DIS/TSS-8 / April 18, 1981
EFFICACY DATA REQUIREMENTS
Products bearing label claims for effectiveness as sanitizers for pre-cleaned carpeting must be tested by a protocol incorporating the basic elements of the attached recommended method. If the product is intended to be represented in labeling as a "one-step" cleaner-sanitizer, the method must be modified by including an appropriate soil with the bacterial inoculum.
- Test requirements.
- Three product samples representing 3 separate batches, one of which is at least 60 days old, must be tested against Staphylococcusaureus ATCC 6538 and Enterobacteraerogenes ATCC 13048 with 2 different types of representative synthetic carpeting, such as acrylic and polypropylene tufted-loop types. If the application is intended for hospitals or medical institutions, the product must also be tested against Pseudomonasaeruginosa ATCC 15442. If the product is also intended for use on wool carpeting, an additional representative sample of wool carpet must be tested; otherwise, the label must bear a disclaimer for use on wool. All carpet samples tested must be fully identified by the pile fiber type, pile yarn weight of finished carpet, pile density, and tuft height. Adequate controls must demonstrate that bacteriostatic agents in the carpet pile or backing do not yield false-negative data which interfere with the test results.
- The amount of solution applied to the sample carpeting in the tests must be determined and extrapolated to obtain the amount of the solution of product to be applied to carpeting (volume per unit area) as stated on the label.
- Performance requirements.
A 99.9% reduction of test bacteria over the scrubbed control count must be demonstrated.
Note: If the product is intended for use in hospitals or medical institutions, a wet vacuum pickup must be specified in the label directions for use. In no case will the so-called "dry shampoo treatment" be considered for use in such areas.
Sanitizers - Carpets
(Proposed method prepared by Registration Division,
Office of Pesticide Programs, EPA, 1976; revised 1981)
- Special equipment and materials.
- Carpet mounting board. Mount a piece of l/8-in. (0.3 cm) tempered hardboard, tempered surface up, on a 16 x 16-in. (40.6 x 40.6 cm) base of 3/4-in. (1.8 cm) thick marine plywood, with 3/4-in. (1.8 cm) brads.
- Cutting equipment. 2 x 2-in. (5.1 x 5.1 cm) squares of 1/4-in. (0.6 cm) acrylic plastic with 3/32-in. (0.24 cm) holes in the center as templates, and a sharp knife with replaceable blade.
- Scrub brushes. 1 1/4 x 3 1/2-in. (4.2 x 8.9 cm) surgical hand brush with 5/8-in. (0.6 cm) nylon bristles.
- 4. Extraction bottles. 8-oz. (236.6 ml), widemouth, round, polypropylene bottles with screw caps (Nalgene 2105 or equivalent) containing 10 stainless steel penicylinders and 100 ml of appropriate neutralizer broth. Similar style glass bottles may be used but care must be taken to prevent breakage during shaking.
- Spray device. Adjustable spray atomizer modified to feed from a calibrated test tube or bottle. A Model 15 DeVilbiss atomizer on a 2-oz. (59.2 ml) bottle graduated with 10-ml marks may be used.
- Carpet. If the product is intended for use on commercial grade carpeting, 2 representative carpets, such as acrylic and polypropylene tufted-loop type must be tested. No carpeting is available to serve as a standard. If the product is intended for use on wool carpeting, a representative wool sample must additionally be tested. All carpet samples tested must be fully identified, and the pile fiber type, pile yarn weight of finished carpet, pile density and tuft height must be reported. Adequate controls must demonstrate that bacteriostatic agents in the carpet pile or backing do not interfere with the test results.
- Test cultures and media.
- Test bacteria. Use Staphylococcusaureus (ATCC 6538) and Enterobacteraerogenes (ATCC 13048). If the product is intended for use in hospitals, Pseudomonasaeruginosa PRD-10 (ATCC 15442) must additionally be tested.
- NutrientagarB. AOAC Methods, sec. 4.023 (a) (2).
- Phosphate buffer dilution water. AOAC Methods, sec. 4.023 (f).
- Double strength neutralizer broth. For phenolic based products, Letheen broth [AOAC Methods, sec. 4.001 (d) (3)] plus an additional 0.7 g lecithin (Azolectin) and 5 g polysorbate 80 (Tween 80) per liter may be used; or a defoaming neutralizer consisting of nutrient broth [AOAC Methods, sec. 4.001 (a)] plus 1.0 % Pluronic 25R2 (Meroxapol 252) has been suggested. In the case of halogen or heavy metal based products, 0.1% sodium thioglycollate and 0.01% isooctylphenoxypolyethoxyethanol (Triton X-100) in phosphate buffer (pH 7.2) may be used.
- Neutralizerplatecountagar. Tryptone glucose extract agar [AOAC Methods, sec. 4.037 (a)] plus 0.7 g lecithin (Azolectin) and 5 g polysorbate 80 (Tween 80) per liter.
- Bacterial inoculum
- Cut the carpet into 8 x 12-in. (20.3 x 30.5 cm) pieces. With the aid of the 2 x 2-in. (5.1 x 5.1 cm) template, cut six 2 x 2-in. squares (2 rows of 3 squares per row) from the backing side of the carpet, leaving at least 4 in. (10.2 cm) between the center of each square. The preferred method is to leave about 1/8 in. (0.32 cm) of backing intact at each corner of each cut square so that the entire piece of carpeting can be sterilized and inoculated without separation. Mark the pile surface in the center of each test square with a waterproof marking pen with the aid of the hole in the center of the template. Cover the pile surface of the carpeting with aluminum foil and fold over edges to secure. Steam sterilize and dry. Only carpet that has been determined to be free of residual bacteriostatic activity on the pile or backing, following autoclaving, shall be used. A seeded agar plate overlay technique should be used for this determination.
- Dilute the standardized bacterial stock suspensions, prepared as in (c) above, with phosphate buffer dilution water containing 0.01% isooctylphenoxypolyethoxyethanol to a concentration 10 x 10 bacteria per ml. Inoculate the previously marked center of each cut square with 0.1 ml of the bacterial suspension. (Retain the bacterial suspensions for determination of inoculation numbers.) Dry inoculated carpet in an incubator at 35-37°C for 60 min. with the foil wrap loosely in place.
- Condition brushes by immersing the bristles in separate containers (15- cm glass petri dishes or equivalent) of diluted test solution and a control solution without the active antimicrobial ingredient(s) for 15 min. (If such a control solution is not available, use sterile distilled water containing 0.01% isooctylphenoxypolyethoxyethanol.) Fasten 2 pieces of inoculated carpet (each containing 6 test squares) onto the carpet mounting board by nailing each corner with upholstery tacks, and with the foil wrapping positioned so as to protect the controls during spraying and scrubbing with the test solution. Place the board in a biological hood or glove box. A simple safety chamber can be constructed from a large plastic bag.
- Determine the amount of test solution intended to be applied to one piece of the carpeting containing 6 spots of dried bacterial inoculum [96 sq. in. or 2/3 sq. ft. (244 sq. cm)] and subtract approximately 15 ml which will be applied later in the brushing procedure. Apply the pre-determined amount of diluted test solution at room temperature uniformly by metered spray to one piece of the test carpet. Shake excess test solution from a conditioned brush and transfer to a fresh dish containing 100 ml of test solution at room temperature. Dip bristles of brush and transfer the retained test solution to an inoculated spot on the sprayed carpet. Scrub the spot for 30 sec. using 30 circular clockwise strokes and 30 circular counterclockwise strokes. A circular area of pile approximately 3 in. (7.6 cm) in diameter around each spot must be covered by this treatment. Moderate to heavy pressure should be applied downward on the brush to work the solution to the base of the pile. Repeat dipping of brush into test solution and scrubbing procedure until each of the 6 spots is treated. The brush dipped into the solution no more than 6 times will deliver about 15 ml of solution to the carpet. Do not exceed this amount. Record the total volume of solution applied by spray and brush. Allow the treated carpet piece to remain at room temperature for 60 min. for partial drying of the treated areas.
- While the piece of carpet treated with the test solution is drying, spray the non-active control solution at room temperature onto half of the other (control) piece of carpet so as to cover 3 of the 6 spots of dried inoculum. Position the aluminum foil over the remainder. Spray an amount equivalent to half of the amount of sprayed test solution. Scrub the 3 wet spots in the same manner as the test carpet. The remaining 3 spots are unscrubbed controls to determine the numbers of bacteria which survived drying of the inoculum. Care must be taken not to wet or scrub over the unscrubbed control area. Allow the scrubbed and unscrubbed controls to remain at room temperature for 60 min. as with the test piece.
- Following the 60-min. drying periods, cut each 2 x 2-in. test square free with flamed forceps and knife. Transfer each square of carpet to a separate extraction bottle of neutralizer broth. Shake each extraction bottle vigorously for at least 1 min. to free the bacteria from the carpet fibers. Determine the number of viable bacteria in each sample bottle by plating duplicate dilutions in neutralizer plate count agar. Similarly determine the number of viable bacteria in 0.1 ml of the suspension used for inoculating the carpet. Also incubate all broth extraction bottles to determine whether neutralization of the test sample was achieved.
- Determine the percent reduction of viable bacteria by the test solution by comparing the number of survivors from each treated test square against the average viable count from the scrubbed control squares. An average viable count of at least 1.0 x 106 bacteria from the extracted unscrubbed control squares is necessary for a valid test.
Prepare French square culture bottles with nutrient agar B and test bacteria (AOAC Methods, sec. 4.026). Prepare standardized bacterial stock suspensions by washing growth from bottles and adjust to a density of 10 x 10 bacteria per ml with phosphate buffer dilution water (AOAC Methods, sec. 4.026).
Horowitz, William, ed. 1975. Official Methods of Analysis of the Association of Official Analytical Chemists, 12th ed. Association of Official Analytical Chemists, Washington, D.C.