision of
the product performance guidelines, including public comment submissions, and the Agency's response
to comments are available at www.regulations.gov, in docket EPA-HQ-QPP-2015-0276.
With the exception of confirmatory testing (described under OCSPP guideline 810.2000, section (B)(7)),
all studies initiated on or after August 28, 2019 should be in compliance with the 2018 revised
guidelines for testing. The study initiation date is defined under 40 CFR Part 160.3 as the date the
protocol is signed by the study director. Studies that were initiated prior to the implementation date but
submitted to the Agency for review after the implementation date may use either the previous version
of the guidelines (2012) or the revised (2018) versions, as appropriate. The Agency intends to address
confirmatory testing through a separate guidance, which will be made available for public comment
prior to finalization. In the interim, applicants should consult with EPA for all confirmatory efficacy
questions with the exception of the examples of formulation changes that do not need confirmatory
efficacy data, identified in section B(7) of the 810.2000 chapter.
This guidance is not binding on EPA or any outside parties, and EPA may depart from the guidance
where circumstances warrant and without prior notice. Registrants and applicants may propose and
submit alternative practices (e.g., modifications to the recommended test methodology) to the Agency
for assessment. The Agency will evaluate any proposed method modifications for appropriateness on a
case-by-case basis. This guidance may be updated in the future.
General Questions:
Question: The "Notice" on the first page of all three test guidelines indicates that the use of the
term "should" means an action is recommended, but not mandatory. In many places
throughout the 810 documents, "should" is used to describe testing requirements and when
testing is required. Could you explain how to interpret the Agency's view of the use of the word
"should" in connection with new or detailed testing situations?
Answer: As indicated in the "Notice" preceding all three referenced guidance documents as
well as in this and other EPA guidance documents, guidance is not binding on EPA or
registrants/applicants. Accordingly, the Agency uses non-mandatory terms in the guidance such
as "should." Through these Product Performance Test Guidelines, the Agency seeks to assist
registrants' understanding of how the Agency interprets the antimicrobial data requirements in
40 CFR Part 158 Subpart W.
Question: When are submissions expected to be in compliance with the revised 2018
guidelines?
Answer: With the exception of confirmatory testing (described under OCSPP guideline
810.2000, section (B)(7)), all studies initiated on or after August 28, 2019 should be in
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compliance with the 2018 revised guidelines for testing. The study initiation date is defined
under 40 CFR Part 160.3 as the date the protocol is signed by the study director. Studies that
were initiated prior to the implementation date but submitted to the Agency for review after
the implementation date may use either the previous version of the guidelines (2012) or the
revised (2018) versions as appropriate.
The Agency intends to address confirmatory testing through a separate guidance, which will be
made available for public comment prior to finalization. In the interim, applicants should consult
with EPA for all confirmatory efficacy questions with the exception of the examples of
formulation changes that do not need confirmatory efficacy data, identified in section B(7) of
the 810.2000 chapter.
Question: What changes can registrants expect for new registration and amendment
applications submitted after the August 28, 2019 implementation date of the revised 810.2000,
810.2100 and 810.2200 Guidelines?
Answer: The revised guidelines generally articulate EPA guidance and interpretations that have
been in effect for some time. Therefore, EPA does not anticipate any substantial changes to
how it processes applications or the ways in which it evaluates and interprets the test data
submitted. The Agency recommends compliance with the guidelines, as applicable, to facilitate
efficient processing and approval of applications. However, EPA evaluates submissions on a
case-by-case basis to determine whether there is adequate support for product registrations or
amendments.
Guideline 810.2000
Section: (B)(7) Confirmatory Data
Question: Section 810.2000 (B)(7) in the second paragraph describes the dye and fragrance
changes where confirmatory efficacy data are not required to be conducted or submitted. In
cases where a combination of dye and fragrance change is proposed, what are the criteria for
data submission?
Answer: Where the total change to fragrances and/or dyes are less than or equal to 1.0% (w/w)
of the total formulation, confirmatory data are not needed.
Section: (B)(7) Confirmatory Data
Question: Please clarify the submission requirements and examples of when confirmatory
testing should be conducted.
Answer: The Agency intends to address confirmatory testing through a separate guidance,
which will be made available for public comment prior to finalization. In the interim, applicants
should consult with EPA concerning all confirmatory efficacy questions with the exception of the
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examples of formulation changes that do not need confirmatory efficacy data under section B(7)
of the 810.2000 chapter as follows:
"Specifically, formulation changes do not need confirmatory efficacy data when:
(i)	Only the concentration of a fragrance or dye is increased, substituted, or decreased in a
formulation, and
(ii)	the concentration of fragrances does not exceed 1.0% (w/w) of the total formulation, or
(iii)	the total percentage of changes in dyes does not exceed 1.0% (w/w) of the total
formulation."
Section: (B)(8) Agency Verification of Efficacy
Question: To make a meaningful determination of efficacy for a product that is registered, how
will the Agency conduct testing for evaluation?
Answer: In general, section (B)(8) refers to products that will be tested as part of the revised
Antimicrobials Testing Program (ATP). The ATP is currently suspended while the Agency
develops a new, risk-based strategy to ensure the effectiveness of public health pesticides used
in hospital settings. This approach will be addressed under the Antimicrobial Performance
Evaluation Program (APEP) strategy.
Section: (C)(2) Antimicrobial Products with Non-Public Health claims
Question: The current link to the "Crosswalk Table for Non-Public Health Guidelines" accesses a
document which states, "EPA's Web Archive: This content is not maintained and may no longer
apply." Please provide an updated table.
Answer: The OCSPP 810 Guidelines primarily focus on public health claims. The crosswalk table
is specific to testing for non-public health claims and is still accessible via the web archive. The
Agency recommends that applicants continue to reference the archived resource and consult
with the Agency until a revised table is available.
Section: (D) Definitions - Biofilm
Question: For the purposes of the 810 test guidelines, OCSPP 810.2000 section D contains a
definition of biofilm that does not distinguish between biofilm products that may include public
health claims and others that do not. The "Response to Comments" document states that all
label claims against biofilm will be considered public health claims unless the label expressly
states the use is non-public health. Did the Agency intend to impose a new definition for biofilm
or new labeling requirements for this claim? Is the "biofilm" claim limited to public health
products?
Answer: Biofilm claims may be made for both public health and non-public health products.
Claims against public health organisms must be supported by appropriate efficacy data. Where
the term biofilm is used on a label to expressly or impliedly make a public health claim, the claim
needs to be supported with efficacy data. The language in the Response to Comments
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document was only intended to illustrate this point. EPA did not intend to impose a new
definition for biofilm or new labeling requirements.
Section: (E)(1) General Testing considerations - Test substance
Question: 810.2000 section E(l)(d): The Agency recognizes the difficulty of formulating products
at the lower certified limit (LCL) for testing and provides acceptable ranges above and below the
LCL based on the product's active ingredient concentration. Will the Agency accept data where
the active ingredient concentration is less than the lower bound for the LCL?
Answer: The Agency strongly encourages that testing be conducted as close to the LCL as
possible. This ensures that products are subject to an even playing field for efficacy testing and
that the test accurately represents the product to be sold. The range around the LCL is to
provide a buffer for formulations where it may be difficult to achieve the target concentration of
active ingredient (A.I.)- In most cases where the concentration of A.I. falls below the lower
bound for the LCL, the data will still be acceptable; applicants should consult with the Agency
prior to testing to confirm that the lower concentration of A.I. (below the LCL) will not impact
product performance evaluation.
Section: (E)(1) General Testing considerations - Test substance
Question: The LCL Testing Guidance (posted by the EPA on December 6, 2013) discusses
reactive and unstable chemistries and the difficulty in achieving the allowable test range by
direct formulation or by product dilution which is omitted from 810.2000 section E(l). What are
the LCL recommendations for reactive and unstable chemistries?
Answer: In the case of products with reactive or unstable chemistries that cannot achieve the
allowable test range, registrants should provide a thorough justification detailing all efforts
made to obtain acceptable test samples, as well as rationale supporting the chemical basis for
product instability. The Agency will address these situations on a case-by-case basis depending
on the supporting information provided by the registrant, existing storage stability data for the
product, and on how close the attained concentration is to the acceptable LCL range.
Section: (E)(6) General Testing considerations - Dilution of Products for Testing - Hard Water Guidance
Question: How is tap water prepared for efficacy testing? How are adjustments to tap water
made if it falls outside of the range?
Answer: A Standard Operating Procedure (SOP) containing specific guidance for how to prepare
and adjust tap water for testing is available on the Agency's webpage: Antimicrobial Testing
Methods & Procedures Developed by EPA's Microbiology Laboratory under MB-30-01.
Section: (E)(6) General Testing considerations - Dilution of Products for Testing - Hard Water Guidance
Question: What are the allowable ranges around the 200 ppm, 400 ppm and 375 ppm hard
water levels cited?
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Answer: EPA recommends the AOAC recommended range of -10% and +5% for each type of
hard water (e.g., AOAC hard water range = 360-420 ppm calcium carbonate; OECD hard water
range = 338-394 ppm calcium carbonate; Tap Water = 180-210 ppm calcium carbonate) as
referenced in AOAC 960.09 and the SOP for preparation of hard water on the Agency's
webpage: Antimicrobial Testing Methods & Procedures Developed by EPA's Microbiology
Laboratory under MB-30-01.
Section: (E)(6) General Testing considerations - Dilution of Products for Testing - Hard Water Guidance
Question: When diluting a concentrated product, what are appropriate label claims (use
directions and claims) for using the various types of water?
Answer: This is determined on a case-by-case basis and individual products may have varying
water dilution scenarios for efficacy testing. Examples of testing scenarios and corresponding
labeling are available in Appendix I.
Section: (E)(8) General Testing considerations - Contact time (Exposure Period)
Question: 810.2000 section (E)(8) indicates that towelette products may use the wetness
determination test from 810.2100 section l(l)(a) to determine the maximum contact time. Is
this wetness determination test required to be conducted? Is this testing required to be
submitted to EPA?
Answer: The Agency recommends conducting wetness determination testing to ensure
appropriate selection of contact times for a towelette product (e.g., surface remains wet and
does not evaporate prior to completion of contact period). Currently, this test is used for
towelette products with claims against Clostridium difficile or Candida auris.
Section: (E)(9) General Testing considerations - Neutralization Confirmation
Question: Could the neutralization confirmation control be performed after testing?
Answer: In general, we recommend that the neutralization control be run on the same day as
the efficacy test. However, we recognize individual methods may have other guidance. For
example, AOAC 955.15 specifies the neutralization confirmation must be performed in advance
or in conjunction with the Use Dilution test. We also recognize that, for certain types of efficacy
studies (e.g., pool field studies, air sanitization, in-tank sanitization, water purification), it is not
practical to conduct daily neutralization controls and neutralization testing is typically conducted
prior to efficacy testing. Certain situations may also justify repeating neutralization confirmation
after the efficacy test. In these cases, a detailed rationale for the neutralization failure should be
provided by the laboratory. If the neutralization failure is due to the inoculum level being too
high or too low (outside of the acceptable range for the neutralization confirmation control
counts), repeat testing of just the neutralization assay is appropriate. For all other failure
scenarios, the efficacy test and the neutralization assay are considered invalid and should be
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repeated. If there is interference in the neutralization assay caused by contamination, up to two
repeat tests may be performed (see Appendix II for information on repeat testing).
Section: (E)10 General Test Considerations - Batch Replication
Question: Batch replication is defined as efficacy testing using a reduced number of product
batches (lots) for certain use modifications of a registered product, e.g., the addition of organic
soil, a change in hard water concentration or testing temperature, or the use of a porous
carrier. Does the performance standard for each of the base organisms tested in the AOAC Use-
Dilution Method remain as described in 810.2200(D) when the batch replication policy is
employed to reduce the number of test batches?
Answer: Yes. The performance standard in the Evaluation of Success Section 810.2200(D) for the
base test organisms (i.e., Salmonella enterica, Staphylococcus aureus, and Pseudomonas
aeruginosa) remains applicable for AOAC Use-Dilution Method studies performed with a
reduced number of test batches as addressed by the Batch Replication Policy outlined in
810.2000(E)(10). The performance standard of up to three positive carriers out of 60 for S.
aureus and up to 6 positive carriers out of 60 for P. aeruginosa remains appropriate when two
test batches are tested on independent test dates. Likewise, at this time, the performance
standard for S. enterica remains at one positive carrier out of 60 and with no need to test each
of the two test batches on separate test dates.
Section: (E)(ll) Repeat Testing
Question: How many times can you repeat test for contamination in a test system?
Answer: Contamination found in the test system may invalidate the assay in certain situations
and applicants have the option to conduct repeat tests up to two times to address
contamination. If multiple contamination events occur, the laboratory may consider a quality
plan to rectify the issue (e.g., performing a facility/equipment cleaning, replacing the stock tube,
purchasing a new lyophilized stock, etc.) as appropriate. Separate laboratory personnel (e.g.,
study director, technical staff, QA auditor) or testing at a different laboratory are not needed for
repeat testing.
Section: (E)(ll) Repeat Testing
Question: How many times can repeat testing be conducted due to contamination on the carrier
control?
Answer: Applicants have the option to conduct repeat tests up to two times to achieve a valid
control. See Appendix II for guidance regarding contaminated carrier control counts and when
repeat testing should be conducted.
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Section: (E)(ll) Repeat Testing
Question: Can a repeat test be conducted for a 60 carrier Qualitative test using S. aureus, S.
enterica or P. aeruginosa, where the total number of positive carriers for growth (including
contamination) exceed the performance standards?
Answer: If the presence of a contaminated carrier results in the performance standard being
exceeded, the test would be invalidated and applicants have the option to conduct repeat tests
up to two times to address contamination. However, if the number of positive carriers due to
growth of the target organism alone exceeds the performance standard, then the test would be
considered to have failed regardless of additional contaminated carriers and repeat testing
should not be conducted. See Appendix III for example scenarios.
Section: (E)(ll) Repeat Testing
Question: If a carrier population control is greater than the acceptance criteria, can a test be
repeated, and if so, how many times?
Answer: Yes, if the carrier population control is greater than the acceptance criteria and the
product fails to achieve the performance standard, applicants have the option to conduct repeat
testing up to two times to address a population control failure. However, if the test substance
meets the performance standard with a carrier population control count greater than the
acceptance criteria, testing does not need to be repeated.
Section: (E)(ll) Repeat Testing
Question: How many times can testing be repeated?
Answer: EPA understands that different issues may arise on each subsequent test date. In cases
of control failures, applicants have the option to conduct up to two repeat tests to address each
type of control failure (e.g., failure to neutralize, soil sterility failure, purity control failure,
carrier control failure) to achieve a valid test. Where repeat testing occurs within the same
study, invalid data due to these control failures should be reported in appendices of the final
report.
Section: (E)(ll) Repeat Testing
Question: Is a repeat test necessary for a 60 carrier Qualitative test using S. aureus, S. enterica
or P. aeruginosa, where the total number of positive carriers for growth (including
contamination) fall within the performance standards?
Answer: In the case that only one carrier is contaminated and the total number of positive
carriers for growth fall within the performance standards, repeat testing is not needed.
However, if multiple carriers are found to be contaminated, applicants should conduct repeat
testing even when the total number of positive carriers fall within the performance standards.
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Applicants have the option to conduct up to two repeat tests to address contamination issues.
Refer to Appendix III for example repeat testing scenarios.
Section: (E)(ll) Repeat Testing
Question: If you have a 10-carrier qualitative test forS. aureus and P. aeruginosa and the
formulation fails with 1 or more positive tubes out of 10, will EPA allow repeat testing using 60
carriers?
Answer: No, the test has failed for the performance standard and applicants should not proceed
with repeat testing using 60 carriers. In these cases, the applicant should report the failure(s)
and has the option to conduct a different test by changing the test conditions (e.g., removing
soil load or using an increased contact time and/or a higher concentration to support the new
efficacy label claim for the product).
Section: (E)(ll) Repeat Testing
Question: If you have a 10-carrier qualitative test for confirmatory testing or for an additional
organism (e.g., MRSA) and it fails 1/10, can testing be repeated on that failing lot with 60
carriers?
Answer: No, the test has failed for the performance standard and applicants should not proceed
with repeat testing using 60 carriers. In these cases, the applicant should report the failure(s)
and has the option to conduct a different test by changing the test conditions (e.g., removing
soil load or using an increased contact time and/or a higher concentration to support the new
efficacy label claim for the product).
Section: (E)(ll) Repeat Testing
Question: For a quantitative assay, is it recommended that a repeat test be conducted when any
level of contamination occurs which interferes with the ability to interpret results, deeming the
test inconclusive?
Answer: For quantitative assays, the following are the Agency's recommended criteria for
repeat testing:
•	A test in which a sporadic, isolated contaminant(s) is observed on a plate that does not
interfere with the reading of results is a valid test. Repeat testing is not needed.
•	A test in which systemic contamination is present (e.g., contamination within an entire
dilution series) is invalid and applicants have the option to repeat the test up to two
times to address contamination.
•	A test in which contaminants inhibit the analyst's ability to accurately read a plate(s) is
invalid and applicants have the option to repeat the test up to two times to address
contamination.
Appendix II and Appendix III provide more information regarding repeat testing for quantitative
tests.
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Guideline 810.2100
Section: (E) Sporicide Claim
Question: Could a product have a disinfectant label claim against the vegetative form of
Clostridium perfringens?
Answer: Consistent with other spore forming organisms (e.g., Clostridium difficile, Bacillus
anthracis), testing should be conducted against the spore form of the organism. Stand-alone
claims against the vegetative form could be potentially misleading to consumers as these are
common spore-formers.
Section: (E) Sporicide Claim
Question: For surface-specific sporicide claims under 810.2100(E), what are the carrier types
and how many test carriers should be tested?
Answer: For surface-specific sporicide claims:
1.	To add a hard, non-porous surface sporicidal claim to a liquid product, perform
testing on the base strains using stainless steel penicylinders (60 carriers/lot/strain,
3 lots). Testing on suture loops or porcelain penicylinders is not necessary.
2.	To add a hard, non-porous and porous surface sporicidal claim to a liquid product,
perform testing on the base strains using the porcelain penicylinders (60
carriers/lot/strain, 3 lots). Testing on suture loops or stainless steel penicylinders is
not necessary.
3.	To add a soft, porous surface sporicidal claim to a liquid product, perform testing on
the base strains using suture loops (60 carriers/lot/strain, 3 lots). Testing on
stainless steel or porcelain penicylinders is not necessary.
Carrier types for testing should be tailored to the label claims and EPA may recommend
alternate surface types. Similar to disinfection testing, for example, sporicidal testing using
stainless steel penicylinders alone generally would support general hard, nonporous surface
claims and use sites on labels. Note, verification testing should be conducted in all of the
scenarios described above as detailed in section (E)(vi) of Guideline 810.2100.
Section: (I) Hospital or Healthcare Disinfectant with Sporicidal Activity against Clostridium difficile
Claim
Question: Is verification testing needed for Clostridium difficile?
Answer: No verification testing is needed at this time.
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Guideline 810.2200
Section: (G)(5) and (6){vii) Virucidal Claims
Question: 810.2200 section (G)(5) and (6)(vii) indicate that the log survivors and log reductions
should be reported per assayed volume and per carrier surface. Reporting per the carrier
surface is new. How should this calculation be performed?
Answer: Please see Appendix IV for sample calculations. Note that most probable number
(MPN) software is no longer necessary for any viral protocols.
Section: (J) Towelette Products and (K)(2) Bridging for Towelettes - Efficacy Testing
Question: What application procedure should be used for bacterial, fungal, tuberculocidal, and
viral testing for towelette disinfection claims?
Answer: For testing bacterial, mycobacterial and fungal disinfection claims for towelettes, EPA
recommends the use of AOAC 961.02 or ASTM E2362. In these methods, one wipe is used to
treat 10 carriers. Typically, each carrier is wiped back and forth 3 times for a total of 6 passes
before moving to the next carrier. A pass is defined as moving from one side of the carrier to the
other with a single motion. For virucidal claims, EPA recommends use of ASTM E1053. When
testing with EPA recommended viral surrogates, please reference the protocols outlined in
810.2000. Due to the large carrier used for viral testing, one towelette is used to wipe one test
carrier. Typically, this testing is also performed with 6 passes across the carrier surface. Due to
the large carrier size and the small folded size of the wipe, consider whether to treat the carrier
in two parallel sections using the same wipe, or whether the wiping technique used in E2896
should be employed for viral testing.
Section: (K)(l) Bridging for EPA-Registered Liquids and Disinfectant Towelettes: Chemical Analysis
Question: 810.2200 section (K)(l) indicates that mechanically expressed liquid from towelettes
should be used for chemical analysis when bridging a disinfectant towelette to an EPA-
registered bulk liquid formulation. What does "mechanically expressed" mean?
Answer: "Mechanically expressed" includes any physical means of extracting liquid from a
towelette product (other than pouring off excess liquid from the bulk towelette container).
"Mechanically expressing" includes but is not limited to:
•	Squeezing using a gloved hand
•	Centrifuging the liquid from the towelette
•	Compressing the towelette using a plunger inside of a syringe
For alternative methods of extraction, applicants should consult with the Agency prior to testing
and provide a justification for why the mechanical methods of extracting or expressing would
not be appropriate.
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APPENDICES
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Appendix I - Label Use-Directions for Dilutable Products
Where the label use-directions require the user to dilute a concentrated product, these are label
examples based on varying types1 of water used in efficacy testing for a given product:
Scenario 1 (mixed use of hard water): A registered product has existing hard water label claims prior to
the implementation of the 2018 guidelines. For example, the organisms on the label were previously
tested and approved at 300 ppm AOAC hard water. New data is submitted for existing or new
microorganisms using 400 ppm AOAC hard water. The following are acceptable label directions:
•	Label: Dilute with [tap] water AND/OR
•	Label: Can be diluted with hard water AND/OR
•	Label: Use up to 300 ppm hard water
Sample Label Language: "For use as a Daily Disinfectant: Dilute 6 ft. oz in water [up to 300 ppm
hardness]. Pre-clean heavily soiled areas. Apply Use Solution by coarse trigger sprayer to hard, non-
porous surfaces. Spray 6-8 inches from the surface, making sure to wet surfaces thoroughly. All surfaces
must remain wet for the reguired time indicated in the directions for use."
Scenario 2 (mixed use of hard water): A registered product has existing hard water label claims prior to
the implementation of the 2018 guidelines. The organisms on the label were tested at a level of water
hardness that is less than the revised hard water recommendations (e.g., 300 ppm AOAC hard water).
Testing is conducted for new microorganisms using 200 ppm hard tap water. The following are
acceptable label directions:
•	Label: Dilute with [tap] water AND/OR
•	Label: Can be diluted with hard water AND/OR
•	Label: Dilute with up to 200 ppm hard water
Sample Label Language:
"For use as a Daily Disinfectant: Dilute 6fl. oz in water [up to 200 ppm hardness]..."
Scenario 3 (mix of different types of hard water, all following the revised 2018 guidelines): A registered
product has hard water label claims prior to the implementation of the 2018 guidelines. The organisms
on the label were tested using any combination of the recommended hard water types specified in the
revised 2018 guidelines (e.g., S. aureus disinfection using 200 ppm tap water, C. auris disinfection using
375 ppm OECD synthetic hard water). The following are acceptable label directions:
•	Label: Dilute with [tap] water AND/OR
1Hard water is defined as:
•	Regular (un-softened) tap water with a minimum of 200 ppm calcium carbonate
•	AOAC Synthetic hard water of 400 ppm calcium carbonate
•	OECD hard water formula of 375 ppm hard water
(OCSPP 810.2000 (E)(6))
Softened water is defined as water with levels of calcium carbonate below the respective concentration indicated
above for each type of water.
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•	Label: Can be diluted with hard water AND/OR
•	Label: Dilute with up to 200 ppm hard water
Sample Label Language:
"For use as a Daily Disinfectant: Dilute 6 ft. oz in water [up to 200 ppm hardness]..."
Scenario 4 (level of water hardness exceeds the revised 2018 guidelines): A registered product has hard
water label claims prior to the implementation of the 2018 guidelines. The microorganisms on the label
were tested using 500 ppm AOAC hard water (or higher). The following are acceptable label directions:
•	Label: Dilute with [tap] water AND/OR
•	Label: Can be diluted with hard water AND/OR
•	Label: Dilute with up to 500 ppm hard water
Sample Label Language:
"For use as a Daily Disinfectant: Dilute 6fl. oz in water [up to 500 ppm hardness]..."
Scenario 5 (mix of hard water consistent with the revised 2018 guidelines and soft water): A registered
product has hard water label claims prior to the implementation of the 2018 guidelines. Organism X was
tested in 400 ppm AOAC hard water and organism Y was tested using sterile deionized water. The
following are acceptable label directions:
•	Label: Dilute using [tap] water for organisms X. Dilute using sterile deionized water for organisms Y
[and X],
•	Label: Can be diluted with hard water for organisms X. Dilute using sterile deionized for organisms Y
[and X],
Sample Label Language:
"For use as a Daily Disinfectant: Dilute 6fl. oz in deionized* water...
	*ideionized water for organism Y and tap water for organism X"
Scenario 6 (mix of hard water consistent with the revised 2018 guidelines and previous testing at below
200 ppm): A registered product with new data against organism X using tap water with 200 ppm
hardness (post implementation of the 2018 guidelines) and old data against organism Y using tap water
with 100 ppm hardness (prior to the implementation of the 2018 guidelines). The following are
acceptable label directions:
•	Label: Dilute using [tap] water for organism X. For organism Y, dilute using water softened to less
than or equal to 100 ppm.
Sample Label Language: "For use as a Daily Disinfectant: Dilute 6fl. oz in water with a minimum water
hardness of 100 ppm...
* water softened to less than or egual to 100 ppm hardness for organism Y and tap water for
organism X"
Scenario 7 (soft water or deionized water): New or registered product tested with soft water.
•	Consult with Agency prior to testing.
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Appendix II - Revised Repeat Testing Policy
Revised language for OCSPP Guideline 810.2000, section (E)(ll): Repeat Testing.
Since the posting of the 2018 guidelines in February 2018, the Agency received additional comments and
concerns from industry regarding approaches for repeat testing. As a result, the Agency has adjusted the
repeat testing guidance to address stakeholder concerns. The new guidance is as follows:
"Repeat Testing. The Agency defines repeat testing as retesting a product lot under the same conditions
as the original test (e.g., same number of carriers, temperature, contact time, soil load, dilution rate, etc).
Repeat testing is appropriate only in certain cases.
The following table provides a general retesting strategy for evaluation of antimicrobial product efficacy.
•	Failed tests are defined as test outcomes in which the product did not meet the performance
standard for the efficacy claim. Performance standards are outlined in the OCSPP 810.2000 series in
the "Evaluation of Success" sections for each efficacy claim.
•	Contaminants are defined as microorganisms which are not the test organism that are present in
the study. Methods such as gram staining, colony morphology, and biochemical assays should be
used to identify the contaminant and presence and/or absence of the test organism. Results should
be provided to EPA.
Outcome
Passed/Failed Test
Lot Retest?
# of Repeats
per Lot
1. Mean control carrier count level above
acceptable range
Failed
Yes
2
2. Mean control carrier count level above
acceptable range
Passed
Not necessary
N/A
3. Mean control carrier count level below
acceptable range
Failed
No
N/A
4. Mean control carrier count level below
acceptable range
Passed
Yes
2
5. Presence of contamination
See Contamination in Subculture Media and
Contamination in Control Carrier Counts or Neutralization
Assays (for qualitative tests) or Quantitative Efficacy
Tests below.
6. Neutralization verification assay failure
Passed or Failed
Yes
2
7. Documented control failure (including
sterility control and test system control)
Passed or Failed
Yes
2
8. Documented operator error or test
system failure which results in an invalid
study as deemed by the Study Director
Passed or Failed
Yes
2
9. Verified product contamination for EPA
registered products
N/A
No, notify EPA
immediately
N/A
Contamination in Subculture Media
• In the case where a contaminant and the test organism are both identified to be present in the
subculture tube, the outcome is considered a positive carrier.
Page 14 of 25

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•	For a 60-carrier qualitative test
o One contaminated carrier (subculture medium tube) may occur per 60-carrier test,
without a retest, if the total number of positive carriers including the contaminant meets
the performance standard,
o A test where the total number of positive carriers, including the contaminant, exceeds
the performance standard by one positive carrier is invalid and may be repeated up to
two times using another 60-carrier test,
o A test with more than one contaminated carrier is invalid and may be repeated up to two
times using another 60-carrier test.
•	For a 10-carrier qualitative test
o A test with one or more contaminated carrier(s) is invalid and may be repeated up to two
times using another 10-carrier test.
Contamination in Control Carrier Counts or Neutralization Assays (for qualitative tests) or Quantitative
Efficacy Tests
•	A test in which a sporadic, isolated contaminant(s) is observed on a plate that does not interfere
with the reading of results is a valid test. Repeat testing is not needed.
•	A test in which systemic contamination is present (e.g., contamination within an entire dilution
series) is invalid and may be repeated up to two times to achieve a valid test.
•	A test in which contaminants inhibit the analyst's ability to accurately read a plate(s) is invalid
and may be repeated up to two times to achieve a valid test.
Repeat Testing Criteria:
1.	All product lots should be tested at the appropriate concentration for the claim (i.e., LCL or
nominal concentration of the A.l.s).
2.	Testing should be performed under the original testing conditions.
3.	A valid rationale to support retesting should be provided in the report.
4.	In case of contamination, run all identification tests necessary to rule out the test system
organism. Results should be included in the final study report.
5.	Report all passing and failing data.
For any exceptional circumstances that fall outside of the scope of this guidance, applicants are
encouraged to consult with the Agency prior to conducting any additional repeat testing.
In cases where repeat testing is not appropriate, and the test conditions are identical to the label
conditions, the applicant should report the failure(s) and may choose to conduct a different test by
changing the test conditions (e.g., removing soil load or using an increased contact time and/or a higher
concentration to support the new efficacy label claim for the product)."
Page 15 of 25

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Appendix III - Repeat Testing Scenarios
Example Repeat Testing Scenarios
Test Parameters
Example outcome
Example Performance
Standard/ (# positive/ total
# carriers)
Conclusion
AOAC UDM
S. aureus 60 carriers
4 total positive tubes; 2
positives test organism and
2 contaminants
3/60
Invalid /
Retest
3 total positive tubes; 1
positive test organism and
2 contaminants
3/60
Invalid /
Retest
3 total positive tubes; 2
positive test organism and
1 contaminant
3/60
Valid / Pass /
No retesting
necessary
5 total positive tubes; 4
positive test organism and
1 contaminant
3/60
Valid / Fail /
No retesting
AOAC Germicidal
Spray Test/ Towelette
S. aureus 60 carriers
2 total positive tubes; 1
positives test organism and
1 contaminant
1/60
Invalid/ Retest
1 total positive tube which
is a contaminant
1/60
Valid / Pass /
No retesting
3 total positive tubes; 2
positive test organism and
1 contaminant
1/60
Valid / Fail /
No retesting
Quantitative Test
Methods (e.g. NFCS,
QCT, FCS, control
counts for qualitative
tests)
Test and or control
contamination
Any isolated contamination
which interferes with the
recording or interpretation
of results OR systemic
contamination in a test
results in an invalid test.
Invalid/
Repeat
Testing
Page 16 of 25

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Appendix IV-Virucidal Calculations
Sample calculation of TCID50 using the Spearman-Karber method
The TCID50 (50% Tissue Culture Infectious Dose) refers to the dilution where 50% of the inoculated cell
cultures exhibit cytopathic effects due to infection by the test virus.
Example 1- No cytotoxicity
Conditions:
200 jiL of virus is dried onto the carrier.
2 mL of media (plate recovery control) or test substance is added to the carrier.
The mixture is immediately passed through a Sephadex or Sephacryl column after the
contact time, with no additional neutralizer volume, and the full 2 mL of filtrate is collected.
This is considered the 10'1 dilution.
The 101 dilution is tenfold serially diluted in a dilution medium.
100 jiL oflO'1 to 10r? dilutions is inoculated into the cell culture well.
The limit of detection for TCID50/O.I mL is 1CPS0-
Results:
Plate Recovery Control
Dilution
Plate Recovery
Control
Percent Infection
Cell Control
0000
-
101
+ + + +
100
10"2
+ + + +
100
10"3
+ + + +
100
10"4
+ + + +
100
10"5
+ + + 0
75
10 s
00 + +
50
10"7
0000
0
TCIDso/100 [il
105.75
-
TCIDso/carrier
io605
-
+) = Positive for the presence of test virus
(0) = No test virus recovered and no cytotoxicity present
Calculations:
Using the Spearman Karber method as follows:
Page 17 of 25

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Negative Log of TCID50/IOO nl = -log of 1st dilution assayed - [((I of % mortality at each dilution/100) -
0.5) x (log of dilution)]
Negative Log of TCID50/IOO \i\ = -1 - [(((100 + 100 + 100 + 100 + 75 + 50J/100) - 0.5) X (1)]
=-1-[((5.25)-(0.5)) xl]
= -1 - 4.75Log of TCID50/IOO \i\ = 5.75
Therefore, the TCID50/100 ^l = 10s 75
To calculate the TCIDso/carrier for the plate recovery control, multiply by 2 [note 2 is used since 200 nl of
virus is applied to the carrier surface]
TCIDso/carrier = (Antilog of 5.75) X 2 = 1,124,682.65
The Logio of 1,124,682.65 = 6.05 so the TCID50/carrier = 106 05
To calculate the TCIDso/carrier for the treated carrier,
Dilution
Virus + Test Substance Lot 1
Percent Infection
Cell Control
0000
-
101
+ + + +
100
10"2
00 + +
50
IO"3
0000
0
IO"4
0000
0
IO"5
0000
0
10-e
0000
0
IO"7
0000
0
TCIDso/100 [il
io200
-
TCIDso/carrier
io2-30
-
Logio Reduction
3.75
-
+) = Positive for the presence of test virus
(0) = No test virus recovered and no cytotoxicity present
Negative log of TCID50/100 \i\ = -1 - [(((100 + 50 + 0 + 0 + 0)/100) - 0.5) X (1)]
= -1 - [((1.5) - (0.5)) x 1]
= -1-1.5
= -2
Log of TCID50/IOO \i\ = 2
TCIDso/100 [i\ = 102 0
To calculate the TCID50/carrier, multiply by 2 [note 2 is used since 200 nl of virus is applied to the carrier
surface]
Page 18 of 25

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TCIDso/carrier for the treated carrier = (Antilog of 2.0) X 2 = 200
The log of 200 = 2.30 Logio, so the TCIDso/carrier = 102 3
The log reduction is:
(LogTCIDso/carrier for the plate recovery control) - (LogTCIDso/carrier for the treated carrier)
6.05-2.3=3.75
Example 2 - No Cytotoxicity
Conditions:
200 jiL of virus is dried onto the carrier.
2 mL of media (plate recovery control) or test substance is added to the carrier.
The mixture is immediately passed through a Sephadex or Sephacryl column after the
contact time, with no additional neutralizer volume, and the full 2 mL of filtrate is collected.
This is considered the 10'1 dilution.
The 10'1 dilution is tenfold serially diluted in a dilution medium.
250 jiL oflO1 to 10r7 dilutions is inoculated into the cell culture well.
The limit of detection for TCIDso/0.25 mL is 1CPS0-
Results:
Plate Recovery Control
Dilution
Cell Control
Plate Recovery
Control
0000
Percent Infection
101
+ + + +
100
10"2
+ + + +
100
10"3
+ + + +
100
10"4
+ + + +
100
10"5
+ + + 0
75
10 s
00 + +
50
10"7
0000
0
TCIDso/250 [il
105-75
-
TCIDso/carrier
105'65
-
+) = Positive for the presence of test virus
(0) = No test virus recovered and no cytotoxicity present
Page 19 of 25

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Calculations:
Using the Spearman Karber equation as follows:
Negative log of TCID50/250 nl = -log of 1st dilution assayed - [((I of % mortality at each dilution/100) -
0.5) x (log of dilution)]
Negative log of TCID50/250 \i\ = -1 - [(((100 + 100 + 100 + 100 + 75 + 50J/100) - 0.5) X (1)]
=-1-[((5.25)-(0.5)) xl]
= -1-4.75
= -5.75
Log ofTCIDso/250 ^ = 5.75
TCIDso/250 [i\ = 105-75
To calculate the TCIDso/carrier for the plate recovery control, multiply by 0.8 [note 0.8 is used since 200
Hi of virus is applied to the carrier surface (200 nl /250 |al)]
TCIDso/carrier = (Antilog of 5.75) X 0.8 = 449,873
The log of 449,873 = 5.65 Logio, so the TCIDso/carrier = 10s 65
The calculations for the treated carriers and the log reduction would be performed consistent with what
is presented in the first example but with the adjustment for the 250 nl inoculation volume.
Example 3 - With Cytotoxicity
Conditions:
200 jiL of virus is dried onto the carrier.
2 mL of media (plate recovery control) or test substance is added to the carrier.
The mixture is immediately passed through a Sephadex or Sephacryl column after the
contact time, with no additional neutralizer volume, and the full 2 mL of filtrate is collected.
This is considered the 101 dilution
The 10'1 dilution is tenfold serially diluted in a dilution medium.
100 jiL oflO'1 to 10r? dilutions is inoculated into the cell culture well.
The limit of detection for TCIDso/0.1 mL is 102 5
Page 20 of 25

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Results:
Dilution
Plate Recovery
Control
Virus
+ Test Substance Lot 1
Percent mortality for
the treated carrier
Cell Control
0000
0000
-
IO1
+ + + +
TTTT
100
IO"2
+ + + +
TTTT
100
IO"3
+ + + +
0000
0
IO"4
+ + + +
0000
0
IO"5
+ + + 0
0000
0
10-e
+ + 00
0000
0
IO"7
0000
0000
0
TCIDso/100 [il
105-75
< io2-5

TCIDso/carrier
IO605
< io2-80

Logio Reduction
N/A
>3.25

(+) = Positive for the presence of test virus
(0) = No test virus recovered and/or no cytotoxicity present
(T) = Cytotoxicity
The plate recovery control is the same as calculated in example 1.
Calculations:
Using the Spearman Karber equation as follows:
Negative log of TCID50/100 nl = -log of 1st dilution assayed - [((I of % mortality at each dilution/100) -
0.5) x (log of dilution)]
Negative log of TCID50/100 \i\ = -1 - [(((100 + 100 + 0 + 0 + 0)/100) - 0.5) X (1)]
= -1 - [((2) - (0.5)) x 1]
= -1-1.5
= -2.5
Log ofTCIDso/100 nl = 2.5
TCIDso/100 nl=<1025
To calculate the TCID50/carrier for the treated carrier, multiply by 2 [note 2 is used since 200 nl of virus is
applied to the carrier surface]
TCID50/treated carrier = (Antilog of 2.5) X 2 = 632.46
The log of 632.46 = 2.8 Logio, so the TCIDso/carrier = < 102 8
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The log reduction is:
(LogTCIDso/carrier for the plate recovery control) - (LogTCIDso/carrier for the treated carrier)
6.05-<2.8= >3.25
Example 4- Use of a Chemical Neutralizer
Conditions:
400 jiL of virus is dried onto the carrier
2 mL of media (plate recovery control) or test substance is added to the carrier.
2 ml of chemical neutralizer is added after the contact time and mixed. This 4 ml of mixture
is considered the 10'1 dilution
The 10'1 dilution is tenfold serially diluted in a dilution medium
100 jiL oflO'1 to 10r? dilutions is inoculated into the cell culture well.
The limit of detection for TCID50/0.1 mL is 10°-5a
Results:
Plate Recovery Control
Dilution
Plate Recovery
Control
Percent mortality
Cell Control
0000
-
101
+ + + +
100
1CT2
+ + + +
100
1CT3
+ + + +
100
10"4
+ + + +
100
icr5
+ + + 0
75
10 s
00 + +
50
10"7
0000
0
TCIDso/mL
105-75
-
TCID5o/carrier
106-35
-
(+) = Positive for the presence of test virus
(0) = No test virus recovered and no cytotoxicity present
Calculations:
Using the Spearman Karber equation as follows:
Page 22 of 25

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Negative log of TCID50/IOO nl = -log of 1st dilution assayed - [((I of % mortality at each dilution/100) -
0.5) x (log of dilution)]
Negative log of TCID50/IOO \i\ = -1 - [(((100 + 100 + 100 + 100 + 75 + 50J/100) - 0.5) X (1)]
=-1-[((5.25)-(0.5)) xl]
= -1-4.75
= -5.75
log of TCID50/IOO nl=5.75
TCIDso/100 [i\ = IO575
To calculate the TCIDso/carrier for the plate recovery control, multiply by 4 [note 4 is used since 400 nl of
virus is applied to the carrier surface]
TCIDso/carrier = (Antilog of 5.75) X 4 = 2,249,365.3
The log of 2,249,365.3= 6.35 Logi0, so the TCID50/carrier = 10s 35
To calculate the TCIDso/carrier for the treated carrier,
Dilution
Virus + Test Substance Lot 1
Percent Infection
Cell Control
0000
-
IO1
+ + + +
100
IO"2
00 + +
50
IO"3
0000
0
IO"4
0000
0
IO"5
0000
0
10 s
0000
0
IO"7
0000
0
TCIDso/mL
IO200
-
TCIDso/carrier
102SO
-
Logio Reduction
3.75
-
(+) = Positive for the presence of test virus
(0) = No test virus recovered and no cytotoxicity present
Negative log of TCIDso/100 \i\ = -1 - [(((100 + 50 + 0 + 0 + 0/100)) - 0.5) X (1)]
= -1 - [((1.5) - (0.5)) x 1]
= -1-1.5
= -2
TCIDso/100 [i\ = IO2 00
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To calculate the TCIDso/carrier for the, multiply by 4 [note 4 is used since 400 nl of virus is applied to the
carrier surface]
TCIDso/carrier for the treated carrier = (Antilog of 2.0) X 4 = 400
The log of 400 = 2.60 Logi0, so the TCID50/carrier = 102 60
The log reduction is:
(LogTCID50/carrier for the plate recovery control) - (LogTCID50/carrier for the treated carrier)
6.35-2.6=3.75
Example 5 - Surrogate virus
Conditions:
200 jiL of virus is dried onto the carrier
2 mL of media (plate recovery control) or test substance is added to the carrier.
The mixture is immediately passed through a Sephadex or Sephacryl column after the
contact time, with no additional neutralizer volume, and the full 2 mL of filtrate is collected.
This is considered the 10'1 dilution
The 10'1 dilution is tenfold serially diluted in a dilution medium
100 jiL oflO'2 to 10r7 dilutions is inoculated into the cell culture well.
The limit of detection for TCID5o/0.1 mL is 1CPS0-
Page 24 of 25

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Results:
Dilution
Plate Recovery Control
Virus
+ Test Substance Lot 1
Virus
+ Test Substance Lot 2

Rep 1
Rep2
Rep 1
Rep2
Rep 1
Rep2
Cell Control
0000
0000
0000
0000
0000
0000

+ + + +
+ + + +
0000
0000
0000
0000
IO"2
+ + + +
+ + + +
0000
0000
0000
0000
IO"3
+ + + +
+ + + +
0000
0000
0000
0000
IO"4
+ + + +
+ + + +
0000
0000
0000
0000
IO"5
+ 0 + +
+ 00 +
0000
0000
0000
0000
10-6
0 + 00
+ 0 + 0
0000
0000
0000
0000
IO"7
0000
0000
0000
0000
0000
0000
TCIDso/100 [il
io5-50
IO5-50
<10150
<10150
<10150
<10150
Average TCID50/100 nL
io5-50

<10150

<10150

Average TCIDso/carrier
io5-80

4.00

>4.00

+) = Positive for the presence of test virus
(0) = No test virus recovered and/or no cytotoxicity present
Calculations:
To calculate the TCIDso/carrier for the plate recovery control,
TCIDso/100 nL = IO550
(Antilog of 5.50) X 2 [note: 2 is used since 200 nl is applied to the carrier surface] = 632,455.532
The log of 632,455.532 = 5.80 Logi0, so the TCID50/carrier = 10s 80
To calculate the TCIDso/carrier for the treated carrier,
TCIDso/100 nL = <101-50
(Antilog of 1.5) X 2 = 63.2456
The log of 63.2456 = 1.8 Logio = TCID50/carrier= < 1018
Logio reduction per carrier surface (200 nL): 5.80 Logio - < 1.80 Logio = > 4.00 Logio reduction in viral
titer
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