dr. william gilbertson food & drug administration, room 1-23

Ecolab Center St. Paul, MN 55102 612/451-5600 Fax: 612/451-4272

Government Technical Affairs

Dr. William Gilbertson % Dockets Management Branch (HFA-305) Food & Drug Administration, Room 1-23 12420 Parklawn Drive Rockvillle MD 20857

December 1, 1995

Subject: Docket 75N - 183H; Topical Antimicrobial Drug Products New Data Submission

Dear Dr. Gilbertson:

Ecolab Inc. develops and markets specialty products, services and systems that enable our industrial and institutional (I&I) customers to meet their cleaning and sanitation needs. These products include topical skin cleansers and sanitizers for healthcare and food handling operations. In response to Note #28 on Page 59 FR 31440 asking for data on products presently marketed in the food handling sector, both food processing and food service, we submit the following information.

I. EFFECTIVENESS OF IODOPHORS

The US Department of Agriculture (USDA) regulates sanitizing dips and skin cleaners used in the food processing establishments inspected by USDA. Consequently, the standard method used by Ecolab and other registrants to demonstrate the effectiveness for these products has been the AOAC Available Chlorine Equivalency Test (AOAC Test) that USDA requires. x.9 r >“I The discussion of iodine and iodophors at pp. 31417-31423 of the Tentative:? Final Monograph (TFM) found iodine itself to be generally safe and effective3 but indicated a lack of effectiveness data for various iodophor products. ThF+; SDA/CTFA new data submission now in preparation presents ample support for the idea that iodine itself is the true active agent for the various iodoph3 complexes. A summary of references and data presented to the USEPA as, part of the pesticide reregistration process made this same argument. In ** those terms, there is not really a basic lack of effectiveness data for the (5 iodophors, therefore, even though not all carriers have been used. Consider the carrier to be in effect the dosage form. The agency said at 59 FR 31407 it was not necessary to consider specific dosage forms unless there were particular reasons to do so.

OTC TFM Data Submission 12/05/95 Ecolab Inc.

The carrier or complexing agent does not matter that much in determining the effectiveness of the iodophor product. If the complex delivers titratable active (electropositive) iodine, the product should work. Virtually all of the iodine-based sanitizing dips or hand cleaners with E2 ratings from USDA are iodophors of various sorts. In documentation submitted for their USDA authorization, aJ of them have been shown to be effective by the AOAC Test.

Some of the formulations are detergent/disinfectant/sanitizer products that at 25 ppm of iodine are authorized as sanitizing dips (E3), just as they are food contact surface sanitizers at that concentration, according to 21 CFR 178.1010. However, in the food handler sector iodine is also used in sanitizing hand cleaners (E2). The iodine is complexed by a surfactant that also assists in soil removal from the hands. The level of iodine needed for antimicrobial efficacy in these hand cleaners may be substantially lower than that of the 5- 10% level in povidone products used as skin antiseptics. A hand cleaner formulated with 700 ppm (0.07%) titratable iodine readily passes the AOAC Available Chlorine Equivalency Test.

We provide as Enclosure 1 the AOAC Test data for such a product, as a demonstration of its effectiveness. In a contact plate test this hand cleaner formulation with 0.07% titratable iodine was found to give a 72% reduction in microorganisms, compared to an average of 48% for simple soaps (Miller et al., 1994). This formulation has been authorized since 1982 by USDA for use in inspected plants.

Included as Enclosures 2 and 3 are AOAC Test data for two iodophor products (with different carriers) that are principally disinfectant/sanitizer products, but which currently have USDA authorization as sanitizing hand dips. Enclosure 3, which uses ten times as many pages as earlier studies, reflects the full application of a Good Laboratory Practices testing regime to obtain the same results as in those earlier tests. In summary, iodophor iodine with various carriers provides adequate AOAC Test performance at as low as 6 ppm titratable iodine.

II. ADDITIONAL ACTIVE INGREDIENTS

A. Benzoic/Sorbic Acids as New Ingredients for Categorization

The mention in Note #28 was the first time the agency addressed explicit attention to products in the food handling sector. Consequently, we wish to add the combination of benzoic acid and sorbic acid to the set of active ingredients being considered for categorization. Ecolab has marketed an antimicrobial skin cleaner for food handler use since 1991.

2

OTC TFM Data Submission 12/05/95 Ecolab Inc.

These two ingredients individually have an ample history of safety, efficacy and regulatory acceptance. Both are Generally Recognized As Safe (GRAS) as direct antimicrobial food additives at about the same concentration in the foodstuff as Ecolab uses in the skin cleaner formulation. In Enclosure 4 are references to existing data on the two ingredients. Enclosure 5 provides the AOAC Test data submitted to USDA to obtain authorization for an antimicrobial skin cleaner and sanitizer using these ingredients in combination.

B. Other Ingredients in USDA Authorized Hand Dips

For the record we also present in Enclosure 6 the AOAC Test data submitted to USDA for authorization of a auaternarv ammonium chloride formulation as a sanitizing hand dip. While 200 ppm quat is the concentration specified for use of this formulation as a hard surface sanitizer, the data show that even a 25 ppm solution is more efficacious than 50 ppm chlorine against the test organisms.

Finally, it seems reasonable to remind the agency that all of these AOAC Test data are compared to 50 ppm available chlorine as the standard for an efficacious sanitizing solution. On that basis USDA has issued authorizations for active chlorine solutions as sanitizing hand dips. The clinical studies for this clearance date to the work of Semmelweiss in 1846 and are not repeated here. Nonetheless, the agency should include active chlorine formulations in any categorization of the active ingredients used in food handling operations.

Sincerely,

ECOLAB INC.

(Z-Q tR*L+--- Jo

v R. Keenan, Ph.D.

Scientist

Enclosures

OTC TFM Data Submission December 5, 1995 Ecolab Inc.

FOOD HANDLER TOPICAL ANTIMICROBIALS

LIST OF ENCLOSURES

1. AOAC Available Chlorine Equivalency Test - Data for KX 6019, a sanitizing hand cleaner formulation; containing 700 ppm available iodine in the concentrate, with ammonium alkylphenoxy- poly(ethyleneoxy) ethanol sulfate as the iodine carrier.

2. AOAC Available Chlorine Equivalency Test - Data for MIKROKLENE DF, a hard surface sanitizer and USDA authorized as a hand dip; containing 1.75% available iodine in the marketed concentrate, with butoxypoly(propyleneoxy/ethyleneoxy)ethanol as the iodine carrier.

3. AOAC Available Chlorine Equivalency Test - Data for BAC-FLUSH, a hard surface sanitizer and USDA authorized as a hand dip; containing 3.5% available iodine in the marketed concentrate, with poly(propyleneoxy/ethyleneoxy)ethanol as the iodine carrier.

4. References for sorbic and benzoic acids: a) 21 CFR 182.3089; GRAS listing for sorbic acid b) 21 CFR 184.1021; GRAS listing for benzoic acid c) Excerpts from Disinfection, Sterilization and Preservation by Seymour

Block, Fourth Edn.; with information on benzic acid and sorbic acid,

5. AOAC Available Chlorine Equivalency Test - Data for KLENZ GEL BLU, a sanitizing hand cleaner formulation; containing 0.2% sorbic acid and 0.1% benzoic acid in the marketed product which is used as is.

6. AOAC Available Chlorine Equivalency Test - Data for STER-BAC KQ-12, a hard surface sanitizer and USDA authorized as a hand dip; containing quaternary ammonium chloride.

Q ECONOMICS LABORATORY, I N c.

OSBORN BUILDING. ST. PAUL, MINNESOTA 55102

RESEARCH AND DEVELOPMENT DEPARTMENT TELEPHONE: (612) 451-5600

June 28, 1982

KX 6019 SANITIZING HANDSCRUB

Microbiological Efficacy Data

PRODUCT IDENTIFICATION:

KX6019, sanitizing handscrub, manufactured by the Klenzade Division of'Economics Laboratory, Inc.

TEST PROTOCOL:

Available Chlorine Germicidal Equivalent Concentration Test (A.O.A,C, Methods of Analysis, 13th Edition, 1980), with

modifications (U,S.D.A.),

The recommended use dilutions of 1:2, 1:3 and 1:4 product in water were prepared in hard water to a final concentration of 400 ppm.

TEST ORGANISMS:

Staphylococcus aureus, ATCC 6538 Salmonella typhi, ATCC 6539

NOTE ADDED 11/28/95 - Composition:

1) Available Iodine by titration = 700 ppm in the formulation

2) Iodine carrier - ammonium salt of a sulfated alkylphenoxy-poly(ethyleneoxy)ethanol

TEST RESULTS:

AOAC AVAILABLE

PRODUCT

JCX6019 Batch #l

KX6019 Batch $2

NaOCl

CHLORINE GERMICIDAL EQUIVALENT CONCENTRATION TEST WITH MODIFICATIONS (U.S.D.A.)

TEST ORGANISM DILUTION

S. aureus -

&typhi

S. aureus

S, typhi

S. aureus 50 ppm ztyphi 50 PPm

1. 2

f +

+ +

6

+ +

z

+ +

8

+ +

NEUTRALIZER:

Thioglycollate broth

A neutraliztion experiment was conducted as follows:

One loopful of buffered water followed by one loopful of culture was added to a single tube of neutralizer. In a second tube of neutralizer, one loopful of test product followed by one loopful of culture was added. The tubes containing neutralizer were then plated on Standard Methods Agar.

S aureus A Buffered Water - 4 x lo4 bacteria/ml Handscrub - 2 x lo4 bacteria/ml

S. - typhi

9 10 --

+ +

+ +

Buffered Water - 7 x lo4 bacteria/ml Handscrub - 9 x lo4 bacteria/ml

The tubes were also placed in the 37'C incubator and read in 48 hours. All tubes were positive.

The results neutralizer Kx6019.

of this neutralization experiment-prove that the thioglycollate broth, , was effective in neutralizing

CONCLUSIONS:

Two different batches of RX6019 were evaluated for their sanitizing effectiveness for U.S.D.A. E-2 classificationagainst Staphylococcus aureus and Salmonella typhi. The dilutions were prepared in 400 ppm synthetic hard water to demonstrate the product's ability to retain antibacterial activity in hard water. Testing revealed that RX6019 was equivalent to 50 ppm available chlorine against both test organisms.

Very truly yours,

ECONOMICS LABORATORY, INC,

Linda J.VCohen, Microbiologist Microbiological Research Research & Development Department

LJC:mjg

LAB Ecolab Center St. Paul, Minnesota 55102

Ecolab Inc.

MIKROKLEKE SIF SANITIZING HAND WASH

MICROBIOIOGY EFFICACY DATA

PRODUCT IDENTIFICATION

MIKRONIXNE DF, EPA Reg. No. 1677-58, manufactured by Ecolab Inc.

ACTIVE INGREDIENTS

Iodine * Phosphoric acid

Inert Ingredients

TOTAL

1.75% 6.50%

91.75%

100.00%

* From Butoxypolypropoxy polyethoxy ethanol-iodine complex.

PROCEDURE

Available Chlorine Equivalency Test, Official Final Action, A.O.A.C. Methods of Analysis, 14th Edition, 1984.

PRODUCT CONCENTRATION

Product was diluted one ounce per five gallons in distilled water to yield a final concentration of 25 ppm titratable iodine.

TEST ORGANISNS

Stanhvlococcus aureus ATCC 6538 Salmonella tvnhi ATCC 6539

Page 1 of 2

RESULTS

Incubation: Subculture tubes incubated 48 hours at 37OC.

Staphvlococcus aureus Subculture Series

chlorine 50 - - - + + + + + + + chlorine 100 - - - - - + c + + + chlorine 200 - - - - - - - - - f Mikroklene DF 25 - - - - - - - - - -

Salmonella tvlshi Subculture Series

chlorine 50 - - - + + + + + + + chlorine 100 - - - - - + + + + + chlorine 200 - - - - - - - - + + Mikroklene DF 25 - - - - - - - - - -

CONCLUSION

MIKROKLENE DF, at one ounce per five gallons of distilled water yielding 25 ppm titratable iodine, is an effective hand sanitizer tested as described above.

Sincerely,

Ecolab Inc.

Sally K. Swart Principal Microbiologist Chemical & Biological Research

SKS:ss:22

Page 2 of 2

ECGMAB Ecolab Inc. Microbiological Services

840 Sibley Memorial Highway Mendata Heights, Minnesota 55118

STUDY TITLE 612/451-5600

BAC-FLUSH AVAILABLE CHLORINE SANITIZING EFFICACY

(EPA REG. NO. 1677-89)

DATA REOUIREMENT

GUIDELINE 91-2

STUDY INITIATED ON

AUGUST 15, 1994

STUDY COMPLETED ON

OCTOBER 18, 1994

STUDY DIRECTOR

JOHN D. HILGREN

SPONSOR

DUANE J. REINHARDT

PERFORMING LABORATORY

ECOIAB INC. 840 SIBLEY MEMORIAL HIGHWAY MENDOTA HEIGHTS, MN 55118

STUDY NUMBER

9400036

Page 1 of 23

STATEMENT OF NJ DATA CONFIDENTIALITY CLAIMS

No claim of confidentiality is made for any information contained in this study on the basis of its falling within the scope of FIFRA Sec. 10(d), il), (A), (B) or CC).

Company: ECOLAB INC.

Company Agent: John D. Hilgren Senior Microbiologist Microbiological Services

Page 2 of 23

,_-..

STATEMENT OF GOOD LABORATORY~PRACTICE STANDARDS

This study has been performed meeting the Good Laboratory Practice Standards outlined in 40 CFR, Part 160.

Company: ECOLAB INC.

Study Director: John D. Hilgren Senior Microbioiogist Microbiologicai Services

Study Sponsor: Duane J. Reinhardt Laboratory Technologist Klenzade Division

Study Submitter: Ann M. Oxford Manager Regulatory Services/ North America

Page 3 of 23

A. TEST SUBSTANCE IDENTIFICATION

Bat-Flush, manufactured by the Klenzade Division of Ecolab Inc. EPA Registration Number 1677-89

Active Ingredients: %

Iodine* 3.50

Phosphoric Acid 25.00

Inert Ingredients:** 71.50

TOTAL 100.00%

* From Polyethoxypolypropoxypolyethoxyethanol-iodine complex

Batches Tested 9358-22 9358-23 9358-14(> 60 day aged batch)

Date Made 07/06/94 07/06/94

05/18/94

5. REFERENCE SUBSTANCE IDENTIFICATION

XY-12, manufactured by the Klenzade Division of Ecolab inc., EPA Registration No. 1677-52.

Active Ingredient:

Sodium Hypochlorite Inert Ingredients:

iJ

8.40 91.60

TOTAL 100.00%

Batch TesTed Date Made

JO60941 06/09/94

Page 4 of 23

C. CHEMICAL CHARACTERIZATION OF TEST AND REFERENCE SUBSTANCES

Chemical characterization information can be found in Appendix I of this report.

D. TEST SYSTEM PROPAGATION

Salmonella tvohi ATCC 6539 was the test system used for this study per USEPA Pesticide Assessment Guidelines Subdivision G, Series 91, Subseries 91-A, § 91-2, (k) (i) (i). The Salmonella tvohi ATCC 6539 was received from the American Type Culture Collection (ATCC) on May 12, 1993, purchase order #P125047. Upon receipt of the culture it was placed in a freezer until it was opened for use on 07/12/94. The opened culture was subcultured to AOAC Nutrient agar and incubated for 48 hours at 37°C. From the AOAC Nutrient agar culture, a Nutrient agar slant (lot date 06/08/94)was inoculated on 07/14/94 and incubated for 48 hours at 37°C. After the 48 hour incubation, the Nutrient agar slant containing the culture was refrigerated until it was needed to make a monthly transfer to a fresh Nutrient agar slant. The freshly inoculated slant was then incubated for 48 hours at 37°C and afterwards refrigerated until it was needed for the next month's transfer. Monthly transfer dates, including the first, were 07/14/94, 08/01/94 and 09/08/94.

From the 09/08/94 Nutrient agar slant (lot date 08/30/94, the growth was gram stained, checked visually for purity and identified using a Biolog bacterial identification panel. Gram stain showed gram negative bacilli, the visual purity check was acceptable and the Biolog MICR02.ID user data base identified the bacteria to be ATCC 6539 with the BIO-NUMBER: 3420-2507-7537-0054-1121-7377-0134-3417.

A subculture of the 09/08/94 Nutrient agar slant was made to AOAC Nutrient broth (lot date 08/29/94) on 09/16/94 and incubated for 24 hours at 37°C. After incubation, the 09/16/94 broth was subcultured to a fresh AOAC Nutrient broth (lot date 08/29/94) on 09/17/94 and incubated for 48 hours at 37°C. After incubation, the 09/1?/94 broth was subcultured to a fresh AOAC Nutrient broth (lot date 08/29/94) on 09/19/94 and incubated for 24 hours at 37°C. These were the three consecutive transfers prior to the test broth. The following table summarizes the test broth preparation:

Page 5 of 23

Date Test Broths Inoculated

Number of Test Broths Inoculated 4

Test Broth Medium AOAC Nutrient Broth Lot Date 08/29/94

Date/Time in Incubator 09/20/94, 8:30 a.m.

Incubator Number I

Initial Incubator Temperature

Date/Time Out of Incubator

Final Incubator Temperature

Total Incubation Time

Incubator Temperature Range

Visual Inspection Result

36.7"C

i

Culture Pooling 11 4 Broths Pooled for Test

E. TEST SUBSTANCE DILUENT PREPARATION

Approximately 17.5 liters of Milli-Q water (17 megaohms - cm) in five 2 liter and two 4 liter vessels were autoclaved for 20 minutes at 121°C on 09/20/94 and were inspected visually on 09/21/94 and appeared acceptable. Preparation and standardization of the 500 ppm synthetic hard water (as CaCO,) was done according to Ecolab Microbiological Services SOP #CB008-06; Synthetic Hard Water Preparation and Standardization. The 500 ppm synthetic hard water was prepared by adding 85 ml of solution A (lot date 09/14/94) and 68 ml of solution B (lot date 08/11/94) per 17 liters of water. The following tables summarize the standardization results of the pooled hard water.

SOP CB008-06 METHOD CHECK

Calcium Carbonate 500 ppm Standard Within Control Standard Lot No. Result (ppm CaCO,) Limits

4179-4 502 Yes

Page 6 of 23

* Hardness Determination Formula:

ppm CaCO, = (ml O.OlM EDTA) x I.00

Standardization procedures completed 8:30 a.m., 09/21/94

F. REFERENCE SUBSTANCE DILUENT

Sterile phosphate buffer (pH 8.0) lot date 09/20/94. Prepared by adding 75.0 ml of a 0.91% solution (0.91 g into iO0 ml) of KH,PO, in water to 2925.0 ml cf a 1.16% solution (34.83 g into 3000 ml) of K2HP0,! in water, mixing well and autoclaving for 20 minutes at 121°C.

pH STANDARDIZATION Initial pH pH Adjustment Final pH

8.54 3.0 ml 1N XC1 7.67 d

WATER HARDNESS DETERMINATION

Initial Final Buret Total Amount Hardness* Buret Reading (ml) of O.OlM (PPm

Reading (ml) EDTA (ml) CaCO, 1

0.00 5.12 5.12 512

G. TEST SUBSTANCE CONCENTRATION

Testing of Bat-Flush at 25 ppm available iodine was performed by diluting 1.0 ml of Bat-Flush into 1663.0 ml of diluent. A titration of each solution was done using Ecolab iodine-chlorine test kit #lOl to determine if adjustments were necessary to achieve 25 ppm available iodine. The following table summarizes the Bat-Flush 25 ppm available iodine test solution preparation:

Page 7 of 23

RAC-FLUSH 25 ___ ------ -- nnrn AVAILABLE ___ --.--_---- IODINE DILUTION PROCEDURE

ppm Available

Adjustment Flush added to

dilution = 25 ppm available

Date/Time

Testing of Bat-Flush at 12.5 ppm available iodine was performed by diluting 1.0 m l of Bat-Flush into 3327.0 m l of diluent. A titration of each solution was done using Ecolab iodine-chlorine test kit #lOl to determ ine if adjustments were necessary to achieve 12.5 ppm available iodine. The following table summarizes the Bat-Flush 12.5 ppm available iodine test solution preparation:

N PROCEDURE

Batch No.

ppm Available Iodine

12.5 12.5 12.5

Date/Time Prepared

09/21/94 9:16 a.m .

09/21/94 9:19 a.m .

09/21/94 9:23 a.m .

Page 8 of 23

Testing of Bat-Flush at 6 ppm available iodine.was performed by diluting 500 ml of each 12.5 pm available iodine Bat-Flush solution into 500 ml of diluent. A titration of each solution was done using Ecolab iodine-chlorine test kit #lo1 to determine if adjustments were necessary to achieve 6 ppm available iodine. The following table summarizes the Bat-Flush 6 ppm available iodine test solution preparation:

BAC-FLUSH 6 PPM AVAILABLE IODINE DILUTION PROCEDURE

Milliliters o Available Iodine

ppm Available Iodine

Date/Time Prepared

6* 6* 6*

09/21/94 09/21/94 09/21/94 9:28 a.m. 9:33 a.m. 9:35 a.m.

* Titration using Ecolab Test Kit #lOl required between 2-3 drops of the sodium thiosulfate N/200 reagent which corresponded to an available iodine between 5 and 7.5 ppm.

H. REFERENCE SUBSTANCE CONCENTRATION

Testing of XY-12 at 200 ppm available chlorine was performed by diluting 2.6 ml of XY-12 into 997.4 ml of diluent. Testing at 100 ppm available chlorine was performed by diluting 50.0 ml of the 200 ppm available chiorine solution XY-12 into 50.0 ml of diluent. Testing at 50 ppm available chlorine was performed by dilu ting 25.0 ml of the 200 ppm available chlorine solution into 75.0 ml of diluent. A titration of each solution was done using Ecolab iodine- chlorine test kit #lOl to determine if adjustments were necessary to achieve their respective concentrations. The following tabie summarizes the XY-12 230, 100 and SO ppm available chlorine test solution preparations:

Page 9 of 23

XY-12 SOLUTION PREPARATION (Batch j-060941)

Amount of Reference Substance

Additional Adjustment and ppm solution ppm Available into 96 ml of

Chlorine available

chlorine = 210 5 ml more

diluent added.

I. NEUTRALIZER/SUBCULTURE MEDIUM

Fluid Thioglycollate Medium (Difco laborataries, Detroit, MI) . Prepared according to manufacturer's directions. 61.09 grams of dehydrated medium (lot 448542C) were dissolved in 2050 ml of Milli-Q water. 10 ml quantities were dispensed into 25 x 150 mm test tubes and capped. Tubes were autoclaved for 20 minutes at 121°C. Lot date of prepared media: 09/20/94, expiration date: 09/27/94.

J. MICROBIOLOGICAL SERVICES EOUIPMENT USED

-. AMSCO Eagle 3000 Autoclave 3 L. Castle Autoclave 3. Baiance B3 4. Hard water buret r 3. Incubators 1 and 11 r c. Micropipetter MP6 7. pH meter Pl 8. Thermocouple THl 9. Water bath WB4

Page 10 of 23

n. 'TEST PROCEDURE

Testing followed Ecolab Microbiological Services SOP CB005- 05, Chlorine (Available) in Disinfectants Germicidal Equivalent Concentration. This SOP was created from AOAC Method 955.16, Available Chlorine Germicidal Equivalent Concentration, 15th edition, 1990.

1. Description of the Operating Procedure

2.

A sterile 25 x 100 mm test tube containing 10 ml of a test or reference substance dilution is placed in a 20°C water bath and left to equilibrate for 10 minutes. After equilibration, 0.05 ml of the test system is added, the test tube is mixed and returned to the water bath. After 1 minute, a one loop subtransfer is made aseptically to a tube of fluid thioglycollate medium using an inoculating loop. At 1.5 minutes another 0.05 ml of test system is added to a test tube, mixed and returned to the water bath. After an additional 1 minute (2.5 minutes total) a second one loop subtransfer is made to a second tube of fluid thioglycollate medium. The operation is repeated until a total of 10 additions have been made (a total time of 14.5 minutes).

Operating Procedure Data

Date/time all test and reference substance tubes put into the 20°C water bath: 09/21/94, lo:05 a.m. The following tables summarize the operating procedure data:

Note: times expressed as hh:mm:ss

Page 11 of 23

XY-12 (Batch 5060941) Available 50 mm 100 ppm 200 ppm Chlorine Concentration

Water bath temperature start of test" II 20.4"C I 20.3"C I 20.3"C

Start time of test

End time of test

Water bath temperature end of test

Test temperature range

Bat-Flush (Batch 9358-22) Available Iodine Concentration

Water bath temperature start of test

Start time of test

End time of test

Water bath temperature end of test


6 pm I

12.5 ppm I

25 PP~ I

20.4"C 20.4"C 20.3"C

10:20:00 10:20:15 10:36:00

10:34:30 10:34:45 10:50:30

20.3"C 1 20.3"C 20.3"C

2O.O"C 20,O"C 2O.O"C I!I 0.4"C I? 0.4"C + 0.4"C

Bat-Flush (Batch 9358-23) Available 6 ppm 12.5 ppm 25 PP~ Iodine Concentration

Water bath temperature start of test 20.3"C 20.5"C 20.5"C

Start time of test [I 10:36:15 110:55:00 1 10:55:1511

End time of test 10:50:45 11:09:30 11:09:45 I II

Water bath temperature end of test 20.3"C 20.4"C 20.4"C II


Page 12 of 23

b. Conclusion

The test system, Salmonella tvmhi ATCC 6539, showed resistance to phenol which exceeded the minimum requirements of growth after a 10 minute exposure to 1:lOO phenol specified in the test method (#CBOOl-04) and was therefore deemed acceptable for use.

2. Neutralization method

a. Description of operating procedure and test data

In duplicate, 0, 10, 50, 100, 500 and 1000 ~1 of 200 ppm available chlorine reference substance and 25 ppm available iodine test substances were transferred to a tube of subculture/neutralizer medium, mixed well and left to rest for 10 minutes. After 10 minutes, a 10 microorganisms per ml inoculum of the 24 hour test system was prepared by diluting 1 ml of the broth to 10s6 in phosphate buffered diluting water (lot date 09/14/94) and t'len adding 0.1 ml to the 10 ml subculture/neutralizer tube. Counts were verified by pour plate technique using Tryptic Soy agar (lot date 09/20/94). The 09/21/94 test data is summarized as follows:

10 minute exposure start time: 12:lO p.m. 10 minute exposure end time: 12:20 p.m.

Incubation Data

Incubator Temperature Range 37.9 k 0.6"C

case 15 of 23

temperature range

3. Incubation

The following table summarizes the incubation data for the test tubes:

INCUBATION DATA

Date/Time Test Tubes In Incubator 09/21/94, 1:30 p.m. ii

L. TEST PROCEDURE CONTROLS

i. Phenol resistance

Testing followed Ecolab Microbiological Services SOP #CBOOl-04, Phenoi Coefficient Methods which was created from AOAC Methods 955.11, 955.12 and 955.13, 15th edition 1990.


Test tubes containing 5 ml of each phenol dilution to be tested were placed in a 20°C water bath for 2 5 minutes. 0.5 ml of a 24 hour broth culture to be tested was added to the first dilution of phenol. 30 seconds later, the second dilution was seeded and so on until 4.5 minutes had passed. After adding the culture, the tubes were agitated gently to distribute the cells. Five minutes after seeding the first test tube, one loopful of the phenol/cuiture mixture was transferred to a subculture broth tube. The procedure was repeated until all tubes had been transferred after 5, 10

Paae 13 of 23

and 15 minutes exposure. Testing was performed on 09/15/94 using 4.99% phenol lot date 09/12/94 and Letheen broth (neutralizer/subculture medium) lot date 09/12/94. The following tables summarize the phenol resistance test data:

4.99% Phenol Dilution Final Dilution

1:90

ml of 4.99% Phenol/100 ml Total*

22.2

u 1:lOO 20.0 II * 4.99% phenol diluted in sterile Milli-Q water.

Note: times expressed as hh:mm:ss

Phenol Resistance Test Note: Test Temperature = 20.0 i- O.4"C

Phenol Time Phenol Start Time 5 Minute 10 Minute 15 Minute Dilution Tubes in of Transfer Transfer Transfer

Water Bath Inoculation Time Time Time

1:90 11:05:00 il:ZZ:OO 11:27:00 11:32:00 11:37:00 a.m. a.m. a.m. a.m. a.m.

1:lOO 11:05:00 a.m.

i1:22:30 a.m. I

11:27:30 11:32:30 11:37:30 a.m. I a.m. I a.m.

Incubation Data

Incubator Temperature Range

Dilution

Pane 14 nf 71

Results t+ = growth, o = no growth)

I Test/Reference Replicate of testjreference substance

9358-2.2) 25 ppm available iodine

Inoculum number = 13, 15, 9 microorganisms/ml Average = 12 microorganisms/ml

b. Conclusion

Growth was observed in all tubes containing at least 1000 ,ul of the 200 ppm available chlorine reference substance and 25 ppm available iodine test substance. Therefore, the neutralization method was effective.

3. Positive growth control


A positive growth control was performed by inoculating one tube of subculture/neutralization medium with a loopful of the 24 hour test system.

cubator Numoer

Results: Growth in tube

P7fTP 16 of 37

The positive growth tube was subcultured to Tryptic Soy agar (lot date 09/20/94) and MacConkey agar (lot date 09/20/94) on 09/23/94 to verify growth as the test system by visual examination.

Incubation Data

Date/Time Test Tubes in Incubator 09/23/94, 2:00 p.m.

Incubator Number 11

Initial Incubator Temperature 37.6"C

Date/Time Test Tubes Out of Incubator 09/25/94, 11:OO a.m.

Total Incubation Time 45 Hours

Final Incubator Temperature 37.6"C

Incubator Temperature Range 37.0 k 1.3"C

Results: Pure cultures of a lactose fermentation negative, gram negative bacilli consistant with Salmonella tvphi.

b. Conclusion

Growth of the test system in the subculture/neutralizer broth demonstrated the ability of the broth to support growth of the test system.

4. Negative growth control


Ten subculture/neutralizer tubes were incubated to check for sterility.

Incubation Data

Incubator Temperature Range 37.0 t 0.6"C

Results: No growth in any of the 10 tubes

?aae 17 of 23

b. Conclusion

No growth in the sterile subcuiture/neutralizer broth demonstrated that the broth was free of bacterial contamination.

5. Reference substance diluent sterility control

a. Description of operat ing procedure and test data

One milliliter of the reference substance diluent was added to a petri plate, then by pour plate technique Tryotic Soy agar (lot date 09/20/94) was added. L

Incubation Data

Date/Time Test Tubes in Incubator 09/21/94, 1:30 p.m.

Incubator Number

Initial Incubator Temper

Results: No growth

b . Conclusion

No growth of microorganisms in the reference substance diluent demonstrated that it was free of bacterial contamination.

6. Positive growth tube control


All positive growth tubes were subcultured to Tryptic Soy agar (lot date 09/23/94) and MacConkey agar (lot date 09/20/94) to verify growth as the test system on 09/23/94.

Pacr~ 18 of 23

Incubation Data

t Tubes Out of

Results: Pure cultures of a lactose fermentation negative, gram negative bacilli conslstant with Salmonella tmhi.

b. Conclusion

Growth of a pure culture of the test system in all positive growth tubes verified that the tubes interpreted as positive for growth contained only growth of the test system and not a contaminating organism.

Page 19 of 23

M. TEST RESULTS ,

1. The following table summarizes the test and reference substance results from the 09/21/94 test:

Test Results (+ = growth 0 = no growth)

200 ppm available chlorine

100 ppm available chlorine ICY-l.2

50 ppm available chlorine XY-12

25 ppm available iodine Bat-Flush 9358-22

12.5 ppm available iodine Bat-Flush 9358-22



12.5 ppm available iodine Bat-Flush 9358-14


Page 20 of 23

N 2 CONCLUSION

Bat-Flush dilutions yielding 6, 12.5 and 2.5 ppm available iodine in 500 ppm synthetic hard water (as CaCO,) were equivalent in antibacterial activity against Salmonella tmhi ATCC 6539 at 20.0 k O.S"C to sodium hypochlorite (XY-121, the reference standard, at concentrations of 50, 100 and 200 ppm available chlorine.

TESTED &ND CERTIFIED BY&:

John D'd Hilgren Senior Microbiologist v Microbiological Services

Page 21 of 23

QUALITY ASSURANCE:

This Study was subjected to Ecolab facility Quality Assurance audits as listed below:

Schedule

Protocol

Laboratory Automated System

Experimental In-Life Chemical Characterization

Analytical Chemistry Data

Efficacy Test

Final Report

Dates Reported to Management

Audit Date 08/.16;94

08/19/94

08/22/94,

08/25/94

08/30/94

09/19/94

QUALITY ASSURANCE STATEMENT:

This report has been reviewed by our company 's Quality Assurance Unit in accordance with United States Environmental Protection Agency's Federal insecticide, Fungicide and Rodenticide Act (FIFRA)'s Good Laboratory Practice Standards (40 CFR Part 160). The report accurately reflects the protocol, methods and standard operating procedures for this study. The reported resuits accurately reflect the raw data of the study.

Company: Ecolab, Inc.

Quality Assurance: Date: Sally K. Swart Quality Assurance Coordinator Microbiological Serv'

Daze: Ic>/&/p’j Douglas G. Anderson GLP Testing Facility Ecolab Inc.

Page 22 of 23

RZCQRDS TO BE MAINTAINED:

The final report of this Study, as well as all raw hata accumulated during this Study, will be maintained in the archives of Ecolab Inc. for the registered lifetime of the test substance.

9

Sincerely,

ECOLAB INC.

Joh%k::"i- Study Director Microbiological Services

Date: JD -(U-v/

Page 23 of 23

APPENDIX 1-a

CHEMICAL CHARACTERIZATION

OBJECTIVE

The objective of the chemical characterization'was to verify that the test and reference substances were of good chemical quality basee on analysi? qf the zrf-;~7e ;n7redients TX- other chemical parameters as indicated. .,

METHODS- Test Substance (Bat-Flush)

QATM #38 Determination of Available Iodine, test date 08/25/94.

A & P Method Determination of Phosphoric Acid in #9200402 Products, test date 08/22/94.

RESULTS

METHODS- Reference Substance (Z-12)

QATM #7 Available Chlorine QATM #44 Excess Alkalinity of Hypochlorites

RESULTS

Batch 80. Test Date % Available Cl,

. .

.

CONCLUSION

Bat-Flush batches 9358-22, 9358-23 and 9358-14 were all verified . to be of good chemical quality. XY-12 batch JO60941 was also Tierifi ed to be of good chemical quality.

Tested, & Certified Bv:

/-- &~$/j$anZ&!~~

Analytical & Physical Chemistry ior Laboratory Technician lytical & Physical Chemistry

Deborah L. Duellman Laboratory Technician Analytical & Physical Chemistry

OTC TFM Data Submission December 5, 1995 Ecolab Inc.

ENCLOSURE 4

a> 21 CFR 182.3089 GRAS Listing for Sorbic Acid

W 21 CFR 184.1021 GRAS Listing for Benzoic Acid

4 Excerpts from: Disinfection, Sterilization and Preservation Seymour Block, Fourth Edition

osphate, ,.,. tr~wAYPho&"

lis substance i s safe when good manu-

3 syrup. .rctose COG e saccharide ‘ximately 52 :osel 43 per- !, and 5 per- ccharides. It ous solution valent corn ial enzymat- dextrose) to dble glucose stration de- ; chapter. ions, or ex- ’ J is general- Len used in srbohydrate exceed cur-

ractice.

ate. 1t. ions, or ex- is generally ;ed in dried

with good

Agents

silicate. :alcium sili-

ons, or ex- is generally ;ed in table d manufac-

tte. and 5 per-

ons, or ex- s generally

Faad and Drug Administration, WHS

recognized as safe when used ?t levels not exceeding 2 percent in table salt and 5 percent in baking powder in accordance with good manufacturing practice.

9 182.2437 Magnesium silicate. (a) Product. Magnesium silicate. (b) Tolerance. 2 percent. (cl Limitations, restrictions, or ex-

planation. This substance is generally recognized as safe when used in table salt in accordance with good manufacturing practice.

g 182.2727 Sodium aluminosilicate. (a) Product. Sodium aluminosilicate

(sodium silicoaluminate). (b) Tolerance. This substance is gen-

erally recognized as safe for use at a level not exceeding 2 percent in accordance with good manufacturing practice.

8 182.2729 Sodium calcium aluminosilicate, hydrated.

(a> Product. Hydrated sodium calcium aluminosilicate (sodium calcium silicoaluminate).

(b) Tolerance. This substance is generally recognized as safe for use at a level not exceeding 2 percent in accordance with good manufacturing practice.

0 182.2906 Tricalcium silicate. (al Product. Tricalcium silicate. (b) Tolerance. 2 percent. (cl Limitations, restrictions, or ex-

planation. This substance is generally recognized as safe when used in table salt in accordance with good manufacturing practice.

Subpart D-Chemical Preservatives

0 182.3013 Ascorbic acid. (a> Product. Ascorbic acid. tb) Conditions of use. This substance

is generally recognized as safe when used in accordance with good manufacturing practice.

8 182.3041 Erythorbic acid. (a) Product. Erythorbic acid. (b) Conditions of use. This substance

is generally recognized as safe when

$182.318

used in accordance with good manufacturing practice.

9 182.3089 Sorbic acid. (a) Product. Sorbic acid. (b) Conditions of use. This substance


g 182.3109 Thiodipropionic acid. (a) Product. Thiodipropionic acid. (b) Tolerance. This substance is gen-

erally recognized as safe for use in food when the total content of anti- oxidants is not over 0.02 percent of fat or oil content, including essential (volatile) oil content of the food, provided the substance is used in accordance with good manufacturing practice.

§ 182.3 149 Ascorbyl palmitate. (a) Product. Ascorbyl palmitate. (b) Conditions of use. This substance


§ 182.3169 Butylated hydroxyanisole. (a) Product. Butylated hydroxyani-

sole. (b) Tolerance. This substance is gen-

erally recognized as safe for use in food when the total content of anti- oxidants is not over 0.02 percent of fat or oil content, including essential (volatile) oil content’ of food, provided the substance is used in accordance with good manufacturing practice.

0 182.3173 Rutylated hydroxytoluene. (a) Product. Butylated hydroxyLo-

luene. (b) Tolerance. This substance is gen-

erally recognized as safe for use in food when the total content of anti- oxidants is not over 0.02 percent of fat or oil content, including essential tvolatile) oil content of food, provided the substance is used in accordance with good manufacturing practice.

D 182.3189 Calcium ascorbate. (a) Product. Calcium ascorbate. (b) Conditions of use. This substance

is generally recognized as safe when

Food ond Drug Ad tration, HHS

si 184.1011 Alginic acid. (a) Alginic acid is a colloidal, hydro-

philic polysaccharide obtained from certain brown algae by alkaline extrac- tion.

(b) The ingredient meets the specifi- cations of the Food Chemicals Codex, 3d Ed. (19811, p. 13, which is incorporated by reference. Copies are available from the National Academy Press, 2101 Constitution Ave. NW., Washington, DC 204I8, or available for inspection at the Office of the Fed- eral Register, 800 North Capitol Street, NW., suite 700, Washington, DC.

(c) In accordance with fi 184.1(b)(Z), the ingredient is used in food only within the following specific Iimita- tions:

Cat22 O’

Soup and souP mixes, 5 170.3(n) (40) of this chapter.

Maximum level of use in food (as

served)

Not to exceed current good manufacturing pracbce.

I

Functional use

Emulsifier, emulsifier salt. 5 170.3(o)(8) of thii chapter; formulation aid. !J 170.3(0)( 14) of this chapter. stabiltzer, thickener, 9 170.3(0)(28) of this chapter.

cd) Prior sanctions for this ingredient different from the use established in this section do not exist or have been waived. 147 FR 47375. Oct. 26.19823

9 184.f021 Benzoic acid. (a) Benzoic acid is the chemical ben-

zenecarboxylic acid (CrHs02), oceur- ring in nature in free and combined forms. Among the foods in which benzoic acid occurs naturally are cranberries, prunes, plums, cinnamon, ripe cloves, and most berries. Benzoic acid is manufactured by treating molten phthalic anhydride with steam in the presence of a zinc oxide catalyst, by the hydrolysis of benzotrichloride, or by the oxidation of toluene with nitric acid or sodium bichromate or with air in the presence of a transition metal salt catalyst.

(b) The ingredient meets the specifi- cations of the “Food Chemicals

.fp42lorcAre $184.1025

Codex,” 3d Ed. (19811, p. 35, which is incorporated by reference. Copies may be obtained from the National Acade- my Press, 2101 Constitution Ave. NW., Washington, DC 20418, or may be ex- amined at the Office of the Federal Register, 800 North Capitol Street, NW., suite 700, Washington, DC.

tc) The ingredient is used as an antimicrobial agent as defined in 5 130.3(o)(Z) of this chapter, and as a flavoring agent and adjuvant as defined in 0 170.3(0X12) of this chapter.

(d) The ingredient is used in food at levels not to exceed good manufacturing practice. Current usage results in a maximum level of 0.1 percent in food. (The Food and Drug Administration has not determined whether signifi- cantly different conditions of use would be GRAS).

(e) Prior sanctions for this ingredient different from those uses established in this section, or different from that set forth in part 181 of this chapter, do not exist or have been waived. E42 FR 14653, Mar. 15. 1977, as amended at 49 FR 5610, Feb. 14, 19843

9 184.1025 Caprylic acid. (al Caprylic acid CCH,(CHZ)BCOOH,

CAS Reg. No. 124-07-21 is the chemical name for octanoic acid. It is considered to be a short or medium chain fatty acid. It occurs normally in various foods and is commercially prepared by oxidation of n-octanol or by fermentation and fractional distilla- tion of the volatile fatty acids present in coconut oil.

(b) The ingredient meets the specifi- cations of the “Food Chemicals Codex,” 3d Ed. (19811, p. 207, which is incorporated by reference. Copies may be obtained from the National Acade- my Press, 2101 Constitution Ave. NW., Washington, DC 20418, or may be ex- amined at the Office of the Federal Register, 800 North Capitol Street, NW., suite 700, Washington, DC.

(c) The ingredient is used as a flavoring agent and adjuvant as defined in p ‘170,3(0X12) of this chapter.

(d) The ingredient is used in foods in accordance with $ 184.1(b)(I), at levels not to exceed good manufacturing practice, Current good manufacturing practices result in maximum levels, as

431

3- hC Id*&xv-4 4-c

Disinfection, S tedixati and Preservation

Fourth Edition

SEYMOUR S. BLOCK, Ph.D. Professor Emeritus of Bioengineering Department of Chemical Engineering Unit;ersity of Florida Gainesoille, Florida

Lea & Febiger Philadelphia l London

primarily of the genera Lactococcus, LactobaciElus, Leu- conostoc, and ~ediococcus. Other bacteria and some mold species also produce lactic acid. Lactic acid is the primary acid produced during microbial fermentation of sugar to produce cheeses, sauerkraut, pickles, sausage, and olives. The acid produced in such fermentations low- ers the pH to levels unfavorable for the growth ofspoilage organisms and inhibitory to Clostridium botulinum growth and toxin production. In the U.S., lactic acid production via fermentation of added sugar is allowed in bacon processing for faster nitrite depletion and botulinal protection due to lowered pH (USDA, 1979). Derivatives of lactic acid may be used as direct food acidulants. Glu- cone-delta-lactone, a permitted acidulant in certain meat products, hydrolyses to release lactic acid and functions identically to added lactic acid. Certain carbonated beverages are preserved by a combination of lactic acid and CO,. Besides being an acidulant and exhibiting preservative action due to decreased pH, it may be used as a flavoring agent in frozen desserts (Gardner, 1972).

Benzoic Acid Benzoic acid (‘benzenecarboxylic acid, phenylformic

acid), C,H,COOH, is a granular or crystalline powder with a sweet or astringent taste. It is usually used in the form of sodium benzoate, because the sodium salt is considerably more water-soluble than the acid. Sodium benzoate, a white powder or flake, dissolves in water at a level of 50 g/100 ml at 25’C, in contrast to the acid form with water solubility of only 0.35 g000 ml. Sodium benzoate is soluble in alcohol at a level of 1.3 g/100 ml. The pK. of benzoic acid and its sodium salt is 4.2, and the molecular weights are 122.2 and 144.11, respectively. Sodium benzoate and benzoic acid are GR4S substances in the U.S. Sodium benzoatc is inespensive and one of the most extensively used food preservatives in the U.S. and other countries. Sodium benzoate is more toxic to rats than sodium sorbate is. Humans have high tolerance to sodium benzoate because of a detoxifjring mechanism whereby benzoate and glycine or glycuronic acid are conjugated and excreted as hippuric acid or benzoyl glu- curonide (Chichester and Tanner, 1972). An intermediate step in the detosification is formation of benzoyl CoA at the expense of ATP. Benzoate is not mutagenic in Drosophila or Salmonella but reportedly interacts with nucleosides and DNA in vitro (Njagi and Gopalan, 1980).

Benzoate inhibits yeast more than it inhibits molds or bacteria. However, osmotolerant yeast resistant to benzoic acid often limit shelf-life of intermediate moisture foods preserved with this acid uermini and Schmidt- Lorenz, 1987). Food-poisoning and spore-forming bacteria are inhibited by undissociated benzoic acid concentrations of 0.01 to 0.02%, whereas many spoilage organisms show higher resistances. Listeria monocytogenes also is susceptible to the action of benzoic acid (El- Shenawy and Marth, 1988b). Rahn and Conn (1944) reported that benzoic, salicylic, and sulfurous acids were approximately 100 times more effective antimicrobial agents in acid than in neutral solution, that the antimi-

CHEMICAL FOOD PRESERVATIVES . 809

crobial activity was due to the undissociated acid, and that yeast multiplication was inhibited by 25 mg/ml undissociated benzoic acid. The undissociated, molecular form is able to pass through the cell membrane more readily than the charged form. Reviews by Bosund (1962) and Chipley (1983) present evidence showing that benzoic acid interferes with many enzymatic processes in microorganisms at concentrations that retard the growth rate. Altered membrane permeability may be another antimicrobial effect of benzoic acid. Eklund (1980), however, showed that inhibition of nutrient uptake could account for only part of the antimicrobial action of benzoate. Zygosaccharomyces baitii is resistant to benzoic and sorbic acids. Resistance of this yeast apparently is due to an inducible, energy-requiring system that trans- ports preservative from the cell (Warth, 1977, 1988).

Benzoate is most effective from pH 2.5 to 4.0 (Cruess and Richert, 1929). At pH values above about 4.5, the antimicrobial activity of benzoate is low, and the addition of an acidulant or another preservative should be considered. Sodium benzoate, the form most widely used, is a common preservative for acid foods, including carbonated and still beverages, fruit juices and salads, syrups, icing, jams, jellies, preserves, margarine, mince- meat, pickles, relishes, pie fillings, and prepared salads. Sodium benzoate is used at concentrations of 0.03 to 0.10% in the U.S. Chipley (1983) tabulated benzoic acid concentrations permitted in foods produced in other countries. It is a natural component of many foods, including cranberries, prunes, plums, cinnamon, ripe cloves, and most berries. Because of the astringent flavor of benzoates, a lower level of benzoate, in combination with a second preservative such as sorbate or a paraben, may be desirable in some food products. Jermini and Schmidt-Lorenz (1987) have proposed concentrations of benzoate plus paraben to preserve intermediate moisture foods at pH values ranging from 3.0 to 4.8. Sulfurous acid may be used in combination with benzoate to avoid discoloration and oxidative changes due to benzoate. Benzoate may be added directly to foods by blending with the dry ingredients of the product, or applied by dipping foods or coating packaging films, especially for fish (Dunn, 1947).

Esters of Parahydroxybenzoic Acid Alkyl (methyl, ethyl, propyl, butyl) esters of p-hy-

droxybenzoic acid (parabens, parasepts, or PHB esters) find use as food, cosmetic, and eharmaceutical preservatives. These esters have properties similar to benzoic acid, yet the modification enhances their utility. The methyl ester is the most water soluble: 0.25 g dissolves in 100 g of water at 25°C. Water solubility decreases as the number of carbon atoms increases, whereas the re- verse is true for ethanol, propylene glycol, and oil solubility. Some salt forms are available for food use to provide higher water solubility. The parabens commonly used in food are white, free-flowing powders with a faint odor or no odor. The methyl and propyl parabens are GRAS in the U.S., with a total addition limit of 0.1%.

Sorbic Acid

Sorbic acid (CH,-CH = CH-CH = CH-COOH), 2,4- hexadienoic acid, is a truns-truns unsaturated fatty acid with a molecular weight of 112.13. It was first manufactured by A. W. Hofmann in 1859 from rowan berry oil. The antimicrobial action of sorbic acid was discovered independently by E. Muller in 1939 and by C. M. Good- ing in 1940 (Lueck, 1980).

Sorbic acid has a pK, of 4.76. The maximum pH for antimicrobial activity of sorbate is about 6.0 to 6.5, whereas for propionate and benzoate, it is 5.0 and 4.5, respectively. Sorbic acid is a white crystalline powder which is only slightly soluble in water (0.16 g dissolves in 100 ml water at 2O’C) with a distinctive odor and sour taste. The acid is more soluble in lipid materials than in water. The sodium, potassium, and calcium salts of sorbic acid are frequently used because of their high water solubilities. For example, potassium sorbate, the most frequently used salt form, is a white, fluffy powder that is soluble at a level of 139 g/l00 ml water at 20°C. When added to acidic foods, the salts equilibrate to the acid form. The esters of sorbic acid with low-molecular- weight, aliphatic alcohols are of no importance in food processing owing to their strong odor.

Sorbic acid is considered nontoxic and is metabolized by beta- and omega-oxidation, as are long-chain fatty acids (Deuel et al., 1954a, b). Thus, feeding studies have reported minor weight gains in animals fed diets containing sorbic acid compared to controls. The LD, for sorbic acid is in the range of 7 to 11 g/kg of body weight and about 6 to 7 g/kg of body weight for the sodium salt (Lueck, 1980). Sorbic acid irritates the mucous membranes in highly sensitive individuals. Potassium and calcium sorbates have no mutagenic action, nor is the potassium salt teratogenic. Sorbic acid and potassium sorbate administered in feed have no carcinogenic action. The FAO acceptable daily intake of sorbic acid and its salts is 25 m&g of body weight, which is the highest acceptable daily intake of the common food preservatives. In foods, sorbic acid and its salts are relatively tasteless and odorless, and they are inexpensive, They were first recommended as food preservatives in 1945 by Gooding and are permitted in nearly all countries of the world for preservation of a wide variety of foods. In the U.S., sorbic acid and sorbates are considered GRAS. The maximum permissible level of sorbic acid is usually between 0.1 to 0.2%.

Components of the antimicrobial action of sorbic acid appear to be due to inhibition of enzymes and nutrient transport. Eklund (1980) reported evidence that inhibition of nutrient transport was responsible for part, but not all, of the antibacterial action of sorbate. Many enzymes reportedly are strongly or weakly inhibited by sorbate. These include enolase (Azukas, 1962), lactate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase (Lueck, 1980), f umarase, and aspartase (York and Vaughn, 1964). Additionally, sorbic acid bonds cova-

CHEMXAL FOOD PRESERVATIVES l 811

lently with sulfhydryl groups, thereby inactivating sulfhydryl enzymes, and it may interact with catalase and peroxidase (Lueck, 1980; Martoadiprawito and Whi- taker, 1963).

Sorbic acid and its salts inhibit yeasts, molds, and some bacteria, primarily c&&se-positive bacteria. Certain molds are more resistant or can metabolize sorbic acid (Melnick et al., 1954a, b; Troller, 1965). Sorbic acid or the potassium salt has bken found to be effective against Staphylococcus aweus in bacon, Vibrio parahemolyticus in fish, yeasts, and molds in ham, Salmonella species in poultry, Listeria monocytogenes in laboratory media, Ba- cillus species in rice pastry filling, Moraxella and Ar- throbactet spp. from seafood, Pseudomonas species (al- though resistant species from seafood have been identified by Chung and Lee, 1982), yeast in Mexican hot sauce, Aspergillus parasiticus and Penicillium com- mune on laboratory media, and molds and &form bacteria in butter (Kaul et d., 1979; Raevuori, 1976; Robach, 1978,197Qa; Robach and Hickey, 1978; Robach and Ivey, 1978; Bullerman, 1979; Kemp et al., 1979; Pierson et al., 1979a; Flores et al., 1988; El-Shenawy and Marth, 1988a). Additionally, sorbate is reported to extend the shelf life of poultry parts and fish sausage (Amano et al., 1968; Wada et al., 1975; Cunningham, 1979; Chung and Lee, 1982).

Early reports indicated that sorbic acid was ineffective against clostridia and could be used to select for such organisms (Emard and Vaughn, 1952; York and Vaughn, 1954, 1955; Hansen and Appleman, 1955). In 1974, Tompkin and coworkers published evidence that potassium sorbate delayed toxin production by Clostridium botulinurn in an uncured sausage product. This report stimulated additional research on sorbate as an antibotulinal agent owing to concern over the carcinogenic properties of products generated by the heating of nitrite and related compounds and the resultant interest in reducing or eliminating nitrite in cured meat products. Several studies on sorbate as an antibotulinal agent in meat products, either alone or in combination with low levels of nitrite (40 to 80 pg/g), h ave demonstrated its effectiveness (Ivey and Robach, 1978; Ivey et al., 1978; Sofos et al., 1979a-d, 198Oa, b; Pierson et al., 1979b, c; Price and Stevenson, 1979) and suggest inhibition of spore germination (Sofos et al., 1979; Sofos and Busta, 1980, 1981), perhaps due to a competitive mechanism (Smoot and Pierson, 1981). Sorbate dissipated the proton motive force of the membrane and inhibited phenylalanine uptake, decreased.the rate of protein synthesis, and altered patterns of phosphorylated nucleotide accumulation, re- sulting in increased cellular concentrations of GTP and ppGpp in Putrefactive Anaerobe 3679 (Ronning and Frank, 1987). The phosphorylated nucleotide concentrations were partially reestablished in the presence of noninhibitory amounts of tetracycline. Thus, Ronning and Frank (1987) concluded that inhibition of PA 3679 by sorbate was due to a stringent-type response induced by the protonophoric activity of sorbate.

812 8 A.~TIMICROBlALS IN FOOD SANITATlON AND PBESERVATlON

A review on botulinal control in cured meats by nitrite and sorbate has been published by Sofos et al. (1979~) and by Sofos and Busta (1983). Meat casings for dry sausages may be dipped in sorbate to prevent growth of molds. Some reports have shown synergistic effects of sorbate with salt and sugar (Kaul et al., 1979; Gooding et al., 1955; Robach, 1979a; Robach and Stateler, 1980). Response by Putrefactive Anaerobe 3679 to combination of sorbate and curing ingredients were used to express caution in modification of curing processes (Ronning and Frank, 1988). Antimicrobial effects of sorbate derivatives such as sorbohydroxamic acid, sorbic aldehyde, and oth- ers have been reported (Dudman, 1963; Troller and Olsen, 1967).

Sorbates are used to inhibit yeasts in applications such as cucumber fermentation, in which growth of the catalase-negative lactic acid bacteria is desirable (Phillips and Mundt, 1950; Jones and Harper, 1952; Costilow et al., 1956, 1957). Sorbates are used for mold and yeast inhibition and general shelf-life extension in cheese products, baked goods, fruits, vegetables, wines, soft drinks, fruit juices, pickles, jams, jellies, syrups, sauerkraut, salads, margarine, meat, and fish products. Reports dealing with such applications have been published by Jones and Harper (1952), Sfelnick and Luckmann (1954), Smith and Rollin (1954), Boyd and Tarr (1955), Salunkhe (1955), Bonner and Harmon (1957), Costilow et al. (1957), Fer- guson and Powrie (1957), Weaver at al. (1957), Auerbach (1959), Geminder (1959), Nury et al. (1960), Perry and Lawrence (lQ60), Pederson et al. (1961), Kaloyereas et al. (lQ61), Seiler (1964), Perry et al. (1964), Harris and Rosenfield (1965), and numerous other investigators. Be- cause sorbate inhibits yeasts, it is not used in yeast-raised bread. Sorbate may be applied by dipping, spraying, dusting, or impregnating wrappers and packaging materials, or it may be added directly to the food.

Formic Acid Formic acid (HCOOH) has a molecular weight of 46.03

and a pK, of 3.75. It is a colorless, transparent liquid with a pungent odor and is m iscible with water. The LD, of formic acid is 1 to 2 g/kg of body weight (Lueck, 1980). In high concentrations it may irritate the skin and mucous membranes. The sodium and potassium salts are less acutely toxic. The daily intake of formic acid in the usual applied concentrations reportedly causes no damage (von Oettingen, 1959). Neither formic acid nor formates are carcinogenic or teratogenic, but formic acid is mutagenic to Drosophila (Tracer- Jitco, Inc., 1974). Formic acid is readily absorbed by the gut or through skin or mucous membranes. It is a normal constituent of human tissue and blood and is metabolically important in transfer of one-carbon substances. The FAO-suggested acceptable daily intake of formic acid is 3 mg/kg of body weight. Formic acid is permitted for food preservation in some countries, but not in the U.S. or the United Kingdom.

Some of the antimicrobial action of formic acid is as an acidulant. Additionally, formic acid inhibits decar- boxylases and heme enzymes, especially catalase (Lueck,

1980), even at pH values at which it is essentially totally dissociated. Formic acid is most effective at pH values below about 3.5. Formic acid is active primarily against yeasts and some bacteria. Lactic acid bacteria and molds are relatively resistant. Organisms that produce formic acid as a byproduct of carbohydrate metabolism through the m ixed acid pathways are more resistant.

Where permitted, formic acid or its salts are used primarily in fish preserves, pickles, and fruit juices. For- mates are frequently used in conjunction with a second preservative, such as benzoic or sorbic acids.

Phosphoric Acid and Hydrochloric Acid Phosphoric acid (H,PO,) and hydrochloric acid (HCl)

are strong acids that rely on low pH, and hence high external hydrogen ion concentration, for antimicrobial action. Phosphoric acid is the acidulant in carbonated beverages. The necessary low pH required for effective antimicrobial activity is usually undesirable in food. These are the only two inorganic acids that are GRAS for use as food acidulants.

Malic Acid Malic acid (COOHCH,CHOHCOOH), hydroxysuc-

cinic acid, is a natural component of and the predominant acid in apples, cherries, apricots, grapes, peaches, or- anges, bananas, broccoli, carrots, peas, potatoes, and rhubarb. It also occurs in citrus fruits, figs, tomatoes, and beans (Gardner, 1972). Its pK, values are 3.4 and 5.1. It is GRAS in the U.S. and is primarily used as an acidifying ingredient and preservative in mayonnaise and other salad dressings, sherbets, fruit preserves, jams, jellies, and beverages. Malic acid is a m icrobiostatic agent against certain bacteria and yeasts (Banwart, 1979).

Tartaric Acid Tartaric acid [COOH(CHOH),COOH] is one of the

primary acids in grapes and is produced from the waste products of the wine industry. Monopotassium tartrate (cream of tartar) is commonly used in baking. Tartaric acid is GRAS in the U.S. It is used in fruits, jams, jellies, preserves, sherbets, and beverages.

Adipic Acid Adipic acid [COOH(CH,),COOH] is a poorly soluble,

nonhygroscopic acid. This feature makes it advantageous for use as an acidulant In &y, powdered food products.

Succinic Acid Succinic acid (COOHCH,CH,COOH) and its anhy-

dride,are used as acidulants primarily in bakery products. Its pK, values are 4.2 and 5.6. Succinic acid effectively inhibited or reduced the m icrobial load on poultry car- casses (Mountney and O’Malley, 1965; Cox et al., 1974). It is GRAS in the U.S.

Caprylic Acid Caprylic acid [CH,(CH&COOH], octanoic acid, is an

eight-carbon fatty acid with a pK, of4.9. It primarily is

E B Ecolab Center St. Paul, Minnesota 55102

Ecolab Inc. Research & Engineering

December 10, 1990 <.

KLFJCZ GEL BLU CD-8058-40

E-2 HAND SANITTZER MICROBIOLOGY EFFICACY DATA

PRODUCT IDENTIFICATION

CD-8058-40, manufactured by Ecolab Inc.

ACTIVE INGRFsDIENTS

Sorbic Acid Benzoic Acid

Inert Ingredients

TOTAL

0.20% 0.10%

99.70%

100.00%

Available Chlorine Equivalency Test, Official Final Action, A.O.A.C. Methods of Analysis, 14th Edition, 1984.

PRODUCT CONCENTRATION

Product was diluted one part product to one part distilled water.

TEST ORGANISMS

Staohvlococcus aureus ATCC 6538 Salmonella tvphi ATCC 6539

Page 1 of 3

RESULTS

Incubation: Subculture tubes incubated 48 hours at 37"~. Test date: December 5, 1990 Results Read: December 7, 1990

Staphylococcus aureus Subculture Series

SamHe

chlorine chlorine chlorine

CD-8058-40

Sanmle

chlorine chlorine chlorine

CD-8058-40

50 - 100 - 200 - 1:l -

50 - 100 - 200 - 1:l -

2 2

+ + + +

Salmonella tvphi Subculture Series

z 8 2 10 -t + + + -i- + + +

c +

+ + + f + + + + + + + +

+ +

Page 2 of 3

CONCLUSION

CD-8058-40, diluted 1:l in distilled water, is an effec-cive hand sanitizer tested as described above.

Sincerely,

Ecolab Inc.

Leanne J.-Adkins Laboratory Technologist Chemical & Biological Research

LJA:ss:40

Page-3 of 3

ECONOMICS LASORATORY, INC.

OSBORN BUILDING. ST. PAUL. MlNNESOTA 55102

RESEAfiCH AND DEVELOPMENT OEPARYMENT TELEPHONE: @X3 45WWJO

August l$; 1985

STER-BAC KQ-12

Product Identification

Ster-Bat K&-12, manufactured by the Klenzade Division of Economics Laboratory, Inc.

Active Ingredients

-n-alkyl (56% Cl4, 40% Cl2, 10% C chloride (50% actxve) provided by

6) h

dimethyl benzyX ammonium -312 (EPA #1677-43AA) manu-

factured by Economics Laboratory, Inc.

DHte '17ested:

Augist 6, 1985

Concentration:

25 ppm, 50 ppm, 100 ppm, 150 ppm, and 200 ppm active quaternary in 400 ppm hard water.

Method: --^-P

A.O.A.C. Available Chlorine Germicidal. Equivalent Concentration A.O.A.C. Nethods of Analysis, 14th Edition, 1984.

Test Organisms:

Stsphyiococcus aureus, ATCC #6538 Phenol Resistance: 1:60

Salmonella typhi, ATCC 56539 Phenol Resistance: 1:90

Results : Staphylococcus aureus

Germicide

NaOCl

Ster-Bat

ppm

Avail. Cl

50 Active Quat

25

50

100

150

200

ATCC 6538

Subculture Series

.-

- - - I- +

- - - - -

- - - - -

- - - w -

- - - - -

- - . - I

lo

+

+

Conclusions : Chlorine Equivalency - Staphylococcus aureus - 400 -ppm hard water

Ster-Bat at 25 ppm active quat is equivalent to somewhat greater than 50 ppm available chlorine. Therefore, Ster-Bat at 25 ppm against S. aureus in the presence of 400 ppm hardness is more effectiveifian 50 ppm available chlorine.

Results : Salmonella typhi ATCC 6539

NaOCl

Ster-Bat

Germicide ppm 1

Avail. Cl

50

Active Quat

25 +

50 +

100

150

200

2.

+

+

+

+

-5

Conclusions : Chlorine Equivalency - Salmonella typhi - 400 -ppm hard water.

Ster-Bat at 150 ppm active quat is equivalent to 50 ppm available chlorine.

Neutralization Verification ’

Tubes snowing negative growth at 50 ppm active quat against S. aureus and 200 ppm active quat against S. typhi werY e subcultured into new letheencbroth tubes and incubated 48 hours x 37OC. All tubes were found to be negative for growth.

Tested and Certified By:

L/zkL-d* As!

Thomas G. Boufford Principal Microbiologist

Sincerely,

ECONOMICS LABORATORY, INC.

Dale L. Fredell. Supervisor Microbiology Group Chemical & Biological Research

dr. william gilbertson food & drug administration, room 1-23

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