United States Prevention, Pesticides EPA712-C-'.m <,rr
Environmental Protection and Tovic Substances ????? 1998 '""
Agency ' (7101)
Product Performance
Test Guidelines
OPPTS810.2100
Products for Use on
Hard Surfaces—Basic
Efficacy Data
Requirements
DRAFT
U.S. Environmental Protection Agency
Region VU
Information Resource Center
901N. 5th Street v *-
Kansas City, KS 66101
September 18. 1998
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INTRODUCTION
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations.
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD).
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U. S. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7U.S.C. I36,etseq.).
Final Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on disks or paper
copies: call (202) 512-0132. This guideline is also available electronically
in PDF (portable document format) from EPA's World Wide Web site
(http://www.epa.gov/epahome/research.htm) under the heading "Research-
ers and Scientists/Test Methods and Guidelines/OPPTS Harmonized Test
Guidelines."
September 18, 1998
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OPPTS 810.2100 Products for use on hard surfaces—basic efficacy
data quirements.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of the Federal Insecticide, Fungicide, and Rodenticide
Act (FIFRA) (7 U.S.C. 136, et seq.}.
(2) Background. The source materials used in developing this har-
monized 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) Sterilizers. The following information applies to all products rep-
resented as spodcidal or sterilizing agents.
(1) Recommended test methods. The Association of Official Analyt-
ical Chemists (AOAC) Sporicidal Test Method (see paragraph (p)(l) of
this guideline). (Refer to OPPTS 810.2000 for general testing requirements
prior to initiating product testing.)
(2) Test standard. Sixty carriers representing each of two types of
surfaces (porcelain penicylinders and silk suture loops) are required to be
tested as outlined in the AOAC Sporicidal Test Method (see paragraph
(p)(l) of this guideline) against spores of both Bacillus subtilis (American
Type Culture Collection (ATCC) 19659) and Clostridium sporogenes
(ATCC 3584) on three samples representing three different batches of
product, one of which is at least 60 days old (240 carriers per sample,
or a total of 720 carriers). Any sterilizing agent (vapor or gas) which is
recommended for use in a specific device must be tested using the AOAC
Sporicidal Test Method in that specific device and according to the direc-
tions for use of that specific device.
(3) Performance standard. The product must kill the test spores on
all of the 720 carriers. No failures are permitted.
(4) Confirmatory testing. Confirmatory validation testing should be
conducted by a second, independent laboratory of the registrant's choice
(other than the laboratory that developed the basic efficacy data).
(i) Recommended confirmatory test method. The AOAC Sporicidal
Test Method (see paragraph (p)(l) of this guideline).
(ii) Confirmatory test standard. Thirty carriers representing each
of the two types of surfaces (porcelain penicylinders and silk suture loops),
are required to be tested against the spores of both B. subtilis and C.
sporogenes on one sample of the product.
(iii) Confirmatory performance standard. The product must kill the
test spores on all 120 carriers. No failures are permitted.
September 18. 1998
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(c) Disinfectants (limited efficacy). When a disinfectant is rec-
ommended in labeling for use against a specific major group of microorga-
nisms (e.g., Gram-negative or Gram-positive bacteria), it is considered to
have only limited effectiveness, and consequently, only limited value as
a disinfectant. The following requirements apply to such products.
(1) Recommended test methods—(i) Water-soluble powders and
non-volatile liquid products. The AOAC Use-Dilution Method (see para-
graph (p)(2) of this guideline) or the AOAC Hard Surface Carrier Method
(distilled water only) (see paragraph (p)(3) of this guideline). (Refer to
OPPTS 810.2000 for general testing requirements prior to initiating prod-
uct testing.)
(ii) Germicidal spray products (aerosol or pump) and volatile liq-
uid products. The AOAC Germicidal Spray Products Test (see paragraph
(p)(3) of this guideline). (Refer to OPPTS 810.2000 for general testing
requirements prior to initiating product testing.)
(2) Test standard. Carriers—60 for each of three samples, represent-
ing three different batches, one of which is at least 60 days old, must
be tested against Salmonella cholerasuis (ATCC 10708) for effectiveness
against Gram-negative bacteria, or Staphylococcus aureus (ATCC 6538)
for effectiveness against Gram-positive bacteria.
(3) Performance standard. For the AOAC Use Dilution Method, the
product must kill the test microorganisms on 59 out of each set of 60
carriers/slides to provide significance at the 95 percent confidence level.
For the AOAC Hard Surface Carrier Method, registrants should contact
the Agency for guidance.
(d) Disinfectants (general or broad spectrum efficacy). When a dis-
infectant is represented in labeling as having a broad spectrum of activity
(e.g., efficacy against both Gram-negative and Gram-positive bacteria),
more extensive testing is required. The following requirements apply to
such products.
(1) Recommended test methods—(i) Water-soluble powders and
non-volatile liquid products. The AOAC Use-Dilution Method (see para-
graph (p)(2) of this guideline) or the AOAC Hard Surface Carrier Method
(distilled water only) (see paragraph (p)(3) of this guideline). (Refer to
OPPTS 810.2000 for general testing requirements prior to initiating prod-
uct testing.)
(ii) Germicidal spray products (aerosol or pump) and volatile liq-
uid products. The AOAC Germicidal Spray Products Test (see paragraph
(p)(3) of this guideline). (Refer to OPPTS 810.2000 for general testing
requirements prior to initiating product testing.)
Seirt9ij.be.;! 3. J9g8
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(2) Test standard. Sixty earners for each of three samples, represent-
ing three different batches, one of which is at least 60 days old, must
be tested against both S: cholerasuis (ATCC 10708) and S. aureits (ATCC
6538).
(3) Performance standard. For the AOAC Use Dilution Method, the
product must kill the test microorganisms on 59 out of each set of 60
carriers/slides to provide significance at the 95 percent confidence level.
For the AOAC Hard Surface Carrier Method, registrants should contact
the Agency for guidance.
(e) Disinfectants (hospital or medical environment efficacy). When
a product is recommended in labeling for use in hospitals, clinics, dental
offices, nursing homes, sickrooms, or any other medical-related facility,
the following requirements apply.
(1) Recommended test methods—(i) Water-soluble powders and
non-volatile liquid products. The AOAC Use-Dilution Method (see para-
graph (p)(2) of this guideline) or the AOAC Hard Surface Carrier Method
(distilled water only) (see paragraph (p)(3) of this guideline). (Refer to
OPPTS 810.2000 for general testing requirements prior to initiating prod-
uct testing.)
(ii) Germicidal spray products (aerosol or pump) and volatile liq-
uid products. The AOAC Germicidal Spray Products Test (see paragraph
(p)(4) of this guideline). (Refer to OPPTS 810.2000 for general testing
requirements prior to initiating product testing.)
(2) Test standard. Sixty carriers for each of three samples, represent-
ing three different batches, one of which is at least 60 days old, must
be tested against each of the following: S. cholerasuis (ATCC 10708),
S. aureus (ATCC 6538) and Pseudomonas aeruginosa (ATCC 15442).
(3) Performance standard. For the AOAC Use Dilution Method, the
product must kill the test microorganisms on 59 out of each set of 60
carriers/slides to provide significance at the 95 percent confidence level.
£or the AOAC Hard Surface Carrier Method, registrants should contact
the Agency for guidance.
(0 Fungicides. The following requirements apply to non-volatile dis-
infectants which bear additional label claims of efficacy against fungi path-
ogenic for man.
(1) Recommended test methods. The AOAC Fungicidal Test Meth-
od (see paragraph (p)(5) of this guideline). (Refer to OPPTS 810.2000
for general testing requirements prior to initiating product testing.)
(2) Test standard. Two samples representing two different batches
of the product must be evaluated for efficacy against Trichophyton
mentagrophytes (ATCC 9533 is suitable).
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(3) Performance standard. Killing of all fungal spores is required.
The highest dilution that kills all fungal spores is the minimum effective
le>
concentration.
(4) Alternative test methods—(i) Water-soluble powders and non-
volatile liquid products. The AOAC Use-Dilution Method (see paragraph
(p)(2) of this guideline). This test may be modified to conform with appro-
priate elements in the AOAC Funicidal Test Method. (Refer to OPPTS
810.2000 for general testing requirements prior to initiating product test-
ing.)
(ii) Germicidal spray products (aerosol or pump) and volatile liq-
uid products. The AOAC Germicidal Spray Products Test (see paragraph
(p)(4) of this guideline). (Refer to OPPTS 810.2000 for general testing
requirements prior to initiating product testing.)
(5) Alternative test standard. Ten carriers for each of two samples
representing two different batches of the product must be evaluated for
efficacy against T. mentagrophytes (ATCC 9533 is suitable). The inoculum
employed in paragraph (f)(4) of this guideline must be modified to provide
a concentration of at least 106 conidia per carrier.
(6) Alternative performance standard. Killing of the fungal spores
on all carriers/slides is required.
(g) Virucides. The following requirements apply to disinfectants
which bear additional label claims of effectiveness against viruses. Most
virucidal products are intended for use on dry inanimate surfaces. For this
reason, acceptable virological data are usually developed by carrier meth-
ods.
_• (1) Recommended test methods. The Agency will accept adequate
data developed by any virological technique which is recognized as tech-
nically sound, and which simulates, to the extent possible in the laboratory,
the conditions under which the product is intended for use. The specific
virus to be tested must be inoculated onto hard, inanimate 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. Each specific virus against
which product effectiveness is claimed must be treated. (Refer to OPPTS
810.2000 for general testing requirements prior to initiating product test-
ing.)
(i) For virucides whose use-directions identify the product as one in-
tended for use upon dry, inanimate, environmental surfaces (e.g., floors,
tables, etc.), carrier methods, which are modifications of either the AOAC
Use-Dilution Method (see paragraph (p)(2) of this guideline) for water-
soluble powders and non-volatile liquid products or, for products with
volatile active ingredients (such as alcohols), the AOAC Germicidal Spray
SejMembgr 18,
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Products Test Method (see paragraph (p)(4) of this guideline) for germi-
cidal spray products (aerosol or pump) must be used in the development
of the virological data.
(ii) To simulate in-use conditions, the specific virus to be treated must
be inoculated onto hard surfaces, allowed to dry, and then treated with
the product according to the directions for use on the product label.
(iii) 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, must be included with the viral inoculum. Addi-
tional organic material need not be incorporated into those procedures
where at least 5 percent blood serum is already present in the viral suspen-
sion used as the inoculum.
(iv) One surface for each of two different batches of disinfectant must
be tested against a recoverable virus titer of at least 104 from the test
surface (e.g., Petri dish, glass slide, cylinder) for a specified exposure pe-
riod at room temperature.
(v) Following treatment of the test virus with the disinfectant product,
the virus is then assayed by an appropriate virological technique. The pro-
tocol for the viral assay must provide the following information:
(A) The virus recovery (titer) from a minimum of four determinations
per each dilution in the assay system (e.g., tissue culture, embryonated
egg, animal infection, etc.).
(B) Cytotoxicity controls. The effect of the germicide on the viral
assay system from a minimum of four determinations per each dilution.
(C) The activity of the germicide against the test virus from a mini-
mum of four determinations per each dilution in the assay system.
(D) Any special methods which are used to increase the virus titer
and to detoxify the residual germicide.
(E) The ID50 values calculated for each assay.
(F) The test results must be reported as the reduction of the virus
titer by the activity of the germicide (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 (for example: Reed, L.J., and H. Muench.
A simple method of estimating 50 percent endpoints. American Journal
of Hygiene 27:493^97 (1938); Litchfield, J.T., and F« Wilcoxon. A sim-
plified method of evaluating dose-effect experiments. Journal of Pharma-
cological Experimental Therapy 96:99-115 (1949).
(G) For virucidal data to be acceptable, the product must demonstrate
complete inactivation of the virus at all dilutions. When cytotoxicity is
September 18, 1998
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evident (as illustrated in the following tables) at least a 3-log reduction
in viral liter must be demonstrated beyond the cytotoxic level. The cal-
culated viral liters must be reported with the test results.
(H) A typical laboratory report of a single test with one virus (recov-
ered from a treated surface) involving a tissue culture assay system would
include the details of the methods employed and the information included
in the following tables:
Table 1.—Example of Hypothetical Test Results Demonstrating Virucidal Activity1
Dilution inoculated
10-1
10-2
10-'
10-4
10-5
10-6
10-7
10-8
Virus — Disinfectant2
TTTT
TTTT
TOGO
0000
0000
0000
0000
0000
Virus— Control2
++++
++++
++++
++++
++++
+++0
+000
0000
Cytotoxicity — Control
TTTT
TTTT
TOOO
0000
0000
0000
0000
0000
1 T = toxic; + = virus recovered; 0 = no virus recovered.
2 Recovery of virus from surfaces demonstrated by cytopathogenic effect, fluorescent antibody,
plaque count, animal response, or other recognized acceptable technique.
Table 2.—Calculation of the Tissue Culture Infective Dose 50 percent (TCID50)1
Dilution inoculated
10-i
10-2 .....;.
10-3
10-»
10-5
10-6 .._,
10-7
TO'8 .,
number
infected/
number
inocu-
lated
4/4
4/4
4/4
4/4
4/4
3/4
1/4
0/4
number
infected
4
4
4
4
4
3
1
0
number
not in-
fected
0
0
0
0
0
1
3
4
Accumulated Values
number
infected
24
20
16
12
8
4
1
0
number
not in-
fected
0
0
0
0
0
1
4
8
number
infected/
number
inocu-
lated
24/24
20/20
16/16
12/12
8/8
4/5
1/5
0/8
Percent
infected
100
100
100
100
100
80
20
0
1TCID50 = 106-5
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Table 3.—Calculation of the Tissue Culture Lethal Dose 50 percent (TCLD50)1
Dilution inoculated
10-'
10-2
10-?
10-"
10-5
10-6
10-7
10-8
number
toxic/
number
inocu-
lated
4/4
4/4
1/4
0/4
0/4
0/4
0/4
0/4
number
toxic
4
4
1
0
0
0
0
0
Accumulated values
number
not toxic
0
0
3
4
4
4
4
4
number
toxic
9
5
1
0
0
0
0
0
number
not toxic
0
0
3
7
11
15
19
23
number
toxic/
number
inocu-
lated
9/9
5/5
1/4
0/7
0/11
0/15
0/19
0/23
Percent
toxic
100
100
25
0
0
0
0
0
1 TCLD50 = 102-7, therefore, virus inactivation = TCID50 - TCLD50 = 103-8 log 10. Claims for virucidal
activity for a product must be restricted to those viruses which have actually been tested.
(2) Test standard. One surface for each of two samples, representing
two different batches of disinfectant, must be tested against a recoverable
virus liter of at least 104 viable viral particles from the test surface for
a specified exposure period at room temperature. (Test surfaces include
Petri dishes, glass slides, stainless steel cylinders, etc.) The presence of
remaining viable virus following treatment with the product is then assayed
by an appropriate virological technique. Separate studies on two batches
of product are required to be conducted using each virus against which
product efficacy is claimed.
(3) Performance standard. Inactivation of virus must be dem-
onstrated at all dilutions when no cytotoxicity is observed in the assay
system, or at all dilutions above the cytotoxic level when it is observed.
The data must demonstrate at least a 3-log reduction in viral liter for both
samples when cytotoxicily is present. The calculated viral liters musl be
reported with the test results.
(h) Tuberculocides: The following requiremenls, presented in the
data call-in notice for tuberculocidal effectiveness dala for all antimicrobial
pesticides with tuberculocidal claims, dated June 13, 1986 (see paragraph
(p)(6) of this guideline), apply to disinfectants which bear additional label
claims of effectiveness as tuberculocides. (Note: Certain chemical classes
are required to undergo validation testing in addition to basic testing.)
(1) Recommended test methods, (i) The AOAC Tuberculocidal Test
Method (see paragraph (p)(7) of this guideline) employing standard tesl
conditions of conlacl time and lemperature. (Refer to«OPPTS 810.2000
for general testing requiremenls prior lo initiating product testing.)
(ii) Tuberculocidal Activity of Disinfectants Test Method with signifi-
cant modification of the standard test conditions of contact time and/or
temperature that are necessary to achieve tuberculocidal efficacy (see para-
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graph (p)(6) of this guideline). (Refer to OPPTS 810.2000 for general test-
ing requirements prior to initiating product testing.)
(iii) Quantitative Tuberculocidal Activity Test Method. The following
test procedure has been published in the Agency's "Data Call-In Notice
for Tuberculocidal Effectiveness-- Data for All Antimicrobial Pesticides
with Tuberculocidal Claims," dated June 13, 1986 (see paragraph (p)(6)
of this guideline). (Refer to OPPTS 810.2000 for general testing require-
ments prior to initiating product testing.)
(A) Stock culture. Mycobacterium bovis (BCG) TMC 1028 (avail-
able from Mycobacterial Culture Collection, National Jewish Hospital and
•Research Center, Denver, CO) (ATCC 35743). Lyophilized culture of M.
bovis BCG is inoculated into 10 mJL of Modified Proskauer-Beck Medium
:(refer to paragraph (h)(l)(iii)(C)(7) of this guideline) and incubated at
37 °C until a pellicle forms. Transfer a loopful of pellicle onto the surface
of 10 mL of Modified Proskauer-Beck Medium with Tween 80 (refer to
paragraph (h)(l)(iii)(C)(2) of this guideline). Incubate at 37 °C until cul-
ture is turbid. Transfer the 10 mL of culture to 100 mL of Modified
Proskauer-Beck Medium with Tween 80 (refer to paragraph
(h)(l)(iii)(C)(2) of this guideline) in a 250-mL flask. Incubate for 5-7
days, shaking the flask daily to aerate. Add 100 mL of culture to a 2-
L roller bottle containing 1 L of Modified Proskauer-Beck Medium with
Tween 80 (refer to paragraph (g)(l)(iii)(C)(2) of this guideline) and incu-
bate for 15-20 days at 37 °C, rolling slowly. Harvest the cells when the
absorbance of the culture, measured at 500 nm, is 0.6 (1-5 xlO8 CPU/
mL). On the day previous to harvesting, add Tween 80 (final concentration
= 0.1 percent) to the culture. Homogenize 10-20 mL aliquots of the culture
with a Teflon tissue homogenizer in a biosafety hood. Dispense 1-2 mL
, of the homogenized culture into vials and freeze at -70 °C.
(B) Test culture. Remove and thaw a vial of frozen stock culture
(refer to paragraph (h)(l)(iii)(A) of this guideline) at room temperature.
'Add an equal volume of buffered gelatin (refer to paragraph
(h)(l)(iii)(C)(4) of this guideline) to the cell suspension and homogenize
with a Teflon tissue grinder for 1 min, maintaining the culture at 0-4 °C
in an ice bath. Dilute the homogenized cell suspension with saline solution
(refer to paragraph (h)(l)(ii.i)(C)(<5) of this guideline) to approximately
107 CFU/mL. To demonstrate minimum culture viability and resistance,
the test culture should be tested against phenol solution (refer to paragraph
(h)(l)(iii)(C)(S) of this guideline) following the procedure described under
paragraph (h)(l)(iii)(F) of this guideline. The test culture should show no
less than 0.5 logio and no more than 1.0 logio kill irr 20 min at 25 °C.
(C) Culture media and solutions. (7) Modified Proskauer-Beck Me-
dium. Dissolve 2.5 g KH2P04, 5.0 g asparagine, 0.6 g MgS04-7H20, 2.5
g magnesium citrate and 20.0 mL glycerol in 1 L distilled water. Adjust
to pH 7.2-7.4 with IN NaOH.
beptamo&r IB, rasa
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(2) Modified Proskauer-Beck Medium with Tween 80. Mix 1 mL
Tween 80 into 1 L Modified Proskauer-Beck Medium (refer to paragraph
(h)(l)(iii)(C)(7) of this guideline).
(3) Mycobacteria 7H11 Agar. Dissolve 21 g Bacto-Mycobacteria
7H11 agar (Difco) in 1 L distilled water containing 0.5 percent glycerol.
Heat to boiling to dissolve medium completely. To each 500 mL sterile
medium, cooled to 50-55 °C, add 50 mL of Bacto-Middlebrook OADC
enrichment under aseptic conditions. Dispense 15-20 mL into 20x60 mm
disposable Petri dishes.
(4) Buffered gelatin. Dissolve 2.8 g NaH2P04 in 100 mL of distilled
water. Dissolve 5.4 g Na2HPO4 7H2O (or 7.2 g Na2HPO4-12H20) in 100
mL distilled water. Mix 33 mL NaH2PO4 solution with 67 mL Na2HPO4
solution and dilute to 200 mL with distilled water (pH 7.1). Dissolve 2.0
g Bacto-gelatin (Difco) in phosphate buffer solution.
(5) Saline solution. Add 8.5 g NaCI to 1 L of distilled water, and
mix thoroughly.
(6) Saline—Tween 80 Solution. Mix 1 mL of Tween 80 in 1 L saline
solution (refer to paragraph (h)(l)(iii)(C)(5) of this guideline).
(7) Phenol stock solution (4 percent). Dissolve 4.0 g of phenol crys-
tals in 100 mL distilled water.
(8) Phenol test solution. Make a 1:5 dilution of phenol stock solution.
This will result in a final 0.8 percent phenol solution.
(D) Neutralizer. A neutralizer appropriate for the active ingredient
in the antimicrobial should be used. Static activity controls should be con-
ducted in order to verify neutralizer capability. Also, it may be required
to demonstrate that the neutralizer is not active against the test microorga-
nism at concentrations employed in this method.
(E) Equipment. (7) Glassware, water bath—see AOAC Method
955.1 IB(a) and (b) (see paragraph (p)(7) of this guideline).
(2) Teflon homogenizer—see AOAC Method 966.04B(e) (see para-
graph (p)(l) of this guideline).
(3) Bacteriologic filters and filter holder—47 mm diameter, 0.45 (irn
pore size.
(4) Roller culture apparatus with 2-L cell culture bottle.
(5) Plastic bags—less than 2 mil thickness.
(F) Procedure. (7) Let 1 tube, containing 9 mL use-dilution
germicide sample to be tested, come to the desired temperature of the test
in a water bath and add 1 mL of test organism (refer to paragraph
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(h)(l)(iii)(B) of this guideline) to the tube containing germicide. Mix by
swirling and, at appropriate time intervals, remove aliquots of germicide-
cell suspension and add directly to equal volume of appropriate neutralize!'
(refer to paragraph (h)(l)(iii)(D) of this guideline). Mix thoroughly. Make
10-fold dilutions of neutralized sample in saline (refer to paragraph
(h)(l)(iii)(C)(5) of this guideline)"dilution blanks. Add 10-20 mL of sterile
saline to the membrane filter holder fitted with a membrane. Pipette 1
mL of appropriate dilution into the liquid on the surface of the bacterio-
logical filter (refer to paragraph (h)(l)(iii)(E)(J) of this guideline). Turn
on vacuum to effect filtration. Wash the filter with at least 50 mL of saline
with the vacuum on. Remove the filter aseptically from the filter holder
and place on the surface of Mycobacteria 7H11 agar (refer to paragraph
(h)(l)(iti)(C)(^) of this guideline). Incubate the plates in plastic bags (para-
graph (h)(l)(iii)(E)(5) of this guideline) for 15-20 days at 37 °C. Colonies
should be counted using a dissecting microscope and lateral lighting to
highlight colonies of M. bovis BCG.
(2) Survival curves should be constructed to determine the
tuberculocidal activity of the solution. Data should be plotted on semilog
paper as S/So vs time. So is calculated by determining the viable count
of the test organism culture (refer to paragraph (h)(l)(iii)(B) of this guide-
line) for each replication, and S is the viable count at each time point
for each replication. Each ratio for time points should then be averaged
to generate data for a survival curve.
(3) Survival curves should be the average of at least four separate
studies so that upper 95 percent confidence limits can be determined for
each point on the curve. The value for each upper 95 percent confidence
limit is calculated by multiplying the standard deviation by 1.96. The mini-
mum time that can be claimed for efficacy is determined by finding the
point at which the average survival curve intersects the S/So line on the
graph which indicates the probability of one survivor (e.g., 1 divided by
So, the average starting count). This time can also be found by inspection
of the raw tabular data, locating the time at which an average of one orga-
nism or less was found. If the data show at least 4 logic kill of the starting
population, but the survivor curve does not intersect the one survivor S/
So line on the graph, the minimum time that can be claimed is determined
by extrapolating the upper 95 percent confidence limit curve to the one
survivor line, using the last two points on the 95 percent confidence limit
curve as a basis for the extrapolation.
(4) The time established for tuberculocidal efficacy claims must be
in 5 min increments. In instances where a 0-survivor pojnt or a 95 percent
confidence limit intercept leads to a time other than that falling on a 5
min increment, the claim will be established by increasing the time up
to the next higher 5 min increment (e.g., if 22 min were a 1-survivor
line intercept, the claim time would be 25 min. If an average 0-point were
found at 20 min, the claim time would be 20 min). If a contact time of
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other than 5 minutes is desired, the testing must be performed by the
AOAC Tuberculocidal Activity of Disinfectants Test Method (refer to
paragraph (h)(l)(ii) of this guideline), the AOAC Germicidal Spray Prod-
ucts Test or the Presaturated or Impregnated Towelette Test (refer to para-
graphs (h)(l)(iv) and (h)(l)(v) of this guideline).
(5) An extrapolation of the upper 95 percent confidence limit interval
curve to determine kill time will be considered valid only if the last data
point shows at least 99.99 percent (4 logio) kill. The 95 percent confidence
limits on the curve should be such that the intersection of the upper 95
percent confidence limit curve with the 1-survivor line (time) should not
be at a value that is 50 percent greater than the value where the survivor
curve, or an extrapolation from the survivor curve, intersects that line.
(iv) AOAC Germicidal Spray Products Test (see paragraph (p)(4) of
this guideline). If the product is a spray or a liquid with volatile active
ingredients, the procedure must be modified to conform with the AOAC
Germicidal Spray Products Test using the media, test culture, and other
elements described in the AOAC Tuberculocidal Activity Method (see
paragraph (p)(7) of this guideline). (Refer to OPPTS 810.2000 for general
testing requirements prior to initiating product testing.)
(v) Presaturated or impregnated towelettes. When the product is
packaged as a towelette (particularly when containing volatile active ingre-
dients such as alcohols), the Presaturated or Impregrated Towelettes Simu-
lated Use Test (refer to paragraph (i)(l) of this guideline) must be used
with the media, test culture, and other elements as described in the AOAC
Tuberculocidal Activity Method (see paragraph (p)(7) of this guideline).
Testing must be performed in an open air environment (not a closed petri
dish).
(2) Test standard, (i) When using the existing or modified AOAC
Tuberculocidal Activity Test Method, the AOAC Germicidal Spray Prod-
ucts Test Method, or the Presaturated or Impregnated Towelettes Method,
10 carriers for each of two samples, representing two different batches
of product must be tested against Mycobacterium tuberculosis var. bovis
(BCG).
(ii) When using the Quantitative Tuberculocidal Activity Method, two
samples, representing two different batches of product must each be uti-
lized in at least four separate studies (a total of at least eight studies),
against M. tuberculosis var. bovis, so that upper 95 percent confidence
limits can be determined for each point on the survival curve.
(3) Performance standard, (i) When using the existing or modified
AOAC Tuberculocidal Activity Test Method, the AOAC Germicidal Spray
Products Test Method, the Presaturated or Impregnated Towelettes Test
Method, killing on all carriers/slides, and no growth in any of the inocu-
lated tubes of two additional media, is required.
September 1C, 1998
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(ii) When using the Quantitative Tuberculocidal Activity Method, an
extrapolation of the upper 95 percent confidence interval curve to deter-
mine kill time will be considered valid only if the last data point shows
at least 99.99 percent (4 log 10) kill (reduction in the original population
number). The 95 percent confidence limits on the curve should be such
that the intersection of the upper 95 percent confidence limit curve with
the one survivor line (time) should not be at a value that is 50 percent
greater than the value where the survivor curve, or an extrapolation from
the survivor curve, intersects that line.
(4) Validation testing requirements for specific chemical classes.
(i) If glutaraldehyde-based products are evaluated using the existing
AOAC Tuberculocidal Activity Method or the AOAC Germicidal Spray
Products Test Method, employing the standard test conditions of contact
time and temperature, validation data will be required. Validation data
must be developed by testing one additional sample of the product by
a laboratory of the registrant's choice (other than the laboratory which
developed the original efficacy data) using the same test conditions as the
original laboratory (10 min contact time and 20 °C). If validation testing
involves stressed (reused) solutions, consultation with the Agency is ad-
vised prior to initiation of testing.
(ii) Products with tuberculocidal claims that are formulated with qua-
ternary ammonium compounds may be evaluated for tuberculocidal effi-
cacy using any one of the test methods listed in paragraph (h)(l) of this
guideline; However, validation data is required for any test method chosen.
Validation data must be developed by testing one additional sample of
the product by a laboratory of the registrant's choice (other than the labora-
tory which developed the original efficacy data) using the same optional
test procedure and test conditions as the original laboratory.
(iii) Products with tuberculocidal claims that are formulated with
chemical groups other than glutaraldehyde and quaternary ammonium
compounds are permitted, at this time, to be evaluated for tuberculocidal
efficacy using any one of the test methods listed in paragraph (h)(l) of
this guideline. Validation data does not need to be submitted.
(i) Presaturated or impregnated towelettes. Presaturated or impreg-
nated towelettes represent a unique combination of antimicrobial chemicals
and applicator prepackaged as a unit in fixed proportions. Therefore, the
complete product, as offered for sale, should be tested according to the
directions for use to ensure its effectiveness in disinfecting hard surfaces.
(1) Recommended simulated-use test — (i) Single-use towelettes.
This product is intended to be removed from the package, used imme-
diately, and discarded after use. (Refer to OPPTS 810.2000 for general
testing requirements prior to initiating product testing.)
kor1Q 1QQB
17
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(A) The standard test methods available for hard-surface disinfectants
(AOAC Use-Dilution Method, the AOAC Hard Surface Carrier Method,
and AOAC Germicidal Spray Products Test Method), if followed exactly,
would not closely simulate the way in which the disinfectant towelette
is used. Of these methods, the AOAC Germicidal Spray Products Test
Method appears to be the one rhost readily modified for this situation.
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.
All remaining liquid should be expressed from the used towelette and
should also be subcultured.
(B) The towelette should be removed from its container and subse-
quently handled with sterile gloves. One towelette should be used to wipe
60 inoculated slides. 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. After wiping the last slide for a particular
towelette, all of the liquid remaining in the material should be expressed
into an empty sterile container by squeezing the towelette; after a specified
holding time (equal to the contact time stated on the product label), an
aliquot from this container (ca. 0.1 mL) should be subcultured in the same
manner as the slides.
(C) For additional test modifications, which may be necessary de-
pending on the intended label claims and directions for use, as well as
for documentation of neutralization, refer to OPPTS 810.2000, paragraph
(c)(4)—Supplemental recommendations (e.g., exposure period, hard water,
organic soil, etc.)
(ii) Simulated re-use protocol for multiple-use towelettes. Multiple-
use towelettes are intended to be unpackaged and used repeatedly for an
extended period until a specified end-point is reached (as determined, for
example, by a visible indicator in the product). Products intended for pat-
terns of repeated use should be tested by a simulated reuse protocol. At
the completion of the simulated reuse test; the used towelettes should be
tested at the specified end-point of their use-life for effectiveness as dis-
infectants as indicated in paragraphs (h)(l)(i) and (h)(2)(i) of this guide-
line. (Refer to OPPTS 810.2000 for general testing requirements prior to
initiating product testing.) The simulated reuse protocol should include,
but is not limited to, the following basic elements:
(A) The cloth should be moistened (in the case of a dry impregnated
towelette) and applied to representative types of surfaces as recommended
on the label and according to the directions for use. The cloth should then
be allowed to partially or completely dry; and the wet-wipe-dry cycle
should be repeated until the claimed use-life or specified end-point is
reached. These cycles should include periodic challenge with micro-
biological "bioburden" (viable test bacteria dried onto surfaces/carriers
September 18, 1998
13
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A:2100.810
which are wiped). The minimum bioburden load should be approximately
equivalent to one glass slide contaminated with at least 106 viable bacteria
(e.g., S. aureus, S. cholerasuis, P. aeruginosa) per each 5 mL of use solu-
tion produced in wetting the towelette.
(B) Periodic chemical monitoring of the active ingredient in the use
solution produced in the cloth should be performed to demonstrate the
adequacy and consistency of the concentration provided. In lieu of chemi-
cal monitoring, microbiological assay of the surfaces/cloth solution ex-
posed to the bioburden must be performed and found to meet the criteria
for acceptable disinfection (refer to paragraphs (c), (d), and (e) of this
guideline).
(C) For additional test modifications, which may be necessary de-
pending on the intended label claims and directions for use, as well as
for documentation of neutralization, refer to OPPTS 810.2000, paragraph
(c)(4)—Supplemental recommendations (e.g., exposure period, hard water,
organic soil, etc.). If claims are made for the use of the product in the
presence of soil ("one-step" cleaning and disinfecting), the reuse protocol
must be conducted with at least 5 percent blood serum added to the bac-
terial inoculum employed as bioburden as well as to the water.
(D) At the completion of the simulated-use protocol, the used
towelettes are tested at the specified end-point of their use-life for effec-
tiveness as disinfectants as indicated in paragraphs (h)(l)(i)(A),
(h)(l)(i)(B), and (h)(l)(i)(C) of this guideline.
(2) Test standard—(i) Single-use towlettes. Refer to paragraphs
(b)(2), (c)(2), and (d)(2) of this guideline for guidance on the test micro-
..organisms and the minimurn number of carriers/slides to be used in the
/simulated-use test for single-use towelettes. Three towelettes from freshly
..opened packages, representing three different batches of product, one of
which is at least 60 days old) should be evaluated for efficacy. Simulated
in-use tests are to be conducted in triplicate. No simulated reuse protocol
is required.
(ii) Multiple-use towelettes. Three towelettes representing three dif-
ferent batches, one of which is at least 60 days old, should be utilized
in the simulated re-use protocol. The tests should be conducted in trip-
licate. The simulated-use test should incorporate, to the extent possible,
the conditions under which the product is reused and to which it could
be exposed, including repeated microbiological challenge, for the.duration
of its intended use-life.
(3) Performance standard—(i) Single-use towelettes. Refer to para-
graphs (b)(3), (c)(3), and (d)(3) of this guideline. Subcultures of the liquid
expressed from the used towelettes should be negative for growth.
A
14
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A.-2100.810
(ii) Multiple-use towelettes. Following reuse testing of the product
according to paragraph (h)(l)(ii) of this guideline and subsequent evalua-
tion of the reused product according to paragraph (h)(l)(i) of this guide-
line, the product should meet the performance standards given in paragraph
(h)(3)(i) of this guideline.
(j) Phenol coefficient. Data from this test are required only when
permitted phenol coefficient claims are made in labeling of disinfectants.
(1) Recommended test methods. The AOAC Phenol Coefficient
Method (see paragraph (p)(8) of this guideline). (Refer to OPPTS 810.2000
for general testing requirements prior to initiating product testing.)
(2) Test standard. Phenol coefficients for Salmonella typhi (ATCC
6539), the only official test organism, and for any other additional Gram-
negative or Gram-positive asporogenous bacteria must be determined on
each of two samples representing two different batches against each bac-
terium.
(3) Performance standard. The phenol coefficient is a numerical
value that compares the bactericidal concentration of a disinfectant to the
bactericidal concentration of pure phenol. This numerical value is obtained
by dividing the greatest dilution of disinfectant killing 5. typhi, or other
bacterium being evaluated, in ten minutes, but not in five minutes, by the
greatest dilution of phenol showing the same results. Since phenol coeffi-
cient values are usually an unreliable index as to the effective use-dilution
of a disinfectant product, the AOAC Use-Dilution Method must be em-
ployed to determine the efficacy of a product, and its effective use-dilution.
(k) Additional microorganisms. The following requirements apply
to disinfectants which bear label claims against specific microorganisms
other than those named in the AOAC Use-Dilution Method, AOAC Hard
Surface Carrier Method, AOAC Germicidal Spray Products Test, AOAC
Fungicidal Test, and AOAC Tuberculocidal Test, but not including viruses
(see paragraph (g) of this guideline for virucides).
(1) Recommended test methods — (i) Water-soluble powders and
non-volatile liquid products. The AOAC Use-Dilution Method (see para-
graph (p)(2) of this guideline). (Refer to OPPTS 810.2000 for general test-
ing requirements prior to initiating product testing.)
(ii) Germicidal spray products (aerosol or pump). The AOAC Ger-
micidal Spray Products Test (see paragraph (p)(4) of this guideline). (Refer
to OPPTS 810.2000 for general testing requirements prior to initiating
product testing.)
(iii) Presaturated or impregnated towelettes. Refer to paragraph
(i)(l) of this guideline.
September 18, 1998
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A:2100.810
(2) Test standard. Ten carriers for each of two product samples rep-
resenting two different batches, must be tested against each additional
microorganism.
(3) Performance standard, (i) Killing of the test microorganisms on
all carriers/slides is required.
(ii) Plate count data, on appropriate culture media, must be submitted
on each test microorganism to demonstrate that a concentration of at least
104 microorganisms survive the carrier-drying step to provide meaningful
results at the 95 percent confidence level (see OPPTS 810.2000, paragraph
(1) Sanitizers—nonfood contact surfaces. The following require-
ments apply to product bearing label claims for effectiveness as sanitizers
for inanimate hard surfaces other than those which come in contact with
food or beverages (e.g., floors, walls, furnishings).
(1) Recommended test methods. The Sanitizer Test for Hard, Inani-
mate Nonfood Contact Surfaces (prepared by Registration Division, Office
of Pesticide Programs, EPA, 1976) (see paragraph (p)(9) of this guideline).
To substantiate the sanitizing claims for a product, data must be submitted
that demonstrate that the product, when used as directed, will substantially
reduce the number of test microorganisms on a treated surface over those
of an untreated control surface. The following protocol may be used.
(Refer to OPPTS 810.2000 for general testing requirements prior to initiat-
ing product testing.)
(i) Determine the bacterial count in an 18-24 h broth culture and
add a 0.01—0.03 mL quantity of the broth culture by spreading on a 1x1
inch square of test surface using a bacteriological loop.
(ii) If the product is intended to be represented as a "one step" clean-
er-sanitizer, an appropriate organic soil load, such as 5"percent blood
serum, must be added to the bacterial inoculum.
(iii) The square of test surface should be dried for 40 min in a bac-
teriological incubator at 30--37 °C.
(iv) A "zero-time" bacterial numbers recovery test must be per-
formed to demonstrate the efficiency of the recovery process and must
be reported. The "zero-time" test shall show the loss in viability that oc-
curred during carrier drying.
(v) Apply the product to the inoculated test surfaces as directed on
the product label.
(vi) Run parallel tests on the formulation with the active ingredients
omitted in an identical manner to serve as the control. If such a control
solution is not suitable, use sterile distilled water to which may be added
*
September 18, 1998 •*•(>
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Ar2100.810
0.01 percent'isooctylphenoxypolyethoxyethanol (9-10 moles oxyethylene,
e.g., Triton X-100).
(vii) After a suitable time interval, recover the test organisms by
washing the squares with adequate agitation in appropriate media or dilu-
tion fluid containing appropriate neutralizers.
(viii) Make plate counts on appropriate nutrient agar containing the
same neutralizers by the pour or spread plate technique.
(ix) Exposure time intervals between 0-time and 5 min must be tested
for the product as well as the untreated controls.
(x) The results must demonstrate a bacterial reduction of at least 99.9
percent over the parallel control within 5 min.
(2) Test standard. The propagation of cultures specified in this para-
graph, and the use of subculture media and other related equipment may
be as specified in Chapter 6, Disinfectants, of the Official Methods of
Analysis of the Association of Official Analytical Chemists. Three product
samples, representing three different batches, one of which is at least 60
days old, should be tested against each test bacterium on each representa-
tive test surface, depending on the uses proposed on the label. The test
microorganisms are: S. aureus (ATCC 6538) and Klebsiella pneumonias
(ATCC 4352). Enterobacter aerogenes (ATCC 13048) can be substituted
for K. pneumoniae. The test surfaces representing the types of surfaces
recommended for treatment on the label include, but are not limited to,
glass, metal, unglazed or glazed ceramic tile, porcelain, or vitreous china.
(3) Performance standard. The results must demonstrate a reduction
of at least 99.9 percent (a 3-log reduction) in the number of each test
microorganism over the parallel control count within 5 min.
(m) Sanitizing rinses for previously cleaned food-contact surfaces.
The following requirements apply to any product with a label rec-
ommendation for the treatment of previously cleaned, nonporous, food-
contact surfaces (e.g., eating and drinking utensils and food processing
equipment) as a terminal sanitizing rinse. Antimicrobial agents applied to
food-contact surfaces are defined as incidental food additives under the
Federal Food, Drug, and Cosmetic Act, as amended (21 U.S.C. 201 et
seq.) and require a food additive tolerance or an exemption from such
tolerance according to 21 CFR 178.1010. Recommendation of a potable
water rinse after treatment is not permitted for products intended for use
as a terminal sanitizing rinse.
'to
September 18, 1998
(1) Halide chemical products. Sanitizing rinses formulated with
iodophors, mixed halides, and chlorine-bearing chemicals.
(i) Recommended test methods. The AOAC Available Chlorine Ger-
micidal Equivalent Concentration Method (see paragraph (p)(10) of this
*
17
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A:2100.810
guideline). (Refer to OPPTS 810.2000 for general testing requirements
prior to initiating product testing.)
(ii) Test standard. Three samples, representing three different
batches, one of which is at least 60 days old, must be evaluated for effi-
cacy against 5. typhi (ATCC 6539). When claims are made for the effec-
tiveness of the product in hard water, all required data must be developed
at the hard water tolerance claimed. (See OPPTS 810.2000, paragraph
(iii) Performance standard. .Test results must demonstrate product
concentrations equivalent in activity to 50, 100, and 200 ppm of available
chlorine. The reference standard is sodium hypochlorite.
(2) Other chemical products. Sanitizing rinses formulated with qua-
ternary ammonium compounds, chlorinated trisodium phosphate, and ani-
onic detergent-acid formulations.
(i) Recommended test methods. The AOAC Germicidal and Deter-
gent Sanitizing Action of Disinfectants Method (see paragraph (p)(ll) of
this guideline). (Refer to OPPTS 810.2000 for general testing requirements
prior to initiating product testing.)
(ii) Test standard. Three samples, representing three different
batches, one of which is at least 60 days old, must be evaluated for effi-
cacy against both Escherichia coli (ATCC 11229) and S. aureus (ATCC
6538). When claims are made for the effectiveness of the product in hard
water, all required data must be developed at the hard water tolerance
claimed. (See OPPTS 810.2000, paragraph (c)(4)(iii).)
(iii) Performance standard. Acceptable results must demonstrate a
'99.999 percent reduction in the number of each test microorganism within
30 sec. The results must be reported according to the actual count and
-percentage reduction over the control. The minimum concentration of the
product which provides the results required is the minimum effective con-
centration.
(n) Residual bacteriostatic activity of .dried chemical residues on
hard inanimate surfaces. Bacteriostatic claims are permitted only against
microorganisms identified as causing economic or aesthetic problems (e.g.,
odor-causing bacteria) in the presence of moisture, but not against micro-
organisms of public health concern. (Refer to OPPTS 810.2000 for general
testing requirements prior to initiating product testing.) ,
(o) Residual self-sanitizing activity of dried chemical residues on
hard-inanimate surfaces. The following requirements apply to products
which bear label claims to provide residual self-sanitizing activity (e.g.,
significant reduction in numbers of infectious microorganisms which may
September 18,«f998
18
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A.-2100.810
be present or subsequently deposited) on treated surfaces that are likely
to become and remain wet under normal conditions of use.
(1) Recommended test methods. Residual self-sanitizing products
for use on hard, inanimate surfaces must be evaluated for efficacy using
a controlled in-use study or simulated in-use study including the elements
outlined under paragraph (o)(2) of this guideline. (Refer to OPPTS
810.2000 for general testing requirements prior to initiating product test-
ing.)
(2) Test standard. Each controlled in-use or simulated in-use study
test must include the following basic elements:
to
(i) The test microorganisms employed in the study must be pathogens
that are likely to be encountered in the environment in which the product
is to be used.
(ii) The starting inocula of the test microorganisms (for initial and
subsequent challenges) must be of sufficient concentration to provide at
least 104 survivors on the parallel control surface.
(iii) The residue on the treated surfaces must be activated by the addi-
tion of moisture in a manner and over an exposure period identical to
the use pattern for which the product is intended.
(iv) Quantitative bacteriological sampling must be conducted at fre-
quent and regular intervals.
(v) The same types of surfaces without the treatment must be em-
ployed in the test and inoculated in a manner and over an exposure period
identical to the use pattern for which the product is intended.
(vi) The environmental conditions employed in the test (e.g., relative
humidity and temperature), must be reported. These conditions must be
the same as those likely to be encountered under normal conditions of
product use. Tests should also include those environmental conditions that
would act to reduce the effective concentration of the product on the inani-
mate surface (e.g., rinsing, abrasion, organic load, repeated challenges by
microorganisms, etc.).
(vii) The length of time the residual activity can be expected to exist
under the expected use conditions must be documented.
(3) Performance standard. For residual self-sanitizing claims, it
must be demonstrated that a product is capable of reducing the number
of test microorganisms on the test surface by 99.9 percent over that of
the parallel control surfaces.
September 18, 1998
(p) References. The following references may be consulted for addi-
A
19
tional background information.
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A:2100.810
(1) Official Methods of Analysis of the Association of Official Analyt-
ical Chemists, Official Method 966.04 Sporicidal Activity of Disinfectants.
Current edition. Association of Official Analytical Chemists (AOAC),
2200 Wilson Boulevard, Arlington, VA 22201.
(2) Official Methods of Analysis of the Association of Official Analyt-
ical. Chemists, Use-Dilution Methods, Chapter 6, Disinfectants. Fifteenth
edition. Association of Official Analytical Chemists (AOAC), 2200 Wilson
Boulevard, Arlington, VA 22201.
(3) Official Methods of Analysis of; the Association of Official Analyt-
ical Chemists, Hard Surface Carrier Methods, Chapter 6, Disinfectants.
Current edition. Association of Official Analytical Chemists (AOAC),
2200 Wilson Boulevard, Arlington, VA 22201.
(4) Official Methods of Analysis of the Association of Official Analyt-
ical. Chemists , Official Method 961.02 Germicidal Spray Products as Dis-
infectants. Fifteenth edition. Association of Official Analytical Chemists
(AOAC), 2200 Wilson Boulevard, Arlington, VA 22201.
(5) Official Methods of Analysis of the Association of Official Analyt-
ical. Chemists, Official Method 955.17 Fungicidal Activity of Disinfect-
ants. Current edition. Association of Official Analytical Chemists (AOAC),
2200 Wilson Boulevard, Arlington, VA 22201.
(6) Environmental Protection Agency, Data Call-in Notice for
Tuberculocidal Effectiveness Data for All Antimicrobial Pesticides with
Tuberculocidal Claims (Registration Division, Office of Pesticide Pro-
grams, June 13, 1986).
(7) Official Methods of Analysis of the Association of Official Analyt-
ical Chemists, Official Method 965.12 Tuberculocidal Activity of Dis-
infectants. Current edition. Association of Official Analytical Chemists
(AOAC), 2200 Wilson Boulevard, Arlington, VA 22201.
(8) Official Methods of Analysis of the Association of Official Analyt-
ical Chemists, Official Method 955.11 Testing Disinfectants Against Sal-
monella typhi. Current edition. Association of Official Analytical Chemists
(AOAC), 2200 Wilson Boulevard, Arlington, VA 22201.
(9) Environmental Protection Agency, Sanitizer Test for Hard, Inani-
mate Nonfood Contact Surfaces Modified to Include Organic Soil. (Reg-
istration Division, Office of Pesticide Programs, 1976). ,
(10) Official Methods of Analysis of the Association of Official Ana-
lytical. Chemists, Official Method 955.16 Chlorine Equivalent Concentra-
tion. Current edition. Association of Official Analytical Chemists (AOAC),
2200 Wilson Boulevard, Arlington, VA 22201.
*
Sepfenfter 18, 1998 20
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A:2100.81D
(11) Official Methods of Analysis of the Association of Official Ana-
lytical Chemists, Official Method 960.09 Germicidal and Detergent Sani-
tizing Action of Disinfectants. Current edition. Association of Official An-
alytical Chemists (AOAC), 2200 Wilson Boulevard, Arlington, VA 22201.
September 18. 1998
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