FRN Revisions to Lab Protocol

712

Federal Register / Vol. 74, No. 4 / Wednesday, January 7, 2009 / Notices

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DATE AND TIME: Thursday, January 8,
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BILLING CODE 6715–01–M

DEPARTMENT OF HEALTH AND
HUMAN SERVICES
Centers for Disease Control and
Prevention
Notice Regarding Revisions to the
Laboratory Protocol To Measure the
Quantity of Nicotine Contained in
Smokeless Tobacco Products
Manufactured, Imported, or Packaged
in the United States
AGENCY: Centers for Disease Control and
Prevention (CDC), Department of Health
and Human Services.
ACTION: Notice and Summary of Public
Comments.

SUMMARY: This notice amends the
uniform protocol for the analysis of
nicotine, total moisture, and pH in
smokeless tobacco products
STATUS: This meeting will be open to the (‘‘Protocol’’). The Protocol, originally
public.
published in the Federal Register in
1999 (64 FR 14086) and revised in the
ITEMS TO BE DISCUSSED:
Federal Register on March 14, 2008 (73
Correction and Approval of Minutes.
FR 13903), implements the requirement
Management and Administrative
of the Comprehensive Smokeless
Matters.
Tobacco Health Education Act
Individuals who plan to attend and
(CSTHEA) of 1986 (15 U.S.C. 4401 et
require special assistance, such as sign
seq., Pub. L. 99–252) that each person
language interpretation or other
manufacturing, packaging, or importing
reasonable accommodations, should
smokeless tobacco products shall
contact Mary Dove, Commission
annually provide the Secretary of Health
Secretary, at (202) 694–1040, at least 72
and Human Services (HHS) with a
hours prior to the hearing date.
specification of the quantity of nicotine
contained in each smokeless tobacco
DATE AND TIME: Friday, January 9, 2009,
product. CDC re-published the notice in
at 10 a.m.
the Federal Register on June 23, 2008
PLACE: 999 E Street, NW., Washington,
(73 FR 35395) concerning the revision of
DC.
the Protocol (1) To make a technical
STATUS: This meeting will be closed to
change to correct the date when the first
the public.
report of information under the revised
Protocol is due and (2) to solicit public
ITEMS TO BE DISCUSSED:
comments concerning a change in the
Compliance matters pursuant to 2
Protocol that increased the volume of
U.S.C. 437g.
water in the pH determination from 10
Audits conducted pursuant to 2
mL to 20 mL, and (3) to solicit public
U.S.C. 437g, § 438(b), and Title 26,
comments concerning the addition of
U.S.C.
the following commercial smokeless
Matters concerning participation in
tobacco product categories: dry snuff
civil actions or proceedings or
portion packs, snus, snus portion packs,
arbitration.
and pellet or compressed. This Notice
also includes a summary of public
Internal personnel rules and
comments and CDC’s response to them.
procedures or matters affecting a
The Protocol as published in the
particular employee.
Federal Register on March 14, 2008 (73
FR 13903), remains in effect with the
* Session Closed—Exempt pursuant to 5 U.S.C.
552b(c)(8) and (9).
technical correction to the date as

999 E Street, NW., Washington,
DC (Ninth Floor).
PLACE:

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described in the Federal Register notice
published on June 23, 2008 (73 FR
35395).
DATES: First report of information due
June 30, 2009, with subsequent
submissions due by March 31 of each
year.
FOR FURTHER INFORMATION, CONTACT:
Matthew McKenna, M.D., Director,
Office on Smoking and Health, Centers
for Disease Control and Prevention,
Telephone: (770) 488–5701.
SUPPLEMENTARY INFORMATION: Since the
implementation of the Protocol in 1999,
several smokeless tobacco product
categories have entered the U.S.
smokeless tobacco market including
snus, low moisture snuff sold in portion
pouches, and smokeless tobacco sold in
a compressed, pellet form. Some of the
new smokeless tobacco product
categories differ physically from
previous smokeless tobacco categories,
prompting a revision to the Protocol to
reflect the current state of the
marketplace.
Through its review of the Protocol,
CDC also determined that an increase in
volume of deionized, distilled water
would facilitate measurements of pH
values. After evaluating information that
was brought to the attention of CDC
regarding low moisture smokeless
tobacco products packaged in portion
pouches, CDC conducted an
independent comparison of pH
measurements in a wide variety of low
and high moisture smokeless tobacco
products. The results of the comparison
indicated an acceptable (less than 2%)
level of change in pH values when
measurements were taken with 20 mL
deionized, distilled water compared to
the volume of deionized, distilled water
specified in the previous Protocol.
Increasing the volume of water in the
mixture ensured that the matrix was
sufficiently fluid to facilitate ease of
measure. Thus, it is anticipated that the
change in the volume of liquid for pH
determination will facilitate the ease of
measure of smokeless tobacco pH for all
currently marketed smokeless tobacco
categories (i.e., plug, twist, moist snuff,
dry snuff, snus, loose leaf, chew, moist
snuff in portion pouches, smokeless
tobacco compressed into a pellet, and
dry snuff in portion pouches).
Summary of Public Comments and
CDC’s Response: On June 23, 2008, a
notice (73 FR 35395) was published
reflecting the above discussed revisions
to the Protocol and to solicit public
comment on these specific changes. Six
comments were received by the CDC, a
majority of which suggested alternative
approaches. A summary of the

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Federal Register / Vol. 74, No. 4 / Wednesday, January 7, 2009 / Notices
comments received and CDC’s response
follows.
One commenter expressed a concern
for the Federal funding and overall
direction of the ‘‘smokeless tobacco
program.’’
The issues raised in this comment
were beyond the scope of the Protocol
and solicitation of public comment.
One commenter, on behalf of several
smokeless tobacco manufacturers,
agreed with the proposed revision of
Section IV(B) (see below for Protocol) of
the Protocol to increase the volume of
deionized, distilled water to be used in
pH measurements from 10mL to 20mL.
One commenter, on behalf of several
smokeless tobacco manufacturers,
suggested that ‘‘some flexibility be
incorporated into Section IV(B) of the
Protocol by providing that, as long as a
minimum of 20 mL of liquid and 2
grams of sample are utilized, then larger
amounts of liquid and sample may be
utilized provided they are in a 10 to 1
ratio.’’
CDC appreciated the suggestion that
there be flexibility in adjusting the
quantity of liquid and sample so long as
the ratio of liquid to sample is 10 to 1.
In evaluating this suggestion, CDC
determined that adopting such a change
would deviate from principles of good
scientific practice as it does not promote
protocol consistency, contrary to the
aims of a uniform analytical protocol.
According to the Cooperative Centre for
Scientific Research Relative to Tobacco
(CORESTA), a central organization
responsible for promoting tobaccorelated cooperative research, ‘‘[t]he
development of standard methods is
critically important in ensuring
consistency and comparability of data
reported by the association members
and as part of regulatory reporting of
data.’’ [Further details on CORESTA’s
viewpoint and its objectives are
available online at http://
www.coresta.org/Home_Page/
PresentationCORESTA(Oct08).pdf.] As
the fundamental purpose of the Protocol
is to implement a multi-site testing
protocol, CDC concluded that the
development of a uniform analytical
protocol is paramount to ensuring
sound scientific efforts.
One commenter, on behalf of several
smokeless tobacco manufacturers, raised
the following point regarding the
categorization of smokeless tobacco
products in Section I(F) of the Protocol:
‘‘* * * many of these separate product
‘categories’ are essentially identical
smokeless tobacco products for the purposes
of sample preparation (e.g., Moist snuff and
snus; Moist snuff portion packs and snus
portion packs) * * * since a number of
smokeless tobacco manufacturers have stated

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that they are developing new or ‘innovative’
smokeless tobacco products, an approach
that creates a new ‘category’ and sample
preparation instruction every time a
smokeless tobacco product is introduced
with a different name or description will
result in a proliferation of smokeless tobacco
product ‘categories’ and a need to constantly
revise the Protocol to add new sample
preparation instructions. Such revisions
would trigger a notice and comment process
under the Administrative Procedure Act.’’

CDC made the determination to
include the four newly listed categories
after having reviewed the number and
types of smokeless tobacco products
that had entered the market since 1999.
In this review, CDC concluded that
several new products would benefit
from a separate categorization to not
only better aid manufacturers in
distinguishing their products in this
protocol, but also reflect the variety of
products being sold to and recognized
by consumers. This review also
determined that in the years since the
implementation of the Protocol in 1999,
the quantities of new products
introduced to market requiring separate
categorization had been fairly limited;
thus, CDC did not believe that constant
revisions to the Protocol would be
necessary. However, CDC will continue
to monitor the introduction of new
smokeless tobacco products and provide
assistance to reporting entities on the
application of the Protocol as needed.
One commenter, on behalf of several
smokeless tobacco manufacturers,
suggested an alternative approach that
would ‘‘eliminate, or at the least
minimize, the need for new ‘categories’
and sample preparation instructions.’’
This alternative proposal suggested
that:
‘‘The alternative approach would be to
define the smokeless tobacco product
categories based on physical characteristics
relevant to sample preparation (essentially
tobacco particle size and whether tobacco
particles are in a pouch), rather than on a
manufacturer’s package label statement or
description * * * ’’

Three product categories were thus
proposed.
If any products did not fall into the
three categories, the proposal suggested
that:
‘‘* * * in the event that a smokeless
tobacco manufacturer or importer believes
that a newly marketed smokeless tobacco
product does not fit within any of the above
categories, then samples should be prepared
in a manner compatible with the above
sample preparation instructions and the
manufacturer or importer should describe the
sample preparation procedures used when
making its submissions to CDC.’’

After an evaluation of this alternative
approach, CDC concluded that the

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current method of categorization is more
appropriate for several reasons. First,
the current method has been in place
since 1999, with no noted difficulties
associated with this product
categorization. Second, CDC noted that
other Federal agencies, such as the
Federal Trade Commission (FTC) and
United States Department of Agriculture
(USDA), receive and review information
on smokeless tobacco, not on the basis
of physical size characteristics, but on
these commonly accepted types of
categories. Examples can be found in
the FTC’s ‘‘Federal Trade Commission
Smokeless Tobacco Report for the Years
2002–2005,’’ available online at http://
www.ftc.gov/reports/tobacco/0205smokeless0623105.pdf, or in the
USDA Economic Research Service’s
‘‘Tobacco Situation and Outlook
Yearbook’’, available online at http://
usda.mannlib.cornell.edu/usda/ers/
TBS-yearbook//2000s/2007/TBSyearbook-01-12-2007.pdf/.
Furthermore, CDC viewed the existing
categorization of products by traditional
‘‘consumer-oriented’’ descriptions as
useful in easily identifying issues that
concern the general consumer and the
overall public’s health. In contrast,
adopting a method of categorization
based solely on physical product
characteristics would not be beneficial
towards that goal.
Finally, during its review of this
alternate approach, CDC noted that
there are only three existing methods to
prepare smokeless tobacco products for
analysis in this protocol, despite the
varied physical characteristics of
currently marketed smokeless tobacco
products.
One commenter, on behalf of several
smokeless tobacco manufacturers,
suggested that ‘‘the reporting provision
of the FRN be amended to provide the
following: (i) The revised Protocol shall
take effect January 1, 2009, and (ii) the
first report of information pursuant to
the revised Protocol is due March 31,
2010, with subsequent submissions due
by March 31 of each year. This
amendment would afford smokeless
tobacco manufacturers a reasonable
amount of time to prepare for the
implementation of the revised Protocol,
and would continue the current practice
of manufacturers submitting a full year
of data based on a consistent
methodology.’’
For the purposes of this comment,
CDC took into consideration a Federal
Register Notice published in March
2008 (73 FR 13903), which served as
public notice about the changes in the
Protocol. CDC regarded this duration of
notice as sufficient for the first report of
information to be due June 30, 2009,

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Federal Register / Vol. 74, No. 4 / Wednesday, January 7, 2009 / Notices

with subsequent submissions due by
March 31 of each year, as laid out in the
June 23, 2008 Federal Register (73 FR
35395).
Collection of Information
This proposed amendment does not
call for any new collection of
information under the Paperwork
Reduction Act of 1995 (44 U.S.C. 3501–
3520).
Dated: December 29, 2008.
James D. Seligman,
Chief Information Officer, Centers for Disease
Control and Prevention.

Revised 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 μm)
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°C. Suggested
dimensions: 18 x 18 x 20inches.
E. Chromatographic Conditions 8 9
1. Detector temperature: 250°C
2. Injector temperature: 250°C
3. Flow rate at 100°C—1.7 mL/min; with
split ratio of 40:1 10
4. Injection volume: 2 μl

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5. Column conditions: 110–185°C at
10°C min¥1; 185–240°C at 6°C min¥1,
hold at final temperature for 10 min.
F. Sample Preparation 11
There are ten different categories of
commercial smokeless tobacco
products:
1. Dry snuff;
2. Moist (wet) snuff;
3. Moist (wet) snuff portion packs;
4. Plug;
5. Twist;
6. Loose leaf;
7. Dry snuff portion packs;
8. Snus;
9. Snus portion packs; and
10. Pellet or Compressed.
Because of their physical
characteristics, some of the ten product
categories must be ground (whole or in
part) 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 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. Freeze- or cryogrinding is also an acceptable grinding
method.
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

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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.
7. Dry snuff portion packs: The
tobacco contents of the dry 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 dry snuff
portion pack and the ground pouch are
combined and thoroughly mixed before
pH analysis.
8. Snus: Snus 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.
9. Snus portion packs: The tobacco
contents of the snus 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 snus
portion pack and the ground pouch are
combined and thoroughly mixed before
pH analysis.
10. Pellet or compressed: Break apart
compressed tobacco pellets 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.

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Federal Register / Vol. 74, No. 4 / Wednesday, January 7, 2009 / Notices

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
(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

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Equation 2:
Recovery = Ni cot inecalculated /Ni cot ineactual
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 smokeless tobacco product is
tested and whenever a change is made
to the product formulation (including a
change to the tobacco blend or cultivar),
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).

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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.
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 Sample each
smokeless tobacco brand name
according to the provided testing
frequency schedule.19 The number of

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EN07JA09.001

A. Calibration Standards

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:

EN07JA09.000

II. Nicotine Analysis 12

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Federal Register / Vol. 74, No. 4 / Wednesday, January 7, 2009 / Notices

Equation 3:18

Ni cot ine (mg/g) =

( Area nicotine /Area IS ) − a
b × Sample Wt × Recovery

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 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, 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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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°C.
B. Accurately weigh 5.00 grams of the
sample (ground to pass ≤ 4 mm
screen) 20 into a weighed moisture dish
and place uncovered dish in oven.21
Sample each smokeless tobacco brand
name according to the provided testing
frequency schedule.19 The number of
products sampled 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 of finished product
that is ready for commercial
distribution. Samples are to be analyzed
in duplicate.
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°C. Remove
from oven, cover, and cool in desiccator
to room temperature (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 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, 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 22
A. Test samples as soon as possible
after they are received. Sample each
smokeless tobacco brand name
according to the provided testing
frequency schedule.19 The number of
products sampled should reflect an
acceptable level of precision.16 The test
material is to be representative of the

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product that is sold to the public and
therefore should consist of sealed,
packaged samples of finished product
that is ready for commercial
distribution. Samples are to be analyzed
in duplicate.
B. Accurately weigh 2.00 grams of the
sample. Place in a 50 mL polypropylene
container with 20 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 twopoint 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 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 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 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, 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 HendersonHassel Balch equation (Equation 4),
based on measured pH and nicotine
content.

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EN07JA09.002

products sampled 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 of finished product
that is ready for commercial
distribution. Samples are to be analyzed
in duplicate.
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

Federal Register / Vol. 74, No. 4 / Wednesday, January 7, 2009 / Notices

717

Equation 4:
pH = pKa + log

[B]
⎡⎣ BH + ⎤⎦

B + H +  BH +
[B]
⎡⎣ BH + ⎤⎦
% un-ionized (free) nicotine =
× 100
[B]
+1
+
⎡⎣ BH ⎤⎦
pKa = 8.02 (CRC Handbook of Chemistry and Physics, 1989-1990)
[B] = amount of un--ionized (free) nicotine
[BH + ] = amount of ionized nicotine
H. Report the final estimated unionized (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 unionized (free) nicotine/gram), to an
accuracy level of two decimal places 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, 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.).
Sample calculation:

Mean total nicotine = 10.30 (mg/g)
Mean pH = 7.50
pKa = 8.02
pH = pKa + log

[ B]
⎡⎣ BH + ⎤⎦

7.50 = 8.02 + log

−0.52 = log
0.302 =

[un-ionized (free) nicotine]
[ionized nicotine]

[un-ionized (free) nicotine]
[ionized nicotine]

[un-ionized (free) nicotine]
[ionized nicotine]

[B]
⎡⎣ BH + ⎤⎦
% un-ionized (free) nicotine =
× 100
[B]
+
1
⎡⎣ BH + ⎤⎦
% un-ionized (free) nicotine =

0.302
× 100
0.302+1

Total free nicotine (mg/g) = 10.30 ×

% un-ionized (free) nicotine
100

23.20
100

Total free nicotine (mg/g) = 2.39

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Total free nicotine (mg/g) = total nicotine ×

EN07JA09.004

% unn-ionized (free) nicotine = 23.20

718

Federal Register / Vol. 74, No. 4 / Wednesday, January 7, 2009 / Notices

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.

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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 a 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

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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 longterm 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:
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.):
1. Product ‘‘category,’’ e.g., loose leaf,
plug, twist, dry snuff, moist (wet) snuff,
etc.
2. Lot number.
3. Lot size.
4. Number of randomly sampled,
sealed, packaged (so as to be
representative of the product that is sold
to the public) smokeless tobacco
products selected (sampling fraction) for
nicotine, moisture, and pH
determination.
5. Documentation of method used for
random sample selection.
6. ‘’’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°C. Room
temperature should not vary more than
1°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

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Federal Register / Vol. 74, No. 4 / Wednesday, January 7, 2009 / Notices
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 testing frequency for each
smokeless tobacco brand name (e.g.,
Skoal Bandits Wintergreen, Skoal Long
Cut Cherry, Skoal Long Cut
Wintergreen, etc.) is based on the
manufacturing duration (refer to table
below). Each smokeless tobacco brand
name will be sampled and tested for
nicotine, total moisture, and pH no
fewer than twice and no more than four
times during a calendar year.

mean of the 4 data points for that smokeless
tobacco brand name are reported.
Example 2: Within a single calendar year
a smokeless tobacco brand name is
manufactured from April 5 to May 3 and
from September 1 to December 15. The
testing frequency for the first manufacturing
interval is 2 and for the second
manufacturing interval is 3. The values for
nicotine, moisture, and pH determinations,
and unionized (free) nicotine calculations
and the mean of the 4 data points for that
smokeless tobacco brand name are reported.
Example 3: Within a single calendar year
a smokeless tobacco brand name is
manufactured from January 1 to January 15
and from September 1 to September 22. The
testing frequency for the first manufacturing
interval is 2 and for the second
manufacturing interval is 2. Four random
sampling dates are selected to fall within the
6 weeks of manufacturing for the smokeless
tobacco brand name. The values for nicotine,
moisture, and pH determinations, and
unionized (free) nicotine calculations and the
mean of the 4 data points for that smokeless
tobacco brand name are reported.

20 The method is a modification of
AOAC Method 966.02 (1990) in that the
Manufacturing duration in
Test
ground tobacco passes through a 4 mm
weeks
frequency*
screen rather than a 1 mm screen.
21 When drying samples, do not dry
up to and including 4 ................
2
up to and including 28 ..............
3 different products (e.g., moist (wet)
up to and including 52 ..............
4 snuff, dry snuff, loose leaf) in the oven
at the same time since this will produce
*Use a statistical program to determine random sampling dates based on the total manu- errors in the moisture determinations.
22 The method is a modification of a
facturing duration during a calendar year.
Sampling dates should fall on actual manufac- method published by Henningfield et al.
turing days for the product when test material (1995).

that is representative of the product that is
sold to the public (consisting of sealed, packaged samples) is available. If a statistically determined sampling date falls on a day that
does not meet this criterion, sample the product on the next date that does meet the
criteria.

For smokeless tobacco brand names
with episodic production during a
calendar year, the total number of
sampling dates is determined by the
sum of the individual test frequencies,
not to exceed four. For the purpose of
the Protocol, episodic production is
defined as manufacturing intervals
separated by periods of 30 or more days
when the smokeless tobacco brand
name is not manufactured.
Example 1: Within a single calendar year
a smokeless tobacco brand name is
manufactured from January 1 to March 31
and from September 1 to December 15. The
testing frequency for the first manufacturing
interval is 3 and for the second
manufacturing interval is 3. The Protocol
allows that each smokeless tobacco brand
name be tested for nicotine, total moisture,
and pH no more than four times during a
calendar year. Therefore, 4 random sampling
dates, as described in the footnote to the
above table, are determined for the smokeless
tobacco brand name. The values for nicotine,
moisture, and pH determinations, and
unionized (free) nicotine calculations and the

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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 Coope´ration
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.

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719

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. E9–19 Filed 1–6–09; 8:45 am]
BILLING CODE 4163–18–P

DEPARTMENT OF HEALTH AND
HUMAN SERVICES
National Institutes of Health
Center for Scientific Review; Notice of
Closed Meetings
Pursuant to section 10(d) of the
Federal Advisory Committee Act, as
amended (5 U.S.C. Appendix 2), notice
is hereby given of the following
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The meetings will be closed to the
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provisions set forth in sections
552b(c)(4) and 552b(c)(6), Title 5 U.S.C.,
as amended. The grant applications and
the discussions could disclose
confidential trade secrets or commercial
property such as patentable material,
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individuals associated with the grant
applications, the disclosure of which
would constitute a clearly unwarranted
invasion of personal privacy.
Name of Committee: Center for Scientific
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Conflict: Auditory Neuroscience.
Date: January 22–23, 2009.
Time: 6 a.m. to 5 p.m.
Agenda: To review and evaluate grant
applications.
Place: National Institutes of Health, 6701
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(Virtual Meeting)
Contact Person: John Bishop, PhD,
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Name of Committee: Center for Scientific
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Agenda: To review and evaluate grant
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Place: National Institutes of Health, 6701
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Contact Person: Fungai F. Chanetsa, PhD,
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