14086 Federal Register / Vol. 64, No. 55 / Tuesday, March 23, 1999 / Notices

DEPARTMENT OF HEALTH AND through programs of information, constituents in a smokeless tobacco HUMAN SERVICES education, and research. product that are volatile at temperatures HHS uses the information collected to of 99 degrees centigrade. pH is defined Centers for Disease Control and exercise its authority under CSTHEA to as the negative logarithm of the molar Prevention conduct research on the addictive concentration of hydrogen ions in an nature of nicotine and general health aqueous solution and is a quantitative Notice Regarding Requirement for effects of using smokeless tobacco. measure of acidity or alkalinity. The Annual Submission of the Quantity of Nicotine data will provide a more degree of nicotine ionization is Nicotine Contained in Smokeless complete picture of the addictive nature calculated from the Henderson- Tobacco Products Manufactured, of smokeless tobacco products. Also, as Hasselbalch equation. Un-ionized Imported, or Packaged in the United authorized in the statute, HHS may nicotine is known to be the form of States report to the Congress information nicotine absorbed most easily in the regarding its current and proposed mouth. This protocol will provide CDC AGENCY: Centers for Disease Control and research relative to nicotine levels in with information on levels of nicotine Prevention (CDC), Department of Health smokeless tobacco products. CSTHEA found in smokeless tobacco products and Human Services. further requires that individuals who manufactured, packaged, or imported ACTION: Notice. manufacture, package, or import during the previous calendar year. The smokeless tobacco products report to schedule for reporting this information SUMMARY: This notice establishes a HHS the list of ingredients added to to CDC corresponds to the reporting of uniform protocol for the analysis of tobacco in the manufacture of such ingredients added to tobacco in the nicotine, total moisture, and pH in products, and this requirement has been manufacture of smokeless tobacco smokeless tobacco products. This implemented by a previous notice (59 products November 8, 1994 (59 FR protocol was designed to implement the FR 4714,). 55670,). requirement of the Comprehensive In 1989 the smokeless tobacco Following public request, on June 2, Smokeless Tobacco Health Education industry submitted a business review 1997, a notice (62 FR 29729) was Act (CSTHEA) of 1986 (15 U.S.C. 4401 letter to the Department of Justice (DOJ), published extending the comment et seq., Pub. L. 99–252), which requires in accordance with 28 CFR 50.6. This period on the proposed protocol by an that each entity manufacturing, letter requested approval of a additional 30 days to July 2, 1997. A packaging, or importing smokeless collaborative industry effort to develop summary of the comments received and tobacco products shall annually provide a uniform analytical protocol for CDC’s response follows. the Secretary of Health and Human determining the nicotine and moisture One respondent, on behalf of several Services (HHS) with a specification of content of smokeless tobacco products. smokeless tobacco manufacturers, had the quantity of nicotine contained in In January 1993, DOJ extended several comments regarding the each smokeless tobacco product. permission to the smokeless tobacco collection of data. The respondent DATES: The first report of information is industry to develop a uniform analytical asserted that the protocols exceeded due June 30, 1999, with subsequent protocol for this purpose. A work group statutory authorization by collecting pH submissions due by March 31 of each representing the 10 major domestic and free base (hereinafter referred to as year. manufacturers of smokeless tobacco was ‘‘un-ionized’’, as reflected in the revised ADDRESSES: The information shall be convened on July 7, 1993. The protocol) nicotine. Furthermore, the submitted to: Michael P. Eriksen, Sc.D., workgroup developed and submitted to respondent felt that the legislative Director, Office on Smoking and Health, CDC for approval the ‘‘Protocol for history of CSTHEA contemplates the National Center for Chronic Disease Analysis of Nicotine in Smokeless reporting of nicotine content alone. Prevention and Health Promotion, Tobacco Products.’’ The protocol was It is CDC’s belief that the collection of Centers for Disease Control and revised by CDC based on individual pH of smokeless tobacco products and Prevention (CDC), 4770 Buford comments from peer reviewers and the the un-ionized nicotine content of each Highway, NE., Atlanta, GA 30341–3724. National Center for Environmental is authorized by section 4 of CSTHEA. Health, CDC. The revised protocol, There is ample scientific evidence that FOR FURTHER INFORMATION CONTACT: ‘‘Protocol for Analysis of Nicotine, Total mere quantity of nicotine alone is Michael P. Eriksen, Sc.D., Director, Moisture, and pH in Smokeless Tobacco insufficient in determining its effect on Office on Smoking and Health, Products,’’ is hereafter referred to as the a user; knowledge of pH and un-ionized telephone: (770) 488–5701. ‘‘protocol.’’ nicotine content of the overall nicotine SUPPLEMENTARY INFORMATION: According On May 2, 1997, a notice (62 FR quantity is essential in determining the to a 1986 report of an Advisory 24115) was published in accordance rate of nicotine absorption. pH and un- Committee to the Surgeon General, with the Paperwork Reduction Act to ionized nicotine content are essential smokeless tobacco represents a solicit public comment on the proposed factors affecting nicotine bioavailability. significant health risk, is not a safe collection of data. A notice was also Furthermore, Congress has never substitute for cigarette smoking, can published (62 FR 24116, May 2, 1997) defined exactly what it meant by cause cancer and a number of to solicit public comment on the ‘‘quantity of nicotine’’ in section 4(b) of noncancerous oral conditions, and can protocol. The protocol consists of CSTHEA. In light of ample scientific lead to nicotine addiction. standard laboratory methods to measure evidence indicating the importance of The Centers for Disease Control and nicotine, moisture, and pH in smokeless pH and un-ionized nicotine content in Prevention’s (CDC) Office on Smoking tobacco products, and an equation assessing the overall quantity of and Health (OSH) has primary (Henderson-Hasselbalch) to calculate nicotine, CDC’s interpretation of its responsibility for the Department of un-ionized nicotine. Nicotine is the statutory authority is clearly Health and Human Services’ (HHS) major alkaloid in tobacco and the drug permissible. tobacco and health program. The overall in tobacco that causes addiction. In the It is CDC’s belief that the legislative goal of this program is to reduce death protocol, moisture is referred to as total history provides support for requiring and disability resulting from cigarette moisture because the method measures the reporting of moisture, pH, and un- smoking and other forms of tobacco use the amount of water and tobacco ionized nicotine content. The Senate

VerDate 17-MAR-99 15:59 Mar 22, 1999 Jkt 183247 PO 00000 Frm 00002 Fmt 4701 Sfmt 4703 E:\FR\FM\23MRN3.XXX pfrm01 PsN: 23MRN3 Federal Register / Vol. 64, No. 55 / Tuesday, March 23, 1999 / Notices 14087

Report accompanying and establishing public with sufficient opportunity to preparation of a standard curve for the Congress’ views on CSTHEA repeatedly comment. Standards Addition Assay that emphasized that ‘‘it is essential that we This respondent also commented that encompasses the range of values inform the public of the health effects of CDC failed to meet the requirements of expected from adding known smokeless tobacco use and continue the Paperwork Reduction Act by failing concentrations of nicotine to the research on such health effects as to provide the public with an accurate smokeless tobacco product. Also, CDC expeditiously as possible’’ (Senate estimate of the burden of compliance. revised the protocol to specify when the Report 99–209, Dec. 4, 1985, p.13). CDC disagrees. CDC based its original Standards Addition Assay is to be Since scientific evidence has estimate on the figures that were conducted by the testing facility. established that knowledge of pH and submitted by the manufacturers Regarding use of a vegetable-based un-ionized nicotine content is essential themselves. Moreover, the 60-day notice matrix, CDC acknowledges that a in determining the health effects of and comment period is designed to nicotine-free tobacco surrogate is not smokeless tobacco, the reporting of solicit comment on the accuracy of the readily available to serve as a vegetable- these elements is supported by the agency’s estimate of the burden of the based matrix; that is why the protocol legislative history. proposed collection of information. 44 thus specifies adding known The respondent also commented that U.S.C. 3506(c)(2)(A)(ii). The tobacco concentrations of nicotine to the CDC failed to comply with procedural manufacturers felt that the estimate of smokeless tobacco product when obligations in violation of 5 USC § 551 burden had changed and commented as performing the Standards Addition et seq., the Administrative Procedure such at that time. CDC then sought Assay. CDC revised the text in Endnote Act (APA). Specifically, the respondent revised estimates from the 1 of the protocol to eliminate the phrase claimed that CDC failed to provide manufacturers. Only one manufacturer ‘‘routine testing of random blind opportunity for advance review and responded to the request for this samples.’’ comment and failed to inform the public information. Based on this response, Regarding triplicate determinations, of the true nature of the proposed CDC conducted new analyses and CDC asserts that the potential sources of protocol. revised its estimate of burden smokeless tobacco product variability necessitate triplicate determinations for CDC feels that the notice given to the accordingly. Thus, CDC clearly evaluation of precision. In response to tobacco manufacturers was clearly complied with the Paperwork Reduction the comment that CDC was attempting adequate under the APA. CDC’s purpose Act’s requirements. CDC also received comments to ‘‘bind the manufacturers’’ to a in publishing the protocol in the regarding the protocol, which raised cigarette testing protocol in the Federal Register was to solicit technical and scientific concerns smokeless tobacco testing protocol, CDC comments from the public. This is the regarding collection and calculation of clarifies that the disputed protocol is a appropriate time for the manufacturers the requested information. Several sampling protocol, not a testing to review the protocol and relay their respondents supported the protocol protocol. Therefore, testing facilities comments to CDC, not before requirement of reporting not only should make use of the document as publication. As a matter of courtesy, moisture and nicotine content but also reference material. However, for CDC has provided the manufacturers pH and unionized nicotine levels. clarification, Endnote 11 of the protocol with a copy of draft protocols before Support for this aspect of the protocol (Endnote 10 of the public comment publication. CDC did that here as well, was based on scientific evidence that version of the protocol) was revised to for the tobacco companies were given an the nicotine-specific effects of a given read—‘‘The testing facility must ensure advance copy of the protocol before amount of smokeless tobacco depend as that samples are obtained through the formal publication. Therefore, CDC did much on the pH of the product as on the use of a survey design protocol for provide the tobacco companies with nicotine content itself. CDC agrees with sampling ‘at one point in time’ at the advance knowledge of the protocol, this scientific observation regarding the factory or warehouse. The survey design even though such notice was not utility of determining smokeless tobacco protocol must address short-, medium- required. pH and calculating unionized nicotine , and long-term smokeless tobacco Furthermore, CDC has not failed to levels in smokeless tobacco products. product variability (e.g., variability over inform the public of the true nature of One respondent, on behalf of several time and from container to container of its proposed protocol. CDC sufficiently smokeless tobacco manufacturers, stated the tobacco product) in a manner apprised the public of the agency’s legal that the protocols for nicotine analysis equivalent to that described for cigarette authority to issue the proposed protocol, and total moisture determination are sampling in Annex C of ISO Protocol for it explicitly states that it is operating scientifically flawed. Specifically, the 8243.’’ under the authority of CSTHEA. Thus, respondent stated that the Standards This respondent also commented that the public is on sufficient notice of the Addition Assay is flawed and the protocol for pH measurement is legal authority under which CDC issued unnecessary, that the protocol specifies scientifically flawed. Specifically, the the proposed protocol, and has had full an unavailable vegetable-based matrix, respondent states that the procedure is opportunity to comment. that triplicate determinations are based on a nonvalidated protocol, that CDC is also not required to lay out unnecessary, and that the protocol an inappropriate volume of liquid is potential criticisms of its scientific requires smokeless tobacco specified, that proper calibration of positions in its notice and request for manufacturers to use a protocol specific instruments has not been incorporated public comment. The purpose of the to cigarettes. in the protocol, that temperature is not notice and comment period is to With respect to the Standards considered in the protocol, and that provide interested parties with the Addition Assay, CDC reaffirms the multiple pH measurements are opportunity to conduct their own function of the Standards Addition unnecessary. analysis of the merits of the protocol, Assay and disputes the inadequacies of CDC disagrees that the protocol for pH and to provide scientific or other the extraction testing offered by the measurement is scientifically flawed. criticisms, if desired. CDC has clearly respondent as a rationale for eliminating The protocol to determine smokeless stated the terms and substance of its the Standards Addition Assay. CDC tobacco pH is based on the validated proposed protocol as to provide the revised the protocol to facilitate protocol published by Henningfield et 14088 Federal Register / Vol. 64, No. 55 / Tuesday, March 23, 1999 / Notices al. (1995), which also provides the than 10% over 15 minutes. Report the conclusions presented in a recent rationale for the quantity of smokeless final pH value.’’ review article that ‘‘pH is a major tobacco and the volume of liquid. This respondent further asserted that determinant of nicotine absorption Parameters that can be standardized the ‘‘smokeless tobacco pH’’ theory has across mucosal tissues’’ in the mouth (sample size, sample preparation, been discredited. In summary, the and that other ‘‘behavioral and quantity and purity of standard and respondent states that calculation of un- biological’’ factors probably have ‘‘little reagents, instrumentation, measurement ionized nicotine is based on a effect on the rate of nicotine absorption’’ time and conditions, etc.) are specified discredited scientific theory and that the (Tomar and Henningfield, Tobacco in the protocol. Of note, the ‘‘smokeless tobacco pH’’ theory ignores Control 1997, 6:219–225). Henningfield et al. (1995) reference was the chemical, biological, and behavioral Information Collection Provisions provided in the version of the protocol factors that govern absorption of that the respondent received for smokeless tobacco. This notice contains information comment. Un-ionized nicotine is known to be collections which have been approved CDC agrees that careful calibration the form of nicotine most easily by the Office of Management and across the range of unknown values to absorbed in the mouth. pH Budget (OMB) under the Paperwork be measured is needed. The protocol determination is a component of the Reduction Act of 1980 and assigned was revised to read—‘‘Measure pH of protocol to allow calculation of un- OMB Action Notice number 0920–0444 sample after a two-point calibration of ionized nicotine. The degree of nicotine (expiration 01/31/2002). The title the pH meter to an accuracy of two ionization is calculated from the description, and respondent information decimal places using standard pH Henderson-Hasselbalch equation. In a are shown below with an estimate of the buffers (4.01 and 7.00 or 7.00 and 10.00) document written at the request of the annualized costs and burden hours. that will encompass the expected pH United States Tobacco Company, Dr. Title: Quantity of Nicotine Contained value of the smokeless tobacco Jeffrey R. Idle recognizes pH as one of in Smokeless Tobacco Products product.’’ the ‘‘chemical factors which determine Manufactured, Imported, or Packaged in the absorption of a substance dissolved the United States. CDC also agrees that conditions such in water across a biological barrier with Description: The Comprehensive as sample preparation, sample size, which the solution is in intimate Smokeless Tobacco Health Education extraction time, volume and purity of contact.’’ In the same document Dr. Idle Act (CSTHEA) of 1986 requires HHS to the water used, and temperature must concludes that ‘‘The concept of a pH for collect this information. HHS is be controlled during determination of snuff depends upon a standardized and authorized under CSTHEA to conduct the pH of a smokeless tobacco product. validated pH assay for aqueous tobacco research on the addictive nature of The protocol was revised to specify suspensions’ and that ‘‘The concept of nicotine and general health effects of room temperature for nicotine pH for a moist solid such as tobacco can using smokeless tobacco. extraction and pH determination. only apply to a solution derived from a Description of Respondents: As described above, CDC recognizes stirred suspension of a standardized Businesses or other For-Profit that there are several potential sources amount of tobacco in a standardized Organizations. of smokeless tobacco product variability volume of water.’’ Estimates of Annualized Burden that necessitate triplicate It is the intent of the protocol to Hours and Costs: The average determinations for evaluation of provide smokeless tobacco annualized total cost to industry is precision. With respect to pH manufacturers with a ‘‘standardized $23,419. This is based on an annualized determination, CDC recognizes the need measurement’’ of pH. Parameters that estimated cost for 11 companies at for multiple measurements to determine can be standardized for pH, moisture, $2,129 per company. Some companies if pH values for the smokeless tobacco and nicotine determination (sample may choose to contract with an product vary systematically with time. size, sample preparation, quantity and independent laboratory while others For edification, the protocol was revised purity of standards and reagents, may elect to complete the to read—‘‘The first time pH values are instrumentation, measurement time and determinations in-house. For those determined for each lot of a smokeless conditions, etc.)—not random companies choosing to conduct the tobacco product, measure the pH of the conditions or circumstances unique to testing in-house, approximately smokeless tobacco product at 5, 15, and each smokeless tobacco user such as US$60,000 would be required to 30 minutes. If there is no systematic ‘‘residues of beverages’’ in the mouth of purchase the necessary equipment, variation in pH values with time, all the smokeless tobacco user (chemical assuming none of the equipment was subsequent pH determinations for the factors), ‘‘surface area of the absorptive previously owned. lot are made at 5 minutes. If there is tissues’’ (biological factors), and The annual response burden to the systematic variation in pH values, ‘‘expectoration’’ or ‘‘swallowing’’ industry is estimated at 170 hours per continue to measure the pH of the (behavioral factors)—are specified in the smokeless tobacco company. Thus, for smokeless tobacco product until the pH protocol. In addition, the protocol’s the 11 respondents, the hour-burden is value is stable and does not vary more methodology is supported by the 1,870 hours.

Avg. number Number of re- of responses Avg. hours per Estimated total Avg. cost per Estimated total Respondents spond-ents per respond- respondent hours respondent cost ent

Tob. Mfrs...... 11 1 170 1,870 $2,129 $23,419

Procedures to maintain moisture data: As provided by CSTHEA, pH, and moisture information as a trade confidentiality of nicotine, pH, and HHS is required to treat the nicotine, secret or confidential in accordance Federal Register / Vol. 64, No. 55 / Tuesday, March 23, 1999 / Notices 14089 with 5 U.S.C. 552(b)(4)and 18 U.S.C. Crosslinked 5% PH ME Siloxane, a powder. The sample must be 1905. CSTHEA also requires HHS to 30 m length x 0.32 mm ID, film thoroughly mixed before weighing for establish written procedures to assure thickness 0.25 or 0.52 µm). nicotine, total moisture, and pH the confidentiality of the information 3. Orion Model EA 940 pH meter analysis. provided. Consistent with these equipped with Orion 8103 Ross 2. Moist (wet) snuff. Moist (wet) snuff statutory provisions, HHS has combination pH electrode. samples do not need to be ground. The sample must be thoroughly mixed developed strict procedures for treating D. Additional Equipment and protecting relevant documents, before weighing for nicotine, total  including secured file storage and Forced-air oven, Fisher Isotemp , moisture, and pH analysis. strictly-limited access to the regulated to 99 ± 1.0°C. Suggested 3. Moist (wet) snuff portion packs. information. The procedures that are dimensions: 18 x 18 x 20.′′ The tobacco contents of the moist (wet) applicable to the nicotine content of E. Chromatographic Conditions 8, 9 snuff portion packs do not need to be smokeless tobacco products comport ground for nicotine, total moisture, or 1. Detector temperature: 250°C pH analysis. The tobacco packaging with those already in place for ° protecting the confidential lists of 2. Injector temperature: 250 C material (the ‘‘pouch’’) should be 3. Flow rate at 100°C—1.7 mL/min; with ingredients in cigarettes and smokeless separated from the tobacco and ground split ratio of 40:1 10 tobacco products. These procedures to obtain particles measuring 4. Injection volume: 2 µl have proven workable, effective, and ° approximately 4 mm for pH analysis. 5. Column conditions: 110–185 C at The tobacco of the moist (wet) snuff acceptable to the companies required to 10°C min ¥1; 185–240°C at 6°C report the confidential information. The ¥1 portion pack and the ground pouch are min , hold at final temperature combined and thoroughly mixed before procedures, Guidelines for Maintaining for 10 min. and Releasing Privileged Information pH analysis. Obtained in Accordance With Sec. F. Sample Preparation 11 4. Plug tobacco. Break or cut apart plugs and add in portions to grinder at 4(b)(2)(a) of Public Law 99–252 (15 There exist six different categories of 2000 RPM. Reduce RPM or stop U.S.C. 4403), were previously published commercial smokeless tobacco grinding if sample bowl becomes warm. in the February 1, 1994, Federal products: Pulse the Robot Coupe, when needed, to Register (59 FR 4714), and are available 1. Dry snuff; from CDC’s Office on Smoking and complete grinding. Grind samples until 2. Moist (wet) snuff; approximately 4 mm in size. The total Health upon request. 3. Moist (wet) snuff portion packs; grinding time should be no more than Dated: March 17, 1999. 4. Plug; 5. Twist; and 3 minutes. Martha Katz, 5. Twist tobacco. Separate twists, add 6. Loose leaf. Acting Director, Centers for Disease Control to grinder and grind at 2000 RPM. and Prevention. Because of their physical Reduce RPM or stop grinding if sample characteristics, samples of three of the Protocol for Analysis of Nicotine, Total bowl becomes warm. Continue grinding six product categories must be ground until sample particles are approximately Moisture, and pH in Smokeless before nicotine, total moisture, and pH Tobacco Products 4 mm in size. The total time for grinding analyses can be conducted. The should be no more than 3 minutes. 1, 2 I. Requirements objective of grinding the samples is to 6. Loose leaf. Grind in the same obtain a homogeneous sample with A. Reagents 3 manner as described in 4 and 5 to obtain particles measuring approximately 4 product with particle size of 1. Sodium hydroxide (NaOH), 2N mm. Grinding to achieve this particle approximately 4 mm. 2. Methyl t-butyl ether (MTBE) size should take no more than 3 3. (-) -Nicotine (Fluka 72290) >99% minutes. To ensure proper grinding and II. Nicotine Analysis 12 purity 4, 5 an adequate amount of the ground A. Calibration Standards 4. Quinoline (Aldrich) sample for analysis, the minimum 5. Standard pH buffers; 4.01, 7.00, and sample size of all commercial products 1. Internal Standard (IS) 10.00 to be ground should not be less than 100 Weigh 10.00 grams of quinoline, 6. Deionized distilled water grams. transfer to a 250 mL volumetric flask B. Glassware and supplies To ensure precision of analyses for and dilute to volume with MTBE. This nicotine, total moisture, and pH, the 1. Volumetric flasks, class A solution will be used for calibration of samples that require grinding should be 2. Culture tubes, 25 mm x 200 mm, with the instrument for the nicotine ground using a Robot Coupe Model RSI Teflon-lined screw caps calibration curve (II.A.2), for the 3. Pasteur pipettes 2V Scientific Batch Processor or its standards addition assay (II.B), and for 4. Repipettors (10 mL and 50 mL) equivalent. This is a variable speed (0 to preparation of the extracting solution 5. Linear shaker (configured to hold 3000 RPM) processor. The variable (II.D). 6, 7 speed motor is required to ensure tubes in horizontal position) 2. Nicotine Calibration Curve 6. Weighing dishes, aluminum proper grinding of the tobacco tissues 7. Teflon-coated magnetic stirring bars (and in the case of pH determination, a. Weigh 1.0000 gram of nicotine into 8. Polypropylene containers, 50 mL the moist (wet) snuff portion pack). a clean, dry 100 mL volumetric flask Elevated temperatures can result in and dilute to volume with MTBE. This C. Instrumentation moisture loss and an underestimated gives a nicotine concentration of 10 mg/ 1. Robot Coupe Model RSI 2V Scientific value for moisture content. Hence, care mL for the stock solution. Batch Processor. must be taken during grinding to avoid b. Accurately pipette 0.5 mL of IS 2. Capillary gas chromatograph, Hewlett elevated temperatures. The bowl should from stock solution (II.A.1) to five clean, Packard, Model 6890, with split/ be cleaned after each grinding to obtain dry 50 mL volumetric flasks. To prepare splitless injector capability, flame accurate results. a nicotine standard corresponding to a ionization detector, and a capillary 1. Dry snuff. Dry snuff samples do not concentration of 0.8 mg/mL, pipette column (Hewlett Packard HP–5, need to be ground since the product is exactly 4.0 mL of the nicotine standard 14090 Federal Register / Vol. 64, No. 55 / Tuesday, March 23, 1999 / Notices

(II.A.2.a) to a 50 mL volumetric flask the homogeneous, prepared tobacco 12. Compare the results of steps containing the internal standard and sample into a culture tube. Repeat this II.A.2. and II.B. If they differ by a factor dilute to volume with MTBE. To obtain five times for a total of 6 culture tubes of 10% or more, the recovery of nicotine nicotine concentrations equivalent to containing the smokeless tobacco from the aqueous matrix is not 0.6, 0.4, 0.2, and 0.1 mg/mL, pipette product. Record the weight of each equivalent to recovery from the precisely 3.0, 2.0, 1.0, and 0.5 mL, sample. vegetable matrix of the smokeless respectively, of the nicotine standard 2. Prepare a five-point standard curve tobacco product. In this instance, the into the four remaining flasks and dilute for the Standards Addition Assay. The nicotine concentration of the smokeless to volume with MTBE. standard curve must consist of nicotine tobacco product must be determined c. Transfer aliquots of the five concentrations that encompass the range from a nicotine calibration curve standards to auto sampler vials and of values expected from adding known prepared from nicotine standards in a determine the detector response for each concentrations of the nicotine standard vegetable-based matrix. standard using gas chromatographic (II.A.2.a) to a measured quantity of the C. Quality Control Pools conditions described in I.E. smokeless tobacco product (1.000 ± d. Calculate least squares line for 0.020 gram, described in II.B.1.). The At least two quality control pools at linear equation from these standards by sixth culture tube is not supplemented the high and low ends of the expected obtaining the ratio of Areanicotine/AreaIS. with nicotine and serves as an analytical nicotine values are recommended to be This ratio will be the Y value and the blank. Allow the samples to equilibrate included in each analytical run. The concentration of nicotine will be the X for 10 minutes. pools should be analyzed in duplicate value for determining the linear 3. Pipette 5 mL of 2 N NaOH into each in every run. The quality control pools equation of the line (Equation 1): tube. Cap each tube. Swirl to wet should be available in sufficient Equation 1: sample and allow to stand 15 minutes.13 quantity to last for all analyses of a Y = a + bX; 4. Pipette 50 mL of extraction solution product lot. Where: (II.D.1) into each tube. Cap each tube D. Sample Extraction Procedure 12 X = Concentration of nicotine in mg and tighten.14 Y =Areanicotine/AreaIS 5. Place tubes in rack(s), place racks 1. Extraction solution is prepared by a = intercept on the ordinate (y axis) in linear shaker in horizontal position pipetting 10 mL of the IS from the stock b = slope of the curve and shake for two hours. solution (II.A.1) to a 1000 mL The final result will be reported in the 6. Remove rack(s) from shaker and volumetric flask and diluting to volume following units: place in vertical position to allow the with MTBE. Concentration of nicotine = mg of phases to separate. 2. Using an analytical balance, nicotine/gram of tobacco sample. 7. Allow the solvent and nicotine accurately weigh 1.000 ± 0.020 gram of e. Determine the recovery of nicotine supplemented samples and the blank to by pipetting 10 mL of the 0.4 mg/mL prepared tobacco sample into culture separate (maximum 2 hours). 15 nicotine standard to a screw capped tube and record weight. The number 8. Transfer aliquots of the five of products sampled per lot should tube containing 1.0 mL of 2 N NaOH. standards and the blank from the Cap the tube. Shake the contents reflect an acceptable level of extraction tubes to sample vials and precision.16 The test material is to be vigorously and allow the phases to determine the detector response for each separate. Transfer an aliquot of the representative of the product that is sold using gas chromatographic conditions to the public and therefore should organic phase to an injection vial and described in I.E. inject. Calculate the concentration of consist of sealed, packaged samples 9. Subtract the Areanicotine/AreaIS of nicotine using the equation of the line from each lot of finished product that is the blank from the Areanicotine/AreaIS of in II.A.2.d above. This should be ready for commercial distribution. each of the standards. repeated two more times to obtain an Triplicate determinations will provide 10. Calculate least squares line for average of the three values. The precision data. linear equation from the corrected recovery of nicotine can be obtained by 3. Pipette 5 mL of 2 N NaOH into the standards as described above (Equation using the following equation: tube. Cap the tube. Swirl to wet sample 1) in II.A.2.d. and allow to stand 15 minutes.13 Equation 2: The final corrected result will be Recovery = Nicotinecalculated/Nicotineactual 4. Pipette 50 mL of extraction solution reported in the following units: into tube, cap tube and tighten.14 B. Standards Addition Assay Concentration of nicotine = mg of 5. Place tubes in rack(s), place racks nicotine/gram of tobacco sample. Prior to analyzing a smokeless tobacco in linear shaker in horizontal position 11. Determine the recovery of nicotine product for nicotine content, the testing and shake for two hours. by pipetting 10 mL of the 0.4 mg/mL facility must validate the system to 6. Remove rack(s) from shaker and verify that matrix bias is not occurring nicotine standard to a screw capped tube containing 1.0 mL of 2 N NaOH place in vertical position to allow the during nicotine extraction. This is done phases to separate. by analyzing the nicotine calibration and 10 mL of extraction solution 7. Allow the solvent and sample to standards in the same vegetable matrix (II.D.1). Cap the tube and tighten. Shake separate (maximum 2 hours). Transfer as the smokeless tobacco. The first time the contents vigorously and allow the an aliquot from the extraction tube to a each lot of a smokeless tobacco product phases to separate. Transfer an aliquot sample vial and cap. is evaluated, the Standards Addition of the organic phase to an injection vial Assay will be performed, and and inject. Calculate the concentration 8. Analyze the extract using GC documentation of its performance and of nicotine using the equation of the line conditions as described above (I.E) and of the nicotine concentrations selected above in II.A.2.d. This should be calculate the concentration of nicotine for the standard curve (II.B.2) will be repeated two more times to obtain an using the linear calibration equation. submitted to the Centers for Disease average of the three values. The Correct percent nicotine values for both Control and Prevention. recovery of nicotine can be obtained by recovery and weight of sample by using 1. Using an analytical balance, using Equation 2: Equation 3.17 accurately weigh 1.000 ± 0.020 gram of Recovery = Nicotinecalculated/Nicotineactual BILLING CODE 4163±18±P Federal Register / Vol. 64, No. 55 / Tuesday, March 23, 1999 / Notices 14091

BILLING CODE 4163±18±C shelf. Dry 3 hr at 99 ± 1.0°C. Remove accuracy of two decimal places using 9. Report the final nicotine from oven, cover, and cool in desiccator standard pH buffers (4.01 and 7.00 or determination as mg of nicotine per to room temp. (about 30 min). Reweigh 7.00 and 10.00) that will encompass the gram of the tobacco product (mg and calculate percent moisture. expected pH value of the smokeless nicotine/gram), to an accuracy level of D. Report the final moisture tobacco product. two decimal places for each lot and for determination as a percentage (%), to an each brand name (e.g., Skoal Bandits accuracy level of one decimal place for E. The first time pH values are Wintergreen, Skoal Long Cut Cherry, each lot and for each brand name (e.g., determined for each lot of a smokeless Skoal Long Cut Wintergreen, etc.). All Skoal Bandits Wintergreen, Skoal Long tobacco product, measure the pH of the data should include the mean value Cut Cherry, Skoal Long Cut smokeless tobacco product at 5, 15, and with a 95% confidence interval, the Wintergreen, etc.). All data should 30 minutes. If there is no systematic range of values, the number of samples include the mean value with a 95% variation in pH values with time, all tested per lot, the number of lots per confidence interval, the range of values, subsequent pH determinations for the brand name, and the estimated the number of samples tested per lot, lot are made at 5 minutes. If there is precision of the mean. Information will the number of lots per brand name, and systematic variation in pH values, be reported for each manufacturer and the estimated precision of the mean. continue to measure the pH of the variety (including brand families and Information will be reported for each smokeless tobacco product until the pH brand variations) and brand name (e.g., manufacturer and variety (including value is stable and does not vary more Skoal Bandits Wintergreen, Skoal Long brand families and brand variations) than 10% over 15 minutes. Report the Cut Cherry, Skoal Long Cut and brand name (e.g., Skoal Bandits final pH value. Wintergreen, etc.). Wintergreen, Skoal Long Cut Cherry, F. Report the final pH determination Skoal Long Cut Wintergreen, etc.). III. Total Moisture Determination to an accuracy level of two decimal A. This procedure is a modification of IV. pH Measurement 12,21 places for each lot and for each brand AOAC Method 966.02 (1990) and is A. Test samples as soon as possible name (e.g., Skoal Bandits Wintergreen, referred to as ‘‘Total Moisture after they are received. The number of Skoal Long Cut Cherry, Skoal Long Cut Determination’’ because it determines products sampled per lot should reflect Wintergreen, etc.). All data should water and tobacco constituents that are an acceptable level of precision.16 The include the mean value with a 95% volatile at temperatures of 99 ± 1.0°C. test material is to be representative of confidence interval, the range of values, B. Accurately weigh 5.00 grams of the the product that is sold to the public the number of samples tested per lot, sample (ground to pass ≤ 4 mm screen) 19 and therefore should consist of sealed, the number of lots per brand name, and into a weighed moisture dish and place packaged samples from each lot of the estimated precision of the mean. uncovered dish in oven.20 The number finished product that is ready for Information will be reported for each of products sampled per lot should commercial distribution. Triplicate manufacturer and variety (including reflect an acceptable level of determinations will provide precision brand families and brand variations) precision.16 The test material is to be data. and brand name (e.g., Skoal Bandits representative of the product that is sold B. Accurately weigh 2.00 grams of the Wintergreen, Skoal Long Cut Cherry, to the public and therefore should sample. Place in a 50 mL polypropylene Skoal Long Cut Wintergreen, etc.). consist of sealed, packaged samples container with 10 mL deionized G. Estimate the un-ionized (free) from each lot of finished product that is distilled water. nicotine content with the Henderson- ready for commercial distribution. C. Place Teflon-coated magnetic Hasselbalch equation (Equation 4), Triplicate determinations will provide stirring bar in container and stir mixture precision data. continuously throughout testing. based on measured pH and nicotine C. Do not exceed 1 sample/10 sq in. D. Measure pH of sample after a two- content. (650 sq cm) shelf space, and use only 1 point calibration of the pH meter to an BILLING CODE 4163±18±P 14092 Federal Register / Vol. 64, No. 55 / Tuesday, March 23, 1999 / Notices

BILLING CODE 4163±18±C each lot and for each brand name (e.g., Information will be reported for each H. Report the final estimated un- Skoal Bandits Wintergreen, Skoal Long manufacturer and variety (including ionized (free) nicotine as a percentage Cut Cherry, Skoal Long Cut brand families and brand variations) (%) of the total nicotine content, to an Wintergreen, etc.). All data should and brand name (e.g., Skoal Bandits accuracy level of two decimal places include the mean value with a 95% Wintergreen, Skoal Long Cut Cherry, and as mg of un-ionized (free) nicotine confidence interval, the range of values, Skoal Long Cut Wintergreen, etc.). per gram of the tobacco product (mg un- the number of samples tested per lot, ionized (free) nicotine/gram), to an the number of lots per brand name, and Billing Code 4163±18±P accuracy level of two decimal places for the estimated precision of the mean. Sample calculation: Federal Register / Vol. 64, No. 55 / Tuesday, March 23, 1999 / Notices 14093

VerDate 17-MAR-99 17:13 Mar 22, 1999 Jkt 183247 PO 00000 Frm 00009 Fmt 4701 Sfmt 4725 E:\FR\FM\23MRN3.XXX pfrm07 PsN: 23MRN3 14094 Federal Register / Vol. 64, No. 55 / Tuesday, March 23, 1999 / Notices

BILLING CODE 4163±18±C Federal Register / Vol. 64, No. 55 / Tuesday, March 23, 1999 / Notices 14095

V. Assay Criteria for Quality Assurance 4. When the mean nicotine survey design protocol for sampling ‘‘at one concentrations in two QC pools are point in time’’ at the factory or warehouse. A. Establishing Limits for Quality different by more than a total of 4 SD, The survey design protocol must address Control Parameters then the run must be rejected. This short-, medium-, and long-term smokeless All quality control parameters must condition may occur, for example, when tobacco product variability (e.g., variability be determined within the laboratory in one QC pool is 2 SD greater than the over time and from container to container of which they are to be used. At least 10 mean, and another is 2 SD less than the the tobacco product) in a manner equivalent to that described for cigarette sampling in within-laboratory runs must be mean. performed to establish temporary Annex C of ISO Protocol 8243. Information confidence intervals for the quality Endnotes accompanying results for each sample should include, but not be limited to: control parameters. Permanent limits The comments and notes listed below can 1. For each product—manufacturer and be described as Good Laboratory Practice should be established after 20 runs and variety (including brand families and brand guidelines; they are described in detail in should be reestablished after each variations) and brand name (e.g., Skoal additional 20 runs. this protocol to ensure minimal interlaboratory variability in the Bandits, Skoal Long Cut Cherry, Skoal Long B. Exclusion of Outliers From the determination of nicotine, total moisture, and Cut Wintergreen, etc.) information. Calibration Curve 18 pH in smokeless tobacco. 2. Product ‘‘category,’’ e.g., loose leaf, plug, 1 This protocol assumes that the testing twist, dry snuff, moist (wet) snuff, etc. The coefficient of determination facility will implement and maintain a 3. Lot number. between Areanicotine/AreaIS and nicotine stringent Quality Assurance/Quality Control 4. Lot size. concentration should be equal to 0.99 or program to include, but not be limited to, 5. Number of randomly sampled, sealed, higher. Any calibration standard having regular interlaboratory comparisons, packaged (so as to be representative of the an estimated concentration computed determination of the quality and purity of product that is sold to the public) smokeless from the regression equation (Equation purchased products, and proper storage and tobacco products selected per lot (sampling handling of all reagents and samples. fraction) for nicotine, moisture, and pH 1) which is different from its actual 2 When a specific product or instrument is determination. concentration by a factor of 10% can be listed, it is the product or instrument that 6. Documentation of method used for excluded from the calibration curve. Up was used in the development of this method. random sample selection. to two concentrations may be excluded, Equivalent products or instruments may also but caution should be used in be used. Use of trade names is for 7. ‘‘Age’’ of product when received by eliminating values, since bias may be identification only and does not constitute testing facility and storage conditions prior to analysis. increased in the calibration curve. If an endorsement by the Public Health Service or 12 Extraction of nicotine and pH outlier value is eliminated, its duplicate the U.S. Department of Health and Human Services. determination must be performed with value must also be discarded to avoid 3 All chemicals, solvents, and gases are to reagents and samples at a room temperature producing a new bias. All unknowns be of the highest purity. of 22–25°C. Room temperature should not must fall within the calibration curve; 4 Companies must ensure that the purity of vary more than 1°C during extraction of therefore, duplicate values excluded at the nicotine base is certified by the vendor nicotine or pH determination. either end of the calibration curve will and that the chemical is properly stored. 13 Use non-glass 10 mL repipette for restrict the useful range of the assay. However, nicotine base oxidizes with storage, transferring NaOH solution. as reflected by the liquid turning brown. If 14 Use 50 mL repipette for transferring C. Quality Control Pools and Run oxidation has occurred, the nicotine base MTBE. Rejection Rules should be distilled prior to use in making a 15 For dry snuff, use 0.500 ± 0.010 gram standard solution. sample. The mean estimated nicotine 5 A suggested method for the determination 16 The testing facility is referred to ISO concentration in a pool should be of nicotine purity is CORESTA compared with the established limits for Recommended Method No. 39. Procedure 8243 for a discussion of sample that pool based on at least 20 6 Horizontal shaking will allow more size and the effect of variability on the consecutive runs. An analytical run intimate contact of this three phase precision of the mean of the sample (ISO 8243, 1991). should be accepted or rejected based extraction. There is a minimal dead volume 17 When analyzing new smokeless tobacco upon the following set of rules adapted in the tube due to the large sample size and extraction volume. This necessitates products, extract product without IS to from Westgard et al. (1981). horizontal shaking. determine if any components co-elute with 1. When the mean of one QC pool 7 ± If linear shaker is not available, a wrist the IS or impurities in the IS. This exceeds the limit of x 3 standard action shaker using 250 mL stoppered interference could artificially lower deviations (SD), then the run is rejected Erlenmeyer flasks can be substituted. Values calculated values for nicotine. as out of control. Here, x and SD for nicotine are equivalent to those obtained 18 The calculated nicotine values for all represent the overall mean and standard from the linear shaker. samples must fall within the low and high deviation of all estimated nicotine 8 After installing a new column, condition nicotine values used for the calibration concentrations for a particular pool in the column by injecting a tobacco sample curve. If not, prepare a fresh nicotine extract on the column, using the described the runs which were used to establish standard solution and an appropriate series column conditions. Injections should be of standard nicotine dilutions. Determine the the control limits. repeated until areas of IS and nicotine are detector response for each standard using 2. When the mean nicotine reproducible. This will require chromatographic conditions described in I.E. concentrations in two QC pools in the approximately four injections. Recondition 19 The method is a modification of AOAC same run exceed the same direction, column when instrument has been used then the run must be rejected. The same infrequently and after replacing glass liner. Method 966.02 (1990) in that the ground 9 Glass liner and septum should be tobacco passes through a 4 mm screen rather direction is the condition in which both than a 1 mm screen. pools exceed either the x + 2 SD or the replaced after every 100 injections. 10 Most older instruments operate at 20 When drying samples, do not dry x ¥2 SD limits. different products (e.g., moist (wet) snuff, dry 3. When the mean nicotine constant pressure. To reduce confusion, it is suggested that the carrier gas flow through snuff, loose leaf) in the oven at the same time concentrations in one or two QC pools since this will produce errors in the moisture ± the column be measured at the initial column exceed their x 2 SD limits in the same temperature. determinations. direction in two consecutive runs, then 11 The testing facility must ensure that 21 The method is based on a method both runs must be rejected. samples are obtained through the use of a published by Henningfield et al. (1995).

VerDate 17-MAR-99 15:59 Mar 22, 1999 Jkt 183247 PO 00000 Frm 00011 Fmt 4701 Sfmt 4703 E:\FR\FM\23MRN3.XXX pfrm01 PsN: 23MRN3 14096 Federal Register / Vol. 64, No. 55 / Tuesday, March 23, 1999 / Notices

References nicotine salts by gravimetric analysis— Sampling. (1991). Second Edition. Prepared Tungstosilic acid method November, 1994. by Technical Committee ISO/TC 126, AOAC (Association of Official Analytical 87–90. Tobacco and tobacco products. International Chemists). Official Methods of Analysis. CRC Handbook of Chemistry and Physics. 966.02: Moisture in Tobacco. (1990) Fifth Organization for Standardization, Case R.C. Weast, D.R. Lide, M.J. Astle, and WH. Postale 56, CH–1211 Gen´ ve 20, Switzerland. Edition. K. Helrich (ed). Association of Beyer (eds). 70th ed. Boca Raton, Florida: Official Analytical Chemists, Inc. Suite 400, CRC Press (1989–1990) D–162. Westgard J.O., Barry P., Hunt M., and Groth 2200 Wilson Boulevard, Arlington, Virginia Henningfield, J.E., Radzius A., Cone E.J. T. (1981). A multi-rule Shewhart chart for 22201 USA. (1995). Estimation of available nicotine quality control in clinical chemistry. Clinical CORESTA (Centre de Coop´ ration pour les content of six smokeless tobacco products. Chemistry 27:493. Recherches Scientifiques relatives au Tabac). Tobacco Control 4:57–61. [FR Doc. 99–7022 Filed 3–22–99; 8:45 am] Recommended Method No. 39: ISO (International Organization for Determination of the purity of nicotine and Standardization). IOS 8243: Cigarettes— BILLING CODE 4163±18±P