United States

Environmental Protection

Agency

	Office of Prevention, Pesticides

And Toxic Substances

(7510P)		EPA 712-C-07-074

 	Product Performance Test Guidelines

		OPPTS 810.2200:

Disinfectants for Use on Hard Surfaces—Efficacy Data Recommendations

Public Review Draft

NOTICE

          This guideline is one of a series of test guidelines
established by the Office of Prevention, Pesticides and Toxic Substances
(OPPTS), United States Environmental Protection Agency for use in
testing pesticides and chemical substances to develop data for
submission to the Agency under the Toxic Substances Control Act (TSCA)
(15 U.S.C. 2601, et seq.), the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.), and section 408 of the
Federal Food, Drug, and Cosmetic (FFDCA) (21 U.S.C. 346a).

	The OPPTS test guidelines serve as a compendium of accepted scientific
methodologies and protocols that are intended to provide data to inform
regulatory decisions under TSCA, FIFRA, and/or FFDCA.  This document
provides guidance for conducting the test, and is also used by EPA, the
public, and the companies that are subject to data submission
requirements under TSCA, FIFRA and/or the FFDCA.  As a guidance
document, these guidelines are not binding on either EPA or any outside
parties, and the EPA may depart from the guidelines where circumstances
warrant and without prior notice.  The procedures contained in this
guideline are strongly recommended for generating the data that are the
subject of the guideline, but EPA recognizes that departures may be
appropriate in specific situations. You may propose alternatives to the
recommendations described in these guidelines, and the Agency will
assess them for appropriateness on a case-by-case basis.

	For additional information about OPPTS harmonized test guidelines and
to access the guidelines electronically, please go to   HYPERLINK
"http://www.epa.gov/oppts"  http://www.epa.gov/oppts  and select “Test
Methods & Guidelines” on the left side navigation menu.  You may also
access the guidelines in   HYPERLINK "http://www.regulations.gov"
http://www.regulations.gov  grouped by Series under Docket ID #s:
EPA-HQ-OPPT-2009-0150 through EPA-HQ-OPPT-2009-0159, and
EPA-HQ-OPPT-2009-0576.

DRAFT DOCUMENT DISCLAIMER:  This draft document is distributed solely
for the purpose of external review.  It has not been formally
disseminated by the EPA and should not be construed to represent any
Agency determination or policy.  The information correction process
under the Agency’s Information Quality Guidelines does not apply until
this document is formally disseminated by the EPA in its final form.
This draft document should only be cited or quoted in the context of
providing comments.

OPPTS 810.2200: Disinfectants for use on hard surfaces - efficacy data
recommendations

(a) Scope.

(1) Applicability.  This guideline is intended to meet testing
requirements of the Federal Insecticide, Fungicide, and Rodenticide Act
(FIFRA)(7U.S.C. 136, et seq.) and the Federal Food, Drug, and Cosmetic
Act (FFDCA) (21 I/S/C. 346a).  It addresses testing to demonstrate
effectiveness of products which are disinfectants, fungicides,
virucides, and tuberculocides.

(2) Background.  The source materials used in developing this OPPTS test
guideline are OPP guidelines 91-2: Products for use on hard surfaces and
91-30: Acceptable methods (Pesticide Assessment Guidelines, Subdivision
G, Product Performance.  EPA report 540/9-82-026, October 1982).

(b) Purpose.  This guideline addresses efficacy testing for
antimicrobial pesticides intended to be used on hard surfaces, namely
disinfectants, fungicides, virucides, and tuberculocides in a variety of
product types (water-soluble powders, liquids, sprays, towelettes,
etc.).

	(c) General considerations

(1)  This guideline recommends tests to be conducted and data to be
submitted which the Agency believes will generally satisfy the
requirements for pesticide registration.  All studies conducted under
this guideline should be completed under EPA’s Good Laboratory
Practice regulations (40 CFR Part 160).  Note:  The Association of
Official Analytical Chemists (AOAC) recommended tests are expected to be
conducted as written.  For deviations (e.g., cultures grown with shaking
instead of static, dilution of culture prior to drying on carriers)
proposed to be used in the conduct of these tests, obtain written
approval from the Agency and document such deviations in the study
reports submitted to the Agency.  The Agency may consult with the AOAC
prior to accepting modifications to their standardized methods.   Refer
to OPPTS Test Guideline 810.2000 for general testing recommendations
prior to initiating tests.

(2)  [Reserved]

(3)  Confirmatory testing.  In certain situations an applicant may rely
on previously submitted efficacy data to support an application or
amendment for registration of a product and submit only confirmatory
efficacy data on his own product to demonstrate his ability to produce
an effective formulation. These situations are as listed in paragraphs
(C)(3)(i) through (C)(3)(iii) of this guideline:

(i)  Duplicated Product Formulations. In this situation, the applicant
manufactures a formulation which duplicates a product that is already
registered with complete supporting efficacy data. The chemical
composition, manufacturing procedure, label claims, and directions for
use are identical in substance to those of the original registration,
and specific references (Master Record ID Numbers [MRIDs]) to the
supporting data developed for the original product are cited by the
applicant.

(ii)  Minor Formulation Change in a Registered Product. In this
situation, the change in the formulation is relatively minor, e.g., a
change of an inert ingredient. The label claims and directions for use
are unchanged from those accepted for the registered formulation, and
specific references (MRIDs) to the supporting data developed for the
original formulation are cited by the applicant.  If the only change in
the formulation is the addition of a fragrance or dye, confirmatory data
does not need to be submitted.  However, when the product is an aerosol
formulation, confirmatory data should be submitted for all formulation
changes, including the addition of fragrances and dyes.

(iii) The confirmatory data must be developed on the applicant's own
finished product. When the test methodology utilized in deriving the
original supporting efficacy data were modified to include additional
elements not specified in the recommended method, such as organic soil,
hard water, longer or shorter contact time, etc., the confirmatory data
should be produced under similarly modified conditions.

(4) Efficacy claims. Table 1 provides a quick reference guide to testing
for basic claims described in this guideline. Consult the text for
detailed testing descriptions.

Table 1.  Testing for basic efficacy claims

Level of Efficacy	Test Methods	Test Organisms	No. of Batches/Carriers
Evaluation of Success

Limited disinfectant/hard non-porous surfaces.	Water soluble
powders/liquids	AOAC Use-Dilution Method or

AOAC Hard Surface Carrier Test (distilled water only)

Staphylococcus aureus (ATCC 6538) or Salmonella enterica (ATCC 10708)
Three batches, one at least 60 days old.  60 carriers against either
organism claimed (180 carriers).	59/60 carriers are negative for each
batch tested for all methods except AOAC Hard Surface Carrier Test,
which is 58/60 carriers are negative for each batch.

	Spray products	AOAC Germicidal Spray Products Test

Towelettes	Modified Germicidal Spray Test

	Broad-spectrum disinfectant/hard non-porous surfaces.	Water soluble
powders/liquids	AOAC Use-Dilution Method or

AOAC Hard Surface Carrier Test (distilled water only)

Staphylococcus aureus and Salmonella enterica	Three batches, one at
least 60 days old.  60 carriers against each organism (360 carriers).
59/60 carriers are negative for each batch tested for all methods except
AOAC Hard Surface Carrier Test, which is 58/60 carriers are negative for
each batch.

	Spray products	AOAC Germicidal Spray Products Test

Towelettes	Modified Germicidal Spray Test

	Hospital disinfectant/hard non-porous surfaces.	Water soluble
powders/liquids	AOAC Use-Dilution Method or

AOAC Hard Surface Carrier Test (distilled water only)

Staphylococcus aureus and Pseudomonas aeruginosa (ATCC 15442)	Three
batches, one at least 60 days old.  60 carriers against each organism
(360 carriers).	59/60 carriers are negative for each batch tested for
all methods except AOAC Hard Surface Carrier Test, which is 58/60
carriers are negative against Staphylococcus aureus for each batch, and
57/60 carriers are negative against Pseudomonas aeruginosa.

	Spray products	AOAC Germicidal Spray Products Test

Towelettes	Modified Germicidal Spray Test

	Fungicidal disinfectant/hard non-porous surfaces.

	Water soluble powders/liquids	AOAC Use-Dilution Test modified for fungi
or

AOAC Fungicidal Test

Trichophyton mentagrophytes (ATCC 9533)	Two batches, ten carriers per
batch for the modified AOAC Use Dilution Test, the modified AOAC
Germicidal Spray Products Test, and the EPA Towelette Test.  Two batches
for the AOAC Fungicidal Test.	All fungal spores on all carriers should
be killed.

	Spray products	AOAC Germicidal Spray Products Test modified for fungi

Towelettes	Modified Germicidal Spray Test

	Virucidal disinfectant/hard non-porous surfaces.	Water soluble
powders/liquids	AOAC Use-Dilution Test modified for viruses or

ASTM E1053-97

Virus claimed on the label or approved surrogate.	Two batches.	Complete
inactivation of the virus.  Where cytotoxicity is present, demonstrate a
3log10 reduction.

	Spray products	AOAC Germicidal Spray Products Test modified for viruses
or ASTM E1053-97

Towelettes	Modified Germicidal Spray Test

	Tuberculocidal disinfectant/hard non-porous surfaces.	Water soluble
powders/liquids	AOAC Tuberculocidal Activity of Disinfectants,
Quantitative Tuberculocidal Activity Test

Mycobacterium bovis BCG	Two batches, ten carriers per batch	10/10
carriers are negative for growth and there is no growth in the
additional test media.

	Spray products	AOAC Germicidal Spray Products Test modified for
tuberculocidal activity

Towelettes	Modified Germicidal Spray Test

	Additional bacteria/hard non-porous surfaces.	Water soluble
powders/liquids	AOAC Use-Dilution Test or

AOAC Hard Surface Carrier Test (distilled water only)

Bacteria claimed on the label.	Two batches, ten carriers for each batch.
All bacteria are killed

	Spray products	AOAC Germicidal Spray Products Test

(d)  Disinfectants

(1) Limited spectrum products.  This section addresses efficacy testing
for disinfectant products with limited efficacy (effective against
Gram-negative or Gram-positive bacteria, but not both).

(i) Water-soluble powders and non-volatile liquid products test
procedure.  The Agency recommends the use of the AOAC International
Use-Dilution Methods (see reference (i)(1) of this guideline) or the
AOAC International Hard Surface Carrier Test Methods (distilled water
only)(Ref. 2).  Sixty carriers for each of three samples, representing
three different batches, one of which should be at least 60 days old,
should be tested against Salmonella enterica (S. enterica)(formerly
designated as Salmonella choleraesuis)(ATCC 10708) for effectiveness
against Gram-negative bacteria, or Staphylococcus aureus (S.
aureus)(ATCC 6538) for effectiveness against Gram-positive bacteria.  If
the product is intended to be represented as bactericidal in the
presence of organic soil (one-step), an appropriate organic soil, such
as 5 percent blood serum, should be included with the bacterial
inoculum.

	(ii) Germicidal spray products (aerosol or pump) and volatile liquid
products test procedure.  The Agency recommends use of the AOAC
International Germicidal Spray Products as Disinfectants test (Ref. 3).
Sixty carriers for each of three samples, representing three different
batches, one of which should be at least 60 days old, should be tested
against S. enterica (ATCC 10708) for effectiveness against Gram-negative
bacteria, or S. aureus (ATCC 6538) for effectiveness against
Gram-positive bacteria.  If the product is intended to be represented as
bactericidal in the presence of organic soil (one-step), an appropriate
organic soil, such as 5 percent blood serum, should be included with the
bacterial inoculum.

(iii) Single-use towelettes test procedure.  The Agency recommends the
use of a modified AOAC Germicidal Spray Products as Disinfectants test.
Sixty carriers for each of three samples, representing three different
batches, one of which should be at least 60 days old, should be tested
against S. enterica (ATCC 10708) for effectiveness against Gram-negative
bacteria, or S. aureus (ATCC 6538) for effectiveness against
Gram-positive bacteria.  If the product is intended to be represented as
bactericidal in the presence of organic soil (one-step), an appropriate
organic soil, such as 5 percent blood serum, should be included with the
bacterial inoculum.  Instead of spraying the inoculated surface of the
glass slide, the product should be tested by wiping the surface of the
glass slide with the saturated towelette, and then subculturing the
slides after the specified holding time.  The towelette should be
removed from its container and subsequently handled with sterile gloves.
 One towelette should be used to wipe a minimum of 10 inoculated slides.
 Alternatively, one carrier with a surface area equivalent to ten 1 x 1
inch carriers can be wiped using one towelette per carrier set per
batch.  The area of the towelette used for wiping should be rotated so
as to expose a maximum amount of its surface in the course of wiping a
set of slides.  Note:  A detailed description of the wiping procedure,
including the towelette folding and rotation process should be included
in the test protocol and documented in the raw data and final report.
The applicant should submit their towelette protocol to the Agency for
review and approval prior to conducting the test.

(iv) Evaluation of limited disinfectant success.   For the AOAC
International Use-Dilution Methods, the Germicidal Spray Products as
Disinfectants test, and single-use towelettes, the product should kill
the test microorganisms on 59 out of each set of 60 carriers/slides
within ten minutes or less.  In addition, per the 2009 revisions for the
Use-Dilution Method, the mean log density for S. aureus must be at least
6.0 (corresponding to a geometric mean density of 1.0 x 106); a mean log
density <6.0 invalidates the test. For the AOAC International Hard
Surface Carrier Test Methods, the product should kill the test
microorganisms on 58 out of each set of 60 carriers for S. enterica or
S. aureus within ten minutes or less.

(2) Confirmatory testing for limited spectrum products

(i) Water-soluble powders and non-volatile liquid products test
procedure. The Agency recommends the use of the AOAC International
Use-Dilution Methods (Ref.1) or the AOAC International Hard Surface
Carrier Test Methods (distilled water only)(Ref. 2).  Ten carriers for
each of two product samples, representing two different batches of the
product, should be tested against either S. aureus or S. enterica
(depending on whether the product is claimed to be effective against
Gram-negative or Gram-positive bacteria).  If the product is intended to
be represented as bactericidal in the presence of organic soil
(one-step), an appropriate organic soil, such as 5 percent blood serum,
should be included with the bacterial inoculum.

(ii) Germicidal spray products (aerosol or pump) and volatile liquid
products test  procedure. The Agency recommends the AOAC International
Germicidal Spray Products as Disinfectants test (Ref. 3).  Ten carriers
for each of two product samples, representing two different batches of
the product, should be tested against either S. aureus or S. enterica
(depending on whether the product is claimed to be effective against
Gram-negative or Gram-positive bacteria).  If the product is intended to
be represented as bactericidal in the presence of organic soil
(one-step), an appropriate organic soil, such as 5 percent blood serum,
should be included with the bacterial inoculum.  For pressurized spray
products, certification should be furnished specifying that all parts
and materials used in manufacturing the container for pressurized spray
disinfectants are identical to those specified by the basic
manufacturer.

(iii) Single-use towelettes test procedure.  The Agency recommends the
use of a modified AOAC Germicidal Spray Products as Disinfectants test.
Ten carriers for each of two product samples, representing two different
batches of the product, should be tested against either S. aureus or S.
enterica (depending on whether the product is claimed to be effective
against Gram-negative or Gram-positive bacteria).  If the product is
intended to be represented as bactericidal in the presence of organic
soil (one-step),  an appropriate organic soil, such as 5 percent blood
serum, should be included with the bacterial inoculum.  Instead of
spraying the inoculated surface of the glass slide, the product should
be tested by wiping the surface of the glass slide with the saturated
towelette, and then subculturing the slides after the specified holding
time.  The towelette should be removed from its container and
subsequently handled with sterile gloves.  One towelette should be used
to wipe a minimum of 10 inoculated slides.  Alternatively, one carrier
with a surface area equivalent to ten 1 x 1 inch carriers can be wiped
using one towelette per carrier set per batch.  The area of the
towelette used for wiping should be rotated so as to expose a maximum
amount of its surface in the course of wiping a set of slides.  Note:  A
detailed description of the wiping procedure, including the towelette
folding and rotation process should be included in the test protocol and
documented in the raw data and final report.  The applicant should
submit their towelette protocol to the Agency for review and approval
prior to conducting the test.

(iv) Evaluation of confirmatory limited disinfectant success.  The
product should kill all the test microorganisms on all carriers/slides
within ten minutes or less.  In addition, per the 2009 revisions for the
Use-Dilution Method, the mean log density for S. aureus must be at least
6.0 (corresponding to a geometric mean density of 1.0 x 106); a mean log
density <6.0 invalidates the test.

(3) General or broad spectrum efficacy products. When a disinfectant is
represented in labeling as having efficacy against both Gram-negative
and Gram-positive bacteria, the product is considered a general or broad
spectrum disinfectant.

(i) Water-soluble powders and non-volatile liquid products test
procedure.  The Agency recommends the use of the AOAC International
Use-Dilution Methods (Ref. 1) or the AOAC International Hard Surface
Carrier Test Methods (distilled water only)(Ref. 2).  Sixty carriers for
each of three samples, representing three different batches, one of
which should be at least 60 days old, should be tested against both S.
enterica (ATCC 10708) and S. aureus (ATCC 6538).  If the product is
intended to be represented as bactericidal in the presence of organic
soil (one-step), an appropriate organic soil, such as 5 percent blood
serum, should be included with the bacterial inoculum.

(ii) Germicidal spray products (aerosol or pump) and volatile liquid
products test procedure.  The Agency recommends the AOAC International
Germicidal Spray Products as Disinfectants test (Ref. 3).  Sixty
carriers for each of three samples, representing three different
batches, one of which should be at least 60 days old, should be tested
against both S. enterica (ATCC 10708) and S. aureus (ATCC 6538).  If the
product is intended to be represented as bactericidal in the presence of
organic soil (one-step), an appropriate organic soil, such as 5 percent
blood serum, should be included with the bacterial inoculum.

(iii) Single-use towelettes test procedure.  The Agency recommends the
use of a modified AOAC Germicidal Spray Products as Disinfectants test.
Sixty carriers for each of three samples, representing three different
batches, one of which should be at least 60 days old, should be tested
against both S. enterica (ATCC 10708) and S. aureus (ATCC 6538).  If the
product is intended to be represented as bactericidal in the presence of
organic soil (one-step), an appropriate organic soil, such as 5 percent
blood serum, should be included with the bacterial inoculum.  Instead of
spraying the inoculated surface of the glass slide, the product should
be tested by wiping the surface of the glass slide with the saturated
towelette, and then subculturing the slides after the specified holding
time.  The towelette should be removed from its container and
subsequently handled with sterile gloves.  One towelette should be used
to wipe a minimum of 10 inoculated slides.  Alternatively, one carrier
with a surface area equivalent to ten 1 x 1 inch carriers can be wiped
using one towelette per carrier set per batch.  The area of the
towelette used for wiping should be rotated so as to expose a maximum
amount of its surface in the course of wiping a set of slides.  Note:  A
detailed description of the wiping procedure, including the towelette
folding and rotation process should be included in the test protocol and
documented in the raw data and final report.  The applicant should
submit their towelette protocol to the Agency for review and approval
prior to conducting the test.

(iv) Evaluation of general or broad spectrum disinfectant success. For
the AOAC International Use-Dilution Methods, the Germicidal Spray
Products as Disinfectants test, and single-use towelettes, the product
should kill the test microorganisms on 59 out of each set of 60
carriers/slides within ten minutes or less. In addition, per the 2009
revisions for the Use-Dilution Method, the mean log density for S.
aureus must be at least 6.0 (corresponding to a geometric mean density
of 1.0 x 106); a mean log density <6.0 invalidates the test. For the
AOAC International Hard Surface Carrier Test Methods, the product should
kill the test microorganisms on 58 out of each set of 60 carriers within
ten minutes or less.

(4) Confirmatory testing for general or broad spectrum products

(i) Water-soluble powders and non-volatile liquid products. test
procedure. The Agency recommends the use of the AOAC International
Use-Dilution Methods (Ref. 1) or the AOAC International Hard Surface
Carrier Test Methods (distilled water only)(Ref. 2).  Ten carriers for
each of two product samples, representing two different batches of the
product, should be tested against both S. aureus and S. enterica.  If
the product is intended to be represented as bactericidal in the
presence of organic soil (one-step), an appropriate organic soil, such
as 5 percent blood serum, should be included with the bacterial
inoculum.

(ii) Germicidal spray products (aerosol or pump) and volatile liquid
products test procedure. The Agency recommends the AOAC International
Germicidal Spray Products as Disinfectants test (Ref. 3).  Ten carriers
for each of two product samples, representing two different batches of
the product, should be tested against both S. aureus and S. enterica.
If the product is intended to be represented as bactericidal in the
presence of organic soil (one-step), an appropriate organic soil, such
as 5 percent blood serum, should be included with the bacterial
inoculum.  For pressurized spray products, certification should be
furnished specifying that all parts and materials used in manufacturing
the container for pressurized spray disinfectants are identical to those
specified by the basic manufacturer.

(iii) Single-use towelettes test procedure.  The Agency recommends the
use of a modified AOAC Germicidal Spray Products as Disinfectants test.
Ten carriers for each of two product samples, representing two different
batches of the product, should be tested against both S. aureus and S.
enterica. If the product is intended to be represented as bactericidal
in the presence of organic soil (one-step), an appropriate organic soil,
such as 5 percent blood serum, should be included with the bacterial
inoculum.  Instead of spraying the inoculated surface of the glass
slide, the product should be tested by wiping the surface of the glass
slide with the saturated towelette, and then subculturing the slides
after the specified holding time.  The towelette should be removed from
its container and subsequently handled with sterile gloves.  One
towelette should be used to wipe a minimum of 10 inoculated slides.
Alternatively, one carrier with a surface area equivalent to ten 1 x 1
inch carriers can be wiped using one towelette per carrier set per
batch.  The area of the towelette used for wiping should be rotated so
as to expose a maximum amount of its surface in the course of wiping a
set of slides.  Note:  A detailed description of the wiping procedure,
including the towelette folding and rotation process should be included
in the test protocol and documented in the raw data and final report.
The applicant should submit their towelette protocol to the Agency for
review and approval prior to conducting the test.

(iv) Evaluation of confirmatory general or broad spectrum disinfectant
success.  The product should kill all the test microorganisms on all
carriers/slides within ten minutes or less.  In addition, per the 2009
revisions for the Use-Dilution Method, the mean log density for S.
aureus must be at least 6.0 (corresponding to a geometric mean density
of 1.0 x 106); a mean log density <6.0 invalidates the test.

(5) Hospital or healthcare disinfectants.  This section addresses
efficacy testing for products recommended for use in hospitals, clinics,
dental offices, nursing homes, sickrooms, or any other
healthcare-related facility.

(i) Water-soluble powders and non-volatile liquid product test
procedure.  The Agency recommends the use of the AOAC International
Use-Dilution Methods (Ref. 1) or the AOAC International Hard Surface
Carrier Test Methods (distilled water only)(Ref. 2).  Sixty carriers for
each of three samples, representing three different batches, one of
which should be at least 60 days old, should be tested against S. aureus
(ATCC 6538), and Pseudomonas aeruginosa (P. aeruginosa)(ATCC 15442).  If
the product is intended to be represented as bactericidal in the
presence of organic soil (one-step), an appropriate organic soil, such
as 5 percent blood serum, should be included with the bacterial
inoculum.

	(ii) Germicidal spray products (aerosol or pump) and volatile liquid
products test procedure.  The Agency recommends the use of the AOAC
International Germicidal Spray Products as Disinfectants test (Ref. 3).
Sixty carriers for each of three samples, representing three different
batches, one of which should be at least 60 days old, should be tested
against: S. aureus (ATCC 6538), and P. aeruginosa (ATCC 15442).  If the
product is intended to be represented as bactericidal in the presence of
organic soil (one-step), an appropriate organic soil, such as 5 percent
blood serum, should be included with the bacterial inoculum.

(iii) Single-use towelettes test procedure.  The Agency recommends the
use of a modified AOAC Germicidal Spray Products as Disinfectants test.
Sixty carriers for each of three samples, representing three different
batches, one of which should be at least 60 days old, should be tested
against S. aureus (ATCC 6538), and P. aeruginosa (ATCC 15442).  If the
product is intended to be represented as bactericidal in the presence of
organic soil (one-step), an appropriate organic soil, such as 5 percent
blood serum, should be included with the bacterial inoculum.  Instead of
spraying the inoculated surface of the glass slide, the product should
be tested by wiping the surface of the glass slide with the saturated
towelette, and then subculturing the slides after the specified holding
time.  The towelette should be removed from its container and
subsequently handled with sterile gloves.  One towelette should be used
to wipe a minimum of 10 inoculated slides.  Alternatively, one carrier
with a surface area equivalent to ten 1 x 1 inch carriers can be wiped
using one towelette per carrier set per batch.  The area of the
towelette used for wiping should be rotated so as to expose a maximum
amount of its surface in the course of wiping a set of slides.  Note:  A
detailed description of the wiping procedure, including the towelette
folding and rotation process should be included in the test protocol and
documented in the raw data and final report.  The applicant should
submit their towelette protocol to the Agency for review and approval
prior to conducting the test.

(iv) Evaluation of hospital or healthcare disinfectant success.  For the
AOAC International Use-Dilution Methods, the Germicidal Spray Products
as Disinfectants test, and single-use towelettes, the product should
kill the test microorganisms on 59 out of each set of 60 carriers/slides
within ten minutes or less. In addition, per the 2009 revisions for the
Use-Dilution Method, the mean log density for S. aureus and P.
aeruginosa must be at least 6.0 (corresponding to a geometric mean
density of 1.0 x 106); a mean log density <6.0 invalidates the test. For
the AOAC International Hard Surface Carrier Test Methods, the product
should kill the test microorganisms on 58 out of each set of 60 carriers
for S. aureus, and 57 out of each set of 60 carriers for P. aeruginosa
within ten minutes or less.

(6) Confirmatory testing for products with hospital or healthcare
disinfectant claim

(i) Water-soluble powders and non-volatile liquid products test
procedure. The Agency recommends the use of the AOAC International
Use-Dilution Methods (Ref. 1) or the AOAC International Hard Surface
Carrier Test Methods (distilled water only)(Ref. 2).  Ten carriers for
each of two product samples, representing two different batches of the
product, should be tested against S. aureus (ATCC 6538) P. aeruginosa
(ATCC 15442).  If the product is intended to be represented as
bactericidal in the presence of organic soil (one-step), an appropriate
organic soil, such as 5 percent blood serum, should be included with the
bacterial inoculum.

(ii) Germicidal spray products (aerosol or pump) and volatile liquid
products test procedure. The Agency recommends the AOAC International
Germicidal Spray Products as Disinfectants test (Ref. 3).  Ten carriers
for each of two product samples, representing two different batches of
the product, should be tested against S. aureus (ATCC 6538) and P.
aeruginosa (ATCC 15442).  If the product is intended to be represented
as bactericidal in the presence of organic soil (one-step), an
appropriate organic soil, such as 5 percent blood serum, should be
included with the bacterial inoculum. For pressurized spray products,
certification should be furnished specifying that all parts and
materials used in manufacturing the container for pressurized spray
disinfectants are identical to those specified by the basic
manufacturer.

(iii) Single-use towelettes test procedure.  The Agency recommends the
use of a modified AOAC Germicidal Spray Products as Disinfectants test.
Ten carriers for each of two product samples, representing two different
batches of the product, should be tested against S. aureus (ATCC 6538)
and P. aeruginosa (ATCC 15442).  If the product is intended to be
represented as bactericidal in the presence of organic soil (one-step),
an appropriate organic soil, such as 5 percent blood serum, should be
included with the bacterial inoculum.  Instead of spraying the
inoculated surface of the glass slide, the product should be tested by
wiping the surface of the glass slide with the saturated towelette, and
then subculturing the slides after the specified holding time.  The
towelette should be removed from its container and subsequently handled
with sterile gloves.  One towelette should be used to wipe a minimum of
10 inoculated slides.  Alternatively, one carrier with a surface area
equivalent to ten 1 x 1 inch carriers can be wiped using one towelette
per carrier set per batch.  The area of the towelette used for wiping
should be rotated so as to expose a maximum amount of its surface in the
course of wiping a set of slides.  Note:  A detailed description of the
wiping procedure, including the towelette folding and rotation process
should be included in the test protocol and documented in the raw data
and final report.  The applicant should submit their towelette protocol
to the Agency for review and approval prior to conducting the test.

(iv) Evaluation of confirmatory hospital or healthcare disinfectant
success.  The product should kill all the test microorganisms on all
carriers/slides within ten minutes or less.  In addition, per the 2009
revisions for the Use-Dilution Method, the mean log density for S.
aureus and P. aeruginosa must be at least 6.0 (corresponding to a
geometric mean density of 1.0 x 106); a mean log density <6.0
invalidates the test.

(7) Bridging for disinfectant towelettes.  In some cases, disinfectant
towelette formulations are identical to registered liquid formulations.
In order to bridge efficacy data from the EPA registered bulk liquid
disinfectant used to saturate a towelette or other related product form,
the studies in paragraphs (d)(7)(i) and (d)(7)(ii) of this guideline
should be conducted and submitted to EPA for review.

(i) Chemical Testing - Comparison of Expressed Liquid from the
Towelette(s) to the EPA Registered Liquid Disinfectant Formulation to
which it is being bridged: All active ingredients in the expressed
liquid should be within the certified limits of the Confidential
Statement of Formula of the liquid formula being referenced/bridged. The
disinfectant towelettes package should be filled according to the
manufacturing specifications.  Excess liquid in the bulk towelette
containers can not be poured off for use in the chemical testing for
bridging of the efficacy data. The liquid used in the chemical testing
should only be that expressed from the towelettes. Three batches (one of
which is 60 days old) should be tested.  Analytical data for the active
ingredients in the expressed liquid should be submitted for review.

(ii) Efficacy Testing:  Efficacy testing should be conducted under the
same testing conditions (e.g. soil load, contact time, temperature) as
used for the bulk liquid testing.  Note:  A detailed description of the
wiping procedure, including the towelette folding and rotation process
should be included in the test protocol and documented in the raw data
and final report.  The applicant should submit their towelette protocol
to the Agency for review and approval prior to conducting the test.  For
limited disinfectants, broad-spectrum disinfectants, and hospital
disinfectants, to bridge bacterial disinfection claims:

(A) Test Procedure.  The Agency recommends the use of the AOAC
Germicidal Spray Products as Disinfectants test modified for towelettes,
using the test organisms specified for limited, broad-spectrum or
hospital disinfectant testing.  Sixty carriers for each organism should
be tested against three different batches of the product (one of which
should be 60 days old).  Instead of spraying the inoculated surface of
the glass slide, the product should be tested by wiping the surface of
the glass slide with the saturated towelette, and then subculturing the
slides after the specified holding time. One towel will be used to treat
10 carriers.  Alternatively, one carrier with a surface area equivalent
to ten 1 x 1 inch carriers can be wiped using one towelette per carrier
set per batch.

(B)  Evaluation of bactericidal towelette success.  The product should
kill the test organism on 59 out of 60 carriers.  This testing is
intended to support bridging of all vegetative bacteria listed on the
EPA registered liquid disinfectant used to saturate the towel to the EPA
registered towelette product.

(8) Disinfectants for Internal Toilet and Urinal Bowl Surfaces Above and
Below the Water Line.  This section addresses efficacy testing for
products bearing label claims as disinfectants (limited, broad-spectrum,
or hospital) for internal toilet and urinal bowl surfaces.  Regarding
water-soluble powders and non-volatile liquid products test procedure,
the Agency recommends the use of the AOAC International Use-Dilution
Methods (see reference (i)(1) of this guideline) modified to include a
5% organic soil challenge. Sixty carriers for each of three samples,
representing three different batches, one of which is at least 60 days
old, should be tested against  Salmonella enterica (ATCC 10708) or
Staphylococcus aureus (ATCC 6538), for limited disinfectant products; S.
enterica and S. aureus, for broad-spectrum disinfectant products; and S.
aureus and  Pseudomonas aeruginosa (ATCC 15442), for hospital
disinfectant products.  The contained bowl water (-96 fl oz) should be
used to calculate the appropriate use dilution for testing.

(i) Evaluation of disinfectant success for internal toilet bowl and
urinal bowl surfaces.  For the AOAC International Use-Dilution Methods
and the Germicidal Spray Products as Disinfectants test, the product
should kill the test microorganisms on 59 out of each set of 60
carriers/slides within ten minutes or less.  In addition, per the 2009
revisions for the Use-Dilution Method, the mean log density for S.
aureus and P. aeruginosa must be at least 6.0 (corresponding to a
geometric mean density of 1.0 x 106); a mean log density <6.0
invalidates the test.

(9) Additional microorganisms.  This section addresses efficacy testing
for broad-spectrum or hospital disinfectants which bear label claims
against bacteria other than Salmonella enterica (ATCC 10708),
Staphylococcus aureus (ATCC 6538) or Pseudomonas aeruginosa (ATCC
15442).

(i) Water-soluble powders and non-volatile liquid products test
procedure.  The Agency recommends the use of the AOAC International
Use-Dilution Methods (Ref. 1) or the AOAC International Hard Surface
Carrier Test Methods (distilled water only)(Ref. 2).  Ten carriers
should be tested against each specific bacterium for each of two samples
representing two different batches.  If the product is intended to be
represented as bactericidal in the presence of organic soil (one-step),
an appropriate organic soil, such as 5 percent blood serum, should be
included with the bacterial inoculum.

	(ii) Germicidal spray products (aerosol or pump) and volatile liquid
products test procedure.  The Agency recommends the use of the AOAC
International Germicidal Spray Products as Disinfectants test (Ref. 3).
Ten carriers should be tested against each specific bacterium for each
of two samples representing two different batches.  If the product is
intended to be represented as bactericidal in the presence of organic
soil (one-step), an appropriate organic soil, such as 5 percent blood
serum, should be included with the bacterial inoculum.

(iii) Single-use towelettes test procedure.  The Agency recommends the
use of a modified AOAC Germicidal Spray Products as Disinfectants test.
Ten carriers should be tested against each specific bacterium for each
of two samples representing two different batches.  If the product is
intended to be represented as bactericidal in the presence of organic
soil (one-step), an appropriate organic soil, such as 5 percent blood
serum, should be included with the bacterial inoculum.  Instead of
spraying the inoculated surface of the glass slide, the product should
be tested by wiping the surface of the glass slide with the saturated
towelette, and then subculturing the slides after the specified holding
time.  The towelette should be removed from its container and
subsequently handled with sterile gloves.  One towelette should be used
to wipe a minimum of 10 inoculated slides.  Alternatively, one carrier
with a surface area equivalent to ten 1 x 1 inch carriers can be wiped
using one towelette per carrier set per batch.  The area of the
towelette used for wiping should be rotated so as to expose a maximum
amount of its surface in the course of wiping a set of slides.  Note:  A
detailed description of the wiping procedure, including the towelette
folding and rotation process should be included in the test protocol and
documented in the raw data and final report.  The applicant should
submit their towelette protocol to the Agency for review and approval
prior to conducting the test.

(iv) Evaluation of disinfectant success for additional microorganisms.
The product should kill all the test microorganisms on all
carriers/slides within ten minutes or less.

(e) Disinfectants with fungicidal claims.  This section addresses
efficacy testing for broad-spectrum or hospital disinfectant products
which bear label claims of efficacy against pathogenic fungi.

(1) Water soluble powders and non-volatile liquid products

(i) Test procedures. The Agency recommends the use of the AOAC
International Fungicidal Activity of Disinfectants test (Ref. 4)  Two
samples representing two different batches of the product should be
evaluated for efficacy against Trichophyton mentagrophytes (T.
mentagrophytes)(ATCC 9533).  The Agency also recommends the use of the
AOAC International Use-Dilution Methods (Ref. 1). This test may be
modified to conform to appropriate elements (e.g., media, growth
conditions, etc.) in the AOAC International Fungicidal Activity of
Disinfectants test.  Ten carriers for each of two samples representing
two different batches of the product should be evaluated against T.
mentagrophytes (ATCC 9533).  The inoculum employed should provide a
concentration of 1 x 104 – 1 x 105 conidia per carrier.  If the
product is intended to be represented as fungicidal in the presence of
organic soil (one-step), an appropriate organic soil, such as 5 percent
blood serum, should be included with the fungal inoculum.

(ii) Evaluation of fungicidal success.  For the AOAC International
Fungicidal Activity of Disinfectants test, all fungal spores in all
replicates for each batch should be killed.  For the AOAC International
Use-Dilution Methods, all fungal spores on all 10 carriers should be
killed within ten minutes or less.

(2) Germicidal spray products (aerosol or pump) and volatile liquid
products—(i) Test procedures.  The Agency recommends the use of the
AOAC International Germicidal Spray Products as Disinfectants test (see
reference (i)(3) of this guideline). This test may be modified to
conform to appropriate elements (e.g., media, growth conditions, etc.)
in the AOAC International Fungicidal Activity of Disinfectants test.
Ten carriers for each of two samples representing two different batches
of the product should be evaluated against T.  mentagrophytes (ATCC
9533).  The inoculum employed should be modified to provide a
concentration of 1 x 104 – 1 x 105 conidia per carrier.  If the
product is intended to be represented as fungicidal in the presence of
organic soil (one-step), an appropriate organic soil, such as 5 percent
blood serum, should be included with the fungal inoculum.

(ii) Evaluation of fungicidal success.  All fungal spores on all 10
carriers/slides should be killed within ten minutes or less.

(3) Single-Use Towelettes

(i) Test Procedure.  The Agency recommends the use of a modified AOAC
Germicidal Spray Products as Disinfectants test.  Ten carriers for each
of two samples representing two different batches of the product should
be evaluated against T.  mentagrophytes (ATCC 9533).  The inoculum
employed should be modified to provide a concentration of 1 x 104 – 1
x 105 conidia per carrier.  If the product is intended to be represented
as fungicidal in the presence of organic soil (one-step), an appropriate
organic soil, such as 5 percent blood serum, should be included with the
fungal inoculum.  Instead of spraying the inoculated surface of the
glass slide, the product should be tested by wiping the surface of the
glass slide with the saturated towelette, and then subculturing the
slides after the specified holding time.  The towelette should be
removed from its container and subsequently handled with sterile gloves.
 One towelette should be used to wipe a minimum of 10 inoculated slides.
 Alternatively, one carrier with a surface area equivalent to ten 1 x 1
inch carriers can be wiped using one towelette per carrier set per
batch.  The area of the towelette used for wiping should be rotated so
as to expose a maximum amount of its surface in the course of wiping a
set of slides.  Note:  A detailed description of the wiping procedure,
including the towelette folding and rotation process should be included
in the test protocol and documented in the raw data and final report.
The applicant should submit their towelette protocol to the Agency for
review and approval prior to conducting the test.

(ii) Evaluation of fungicidal towelette success.  All fungal spores on
all 10 carriers/slides should be killed within ten minutes or less.

(4) Bridging for disinfectant towelettes.  In some cases, disinfectant
towelette formulations are identical to registered liquid formulations.
In order to bridge efficacy data from the EPA registered bulk liquid
disinfectant used to saturate a towelette or other related product form,
the studies in paragraphs (e)(4)(1) and (e)(4)(ii) of this guideline
should be conducted and submitted to EPA for review:

(i) Chemical Testing - Comparison of Expressed Liquid from the
Towelette(s) to the EPA Registered Liquid Disinfectant Formulation to
which it is being bridged: All active ingredients in the expressed
liquid should be within the certified limits of the Confidential
Statement of Formula of the liquid formula being referenced/bridged. The
disinfectant towelettes package should be filled according to the
manufacturing specifications.  Excess liquid in the bulk towelette
containers can not be poured off for use in the chemical testing for
bridging of the efficacy data. The liquid used in the chemical testing
should only be that expressed from the towelettes. Three batches (one of
which is 60 days old) should be tested.  Analytical data for the active
ingredients in the expressed liquid should be submitted for review.

(ii) Efficacy Testing:  Efficacy testing should be conducted under the
same testing conditions (e.g. soil load, contact time, temperature) as
used for the bulk liquid testing.  This testing allows bridging of data
from the registered bulk liquid used to saturate the towel for each type
of organism in this paragraph.  Note:  A detailed description of the
wiping procedure, including the towelette folding and rotation process
should be included in the test protocol and documented in the raw data
and final report.  The applicant should submit their towelette protocol
to the Agency for review and approval prior to conducting the test.  For
fungicidal test procedure, the Agency recommends the use of the AOAC
International Germicidal Spray Products as Disinfectants (Ref. 3)
modified for fungicidal towelette testing.  The test should be modified
to conform to appropriate elements (e.g. media, growth conditions) in
the AOAC International Fungicidal Activity of Disinfectants test.  Ten
carriers for each of two samples, representing two batches of the
product should be evaluated against T. mentagrophytes (ATCC 9533) for
the label recommended contact time.  The inoculum employed should be at
a count to achieve 1 x 104 – 1 x 105 conidia per carrier.  Instead of
spraying the inoculated surface of the glass slide, the product should
be tested by wiping the surface of the glass slide with the saturated
towelette, and then subculturing the slides after the specified holding
time.  One towelette should be used to wipe a minimum of 10 inoculated
slides.  Alternatively, one carrier with a surface area equivalent to
ten 1 x 1 inch carriers can be wiped using one towelette per carrier set
per batch.

(A) Evaluation of Fungicidal towelette success.  The product should kill
the test organism on all 10 carriers.

(B)  Bridging. This testing is intended to support bridging of all
fungal test organisms from the EPA registered bulk liquid disinfectant
used to saturate the towel to the EPA registered towelette product.

(f)  Disinfectants with virucidal claims.  This section addresses
efficacy testing for broad-spectrum or hospital disinfectant products
that bear label claims of effectiveness against viruses.  Virucidal
products are intended for use on dry inanimate surfaces; therefore,
virological data are usually developed by carrier methods.  Each
specific virus listed on the label should be tested.  For label claims
against Hepatitis B virus, Hepatitis C virus, and Norovirus, the Duck
Hepatitis B virus, Bovine Viral Diarrhea virus, and Feline Calicivirus,
respectively, are considered acceptable surrogates for testing.
Additional guidance and protocols for surrogate virus testing can be
found at   HYPERLINK "http://www.epa.gov/oppad001/regpolicy.htm"
http://www.epa.gov/oppad001/regpolicy.htm .  To simulate in-use
conditions, the specific virus to be treated (or surrogate as noted in
this paragraph) should be inoculated onto hard surfaces (e.g., Petri
dishes, glass slides, stainless steel penicylinders, or other
appropriate test surface), allowed to dry, and then treated with the
product according to the directions for use on the product label.

(1) Water soluble powders and non-volatile liquid products test
procedures.  The Agency recommends the use of either the AOAC
International Use-Dilution Methods (see reference (i)(1) of this
guideline) modified for virucidal testing or the ASTM E1053-97 Virucidal
Test Method (Ref. 5).  One surface for each of two samples, representing
two different batches of disinfectant, should be tested against a
recoverable virus titer of at least 104 viable viral particles from the
test surface for a specified exposure period at room temperature.  If
the product is intended to be represented as virucidal in the presence
of organic soil (one-step), an appropriate organic soil, such as 5
percent blood serum, should be included with the viral inoculum.  When
viral suspensions are grown in the presence of at least 5% serum,
addition of serum to the inoculum is not expected as part of a study to
support a one-step label claim.

	(2)  Germicidal spray products (aerosol or pump) and volatile liquid
products test procedure.  The Agency recommends the use of a AOAC
International Germicidal Spray Products as Disinfectants test (Ref. 3)
modified for virucidal testing or the ASTM E1053-97 Virucidal Test
Method (Ref. 5).  One surface for each of two samples, representing two
different batches of disinfectant, should be tested against a
recoverable virus titer of at least 104 viable viral particles from the
test surface for the exposure period specified on the label.  If the
product is intended to be represented as virucidal in the presence of
organic soil (one-step), an appropriate organic soil, such as 5 percent
blood serum, should be included with the viral inoculum.  When viral
suspensions are grown in the presence of at least 5% serum, addition of
serum to the inoculum is not expected as part of a study to support a
one-step label claim.

(3) Single-use towelettes test procedure.  The Agency recommends the use
of the modified AOAC Germicidal Spray Products as Disinfectants test.
One surface for each of two samples, representing two different batches
of disinfectant, should be tested against a recoverable virus titer of
at least 104 viable viral particles from the test surface for a
specified exposure period at room temperature.  If the product is
intended to be represented as virucidal in the presence of organic soil
(one-step), an appropriate organic soil, such as 5 percent blood serum,
should be included with the viral inoculum.  When viral suspensions are
grown in the presence of at least 5% serum, addition of serum to the
inoculum is not expected as part of a study to support a one-step label
claim.  Instead of spraying the inoculated surface of the glass slide,
the product should be tested by wiping the surface of the glass slide
with the saturated towelette, and then subculturing the slides after the
specified holding time.  The towelette should be removed from its
container and subsequently handled with sterile gloves.  One towelette
should be used to wipe a minimum of 10 inoculated slides.
Alternatively, one carrier with a surface area equivalent to ten 1 x 1
inch carriers can be wiped using one towelette per carrier set per
batch.  The area of the towelette used for wiping should be rotated so
as to expose a maximum amount of its surface in the course of wiping a
set of slides.  Note:  A detailed description of the wiping procedure,
including the towelette folding and rotation process should be included
in the test protocol and documented in the raw data and final report.
The applicant should submit their towelette protocol to the Agency for
review and approval prior to conducting the test.

(4) Evaluation of virucidal success.  Following treatment of the test
virus with the disinfectant product, the presence of remaining viable
virus should then be assayed using an appropriate virological technique
(e.g., cytopathogenic effect, fluorescent antibody, plaque count, or
animal response).  The protocol for the viral assay should provide the
information in paragraphs (f)(4)(i)through (f)(4)(ix) of this guideline.

(i) The virus recovery (titer) should include a minimum of four
determinations per each dilution in the assay system (e.g., tissue
culture, embryonated egg, animal infection, etc.).

(ii) Cytotoxicity controls.  The effect of the disinfectant on the viral
assay system should include a minimum of four determinations per each
dilution.

(iii) The activity of the disinfectant against the test virus should
include a minimum of four determinations per dilution in the assay
system.

(iv) Neutralization controls.  Neutralization controls should be
performed (see reference (i)(6)) and should include a minimum of four
determinations per each dilution.

(v) Any special methods which are used to increase the virus titer and
to detoxify the residual disinfectant should be described.

(vi) The ID50 values calculated for each assay should be provided.

(vii) The test results should be reported as the reduction of the virus
titer by the activity of the disinfectant (ID50 of the virus control
less the ID50 of the test system) expressed as the logarithm to the base
10 and calculated by a statistical method (e.g., Reed and Munch, Most
Probable Number).

(viii) The product should demonstrate complete inactivation of the virus
at all dilutions.  If cytotoxicity is present, at least a 3-log10
reduction in viral titer should be demonstrated beyond the cytotoxic
level.  Table 1 provides an example of a typical laboratory report of a
single test with one virus, assayed in a tissue culture system.

(ix) A typical laboratory report of a single test with one virus
(recovered from a treated surface) involving a tissue culture assay
system would include the details of the methods employed and the
information included in Tables 2-1, 2-2 and 2-3:

Table 2-1: Test Results

Dilution of Virus

Virus - Disinfectant*

Virus - Control*

Cytotoxic - Control

10-1

T  T  T  T

+  +  +  +

T  T  T  T

10-2

T  T  T  T

+  +  +  +

T  T  T  T

10-3

T  0  0  0

+  +  +  +

T  0  0  0

10-4

0  0  0  0

+  +  +  +

0  0  0  0

10-5

0  0  0  0

+  +  +  +

0  0  0  0

10-6

0  0  0  0

+  +  +  0

0  0  0  0

10-7

0  0  0  0

+  0  0  0

0  0  0  0

10-8

0  0  0  0

0  0  0  0

0  0  0  0

Note:  T = toxic; + = virus recovered; 0 = no virus recovered

Table 2-2: Calculation of the Tissue Culture Infective Dose 50 (TCID50)

Values

Accumulated Values

Virus Dilution Inoculated

No. Infected / No. Inoculated

No. Infected

No. not Infected

No. Infected

No. not Infected

No. Infected / No. Inoculated

% Infected

10-1

4/4

4

0

24

0

24/24

100

10-2

4/4

4

0

20

0

20/20

100

10-3

4/4

4

0

16

0

16/16

100

10-4

4/4

4

0

12

0

12/12

100

10-5

4/4

4

0

8

0

8/8

100

10-6

3/4

3

1

4

1

4/5

80

10-7

1/4

1

3

1

4

1/5

20

10-8

0/4

0

4

0

8

0/8

0

TCID50 = 106.5

Table 2-3: Calculations of the Tissue Culture Lethal Dose 50 (TCLD50)

Values

Accumulated Values

Virus Dilution Inoculated

No. Toxic / No. Inoculated

No. Toxic

No. not Toxic

No. Toxic

No. not Toxic

No. Toxic / No. Inoculated

% Toxic

10-1

4/4

4

0

9

0

9/9

100

10-2

4/4

4

0

5

0

5/5

100

10-3

1/4

1

3

1

3

1/4

25

10-4

0/4

0

4

0

7

0/7

0

10-5

0/4

0

4

0

11

0/11

0

10-6

0/4

0

4

0

15

0/15

0

10-7

0/4

0

4

0

19

0/19

0

10-8

0/4

0

4

0

23

0/23

0

TCLD50 = 102.7 Therefore: Virus inactivation = TCID50 - TCLD50 = 103.8
log 10

(5)  Bridging for disinfectant towelettes.  In some cases, disinfectant
towelette formulations are identical to registered liquid formulations.
In order to bridge efficacy data from the EPA registered bulk liquid
disinfectant used to saturate a towelette or other related product form,
the studies in paragraphs (f)(5)(1) and (f)(5)(ii) of this guideline
should be conducted and submitted to EPA for review.

(i)  Chemical Testing—Comparison of Expressed Liquid from the
Towelette(s) to the EPA Registered Liquid Disinfectant Formulation to
which it is being bridged: All active ingredients in the expressed
liquid should be within the certified limits of the Confidential
Statement of Formula of the liquid formula being referenced/bridged. The
disinfectant towelettes package should be filled according to the
manufacturing specifications.  Excess liquid in the bulk towelette
containers can not be poured off for use in the chemical testing for
bridging of the efficacy data. The liquid used in the chemical testing
should only be that expressed from the towelettes. Three batches (one of
which is 60 days old) should be tested.  Analytical data for the active
ingredients in the expressed liquid should be submitted for review.

(ii)  Efficacy Testing:  Efficacy testing should be conducted under the
same testing conditions (e.g. soil load, contact time, temperature) as
used for the bulk liquid testing.  This testing allows bridging of data
from the registered bulk liquid used to saturate the towel for each type
of virus in paragraphs (f)(5)(ii)(A)(1) through (f)(5)(ii)(A)(3) of this
guideline.  Note:  A detailed description of the wiping procedure,
including the towelette folding and rotation process should be included
in the test protocol and documented in the raw data and final report.
The applicant should submit their towelette protocol to the Agency for
review and approval prior to conducting the test.

(A)  Virucidal Test Procedure.  The Agency recommends the use of either
the AOAC International Germicidal Spray Products as Disinfectants (see
reference (i)(3) of this guideline) modified for virucidal towelette
testing or the ASTM E1053 / current edition Virucidal Test Method (see
reference (i)(5)) modified for virucidal towelette testing.

(1)  To support bridging of all viral claims, the most difficult to
inactivate small-sized non-enveloped virus, from the viral strains
registered for the bulk liquid, should be selected for testing.
Examples of small-sized non-enveloped viral families include members of
the Picornaviridae family (e.g., poliovirus, enterovirus, hepatitis A
virus, rhinovirus), and Parvoviridae family (e.g., parvovirus).

(2)  To support bridging of viral claims for large-sized non-enveloped
and enveloped viral strains, the most difficult to inactivate
large-sized non-enveloped virus, from the viral strains registered for
the bulk liquid, should be selected for testing.  Examples of
large-sized non-enveloped viral families include members of the
Adenoviridae family (e.g., adenovirus), Reoviridae family (e.g.,
rotavirus), and Papillomaviridae family (e.g., papillomavirus).

(3)  To support bridging of viral claims for enveloped viral strains,
the most difficult to inactivate enveloped virus, from the viral strains
registered for the bulk liquid, should be selected for testing.
Examples of enveloped viral families include members of the
Coronaviridae family (e.g., coronavirus), Flaviviridae family (e.g.,
hepatitis C virus), Herpesviridae family (e.g., herpesvirus), Poxviridae
family (e.g., vaccinia), Hepadnaviridae family (e.g., hepatitis B
virus), Orthomyxoviridae family (e.g., Influenza), Paramyxoviridae
family (e.g., parainfluenza) and Retroviridae family (e.g., human
immunodeficiency virus).

(B)  Ten carriers for each of two samples, representing two batches of
disinfectant, should be tested against a recoverable dried virus titer
of at least 104 viral particles from the test surface for a specified
exposure period at room temperature.  Instead of spraying the inoculated
surface of the glass slide, the product should be tested by wiping the
surface of the glass slide with the saturated towelette, and then
subculturing the slides after the specified holding time.  One towelette
should be used to wipe a minimum of 10 inoculated slides.
Alternatively, one carrier with a surface area equivalent to ten 1 x 1
inch carriers can be wiped using one towelette per carrier set per
batch.

The protocol for the viral assay should provide the information in
paragraphs (f)(5)(ii)(B)(1) through (f)(5)(ii)(B)(7) of this guideline:

(1)  The virus recovery (titer) should include a minimum of four
determinations for each dilution in the assay system (e.g., cell
culture, embryonated egg, animal infection).

(2)  Cytotoxicity controls.  The effect of the test substance on the
viral assay system should include a minimum of four determinations for
each dilution.

(3)  The activity of the test substance against the test virus should
include a minimum of four determinations for each dilution in the assay
system.

(4)   Neutralization controls.  Neutralization controls should be
performed (see reference (i)(6)) and should include a minimum of four
determinations per each dilution.

(5)  Any special methods which are used to increase the virus titer and
to detoxify the residual test substance should be described.

(6)  The LD50 values calculated for each assay should be provided.

(7)   The test results should be reported as the reduction of the virus
titer by the activity of the test substance (LD50 of the virus control
less the LD50 of the test system) expressed as the logarithm to the base
10 and calculated by a statistical method (e.g., Reed and Munch, Most
Probable Number).

(C)  Evaluation of virucidal success.  The product should demonstrate
complete inactivation of the virus at all dilutions.  If cytotoxicity is
present, at least a 3-log reduction in viral titer should be
demonstrated beyond the cytotoxic level recovered from the carrier
surface.

 (g)  Disinfectants with tuberculocidal claims. This section addresses
efficacy testing for broad-spectrum or hospital disinfectant products
which bear label claims of effectiveness as tuberculocides.  In the
Agency’s “Data Call-In Notice for Tuberculocidal Effectiveness for
All Antimicrobial Pesticides with Tuberculocidal Claims,” dated June
13, 1986 (Ref. 8), applicants were given the option of choosing from one
of three test methods (AOAC Tuberculocidal Activity of Disinfectants
test, a modified AOAC Tuberculocidal Activity of Disinfectants test, or
the Quantitative Tuberculocidal Activity Test) for conducting
tuberculocidal efficacy tests.  In general, the Agency does not believe
that a suspension test is appropriate for disinfectant formulations used
on hard surfaces.   An exception to this is for glutaraldehyde-based
products, which have never been validated in the AOAC Tuberculocidal
Activity of Disinfectants test (a carrier based test).  Therefore, the
Quantitative Tuberculocidal Activity Test should only be used for
glutaraldehyde-based products.  The Agency strongly recommends all other
formulations to use the carrier-based AOAC Tuberculocidal Activity of
Disinfectants test.

(1) Water-soluble powders and non-volatile liquid products test
procedures. The Agency recommends the test procedures in paragraphs
(g)(1)(i) through (g)(1)(iv) of this guideline.

(i) AOAC International Tuberculocidal Activity of Disinfectants test.
The AOAC International Tuberculocidal Activity of Disinfectants test
(see reference (i)(8) of this guideline) employing a 10 minute contact
time and 20˚C temperature. Ten carriers for each of two samples
representing two different batches of the product should be tested
against Mycobacterium bovis (BCG)(M.bovis).  If the product is intended
to be represented as tuberculocidal in the presence of organic soil
(one-step), an appropriate organic soil, such as 5 percent blood serum,
should be included with the bacterial inoculum.

 20˚C temperature (see reference (i)(7) of this guideline). Ten
carriers for each of two samples representing two different batches of
the product should be tested against M. bovis (BCG).  If the product is
intended to be represented as tuberculocidal in the presence of organic
soil (one-step), an appropriate organic soil, such as 5 percent blood
serum, should be included with the bacterial inoculum.

(iii) Evaluation of tuberculocide success.  For the AOAC International
Tuberculocidal Activity of Disinfectants test, all organisms on all
carriers should be killed, and there should be no growth in any of the
inoculated subculture media.

(iv) Validation testing for Quaternary Ammonium Compounds.  Products
formulated solely with quaternary ammonium compounds as the active
ingredient(s) should be supported with validation testing to confirm
their tuberculocidal label claim.  One additional product sample should
be tested in a different laboratory from the original one, using the
same test procedure and conditions as used in the first laboratory test.

(2) Glutaraldehyde formulations

(i) Test Procedure.  For glutaraldehyde formulations, the Agency
recommends the Quantitative Tuberculocidal Activity Test.  This test has
been published in the Agency’s  “Data Call-In Notice for
Tuberculocidal Effectiveness for All Antimicrobial Pesticides with
Tuberculocidal Claims”, dated June 13, 1986 (Ref. 8).  Two samples,
representing two different batches of the product should each be
utilized in at least four separate studies (a total of at least eight
studies), against M. bovis, so that upper 95 percent confidence limits
can be determined for each point on the survival curve.  If the product
is intended to be represented as tuberculocidal in the presence of
organic soil (one-step), an appropriate organic soil, such as 5 percent
blood serum, should be included with the bacterial inoculum.

(ii) Evaluation of tuberculocide success.  For the Quantitative
Tuberculocidal Activity Method, survival curves should be constructed
from the average of four separate replicates so that the upper 95%
Confidence Limit can be determined for each point on the curve. The
minimum time claimed for efficacy is determined by finding the point
where the average survival curve intersects the probability of one
survivor.  If the data show a four-log reduction, but the survivor curve
does not intersect the one-survivor line, the minimal time is found by
extrapolating the upper 95% confidence limit curve such that the value
where it intersects the one survivor line is not 50% greater that when
the survivor curve intersects the one survivor line.

(3) Germicidal spray products and volatile liquid products—(i) Test
procedure.  The Agency recommends the AOAC International Germicidal
Spray Products as Disinfectants test (Ref. 3), using the media, test
culture, and other elements described in the AOAC International
Tuberculocidal Activity of Disinfectants test.  Ten carriers for each of
two samples representing two different batches of the product should be
tested against M. bovis (BCG).  If the product is intended to be
represented as tuberculocidal in the presence of organic soil
(one-step), an appropriate organic soil, such as 5 percent blood serum,
should be included with the bacterial inoculum.

(ii) Evaluation of tuberculocide success.  When using the AOAC
International Germicidal Spray Products as Disinfectants test, all
organisms on all carriers/slides should be killed, and there should be
no growth in any of the inoculated subculture media.

(4)  Single-Use Towelettes

(i) Test Procedure.  The Agency recommends the use of a modified AOAC
Germicidal Spray Products as Disinfectants test.  Ten carriers for each
of two samples representing two different batches of the product should
be evaluated against M. bovis (BCG).  If the product is intended to be
represented as tuberculocidal in the presence of organic soil
(one-step), an appropriate organic soil, such as 5 percent blood serum,
should be included with the bacterial inoculum.  Instead of spraying the
inoculated surface of the glass slide, the product should be tested by
wiping the surface of the glass slide with the saturated towelette, and
then subculturing the slides after the specified holding time.  The
towelette should be removed from its container and subsequently handled
with sterile gloves.  One towelette should be used to wipe a minimum of
10 inoculated slides.  Alternatively, one carrier with a surface area
equivalent to ten 1 x 1 inch carriers can be wiped using one towelette
per carrier set per batch.  The area of the towelette used for wiping
should be rotated so as to expose a maximum amount of its surface in the
course of wiping a set of slides.  Note:  A detailed description of the
wiping procedure, including the towelette folding and rotation process
should be included in the test protocol and documented in the raw data
and final report.  The applicant should submit their towelette protocol
to the Agency for review and approval prior to conducting the test.

(ii) Evaluation of tuberculocidal towelette success.  All organisms on
all carriers/slides should be killed, and there should be no growth in
any of the inoculated subculture media.

(5) Bridging for disinfectant towelettes.  In some cases, disinfectant
towelette formulations are identical to registered liquid formulations.
In order to bridge efficacy data from the EPA registered bulk liquid
disinfectant used to saturate a towelette or other related product form,
the studies in paragraphs (g)(5)(i) and (g)(5)(ii) of this guideline
should be conducted and submitted to EPA for review.

(i) Chemical Testing - Comparison of Expressed Liquid from the
Towelette(s) to the EPA Registered Liquid Disinfectant Formulation to
which it is being bridged: All active ingredients in the expressed
liquid should be within the certified limits of the Confidential
Statement of Formula of the liquid formula being referenced/bridged. The
disinfectant towelettes package should be filled according to the
manufacturing specifications.  Excess liquid in the bulk towelette
containers can not be poured off for use in the chemical testing for
bridging of the efficacy data. The liquid used in the chemical testing
should only be that expressed from the towelettes. Three batches (one of
which is 60 days old) should be tested.  Analytical data for the active
ingredients in the expressed liquid should be submitted for review.

(ii) Efficacy Testing:  Efficacy testing should be conducted under the
same testing conditions (e.g. soil load, contact time, temperature) as
used for the bulk liquid testing.  This testing allows bridging of data
from the registered bulk liquid used to saturate the towel for each type
of organism in paragraph (g)(5)(ii)(A) of this guideline.  Note:  A
detailed description of the wiping procedure, including the towelette
folding and rotation process should be included in the test protocol and
documented in the raw data and final report.  The applicant should
submit their towelette protocol to the Agency for review and approval
prior to conducting the test.

(A) Test Procedure.  The Agency recommends the use of the AOAC
Germicidal Spray Products as Disinfectants test, modified for towelette
testing.  The test should be modified to conform to appropriate elements
(e.g., media, growth conditions, etc) in the AOAC International
Tuberculocidal Activity of Disinfectants test. Ten carriers for each of
two samples, representing two batches of the product, should be tested
against M. bovis BCG.  Instead of spraying the inoculated surface of the
glass slide, the product should be tested by wiping the surface of the
glass slide with the saturated towelette, and then subculturing the
slides after the specified holding time.  One towelette should be used
to wipe a minimum of 10 inoculated slides.  Alternatively, one carrier
with a surface area equivalent to ten 1 x 1 inch carriers can be wiped
using one towelette per carrier set per batch.

(B) Evaluation of tuberculocidal towelette success.  All organisms on
all carriers/slides should be killed, and there should be no growth in
any of the inoculated subculture media.

(C)  This testing is intended to support bridging of all mycobacteria
listed on the EPA registered liquid disinfectant used to saturate the
towel to the EPA registered towelette product.

(h)  Data collection and reporting

(1) General.  To assist in the proper review and evaluation of product
performance, complete descriptions of the test employed and the results
obtained should be submitted to the Agency.  All test reports should
include, at the least, the information in paragraphs (h)(1)(i) through
(h)(i)(xiv) of this guideline:

(i) Study title;

(ii) Product identity;

(iii) Guideline number;

(iv) Identification of the testing laboratory or organization;

(v) Location where the test was performed;

(vi) Name(s) of the person(s) responsible for the test;

(vii) 40 CFR Part 160 Good Laboratory Practice compliance;

(viii) Purpose of the study;

(ix) Date and time of the start and end of the test;

(x) Statistical treatment of the data;

(xi) Conclusions;

(xii) References;

(xiii) Appendices;

(xiv) Certification

The applicant is encouraged to use the EPA’s standard efficacy report
format, which may be found at   HYPERLINK
"http://www.epa.gov/oppad001/efficacystudystandards.htm"
http://www.epa.gov/oppad001/efficacystudystandards.htm .

(2) Data for recommended methods.  When recommended methods from the
Official Methods of Analysis of AOAC International; the Annual Book of
Standards of the American Society for Testing and Materials; or, EPA
methods are used to develop efficacy data, certain minimal information,
in addition to that described in this guideline, should be provided in
the test report.  The report should include, at the least, the
information in paragraphs (h)(2)(i) through (h)(2)(xii) of this
guideline:

(i) Test employed, and any significant modifications thereto (e.g.,
organic load, hard water);

(ii) Test microorganisms employed, including identification of the
specific strain (ATCC or other);

(iii) Description of the test substance, including the percent of active
ingredient;

(iv) Concentration or dilution of the product tested and how prepared;

(v) Number of samples, batches and replicates tested;

(vi) Preparation dates of each product batch (individually formulated
preparation of the product);

(vii) Identification of all material or procedural options employed,
where such choice is permitted or recommended in the test method
selected (e.g., growth media, drying  time for inoculated carriers,
neutralization confirmation and/or subculture media, secondary
subculturing);

(viii) Test exposure conditions (e.g., contact time, temperature, and
relative humidity);

(ix) Complete reports of results obtained for each replication;

(x) Any control data essential to establish the validity of the test;

(xi) Carrier counts;

(xii) Any additional data pertinent for specific tests described in this
guideline.

(3) Data for modifications of recommended methods.  When recommended
methods are modified to support specific claims and/or use patterns for
a product, the protocol, identifying and describing each modification,
should be provided with the study report.  The applicant should submit
the proposed modification to the Agency for review and evaluation prior
to initiation of the test.

(4) Data for other methods.  When recommended methods, or modifications
thereto, are not employed to develop efficacy data (such as actual
in-use or many kinds of simulated-use testing), complete testing
protocols should be submitted with the test reports.  All materials and
procedures employed in testing should be described in a manner
consistent with original research reports published in technical or
scientific journals.  Where references to published reports or papers
are made, copies or reprints of such references should be provided with
the test reports.  The applicant should submit the proposed testing
protocols for in-use or simulated-use studies (with a proposed label to
show the claims to be supported by the protocol) to the Agency for
review and evaluation prior to initiation of the test.

(i) References.  The references in this paragraph may be consulted for
additional background information:

(1) Official Methods of Analysis of the AOAC International, Chapter 6,
Disinfectants, Use-Dilution Methods (955.14, 955.15, & 964.02),
Eighteenth edition.   AOAC International, Suite 500, 481 North Frederick
Avenue, Gaithersburg, MD 20877-2417.

(2) Official Methods of Analysis of the AOAC International, Chapter 6,
Disinfectants, Hard Surface Carrier Test Methods,  Eighteenth edition.
AOAC International, Suite 500, 481 North Frederick Avenue, Gaithersburg,
MD 20877-2417.

(3) Official Methods of Analysis of the AOAC International, Chapter 6,
Disinfectants, Official Method 961.02 Germicidal Spray Products as
Disinfectants,   Eighteenth edition. AOAC International, Suite 500, 481
North Frederick Avenue, Gaithersburg, MD 20877-2417.

(4) Official Methods of Analysis of the AOAC International, Chapter 6,
Disinfectants, Official Method 955.17 Fungicidal Activity of
Disinfectants. Eighteenth edition.  AOAC International, Suite 500, 481
North Frederick Avenue, Gaithersburg, MD 20877-2417.

(5)  Annual Book of ASTM Standards, Test Method for Efficacy of
Virucidal Agents Intended for Inanimate Environmental Surfaces,
Designation E1053-97.  American Society for Testing and Materials, 100
Barr Harbor Drive, West Conshohocken, PA 19428, current edition.

(6) Annual Book of ASTM Standards, Standard Test Method for
Neutralization of Virucidal Agents in Virucidal Efficacy Evaluations,
Designation E1483-04.  American Society for Testing and Materials, 100
Barr Harbor Drive, West Conshohocken, PA 19428, current edition.

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(8) Environmental Protection Agency, Data Call-in Notice for
Tuberculocidal Effectiveness Data for All Antimicrobial Pesticides with
Tuberculocidal Claims (Registration Division, Office of Pesticide
Programs, June 13, 1986).

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