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
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A:21'00.810'
                                        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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A:2i00.810
             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
                                               1

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A:2100.810
                 (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.)
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A:2100.81Q
                 (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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A:2-100.310
                 (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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A:2100.810
             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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A:2100.810
              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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A:2l00.810
             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.
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A:2100.810
                 (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
September 18, 1998

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A:2100.810
             (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
September 18, 1998
                                               10

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A:2100.810
             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
                                               n

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A:2100.810
                 (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:2100.810
                 (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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