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A Method to Amend Cigarettes with Menthol for use in Research

Kia J. Jackson, PhD , Megan J. Schroeder, PhD

, Jiu Ai, PhD

, Kenneth M. Taylor, PhD

Olusola Oniyide, PhD ,Lauren C. Viray, BS

, Carson Smith, MPA, MPH

, Bartosz

Koszowski, PhD , Jenny M. Butler

, Wallace B. Pickworth, PhD

Food and Drug Administration, Center for Tobacco Products, Office of Science, 11785

Beltsville Drive, Calverton, MD 20705;

Food and Drug Administration, Center for

Veterinary Medicine, Office of Research, 8401 Muirkirk Road, Laurel, MD 20708;

Battelle

Center for Public Health Research, 6115 Falls Road, Suite 200, Baltimore, MD 21209;

Battelle Center for Public Health Research, 505 King Avenue, Columbus, OH 43201

Corresponding author: Kia J. Jackson; kia.jackson@fda.hhs.gov

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Abstract

Introduction: Studies have evaluated the role of menthol cigarettes on various addiction-related outcomes; however, the effect of varying menthol content on these outcomes has not been evaluated. We developed a method to amend non-menthol SPECTRUM Research

Cigarettes to contain menthol at 4 different levels. Methods: SPECTRUM Research

Cigarettes, NRC 600 (0.8 mg nicotine; 10 mg tar), were modified to contain target menthol amounts at 3, 6, and 12 mg/cigarette by injecting 25 µL ethanol/triacetin/menthol solutions of varying concentrations (120 mg menthol/mL, 240 mg/mL, and 480 mg/mL) into 4 distinct locations in the filter and tobacco rod. Menthol content was tested in triplicate in the whole cigarette and in the tobacco rod and filter at 1, 24, 48, and 72 hr for each target menthol level using an extraction solution of quinoline in methyl-tert-butyl ether and measured using gas chromatography with flame ionization detection. Results: Injections into the filter and tobacco rod (12.5 µL each) yielded equal menthol distribution up to 72 hr. However, total menthol content decreased from an average of 90.3% of the target menthol concentration at 1 hr to 80.7% at 72 hr in cigarettes stored individually in glass tubes at room temperature.

Analysis of urinary menthol glucuronide confirmed that amended cigarettes used within 24 hr of injection delivered dose-related menthol levels to participants in a clinical laboratory setting. Conclusion: This method can be used to modify cigarettes with a range of reliable menthol levels in both filter and tobacco rod for use in laboratory and clinical research.

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Implications: This study presents a technique for modifying cigarettes with different levels of menthol that can reliably deliver dose-related menthol levels to participants when smoked in a clinical study. The technique can be used to quickly amend cigarettes to examine the independent effects of varying flavor and additive levels on smoking behavior, nicotine pharmacokinetics, mainstream smoke emissions, and other laboratory or clinical research outcomes.

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INTRODUCTION

Menthol is a widely used compound found in drug products, food, and cosmetics, and is a distinctive flavor additive in tobacco products. Mentholated cigarettes have been part of the

U.S. tobacco marketplace since the 1920s and are disproportionately used by youth, young adults, and other at-risk populations such as African Americans and other racial ethnic groups.

The range of menthol content in commercially available cigarettes labeled as menthol varies by brand, and menthol may be present in small quantities in non-menthol cigarettes.

Menthol imparts a minty taste and cooling sensation when inhaled.

In cigarettes, menthol’s sensory effects reduce dryness, irritation, unpleasant tobacco taste, and other negative sensory attributes of smoking, thus promoting cigarette smoking.

5-7

However, because high concentrations of menthol can produce tingling, stinging, and burning in the mouth and throat, cigarette menthol levels are adjusted to achieve the desired subjective response.

5, 8, 9

Studies have evaluated the effects of menthol cigarettes (compared to non-menthol cigarettes) on addiction-related outcomes, including nicotine pharmacokinetics and exposure, 10, 11 subjective measures, 12, 13 and use behaviors (e.g., smoking topography).

However, studies have not evaluated the effect of varying menthol content on these outcomes. Tobacco industry documents indicate that the varying menthol levels in cigarettes appeal to different populations of menthol smokers. For example, cigarettes with lower menthol content appeal to new, younger smokers by reducing negative sensory effects of smoking, while cigarettes with higher menthol content produce stronger sensations suited for longer-term menthol smokers.

5, 15

Therefore, understanding how different menthol levels may influence sensory effects and smoking behavior is an important consideration.

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In cigarette manufacturing, menthol may be applied directly to the tobacco rod, filter (e.g.

menthol beads), or packaging, but migrates to other parts of the cigarette irrespective of the original application. In the lab, injection of alcohol-based menthol solutions to add menthol to cigarettes was mentioned by Miller et al.

MacGregor et al.

described a passive absorption technique to mentholate Camel full flavor cigarettes using a direct vapor deposition procedure. Desired menthol levels that were consistent with commercial cigarette menthol levels were primarily achieved after 72 hr of vapor exposure.

In this study, we describe a method to amend cigarettes by directly injecting menthol into the tobacco rod and filter. Cigarettes were successfully prepared at various menthol levels, including within the range of menthol commercial cigarettes. This injection method was chosen because the desired maximum menthol level (12 mg/cigarette) was higher than the maximum menthol levels achieved with the passive absorption method.

Following the amendment process, we measured menthol levels to assess stability and determine menthol loss with time. We further verified the utility of the amended cigarettes by measuring menthol glucuronide levels in urine following use by participants in a research study.

METHODS

Cigarettes

The National Institute of Drug Abuse SPECTRUM NRC 600 cigarettes (non-menthol, filtered 85 mm) were chosen to amend with menthol because they deliver an intermediate level of tar (10 mg) and nicotine (0.8 mg). Additionally, the cigarettes are not commercially available and would be novel products for all participants in a clinical study.

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Preparation of stock menthol solutions

The menthol ((1R,2S,5R)-2-Isopropyl-5-methylcyclohexanol; Alfa Aesar A 10474-18) solutions were prepared by dissolving the weighed amount of menthol with vehicle solvent in

100 mL volumetric flasks (accuracy: ±0.08 mL at 20°C). The vehicle solvent was prepared by adding 99.5% ethanol to 10 mL of triacetin solution to make approximately 1000 mL of vehicle. Menthol solutions were labeled with concentration, contents, and date of preparation and stored in four 25 mL amber storage bottles at ambient room temperature.

Cigarette menthol addition

Each cigarette was injected (by a blinded staff member using a 50 μL Hamilton syringe) with

25μL of solutions containing 0, 120, 240, and 480 mg/mL l-menthol to yield target menthol contents of approximately 0 mg, 3 mg, 6 mg, and 12 mg of menthol/cigarette, respectively.

These menthol levels were selected to bracket the published menthol contents in U.S.

marketed cigarettes (i.e., 2.9- 6.7 mg/cigarette of menthol) and a higher content for experimental purposes. Solutions were injected into the filter end (with 6.25 μL of solution each deposited at depths of 20 mm and 10 mm, respectively) and the tobacco rod end (with

6.25 μL of solution injected each at depths of 50 mm and 25 mm, respectively). Four injections were used to minimize migration outside the cigarette and promote uniform menthol distribution within the tobacco rod and filter, thereby ensuring a consistent transfer of menthol to the smoke during a subsequent clinical study. Amended cigarettes were maintained individually in sealed glass vials at ambient room temperature up to 72 hr, and all were labeled with an identifier, date, and time of preparation.

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Verification of stock menthol solutions

Menthol was quantitated against 7-point calibration curves covering 5 to 1000 μg/mL, which is approximately equivalent to 0.1 to 20 mg nicotine and menthol/cigarette. Menthol solutions were prepared by spiking calculated volumes of each menthol solution into a 10 mL volumetric flask and then bringing it to volume with extraction solvent (methyl-tert-butyl ether [MTBE] spiked with quinoline [100 μg/mL]). An aliquot of the extraction solvent was then removed and transferred to an autosampler vial for analysis on an Agilent 6890 gas chromatograph with flame ionization detection (GC-FID) using a capillary, fused silica, 15 m x 0.53 mm ID DB-WAX analytical column (Agilent 125-7012). The data acquisition/reduction software was an online data system EXChrom used for data acquisition, peak integration, post-acquisition data processing, and reporting. The predetermined acceptable range for menthol stock solutions was within 20% of the target (Timepoint 0) concentrations.

Determination of menthol in the whole cigarette

Analyses were performed on cigarettes that were broken down to tobacco rod and filter in a single assay vial. Each portion was weighed, and the tobacco rod and filter were placed into a

40 mL glass extraction vial. Extraction solvent was then added, and the sample was placed on an orbital shaker at 160 rpm for 4 hours at ambient laboratory conditions. Following extraction, an aliquot of the extract was transferred to an autosampler vial for analysis by GC-

FID.

Menthol was quantitated against 7-point calibration curves covering 5 to 1000 μg/mL, which is approximately equivalent to 0.1 to 20 mg menthol/cigarette. Samples were extracted using

MTBE and IPA in aqueous base and the extracts were analyzed via GC-FID (specifications as described). Cigarettes were also analyzed for nicotine content in the whole cigarette.

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Details for nicotine and menthol analysis are as described.

17, 18

The accetable range for menthol content in amended cigarettes was within 20% of the Timepoint 0 concentrations.

Amended cigarette clinical urinary analysis

Amended cigarettes were used in a clinical study approved by Battelle Institutional Review

Board (Federalwide Assurance: FWA00004696). Menthol glucuronide was measured in urine collected from 48 healthy, adult (age 21-65), daily menthol smokers who were not interested in quitting, recruited for a multi-visit clinical study to assess differences in cigarette menthol content on various clinical outcomes. Informed consent was obtained from all participants prior to screening. Participants were randomly assigned to smoke the non-menthol

SPECTRUM cigarettes and the three amended cigarettes in separate visits. Each visit was composed of a prescribed smoking session and a 1 hr ad libitum use session; only one cigarette type was smoked during each study visit. For prescribed use, participants were directed to smoke the assigned cigarette for 10, 2-second puffs at 30 second intervals. At 150 minutes following initiation of prescribed use, participants were directed to smoke ad libitum for 1 hr. Urine samples were collected prior to the prescribed use session and at 145 minutes

(i.e., prior to ad libitum smoking) and 210 minutes after initiating prescribed use (i.e., 60 minutes after initiation of ad libitum smoking) to assess menthol glucuronide levels.

Measured menthol glucuronide was analyzed as described in Benowitz et al.

and concentrations were normalized for creatinine. Statistical analysis was conducted using a mixed-model approach to investigate concentration-time profiles and dose-response relationships. Bonferroni procedure was used to adjust for multiple testing.

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RESULTS

Menthol solution stability

The stock menthol solutions were tested throughout this study. Menthol solutions were analyzed when initially prepared (Timepoint 0) and approximately every 3 months thereafter

(i.e., Timepoint 0 and Months 3, 6, 9, 14, 17, and 20). Overall, none of the reported stock solutions’ menthol concentrations were outside the 20% acceptable limit at any timepoint.

Over 20 months, the percent difference from Timepoint 0 ranged as follows: -8.4 % to 4.0 % for the 120 mg/mL solution (3 mg menthol/cigarette); -8.6 % to 4.1 % for the 240 mg/mL solution (6 mg menthol/cigarette); and -6.2 % to 5.8 % for the 480 mg/mL solution (12 mg menthol/cigarette). No menthol was detected in the 0 mg/mL menthol solution.

Cigarette modification

The tobacco rod, filter, and whole cigarette were analyzed at 1, 24, 48, and 72 hr following menthol addition. Menthol is reported as content in the whole cigarette. Injections into the filter and tobacco rod yielded equal and uniform menthol distribution throughout the cigarette that steadily decreased over 72 hr (Figure 1). At 1 hr, the menthol concentration was an average of 90.3% of the target concentration for all cigarettes (2.59, 5.61, and 11.08 mg menthol/cigarette for 3, 6, and 12 mg menthol/cigarette target concentrations, respectively).

The menthol percentage further decreased to an average 86.3% of the target menthol/cigarette at 24 hr, 83.0% at 48 hr, and 80.7% at 72 hr. Cigarette nicotine content was constant (~10

mg) at all menthol levels.

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Urinary analysis following amended cigarette smoking

The majority of participants were male (56%), African American (69%), aged 30-44 (46%), and smoked an average of 17.1 cigarettes per day. Amended cigarettes were administered to participants within 24 hr of menthol injection. Urinary menthol glucuronide levels significantly increased in a dose-related manner following prescribed and ad libitum smoking sessions (Figure 2). Significant differences between concentrations (3, 6, and 12 mg menthol) were also identified (Supplemental Table 1).

DISCUSSION

The menthol solution injection method was successfully used to modify non-menthol

SPECTRUM cigarettes to include non-overlapping menthol levels that bracket menthol content in the U.S. and a higher dose for experimental purposes. The method allows for preparing cigarettes with approximately equal amounts of menthol in the filter and tobacco rod. The menthol stock solution stability demonstrates that they can be reliably used to prepare amended cigarettes throughout the course of extended duration research, such as clinical studies. Participants also found the amended cigarettes to be acceptable for smoking in the study. However, cigarette menthol content was not stable, as the concentration steadily decreased in the rod and filter over the course of 72 hr. Thus, menthol should be injected into cigarettes within 24 hr of testing or administering to study participants and discarded 24 hr after preparation to ensure target menthol levels. In this regard, studies may be limited to inpatient studies as opposed to the participant’s natural environment. Testing beyond 72 hr would be necessary to further characterize the extent of the decrease in cigarette menthol content and the feasibility of this method for use with longer term studies.

Analysis of urinary menthol glucuronide confirmed that amended cigarettes delivered dose-related menthol levels to participants in a clinical laboratory setting. These data provide

D ow nloaded from https://academ ic.oup.com ted M an us evidence that the procedure adopted for cigarette mentholation exposed participants to levels of menthol that were measurable in urine up to 210 min after initiating smoking. These amended cigarettes can be used in clinical studies to understand how different levels of menthol affect nicotine pharmacokinetics, use behavior, and subjective effects or to determine levels at which menthol is perceptible to consumers in cigarettes. The method to amend these cigarettes is not onerous. A trained laboratory technician was able to prepare six cigarettes in less than 15 minutes. It is possible the technique used to amend these cigarettes could be used for other flavors, additives, or components to independently assess their isolated effect on smoking behavior, mainstream smoke emissions, or product appeal. The appropriate stability and verification procedures would be necessary to demonstrate the success of the technique for flavors, additives, or components other than menthol.

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Data Availability Statement: The data underlying this article will be shared on reasonable request to the corresponding author.

Funding: This research was funded by a contract with the Food and Drug Administration

(FDA) (HHSF223201310030I/ HHSF22301005T).

Declaration of Interests: The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Food and Drug Administration.

The authors declare no conflicts of interest.

ACKNOWLEDGEMENTS

The authors wish to acknowledge Meredith Fritz and Joel Vincent for their technical assistance in preparing the amended cigarettes.

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Figure Legends

Figure 1. Menthol concentrations in the whole cigarette 1, 24, 48, and 72 hr following injection into the tobacco rod and filter.

Figure 2. Mean difference from 0 mg menthol in creatinine adjusted urinary menthol glucuronide following prescribed (0-150 minutes) and ad libitum (150-210 minutes) smoking by menthol cigarette condition. Urine levels at 150 min represent smoking one cigarette at time= 0. Urine levels at 210 min represent smoking one cigarette at time= 0 and generally one (for some participants, two) cigarette(s) between 150 and 210 min. * denotes p<0.005 vs.

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