Notice Regarding Requirement for Annual Submission of the Quantity of Nicotine Contained in Smokeless Tobacco Products Manufactured, Imported, or Packaged in the United States

SUMMARY: This notice establishes a uniform protocol for the analysis of

nicotine, total moisture, and pH in smokeless tobacco products. This

protocol was designed to implement the requirement of the Comprehensive

Smokeless Tobacco Health Education Act (CSTHEA) of 1986 (15 U.S.C. 4401

et seq., Pub. L. 99-252), which requires that each entity

manufacturing, packaging, or importing smokeless tobacco products shall

annually provide the Secretary of Health and Human Services (HHS) with

a specification of the quantity of nicotine contained in each smokeless

tobacco product.

DATES: The first report of information is due June 30, 1999, with

subsequent submissions due by March 31 of each year.

ADDRESSES: The information shall be submitted to: Michael P. Eriksen,

Sc.D., Director, Office on Smoking and Health, National Center for

Chronic Disease Prevention and Health Promotion, Centers for Disease

Control and Prevention (CDC), 4770 Buford Highway, NE., Atlanta, GA

30341-3724.

FOR FURTHER INFORMATION CONTACT: Michael P. Eriksen, Sc.D., Director,

Office on Smoking and Health, telephone: (770) 488-5701.

SUPPLEMENTARY INFORMATION: According to a 1986 report of an Advisory

Committee to the Surgeon General, smokeless tobacco represents a

significant health risk, is not a safe substitute for cigarette

smoking, can cause cancer and a number of noncancerous oral conditions,

and can lead to nicotine addiction.

The Centers for Disease Control and Prevention's (CDC) Office on

Smoking and Health (OSH) has primary responsibility for the Department

of Health and Human Services' (HHS) tobacco and health program. The

overall goal of this program is to reduce death and disability

resulting from cigarette smoking and other forms of tobacco use through

programs of information, education, and research.

HHS uses the information collected to exercise its authority under

CSTHEA to conduct research on the addictive nature of nicotine and

general health effects of using smokeless tobacco. Nicotine data will

provide a more complete picture of the addictive nature of smokeless

tobacco products. Also, as authorized in the statute, HHS may report to

the Congress information regarding its current and proposed research

relative to nicotine levels in smokeless tobacco products. CSTHEA

further requires that individuals who manufacture, package, or import

smokeless tobacco products report to HHS the list of ingredients added

to tobacco in the manufacture of such products, and this requirement

has been implemented by a previous notice (59 FR 4714,).

In 1989 the smokeless tobacco industry submitted a business review

letter to the Department of Justice (DOJ), in accordance with 28 CFR

50.6. This letter requested approval of a collaborative industry effort

to develop a uniform analytical protocol for determining the nicotine

and moisture content of smokeless tobacco products.

In January 1993, DOJ extended permission to the smokeless tobacco

industry to develop a uniform analytical protocol for this purpose. A

work group representing the 10 major domestic manufacturers of

smokeless tobacco was convened on July 7, 1993. The workgroup developed

and submitted to CDC for approval the ``Protocol for Analysis of

Nicotine in Smokeless Tobacco Products.'' The protocol was revised by

CDC based on individual comments from peer reviewers and the National

Center for Environmental Health, CDC. The revised protocol, ``Protocol

for Analysis of Nicotine, Total Moisture, and pH in Smokeless Tobacco

Products,'' is hereafter referred to as the ``protocol.''

On May 2, 1997, a notice (62 FR 24115) was published in accordance

with the Paperwork Reduction Act to solicit public comment on the

proposed collection of data. A notice was also published (62 FR 24116,

May 2, 1997) to solicit public comment on the protocol. The protocol

consists of standard laboratory methods to measure nicotine, moisture,

and pH in smokeless tobacco products, and an equation (Henderson-

Hasselbalch) to calculate un-ionized nicotine. Nicotine is the major

alkaloid in tobacco and the drug in tobacco that causes addiction. In

the protocol, moisture is referred to as total moisture because the

method measures the amount of water and tobacco constituents in a

smokeless tobacco product that are volatile at temperatures of 99

degrees centigrade. pH is defined as the negative logarithm of the

molar concentration of hydrogen ions in an aqueous solution and is a

quantitative measure of acidity or alkalinity. The degree of nicotine

ionization is calculated from the Henderson-Hasselbalch equation. Un-

ionized nicotine is known to be the form of nicotine absorbed most

easily in the mouth. This protocol will provide CDC with information on

levels of nicotine found in smokeless tobacco products manufactured,

packaged, or imported during the previous calendar year. The schedule

for reporting this information to CDC corresponds to the reporting of

ingredients added to tobacco in the manufacture of smokeless tobacco

products November 8, 1994 (59 FR 55670,).

Following public request, on June 2, 1997, a notice (62 FR 29729)

was published extending the comment period on the proposed protocol by

an additional 30 days to July 2, 1997. A summary of the comments

received and CDC's response follows.

One respondent, on behalf of several smokeless tobacco

manufacturers, had several comments regarding the collection of data.

The respondent asserted that the protocols exceeded statutory

authorization by collecting pH and free base (hereinafter referred to

as ``un-ionized'', as reflected in the revised protocol) nicotine.

Furthermore, the respondent felt that the legislative history of CSTHEA

contemplates the reporting of nicotine content alone.

It is CDC's belief that the collection of pH of smokeless tobacco

products and the un-ionized nicotine content of each is authorized by

section 4 of CSTHEA. There is ample scientific evidence that mere

quantity of nicotine alone is insufficient in determining its effect on

a user; knowledge of pH and un-ionized nicotine content of the overall

nicotine quantity is essential in determining the rate of nicotine

absorption. pH and un-ionized nicotine content are essential factors

affecting nicotine bioavailability. Furthermore, Congress has never

defined exactly what it meant by ``quantity of nicotine'' in section

4(b) of CSTHEA. In light of ample scientific evidence indicating the

importance of pH and un-ionized nicotine content in assessing the

overall quantity of nicotine, CDC's interpretation of its statutory

authority is clearly permissible.

It is CDC's belief that the legislative history provides support

for requiring the reporting of moisture, pH, and un-ionized nicotine

content. The Senate

[[Page 14087]]

Report accompanying and establishing Congress' views on CSTHEA

repeatedly emphasized that ``it is essential that we inform the public

of the health effects of smokeless tobacco use and continue research on

such health effects as expeditiously as possible'' (Senate Report 99-

209, Dec. 4, 1985, p.13). Since scientific evidence has established

that knowledge of pH and un-ionized nicotine content is essential in

determining the health effects of smokeless tobacco, the reporting of

these elements is supported by the legislative history.

The respondent also commented that CDC failed to comply with

procedural obligations in violation of 5 USC Sec. 551 et seq., the

Administrative Procedure Act (APA). Specifically, the respondent

claimed that CDC failed to provide opportunity for advance review and

comment and failed to inform the public of the true nature of the

proposed protocol.

CDC feels that the notice given to the tobacco manufacturers was

clearly adequate under the APA. CDC's purpose in publishing the

protocol in the Federal Register was to solicit comments from the

public. This is the appropriate time for the manufacturers to review

the protocol and relay their comments to CDC, not before publication.

As a matter of courtesy, CDC has provided the manufacturers with a copy

of draft protocols before publication. CDC did that here as well, for

the tobacco companies were given an advance copy of the protocol before

formal publication. Therefore, CDC did provide the tobacco companies

with advance knowledge of the protocol, even though such notice was not

required.

Furthermore, CDC has not failed to inform the public of the true

nature of its proposed protocol. CDC sufficiently apprised the public

of the agency's legal authority to issue the proposed protocol, for it

explicitly states that it is operating under the authority of CSTHEA.

Thus, the public is on sufficient notice of the legal authority under

which CDC issued the proposed protocol, and has had full opportunity to

comment.

CDC is also not required to lay out potential criticisms of its

scientific positions in its notice and request for public comment. The

purpose of the notice and comment period is to provide interested

parties with the opportunity to conduct their own analysis of the

merits of the protocol, and to provide scientific or other criticisms,

if desired. CDC has clearly stated the terms and substance of its

proposed protocol as to provide the public with sufficient opportunity

to comment.

This respondent also commented that CDC failed to meet the

requirements of the Paperwork Reduction Act by failing to provide the

public with an accurate estimate of the burden of compliance. CDC

disagrees. CDC based its original estimate on the figures that were

submitted by the manufacturers themselves. Moreover, the 60-day notice

and comment period is designed to solicit comment on the accuracy of

the agency's estimate of the burden of the proposed collection of

information. 44 U.S.C. 3506(c)(2)(A)(ii). The tobacco manufacturers

felt that the estimate of burden had changed and commented as such at

that time. CDC then sought revised estimates from the manufacturers.

Only one manufacturer responded to the request for this information.

Based on this response, CDC conducted new analyses and revised its

estimate of burden accordingly. Thus, CDC clearly complied with the

Paperwork Reduction Act's requirements.

CDC also received comments regarding the protocol, which raised

technical and scientific concerns regarding collection and calculation

of the requested information. Several respondents supported the

protocol requirement of reporting not only moisture and nicotine

content but also pH and unionized nicotine levels. Support for this

aspect of the protocol was based on scientific evidence that the

nicotine-specific effects of a given amount of smokeless tobacco depend

as much on the pH of the product as on the nicotine content itself. CDC

agrees with this scientific observation regarding the utility of

determining smokeless tobacco pH and calculating unionized nicotine

levels in smokeless tobacco products.

One respondent, on behalf of several smokeless tobacco

manufacturers, stated that the protocols for nicotine analysis and

total moisture determination are scientifically flawed. Specifically,

the respondent stated that the Standards Addition Assay is flawed and

unnecessary, that the protocol specifies an unavailable vegetable-based

matrix, that triplicate determinations are unnecessary, and that the

protocol requires smokeless tobacco manufacturers to use a protocol

specific to cigarettes.

With respect to the Standards Addition Assay, CDC reaffirms the

function of the Standards Addition Assay and disputes the inadequacies

of the extraction testing offered by the respondent as a rationale for

eliminating the Standards Addition Assay. CDC revised the protocol to

facilitate preparation of a standard curve for the Standards Addition

Assay that encompasses the range of values expected from adding known

concentrations of nicotine to the smokeless tobacco product. Also, CDC

revised the protocol to specify when the Standards Addition Assay is to

be conducted by the testing facility.

Regarding use of a vegetable-based matrix, CDC acknowledges that a

nicotine-free tobacco surrogate is not readily available to serve as a

vegetable-based matrix; that is why the protocol thus specifies adding

known concentrations of nicotine to the smokeless tobacco product when

performing the Standards Addition Assay. CDC revised the text in

Endnote 1 of the protocol to eliminate the phrase ``routine testing of

random blind samples.''

Regarding triplicate determinations, CDC asserts that the potential

sources of smokeless tobacco product variability necessitate triplicate

determinations for evaluation of precision. In response to the comment

that CDC was attempting to ``bind the manufacturers'' to a cigarette

testing protocol in the smokeless tobacco testing protocol, CDC

clarifies that the disputed protocol is a sampling protocol, not a

testing protocol. Therefore, testing facilities should make use of the

document as reference material. However, for clarification, Endnote 11

of the protocol (Endnote 10 of the public comment version of the

protocol) was revised to read--``The testing facility must ensure that

samples are obtained through the use of a survey design protocol for

sampling `at one point in time' at the factory or warehouse. The survey

design protocol must address short-, medium-, and long-term smokeless

tobacco product variability (e.g., variability over time and from

container to container of the tobacco product) in a manner equivalent

to that described for cigarette sampling in Annex C of ISO Protocol

8243.''

This respondent also commented that the protocol for pH measurement

is scientifically flawed. Specifically, the respondent states that the

procedure is based on a nonvalidated protocol, that an inappropriate

volume of liquid is specified, that proper calibration of instruments

has not been incorporated in the protocol, that temperature is not

considered in the protocol, and that multiple pH measurements are

unnecessary.

CDC disagrees that the protocol for pH measurement is

scientifically flawed. The protocol to determine smokeless tobacco pH

is based on the validated protocol published by Henningfield et

[[Page 14088]]

al. (1995), which also provides the rationale for the quantity of

smokeless tobacco and the volume of liquid. Parameters that can be

standardized (sample size, sample preparation, quantity and purity of

standard and reagents, instrumentation, measurement time and

conditions, etc.) are specified in the protocol. Of note, the

Henningfield et al. (1995) reference was provided in the version of the

protocol that the respondent received for comment.

CDC agrees that careful calibration across the range of unknown

values to be measured is needed. The protocol was revised to read--

``Measure pH of sample after a two-point calibration of the pH meter to

an accuracy of two decimal places using standard pH buffers (4.01 and

7.00 or 7.00 and 10.00) that will encompass the expected pH value of

the smokeless tobacco product.''

CDC also agrees that conditions such as sample preparation, sample

size, extraction time, volume and purity of the water used, and

temperature must be controlled during determination of the pH of a

smokeless tobacco product. The protocol was revised to specify room

temperature for nicotine extraction and pH determination.

As described above, CDC recognizes that there are several potential

sources of smokeless tobacco product variability that necessitate

triplicate determinations for evaluation of precision. With respect to

pH determination, CDC recognizes the need for multiple measurements to

determine if pH values for the smokeless tobacco product vary

systematically with time. For edification, the protocol was revised to

read--``The first time pH values are determined for each lot of a

smokeless tobacco product, measure the pH of the smokeless tobacco

product at 5, 15, and 30 minutes. If there is no systematic variation

in pH values with time, all subsequent pH determinations for the lot

are made at 5 minutes. If there is systematic variation in pH values,

continue to measure the pH of the smokeless tobacco product until the

pH value is stable and does not vary more than 10% over 15 minutes.

Report the final pH value.''

This respondent further asserted that the ``smokeless tobacco pH''

theory has been discredited. In summary, the respondent states that

calculation of un-ionized nicotine is based on a discredited scientific

theory and that the ``smokeless tobacco pH'' theory ignores the

chemical, biological, and behavioral factors that govern absorption of

smokeless tobacco.

Un-ionized nicotine is known to be the form of nicotine most easily

absorbed in the mouth. pH determination is a component of the protocol

to allow calculation of un-ionized nicotine. The degree of nicotine

ionization is calculated from the Henderson-Hasselbalch equation. In a

document written at the request of the United States Tobacco Company,

Dr. Jeffrey R. Idle recognizes pH as one of the ``chemical factors

which determine the absorption of a substance dissolved in water across

a biological barrier with which the solution is in intimate contact.''

In the same document Dr. Idle concludes that ``The concept of a pH for

snuff depends upon a standardized and validated pH assay for aqueous

tobacco suspensions' and that ``The concept of pH for a moist solid

such as tobacco can only apply to a solution derived from a stirred

suspension of a standardized amount of tobacco in a standardized volume

of water.''

It is the intent of the protocol to provide smokeless tobacco

manufacturers with a ``standardized measurement'' of pH. Parameters

that can be standardized for pH, moisture, and nicotine determination

(sample size, sample preparation, quantity and purity of standards and

reagents, instrumentation, measurement time and conditions, etc.)--not

random conditions or circumstances unique to each smokeless tobacco

user such as ``residues of beverages'' in the mouth of the smokeless

tobacco user (chemical factors), ``surface area of the absorptive

tissues'' (biological factors), and ``expectoration'' or ``swallowing''

(behavioral factors)--are specified in the protocol. In addition, the

protocol's methodology is supported by the conclusions presented in a

recent review article that ``pH is a major determinant of nicotine

absorption across mucosal tissues'' in the mouth and that other

``behavioral and biological'' factors probably have ``little effect on

the rate of nicotine absorption'' (Tomar and Henningfield, Tobacco

Control 1997, 6:219-225).

Information Collection Provisions

This notice contains information collections which have been

approved by the Office of Management and Budget (OMB) under the

Paperwork Reduction Act of 1980 and assigned OMB Action Notice number

0920-0444 (expiration 01/31/2002). The title description, and

respondent information are shown below with an estimate of the

annualized costs and burden hours.

Title: Quantity of Nicotine Contained in Smokeless Tobacco Products

Manufactured, Imported, or Packaged in the United States.

Description: The Comprehensive Smokeless Tobacco Health Education

Act (CSTHEA) of 1986 requires HHS to collect this information. HHS is

authorized under CSTHEA to conduct research on the addictive nature of

nicotine and general health effects of using smokeless tobacco.

Description of Respondents: Businesses or other For-Profit

Organizations.

Estimates of Annualized Burden Hours and Costs: The average

annualized total cost to industry is $23,419. This is based on an

annualized estimated cost for 11 companies at $2,129 per company. Some

companies may choose to contract with an independent laboratory while

others may elect to complete the determinations in-house. For those

companies choosing to conduct the testing in-house, approximately

US$60,000 would be required to purchase the necessary equipment,

assuming none of the equipment was previously owned.

The annual response burden to the industry is estimated at 170

hours per smokeless tobacco company. Thus, for the 11 respondents, the

hour-burden is 1,870 hours.

--------------------------------------------------------------------------------------------------------------------------------------------------------

Avg. number of

Respondents Number of responses per Avg. hours per Estimated total Avg. cost per Estimated total

respond-ents respondent respondent hours respondent cost

--------------------------------------------------------------------------------------------------------------------------------------------------------

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

--------------------------------------------------------------------------------------------------------------------------------------------------------

Procedures to maintain confidentiality of nicotine, pH, and

moisture data: As provided by CSTHEA, HHS is required to treat the

nicotine, pH, and moisture information as a trade secret or

confidential in accordance

[[Page 14089]]

with 5 U.S.C. 552(b)(4)and 18 U.S.C. 1905. CSTHEA also requires HHS to

establish written procedures to assure the confidentiality of the

information provided. Consistent with these statutory provisions, HHS

has developed strict procedures for treating and protecting relevant

documents, including secured file storage and strictly-limited access

to the information. The procedures that are applicable to the nicotine

content of smokeless tobacco products comport with those already in

place for protecting the confidential lists of ingredients in

cigarettes and smokeless tobacco products. These procedures have proven

workable, effective, and acceptable to the companies required to report

the confidential information. The procedures, Guidelines for

Maintaining and Releasing Privileged Information Obtained in Accordance

With Sec. 4(b)(2)(a) of Public Law 99-252 (15 U.S.C. 4403), were

previously published in the February 1, 1994, Federal Register (59 FR

4714), and are available from CDC's Office on Smoking and Health upon

request.

Dated: March 17, 1999.

Martha Katz,

Acting Director, Centers for Disease Control and Prevention.

Protocol for Analysis of Nicotine, Total Moisture, and pH in

Smokeless Tobacco Products

I. Requirements 1, 2

A. Reagents 3

1. Sodium hydroxide (NaOH), 2N

2. Methyl t-butyl ether (MTBE)

3. (-) -Nicotine (Fluka 72290) >99% purity 4, 5

4. Quinoline (Aldrich)

5. Standard pH buffers; 4.01, 7.00, and 10.00

6. Deionized distilled water

B. Glassware and supplies

1. Volumetric flasks, class A

2. Culture tubes, 25 mm x 200 mm, with Teflon-lined screw caps

3. Pasteur pipettes

4. Repipettors (10 mL and 50 mL)

5. Linear shaker (configured to hold tubes in horizontal position)

6, 7

6. Weighing dishes, aluminum

7. Teflon-coated magnetic stirring bars

8. Polypropylene containers, 50 mL

C. Instrumentation

1. Robot Coupe Model RSI 2V Scientific Batch Processor.

2. Capillary gas chromatograph, Hewlett Packard, Model 6890, with

split/splitless injector capability, flame ionization detector, and a

capillary column (Hewlett Packard HP-5, Crosslinked 5% PH ME Siloxane,

30 m length x 0.32 mm ID, film thickness 0.25 or 0.52 m).

3. Orion Model EA 940 pH meter equipped with Orion 8103 Ross

combination pH electrode.

D. Additional Equipment

Forced-air oven, Fisher Isotemp, regulated to 99

1.0 deg.C. Suggested dimensions: 18 x 18 x 20.''

E. Chromatographic Conditions 8, 9

1. Detector temperature: 250 deg.C

2. Injector temperature: 250 deg.C

3. Flow rate at 100 deg.C--1.7 mL/min; with split ratio of 40:1

10

4. Injection volume: 2 l

5. Column conditions: 110-185 deg.C at 10 deg.C min -1; 185-

240 deg.C at 6 deg.C min -1, hold at final temperature for

10 min.

F. Sample Preparation 11

There exist six different categories of commercial smokeless

tobacco products:

1. Dry snuff;

2. Moist (wet) snuff;

3. Moist (wet) snuff portion packs;

4. Plug;

5. Twist; and

6. Loose leaf.

Because of their physical characteristics, samples of three of the

six product categories must be ground before nicotine, total moisture,

and pH analyses can be conducted. The objective of grinding the samples

is to obtain a homogeneous sample with particles measuring

approximately 4 mm. Grinding to achieve this particle size should take

no more than 3 minutes. To ensure proper grinding and an adequate

amount of the ground sample for analysis, the minimum sample size of

all commercial products to be ground should not be less than 100 grams.

To ensure precision of analyses for nicotine, total moisture, and

pH, the samples that require grinding should be ground using a Robot

Coupe Model RSI 2V Scientific Batch Processor or its equivalent. This

is a variable speed (0 to 3000 RPM) processor. The variable speed motor

is required to ensure proper grinding of the tobacco tissues (and in

the case of pH determination, the moist (wet) snuff portion pack).

Elevated temperatures can result in moisture loss and an underestimated

value for moisture content. Hence, care must be taken during grinding

to avoid elevated temperatures. The bowl should be cleaned after each

grinding to obtain accurate results.

1. Dry snuff. Dry snuff samples do not need to be ground since the

product is a powder. The sample must be thoroughly mixed before

weighing for nicotine, total moisture, and pH analysis.

2. Moist (wet) snuff. Moist (wet) snuff samples do not need to be

ground. The sample must be thoroughly mixed before weighing for

nicotine, total moisture, and pH analysis.

3. Moist (wet) snuff portion packs. The tobacco contents of the

moist (wet) snuff portion packs do not need to be ground for nicotine,

total moisture, or pH analysis. The tobacco packaging material (the

``pouch'') should be separated from the tobacco and ground to obtain

particles measuring approximately 4 mm for pH analysis. The tobacco of

the moist (wet) snuff portion pack and the ground pouch are combined

and thoroughly mixed before pH analysis.

4. Plug tobacco. Break or cut apart plugs and add in portions to

grinder at 2000 RPM. Reduce RPM or stop grinding if sample bowl becomes

warm. Pulse the Robot Coupe, when needed, to complete grinding. Grind

samples until approximately 4 mm in size. The total grinding time

should be no more than 3 minutes.

5. Twist tobacco. Separate twists, add to grinder and grind at 2000

RPM. Reduce RPM or stop grinding if sample bowl becomes warm. Continue

grinding until sample particles are approximately 4 mm in size. The

total time for grinding should be no more than 3 minutes.

6. Loose leaf. Grind in the same manner as described in 4 and 5 to

obtain product with particle size of approximately 4 mm.

II. Nicotine Analysis 12

A. Calibration Standards

1. Internal Standard (IS)

Weigh 10.00 grams of quinoline, transfer to a 250 mL volumetric

flask and dilute to volume with MTBE. This solution will be used for

calibration of the instrument for the nicotine calibration curve

(II.A.2), for the standards addition assay (II.B), and for preparation

of the extracting solution (II.D).

2. Nicotine Calibration Curve

a. Weigh 1.0000 gram of nicotine into a clean, dry 100 mL

volumetric flask and dilute to volume with MTBE. This gives a nicotine

concentration of 10 mg/mL for the stock solution.

b. Accurately pipette 0.5 mL of IS from stock solution (II.A.1) to

five clean, dry 50 mL volumetric flasks. To prepare a nicotine standard

corresponding to a concentration of 0.8 mg/mL, pipette exactly 4.0 mL

of the nicotine standard

[[Page 14090]]

(II.A.2.a) to a 50 mL volumetric flask containing the internal standard

and dilute to volume with MTBE. To obtain nicotine concentrations

equivalent to 0.6, 0.4, 0.2, and 0.1 mg/mL, pipette precisely 3.0, 2.0,

1.0, and 0.5 mL, respectively, of the nicotine standard into the four

remaining flasks and dilute to volume with MTBE.

c. Transfer aliquots of the five standards to auto sampler vials

and determine the detector response for each standard using gas

chromatographic conditions described in I.E.

d. Calculate least squares line for linear equation from these

standards by obtaining the ratio of Areanicotine/

AreaIS. This ratio will be the Y value and the concentration

of nicotine will be the X value for determining the linear equation of

the line (Equation 1):

Equation 1:

Y = a + bX;

Where:

X = Concentration of nicotine in mg

Y =Areanicotine/AreaIS

a = intercept on the ordinate (y axis)

b = slope of the curve

The final result will be reported in the following units:

Concentration of nicotine = mg of nicotine/gram of tobacco sample.

e. Determine the recovery of nicotine by pipetting 10 mL of the 0.4

mg/mL nicotine standard to a screw capped tube containing 1.0 mL of 2 N

NaOH. Cap the tube. Shake the contents vigorously and allow the phases

to separate. Transfer an aliquot of the organic phase to an injection

vial and inject. Calculate the concentration of nicotine using the

equation of the line in II.A.2.d above. This should be repeated two

more times to obtain an average of the three values. The recovery of

nicotine can be obtained by using the following equation:

Equation 2:

Recovery = Nicotinecalculated/Nicotineactual

B. Standards Addition Assay

Prior to analyzing a smokeless tobacco product for nicotine

content, the testing facility must validate the system to verify that

matrix bias is not occurring during nicotine extraction. This is done

by analyzing the nicotine calibration standards in the same vegetable

matrix as the smokeless tobacco. The first time each lot of a smokeless

tobacco product is evaluated, the Standards Addition Assay will be

performed, and documentation of its performance and of the nicotine

concentrations selected for the standard curve (II.B.2) will be

submitted to the Centers for Disease Control and Prevention.

1. Using an analytical balance, accurately weigh 1.000

0.020 gram of the homogeneous, prepared tobacco sample into a culture

tube. Repeat this five times for a total of 6 culture tubes containing

the smokeless tobacco product. Record the weight of each sample.

2. Prepare a five-point standard curve for the Standards Addition

Assay. The standard curve must consist of nicotine concentrations that

encompass the range of values expected from adding known concentrations

of the nicotine standard (II.A.2.a) to a measured quantity of the

smokeless tobacco product (1.000 0.020 gram, described in

II.B.1.). The sixth culture tube is not supplemented with nicotine and

serves as an analytical blank. Allow the samples to equilibrate for 10

minutes.

3. Pipette 5 mL of 2 N NaOH into each tube. Cap each tube. Swirl to

wet sample and allow to stand 15 minutes.\13\

4. Pipette 50 mL of extraction solution (II.D.1) into each tube.

Cap each tube and tighten.\14\

5. Place tubes in rack(s), place racks in linear shaker in

horizontal position and shake for two hours.

6. Remove rack(s) from shaker and place in vertical position to

allow the phases to separate.

7. Allow the solvent and nicotine supplemented samples and the

blank to separate (maximum 2 hours).

8. Transfer aliquots of the five standards and the blank from the

extraction tubes to sample vials and determine the detector response

for each using gas chromatographic conditions described in I.E.

9. Subtract the Areanicotine/AreaIS of the

blank from the Areanicotine/AreaIS of each of the

standards.

10. Calculate least squares line for linear equation from the

corrected standards as described above (Equation 1) in II.A.2.d.

The final corrected result will be reported in the following units:

Concentration of nicotine = mg of nicotine/gram of tobacco sample.

11. Determine the recovery of nicotine by pipetting 10 mL of the

0.4 mg/mL nicotine standard to a screw capped tube containing 1.0 mL of

2 N NaOH and 10 mL of extraction solution (II.D.1). Cap the tube and

tighten. Shake the contents vigorously and allow the phases to

separate. Transfer an aliquot of the organic phase to an injection vial

and inject. Calculate the concentration of nicotine using the equation

of the line above in II.A.2.d. This should be repeated two more times

to obtain an average of the three values. The recovery of nicotine can

be obtained by using Equation 2:

Recovery = Nicotinecalculated/Nicotineactual

12. Compare the results of steps II.A.2. and II.B. If they differ

by a factor of 10% or more, the recovery of nicotine from the aqueous

matrix is not equivalent to recovery from the vegetable matrix of the

smokeless tobacco product. In this instance, the nicotine concentration

of the smokeless tobacco product must be determined from a nicotine

calibration curve prepared from nicotine standards in a vegetable-based

matrix.

C. Quality Control Pools

At least two quality control pools at the high and low ends of the

expected nicotine values are recommended to be included in each

analytical run. The pools should be analyzed in duplicate in every run.

The quality control pools should be available in sufficient quantity to

last for all analyses of a product lot.

D. Sample Extraction Procedure \12\

1. Extraction solution is prepared by pipetting 10 mL of the IS

from the stock solution (II.A.1) to a 1000 mL volumetric flask and

diluting to volume with MTBE.

2. Using an analytical balance, accurately weigh 1.000

0.020 gram of prepared tobacco sample into culture tube and record

weight.\15\ The number of products sampled per lot should reflect an

acceptable level of precision.\16\ The test material is to be

representative of the product that is sold to the public and therefore

should consist of sealed, packaged samples from each lot of finished

product that is ready for commercial distribution. Triplicate

determinations will provide precision data.

3. Pipette 5 mL of 2 N NaOH into the tube. Cap the tube. Swirl to

wet sample and allow to stand 15 minutes.\13\

4. Pipette 50 mL of extraction solution into tube, cap tube and

tighten.\14\

5. Place tubes in rack(s), place racks in linear shaker in

horizontal position and shake for two hours.

6. Remove rack(s) from shaker and place in vertical position to

allow the phases to separate.

7. Allow the solvent and sample to separate (maximum 2 hours).

Transfer an aliquot from the extraction tube to a sample vial and cap.

8. Analyze the extract using GC conditions as described above (I.E)

and calculate the concentration of nicotine using the linear

calibration equation. Correct percent nicotine values for both recovery

and weight of sample by using Equation 3.\17\

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9. Report the final nicotine determination as mg of nicotine per

gram of the tobacco product (mg nicotine/gram), to an accuracy level of

two decimal places for each lot and for each brand name (e.g., Skoal

Bandits Wintergreen, Skoal Long Cut Cherry, Skoal Long Cut Wintergreen,

etc.). All data should include the mean value with a 95% confidence

interval, the range of values, the number of samples tested per lot,

the number of lots per brand name, and the estimated precision of the

mean. Information will be reported for each manufacturer and variety

(including brand families and brand variations) and brand name (e.g.,

Skoal Bandits Wintergreen, Skoal Long Cut Cherry, Skoal Long Cut

Wintergreen, etc.).

III. Total Moisture Determination

A. This procedure is a modification of AOAC Method 966.02 (1990)

and is referred to as ``Total Moisture Determination'' because it

determines water and tobacco constituents that are volatile at

temperatures of 99 1.0 deg.C.

B. Accurately weigh 5.00 grams of the sample (ground to pass

4 mm screen) \19\ into a weighed moisture dish and place

uncovered dish in oven.\20\ The number of products sampled per lot

should reflect an acceptable level of precision.\16\ The test material

is to be representative of the product that is sold to the public and

therefore should consist of sealed, packaged samples from each lot of

finished product that is ready for commercial distribution. Triplicate

determinations will provide precision data.

C. Do not exceed 1 sample/10 sq in. (650 sq cm) shelf space, and

use only 1 shelf. Dry 3 hr at 99 1.0 deg.C. Remove from

oven, cover, and cool in desiccator to room temp. (about 30 min).

Reweigh and calculate percent moisture.

D. Report the final moisture determination as a percentage (%), to

an accuracy level of one decimal place for each lot and for each brand

name (e.g., Skoal Bandits Wintergreen, Skoal Long Cut Cherry, Skoal

Long Cut Wintergreen, etc.). All data should include the mean value

with a 95% confidence interval, the range of values, the number of

samples tested per lot, the number of lots per brand name, and the

estimated precision of the mean. Information will be reported for each

manufacturer and variety (including brand families and brand

variations) and brand name (e.g., Skoal Bandits Wintergreen, Skoal Long

Cut Cherry, Skoal Long Cut Wintergreen, etc.).

IV. pH Measurement 12,21

A. Test samples as soon as possible after they are received. The

number of products sampled per lot should reflect an acceptable level

of precision.\16\ The test material is to be representative of the

product that is sold to the public and therefore should consist of

sealed, packaged samples from each lot of finished product that is

ready for commercial distribution. Triplicate determinations will

provide precision data.

B. Accurately weigh 2.00 grams of the sample. Place in a 50 mL

polypropylene container with 10 mL deionized distilled water.

C. Place Teflon-coated magnetic stirring bar in container and stir

mixture continuously throughout testing.

D. Measure pH of sample after a two-point calibration of the pH

meter to an accuracy of two decimal places using standard pH buffers

(4.01 and 7.00 or 7.00 and 10.00) that will encompass the expected pH

value of the smokeless tobacco product.

E. The first time pH values are determined for each lot of a

smokeless tobacco product, measure the pH of the smokeless tobacco

product at 5, 15, and 30 minutes. If there is no systematic variation

in pH values with time, all subsequent pH determinations for the lot

are made at 5 minutes. If there is systematic variation in pH values,

continue to measure the pH of the smokeless tobacco product until the

pH value is stable and does not vary more than 10% over 15 minutes.

Report the final pH value.

F. Report the final pH determination to an accuracy level of two

decimal places for each lot and for each brand name (e.g., Skoal

Bandits Wintergreen, Skoal Long Cut Cherry, Skoal Long Cut Wintergreen,

etc.). All data should include the mean value with a 95% confidence

interval, the range of values, the number of samples tested per lot,

the number of lots per brand name, and the estimated precision of the

mean. Information will be reported for each manufacturer and variety

(including brand families and brand variations) and brand name (e.g.,

Skoal Bandits Wintergreen, Skoal Long Cut Cherry, Skoal Long Cut

Wintergreen, etc.).

G. Estimate the un-ionized (free) nicotine content with the

Henderson-Hasselbalch equation (Equation 4), based on measured pH and

nicotine content.

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H. Report the final estimated un-ionized (free) nicotine as a

percentage (%) of the total nicotine content, to an accuracy level of

two decimal places and as mg of un-ionized (free) nicotine per gram of

the tobacco product (mg un-ionized (free) nicotine/gram), to an

accuracy level of two decimal places for each lot and for each brand

name (e.g., Skoal Bandits Wintergreen, Skoal Long Cut Cherry, Skoal

Long Cut Wintergreen, etc.). All data should include the mean value

with a 95% confidence interval, the range of values, the number of

samples tested per lot, the number of lots per brand name, and the

estimated precision of the mean. Information will be reported for each

manufacturer and variety (including brand families and brand

variations) and brand name (e.g., Skoal Bandits Wintergreen, Skoal Long

Cut Cherry, Skoal Long Cut Wintergreen, etc.).

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Sample calculation:

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V. Assay Criteria for Quality Assurance

A. Establishing Limits for Quality Control Parameters

All quality control parameters must be determined within the

laboratory in which they are to be used. At least 10 within-laboratory

runs must be performed to establish temporary confidence intervals for

the quality control parameters. Permanent limits should be established

after 20 runs and should be reestablished after each additional 20

runs.

B. Exclusion of Outliers From the Calibration Curve \18\

The coefficient of determination between Areanicotine/

AreaIS and nicotine concentration should be equal to 0.99 or

higher. Any calibration standard having an estimated concentration

computed from the regression equation (Equation 1) which is different

from its actual concentration by a factor of 10% can be excluded from

the calibration curve. Up to two concentrations may be excluded, but

caution should be used in eliminating values, since bias may be

increased in the calibration curve. If an outlier value is eliminated,

its duplicate value must also be discarded to avoid producing a new

bias. All unknowns must fall within the calibration curve; therefore,

duplicate values excluded at either end of the calibration curve will

restrict the useful range of the assay.

C. Quality Control Pools and Run Rejection Rules

The mean estimated nicotine concentration in a pool should be

compared with the established limits for that pool based on at least 20

consecutive runs. An analytical run should be accepted or rejected

based upon the following set of rules adapted from Westgard et al.

(1981).

1. When the mean of one QC pool exceeds the limit of x

3 standard deviations (SD), then the run is rejected as out of control.

Here, x and SD represent the overall mean and standard deviation of all

estimated nicotine concentrations for a particular pool in the runs

which were used to establish the control limits.

2. When the mean nicotine concentrations in two QC pools in the

same run exceed the same direction, then the run must be rejected. The

same direction is the condition in which both pools exceed either the x

+ 2 SD or the x -2 SD limits.

3. When the mean nicotine concentrations in one or two QC pools

exceed their x 2 SD limits in the same direction in two

consecutive runs, then both runs must be rejected.

4. When the mean nicotine concentrations in two QC pools are

different by more than a total of 4 SD, then the run must be rejected.

This condition may occur, for example, when one QC pool is 2 SD greater

than the mean, and another is 2 SD less than the mean.

Endnotes

The comments and notes listed below can be described as Good

Laboratory Practice guidelines; they are described in detail in this

protocol to ensure minimal interlaboratory variability in the

determination of nicotine, total moisture, and pH in smokeless

tobacco.

\1\ This protocol assumes that the testing facility will

implement and maintain a stringent Quality Assurance/Quality Control

program to include, but not be limited to, regular interlaboratory

comparisons, determination of the quality and purity of purchased

products, and proper storage and handling of all reagents and

samples.

\2\ When a specific product or instrument is listed, it is the

product or instrument that was used in the development of this

method. Equivalent products or instruments may also be used. Use of

trade names is for identification only and does not constitute

endorsement by the Public Health Service or the U.S. Department of

Health and Human Services.

\3\ All chemicals, solvents, and gases are to be of the highest

purity.

\4\ Companies must ensure that the purity of the nicotine base

is certified by the vendor and that the chemical is properly stored.

However, nicotine base oxidizes with storage, as reflected by the

liquid turning brown. If oxidation has occurred, the nicotine base

should be distilled prior to use in making a standard solution.

\5\ A suggested method for the determination of nicotine purity

is CORESTA Recommended Method No. 39.

\6\ Horizontal shaking will allow more intimate contact of this

three phase extraction. There is a minimal dead volume in the tube

due to the large sample size and extraction volume. This

necessitates horizontal shaking.

\7\ If linear shaker is not available, a wrist action shaker

using 250 mL stoppered Erlenmeyer flasks can be substituted. Values

for nicotine are equivalent to those obtained from the linear

shaker.

\8\ After installing a new column, condition the column by

injecting a tobacco sample extract on the column, using the

described column conditions. Injections should be repeated until

areas of IS and nicotine are reproducible. This will require

approximately four injections. Recondition column when instrument

has been used infrequently and after replacing glass liner.

\9\ Glass liner and septum should be replaced after every 100

injections.

\10\ Most older instruments operate at constant pressure. To

reduce confusion, it is suggested that the carrier gas flow through

the column be measured at the initial column temperature.

\11\ The testing facility must ensure that samples are obtained

through the use of a survey design protocol for sampling ``at one

point in time'' at the factory or warehouse. The survey design

protocol must address short-, medium-, and long-term smokeless

tobacco product variability (e.g., variability over time and from

container to container of the tobacco product) in a manner

equivalent to that described for cigarette sampling in Annex C of

ISO Protocol 8243. Information accompanying results for each sample

should include, but not be limited to:

1. For each product--manufacturer and variety (including brand

families and brand variations) and brand name (e.g., Skoal Bandits,

Skoal Long Cut Cherry, Skoal Long Cut Wintergreen, etc.)

information.

2. Product ``category,'' e.g., loose leaf, plug, twist, dry

snuff, moist (wet) snuff, etc.

3. Lot number.

4. Lot size.

5. Number of randomly sampled, sealed, packaged (so as to be

representative of the product that is sold to the public) smokeless

tobacco products selected per lot (sampling fraction) for nicotine,

moisture, and pH determination.

6. Documentation of method used for random sample selection.

7. ``Age'' of product when received by testing facility and

storage conditions prior to analysis.

\12\ Extraction of nicotine and pH determination must be

performed with reagents and samples at a room temperature of 22-

25 deg.C. Room temperature should not vary more than 1 deg.C during

extraction of nicotine or pH determination.

\13\ Use non-glass 10 mL repipette for transferring NaOH

solution.

\14\ Use 50 mL repipette for transferring MTBE.

\15\ For dry snuff, use 0.500 0.010 gram sample.

\16\ The testing facility is referred to ISO Procedure 8243 for

a discussion of sample size and the effect of variability on the

precision of the mean of the sample (ISO 8243, 1991).

\17\ When analyzing new smokeless tobacco products, extract

product without IS to determine if any components co-elute with the

IS or impurities in the IS. This interference could artificially

lower calculated values for nicotine.

\18\ The calculated nicotine values for all samples must fall

within the low and high nicotine values used for the calibration

curve. If not, prepare a fresh nicotine standard solution and an

appropriate series of standard nicotine dilutions. Determine the

detector response for each standard using chromatographic conditions

described in I.E.

\19\ The method is a modification of AOAC Method 966.02 (1990)

in that the ground tobacco passes through a 4 mm screen rather than

a 1 mm screen.

\20\ When drying samples, do not dry different products (e.g.,

moist (wet) snuff, dry snuff, loose leaf) in the oven at the same

time since this will produce errors in the moisture determinations.

\21\ The method is based on a method published by Henningfield

et al. (1995).

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References

AOAC (Association of Official Analytical Chemists). Official

Methods of Analysis. 966.02: Moisture in Tobacco. (1990) Fifth

Edition. K. Helrich (ed). Association of Official Analytical

Chemists, Inc. Suite 400, 2200 Wilson Boulevard, Arlington, Virginia

22201 USA.

CORESTA (Centre de Coopration pour les Recherches Scientifiques

relatives au Tabac). Recommended Method No. 39: Determination of the

purity of nicotine and nicotine salts by gravimetric analysis--

Tungstosilic acid method November, 1994. 87-90.

CRC Handbook of Chemistry and Physics. R.C. Weast, D.R. Lide,

M.J. Astle, and WH. Beyer (eds). 70th ed. Boca Raton, Florida: CRC

Press (1989-1990) D-162.

Henningfield, J.E., Radzius A., Cone E.J. (1995). Estimation of

available nicotine content of six smokeless tobacco products.

Tobacco Control 4:57-61.

ISO (International Organization for Standardization). IOS 8243:

Cigarettes--Sampling. (1991). Second Edition. Prepared by Technical

Committee ISO/TC 126, Tobacco and tobacco products. International

Organization for Standardization, Case Postale 56, CH-1211 Genve 20,

Switzerland.

Westgard J.O., Barry P., Hunt M., and Groth T. (1981). A multi-

rule Shewhart chart for quality control in clinical chemistry.

Clinical Chemistry 27:493.

[FR Doc. 99-7022 Filed 3-22-99; 8:45 am]

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