Levels of mint and wintergreen flavorants: Smokeless tobacco products vs. confectionery products

Food and Chemical Toxicology 48 (2010) 755–763

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Levels of mint and wintergreen flavorants: Smokeless tobacco products vs. confectionery products C. Chen a, L.M. Isabelle a,b, W.B. Pickworth c, J.F. Pankow a,b,* a

Department of Chemistry, Portland State University, Portland, OR 97207, United States Department of Civil and Environmental Engineering, Portland State University, Portland, OR 97207, United States c Battelle, Centers for Public Health Research and Evaluation, Baltimore, MD 21209, United States b

a r t i c l e

i n f o

Article history: Received 30 July 2009 Accepted 16 December 2009

Keywords: Tobacco Smokeless tobacco Flavorant compounds Menthol Methyl salicylate Confectionery

a b s t r a c t Nicotine and flavorant compound levels were measured in 10 ‘‘mint”-related sub-brands and 8 ‘‘wintergreen” sub-brands of smokeless tobacco (SLT). Also analyzed were ‘‘mint”-related and ‘‘wintergreen” confectionery products. Of the ‘‘mint” SLT, ‘‘Timberwolf Packs Mint Pouches” contained the highest menthol level (5.3 mg/g); the average for the five most-highly mentholated SLT products was 4.3 mg/g. The average for the most five most-highly mentholated confectionery products was 3.5 mg/g. For hard candy, a reported average of maximum use levels is 2.1 mg/g (Burdock, 2009). Of the ‘‘wintergreen” SLT, ‘‘Hawken Wintergreen” was found to contain the highest methyl salicylate (MS) level (29.7 mg/g). The average of the five highest SLT MS levels was 23.8 mg/g, i.e., 5 higher than the level found in the confectionery product with the highest MS level (LifeSavers Wint O Green Sugar Free, 4.6 mg/g). For hard candy, a reported average of maximum use levels is 2.0 mg/g (Burdock, 2009). Assuming 23.8 mg/g MS in SLT, SLT use at 15 g/day, 100% bodily absorption of the MS, and 60 kg body weight, the average daily intake would be 6.0 mg/kg-day, i.e., 12 the acceptable daily intake (ADI) of 0.5 mg/kg-day established for this compound by a joint FAO/WHO committee. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction Tobacco has been cultivated by human beings for thousands of years, and is now grown worldwide (Kessler et al., 1996; Mackay and Eriksen, 2002). In developed countries, both the production and use of tobacco are in decline (FAO, 2003). World production of tobacco, however, has more than doubled since 1960s (Mackay and Eriksen, 2002), and is projected to be 7.1 million tons in 2010 (FAO, 2003). This overall increase has therefore been due to increasing production and use within developing countries, for which expanding tobacco cultivation has negative environmental effects including loss of natural vegetation, degradation of soils, and consequences of pesticide use (WHO, 2008a). In terms of its social effects, worldwide, tobacco is currently used by about 48% of men and 10% of women (WHO, 2006), and leads to the premature death of 1/3 to 1/2 of its users; the average loss in longevity is 15 years (WHO, 2008b). In the US, from 2000 to 2007, the sales of all tobacco products declined 11%, and the sales of packs of cigarettes declined 18% (Connolly and Alpert, 2008). For the same period, however, sales of non-cigarette tobacco products increased 43%, and for moist * Corresponding author. Address: Department of Chemistry, Portland State University, 1930 SW 4th Avenue, Portland, OR 97207, United States. E-mail address: [email protected] (J.F. Pankow). 0278-6915/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2009.12.015

snuff, the major form of smokeless tobacco (SLT) sold in the US market, sales increased 33% (Connolly and Alpert, 2008). In 2007, of the 250 million Americans aged 12 or older, an estimated 8 million (or 3.2%) were users of SLT (SAMHSA, 2008). This rise in SLT consumption, even as cigarette consumption has declined, is considered to be due to increased public awareness of the harm associated with smoking cigarettes, smoking restrictions in the workplace, and lower taxes on SLT products (Alpert et al., 2008; Connolly and Alpert, 2008). In addition, recent marketing data suggest that the availability of a wide range of flavored SLT products has broadened the appeal of SLT (Convenience Store News, 2007). Alpert et al. (2008) have argued that manufacturers may be seeking to ‘‘feature an array of tobacco leaf cuts and flavoring categories to attract young and/or inexperienced users in addition to more experienced long term users”. Historically, hundreds of natural and synthetic organic flavorant compounds have been used as additives in both smoked and smokeless tobacco products (B&W, 1978; B&W et al., 1994; Crouse et al., 1983; Hertz, 1982; Klein et al., 2008; Lewis and Wackowski, 2006; Lorillard, 1985; RJR, 1972; Samfield, 1984). Giovino et al. (2004) stated that ‘‘menthol is added to about 90% of commercial cigarettes sold in the United States”, and contrasted this value with Federal Trade Commission data indicating that the US market share of cigarettes actually advertised as being mentholated during 1998–2001 was only 26%. Similarly, industry documents (e.g.,

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Lorillard, 1985; Vora, 1986) indicate that licorice extract has frequently been used in cigarette tobacco and in SLT products, yet tobacco products identified as being ‘‘licorice” flavored have been rare. Of the cigarette sub-brands currently marketed in the US, about 32% are advertised as being flavored, with menthol being by far the most important flavorant compound (Giovino et al., 2004). In contrast, of the approximately 100 SLT sub-brands in the US market, about 75 are flavored (Alpert et al., 2008) (data for the total market share of flavored SLT are not readily available). Of those flavored sub-brands of SLT, about 15 are marketed as ‘‘mint”, ‘‘ice”, or ‘‘frost”, and about 30 are marketed as ‘‘wintergreen” (Alpert et al., 2008). A study conducted in the state of New Jersey reports that: ‘‘flavored SLT products accounted for nearly two-thirds (62.1%) of all smokeless tobacco sold in each year between 2003 and 2005” (UMDNJ – School of Public Health, 2006). The most popular SLT flavors in New Jersey in 2005 were ‘‘mint” and ‘‘wintergreen”, accounting for 21% and 48% of total sales respectively; other flavors such as ‘‘cherry”, ‘‘berry”, and ‘‘apple” were also found to possess significant SLT market share (UMDNJ – School of Public Health, 2006). Just as the tobacco industry has been criticized for having offered fruity, chocolate, and otherwise flavored cigarette products (Carpenter et al., 2005; Prokhorov et al., 2006; Wayne and Connolly, 2002), criticisms have been made that the use of flavorant compounds in SLT products may entice children and young adults into early nicotine addiction (Connolly, 1995). Relevant flavorant-related documents internal to the tobacco industry were obtained as a consequence of litigation against the tobacco industry in the 1990s. For SLT, such documents indicate methyl salicylate values of 8.4, 11.6, and 18.3 mg/g in ‘‘wintergreen”-flavored Skoal ‘‘Bandits” (Lorillard, 1983a), Skoal Regular (Lorillard, 1983b), and Hawken ‘‘Rough Cut” (Lorillard, 1980a), respectively. In addition, one of the industry documents (Lorillard, 1980b) reports 0.57 mg/g of ethyl salicylate in Skoal ‘‘Straight”. In the open scientific literature, Sharma et al. (1991) reported a value of 21.7 mg/g of methyl salicylate in an unnamed commercial SLT brand. Reviews of the animal toxicities of two compounds of interest here are available, namely menthol and methyl salicylate (Bhatia et al., 2008; Lapczynski et al., 2007). In response to general public concerns about additives, RJR (2001) has argued that ‘Most of these ingredients are commonly used in foods and beverages, or permitted for use in foods by the US Food and Drug Administration (FDA), or have been given the status ‘‘Generally Recognized as Safe in Foods” (GRAS) by FDA, the Flavor and Extract Manufacturers Association (FEMA) or other expert committees.’ Other major tobacco companies have made similar statements (Doull et al., 1994, 1998; Philip Morris USA, 2000). In any case, ‘‘safe when used in food” does not equate with ‘‘safe when used in tobacco” (Hallagan and Hall, 2009; Pauly, 1994, 1998; Philip Morris USA, 2000). This is clearly true for cigarette additives (cigarettes are burned), but it is also likely true for additives in SLT because of potential effects of additives on the chemistry and behavior on nicotine and tobacco toxicants. An example of a potentially important effect due to flavorant compounds in SLT would be enhanced permeation of nicotine and toxicants into oral tissue. Indeed, permeation enhancement by menthol and other organic compounds has been investigated as a means to accelerate transdermal delivery of drugs (Aungst and Rogers, 1988; Rachakonda et al., 2008; Shojaei, 1998; Shojaei et al., 1999, 2001; Williams and Barry, 2004). For example, Shojaei et al. (1999) reported that menthol increases the absorption of dideoxycytidine, and Aungst and Rogers (1988) studied the effects of sodium salicylate on absorption of insulin by rat buccal tissue. Two mechanisms by which one chemical can enhance the transport of another into and through tissue are: (1) increased local

blood flow (Ryatt et al., 1986); and (2) increased permeability of biological membranes (Nicolazzo et al., 2005). [Skin and buccal tissue may be affected differently (Nicolazzo et al., 2005) by a given permeation enhancing chemical.] A clinical study by Hong and Shellock (1991) found increased blood flow following topical applications of the menthol-containing ointment EucalyptamintÒ which is marketed for its pain masking (i.e., ‘‘counterirritant”) effects. Cremes that contain both methyl salicylate and menthol are Ben GayÒ, Icy HotÒ, and Arthritis HotÒ. Despite the extensive use of flavorant compounds in SLT products, little is known about either their levels in contemporary products or their effects on the user. Menthol in cigarettes has, in contrast, received considerable study (e.g., B&W, 1986; Celebucki et al., 2005). Here we report on the levels of major flavorant compounds, nicotine, and water in selected SLT products. For comparison, the levels of the major flavorant compounds in selected confectionary products are also discussed.

2. Materials and methods 2.1. Analyses of SLT products Samples of 25 sub-brands of flavored and unflavored SLT products were purchased in the Portland, OR market (Tables 1–3). In addition, two unflavored reference SLT products (1S3 and 2S3) were obtained from North Carolina State University (2008). Each SLT sample was analyzed for nicotine, water, and seven flavorant compounds, namely menthol*, menthone*, methyl salicylate, ethyl salicylate, menthyl acetate*, carvone*, and limonene*. Each

Table 1 Nicotine and water levels in 25 commercial sub-brands of smokeless tobacco (SLT) products, and two types of standard reference SLT. All values are given ±1 standard deviation (sd) for three replicates.

a

Nicotine (mg/g)a

Water (%)

Unflavored Copenhagen Long Cut Husky Long Cut Natural Longhorn Long Cut Natural Redman Fine Cut Natural Skoal Long Cut Classic Average:

11.24 ± 0.38 11.53 ± 0.38 13.30 ± 0.38 13.16 ± 0.49 12.28 ± 0.20 12.30

54.03 ± 0.26 49.01 ± 0.22 45.68 ± 0.60 46.59 ± 0.22 51.48 ± 0.43 49.36

Flavored Camel Snus Frost Grizzly Long Cut Mint Grizzly Long Cut Straight Grizzly Long Cut Wintergreen Hawken WinterGreen Husky Long Cut Wintergreen Husky Long Cut Mint Longhorn Long Cut Wintergreen Longhorn Long Cut Mint Kodiak Long Cut Ice Kodiak Premium Mint Kodiak Premium Straight Kodiak Premium Wintergreen Redman Long Cut Wintergreen Rooster Bold Wintergreen Skoal Pouches Mint Skoal Bandit Pouches Mint Skoal Long Cut Spearmint Skoal Pouches Wintergreen Timberwolf Packs Mint Pouches Average:

10.54 ± 0.29 12.04 ± 0.20 11.58 ± 0.33 9.63 ± 0.08 1.55 ± 0.13 9.63 ± 0.28 9.84 ± 0.46 11.58 ± 0.27 13.11 ± 0.66 7.24 ± 0.27 8.56 ± 0.18 8.74 ± 0.55 8.42 ± 0.25 12.48 ± 0.43 9.76 ± 0.22 9.73 ± 0.05 10.89 ± 0.66 12.16 ± 0.79 9.39 ± 0.19 11.75 ± 0.40 9.93

28.09 ± 0.22 52.29 ± 0.33 52.07 ± 0.45 48.47 ± 0.53 22.56 ± 0.15 53.81 ± 0.45 54.25 ± 0.13 52.26 ± 0.62 50.17 ± 0.25 51.72 ± 0.50 51.00 ± 0.19 50.59 ± 0.25 50.49 ± 0.30 51.98 ± 0.28 52.62 ± 0.27 51.35 ± 1.21 50.72 ± 0.56 50.62 ± 0.30 52.64 ± 0.19 46.68 ± 0.53 48.72

Standard reference SLT Standard Reference 1S3 Standard Reference 2S3 Average:

7.05 ± 0.81 14.39 ± 0.42 10.72

47.64 ± 0.20 51.44 ± 0.10 49.54

All values calculated per g wet weight of smokeless tobacco.

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Table 2 Levels of major flavorant compounds in 10 mint-type sub-brands of smokeless tobacco (SLT). Products are listed in order of decreasing menthol level. All values are given ±1 standard deviation (sd) for three replicates.

a

Sub-brand

Menthol (mg/g)

Menthone (mg/g)

Methyl salicylate (mg/g)

Ethyl salicylate (mg/g)

Menthyl acetate (mg/g)

Carvone (mg/g)

Limonene (mg/g)

Total (mg/g)

Timberwolf Packs Mint Pouches Husky Long Cut Mint Longhorn Long Cut Mint Skoal Bandit Pouches Mint Skoal Pouches Mint Average for menthol top five:

5.25 ± 0.50 4.66 ± 0.27 4.25 ± 0.32 3.82 ± 0.37 3.69 ± 0.09 4.34

1.65 ± 0.09 2.45 ± 0.17 1.57 ± 0.10 1.52 ± 0.09 1.56 ± 0.12 1.75

nda nd nd nd nd nd

0.23 ± 0.01 nd 0.17 ± 0.15 2.63 ± 0.31 4.14 ± 0.18 1.43

0.60 ± 0.02 0.72 ± 0.08 0.36 ± 0.03 0.60 ± 0.06 0.55 ± 0.03 0.56

nd nd nd nd nd nd

0.26 ± 0.24 0.58 ± 0.08 0.47 ± 0.15 nd 0.17 ± 0.02 0.30

7.99 ± 0.44 8.41 ± 0.51 6.82 ± 0.37 8.57 ± 0.67 10.11 ± 0.42 8.38

Camel Snus Frost Grizzly Long Cut Mint Kodiak Premium Mint Kodiak Long Cut Ice Skoal Long Cut Spearmint Overall average:

3.18 ± 0.23 3.17 ± 0.07 3.09 ± 0.18 0.85 ± 0.02 nd 3.20

0.17 ± 0.15 1.55 ± 0.17 1.42 ± 0.11 0.26 ± 0.03 nd 1.22

nd nd nd nd nd nd

nd 5.78 ± 0.16 2.21 ± 0.06 4.22 ± 0.34 3.27 ± 0.24 2.27

nd 0.44 ± 0.02 0.35 ± 0.01 nd nd 0.36

3.35 ± 0.24 nd nd nd 4.57 ± 0.25 0.79

1.11 ± 0.22 0.27 ± 0.25 0.36 ± 0.02 nd nd 0.32

7.81 ± 0.25 11.21 ± 0.30 7.43 ± 0.33 5.33 ± 0.37 7.84 ± 0.49 8.15

nd = not detected.

Table 3 Levels of methyl salicylate and ethyl salicylate in 10 commercial sub-brands of smokeless tobacco (SLT) labeled as ‘‘wintergreen” and ‘‘straight”. All values are given ±1 standard deviation (sd) for three replicates. Sub-brand

a b

Methyl salicylate (mg/g)a

Ethyl salicylate (mg/g)a

Wintergreen group Hawken Wintergreen Grizzly Long Cut Wintergreen Husky Long Cut Wintergreen Skoal Pouches Wintergreen Longhorn Long Cut Wintergreen Average for methyl salicylate top five:

29.67 ± 2.64 24.74 ± 0.62 22.97 ± 0.62 21.59 ± 1.27 20.06 ± 0.38 23.81

ndb nd nd nd nd

Rooster Bold Wintergreen Redman Long Cut Wintergreen Kodiak Premium Wintergreen Overall average for methyl salicylate:

19.11 ± 0.82 18.83 ± 0.53 18.52 ± 0.89 21.94

nd nd nd

‘‘Straight” group Grizzly Long Cut Straight Kodiak Premium Straight Average:

0.48 ± 0.07 0.40 ± 0.01 0.44

15.98 ± 1.26 13.07 ± 1.82 14.52

All values calculated per g wet weight of smokeless tobacco. nd = not detected.

compound marked with an asterisk has multiple enatiomeric forms; in this work, only total levels were determined. For each replicate analysis of SLT for nicotine and the flavorant compounds, 1 g of SLT was weighed into a 40-mL glass ‘‘VOA” vial (I-Chem, New Castle, DE) fitted with a Teflon-lined screw-cap lid. 20 mL of isopropanol was then added as the extraction solvent, followed by 4 mg of the surrogate standard 4-tert-butylcyclohexanol (in isopropanol). Extraction in the dark for 24 h (with shaking) was then carried out. Quiescent settling for 3 h allowed formation of a clear supernatant layer. After removal of that layer, the process was repeated with a second 20 mL of isopropanol to verify the high extraction efficiency of the first extraction. Each clear extract was analyzed using a Perkin–Elmer (Norwalk, CT) TurboMass Gold gas chromatograph/mass spectrometer (GC/MS) with a Restek (Bellefonte, PA) Rtx-5 amine column (30 m, 0.32 mm i.d., 0.25 lm film thickness). The temperature program for each run was: 60 °C for 1 min, then 10 °C/min to 300 °C. Because different dilution factors were needed, the analyses for nicotine and the flavorant compounds utilized separate GC runs. Nicotine-d3 was the internal standard for determination of nicotine; naphthalened8 was the internal standard for the flavorant compounds. For each SLT sample, the mass amounts found in the two extractions were summed.

For each replicate determination of water, 1 g of SLT was weighed into a polystyrene weighing dish, then allowed to dry at room temperature for 3–5 days at 50% humidity until a stable weight was attained. Percent water was determined by difference. The moisture determination method of CDC (2008) involves drying at 99 °C for 3 h, and was not used here because of a concern that such conditions would lead to significant volatilization of compounds other than water (e.g., flavorant compounds, humectants, etc.). 2.2. Analyses of confectionery products Samples of 16 sub-brands of flavored confectionery products were purchased in the Portland, OR market (Table 4). The levels of flavorant compounds in these products were determined in a manner similar to that described by Schober and Peterson (2004). For each replicate sample, 1–2 g of product was weighed into a 40-mL glass ‘‘VOA” vial. After addition of 15 mL of distilled water, the vial was sealed with a Teflon-lined screw-cap lid and left in the dark at room temperature with shaking for 18 h. In each case, most or all of the product was dissolved during that period. Four milligrams of the surrogate standard 4-tert-butylcyclohexanol (in isopropanol) was then added, followed by 20 mL of a mixed hexanes solvent (Optima grad, Fisher Scientific, Pittsburg, PA). Extraction for 20 min (with shaking) followed. Quiescent settling for 1 h allowed formation of a clear supernatant layer. After removal of that layer, the extraction with a second 10 mL of mixed hexanes was carried out to verify the high extraction efficiency of the first extraction. Each clear extract was analyzed for flavorant compounds using: (1) a LECO (St. Joseph, MI) Pegasus IV GC-time of flight (TOF) MS; (2) a Restek Rxi-5MS column (30 m, 0.25 mm i.d., and 0.5 lm film thickness); (3) the same GC temperature program used in analyses of SLT; and (4) naphthalene-d8 as the internal standard compound. For each confectionery sample, the mass amounts found in the two extractions were summed. 3. Results 3.1. SLT products Nicotine and water levels for the SLT products are given in Table 1. For nicotine levels, most of the commercial brands were found to be in the range of 7–13 mg/g; the one exception was ‘‘Hawken Wintergreen” (1.55 mg/g). The sub-brand-average nicotine levels for the unflavored and flavored SLT products were 12.3 and 9.93 mg/g, respectively. Under the assumption that the

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Table 4 Levels of menthol and methyl salicylate measured in 16 selected commercial sub-brands of confectionery products. All values are given ± 1 standard deviation (sd) for three replicates. Sub-brand

a

Menthol (mg/g)

Menthone (mg/g)

Methyl salicylate (mg/g)

Ethyl salicylate (mg/g)

Menthyl acetate (mg/g)

Carvone (mg/g)

Limonene (mg/g)

Total (mg/g)

‘‘Mint group” Tic Tac Bold Mint Altoids Spearmint Icebreakers Coolmint Altoids Peppermint Eclipse Winterfrost Average for menthol top five:

5.95 ± 0.28 3.63 ± 0.12 3.50 ± 0.27 2.32 ± 0.31 1.98 ± 0.07 3.48

0.08 ± 0.01 0.11 ± 0.00 0.95 ± 0.06 0.41 ± 0.06 0.14 ± 0.04 0.34

nd a nd nd nd 0.35 ± 0.06 0.07

nd nd nd nd nd

0.04 ± 0.00 0.04 ± 0.00 0.20 ± 0.01 0.10 ± 0.01 0.09 ± 0.01 0.09

nd 4.14 ± 0.25 nd nd nd 0.83

nd 0.30 ± 0.06 0.04 ± 0.00 0.04 ± 0.00 nd 0.08

6.07 ± 0.29 8.22 ± 0.39 4.69 ± 0.34 2.88 ± 0.37 2.56 ± 0.16 4.88

Eclipse Spearmint Tic Tac Freshmints LifeSavers Cryst O Mint Tic Tac Spearmint LifeSavers Sweet Mint Overall average:

1.91 ± 0.13 1.03 ± 0.09 0.52 ± 0.03 0.35 ± 0.03 0.22 ± 0.01 2.14

nd 0.12 ± 0.01 0.23 ± 0.01 0.06 ± 0.00 0.04 ± 0.00 0.22

nd nd nd nd nd 0.03

nd nd nd nd nd nd

nd 0.07 ± 0.01 0.07 ± 0.00 0.03 ± 0.00 0.02 ± 0.00 0.07

0.92 ± 0.03 nd nd 1.65 ± 0.10 nd 0.67

0.07 ± 0.00 nd nd 0.07 ± 0.01 nd 0.05

2.90 ± 0.16 1.22 ± 0.12 0.83 ± 0.05 2.16 ± 0.13 0.29 ± 0.01 3.18

‘‘Wintergreen group” LifeSavers Wint O Green Sugar Free Tic Tac Wintergreen LifeSavers Wint O Green Icebreaker Wintergreen Altoids Wintergreen Average for methyl salicylate top five:

0.02 ± 0.00 0.13 ± 0.00 nd 0.90 ± 0.04 0.81 ± 0.05 0.37

nd nd nd 0.02 ± 0.00 0.07 ± 0.01 0.02

4.58 ± 0.37 3.90 ± 0.35 2.59 ± 0.48 2.21 ± 0.05 1.75 ± 0.14 3.01

nd nd nd nd nd nd

nd nd nd nd 0.08 ± 0.01 0.02

nd nd nd nd nd nd

nd nd nd nd nd nd

4.60 ± 0.37 4.06 ± 0.36 2.59 ± 0.48 3.13 ± 0.08 2.71 ± 0.17 3.42

Breathsavers Wintergreen Overall average:

nd 0.31

nd 0.01

1.60 ± 0.45 2.77

nd nd

nd 0.01

nd nd

nd nd

1.61 ± 0.44 3.12

nd = not detected.

underlying coefficient of variation was approximately the same for the analytical measurements of nicotine for each sub-brand, the difference between the unflavored vs. flavored averages is statistically significant (p < 0.001). For water, the moisture levels of most sub-brands, including the two reference products, were found to be in the range of 45.7– 54.2%; the two exceptions were ‘‘Camel Snus Frost” (28.1%) and ‘‘Hawken Wintergreen” (22.6%). No significant difference was found between the sub-brand averages for water for unflavored vs. flavored. The levels of six primary flavorants in 10 commercial SLT products labeled as ‘‘mint”, ‘‘spearmint”, ‘‘ice”, or ‘‘frost” are given in Table 2. Hereinafter these 10 sub-brands are referred to as the ‘‘mint group”. Menthol was found to be an important flavorant compound for the ‘‘mint group”, with most values in the range 3.1– 5.3 mg/g. The levels of methyl salicylate in eight commercial ‘‘wintergreen” SLT products are given in Table 3; the levels found ranged from 18.5 to 29.7 mg/g. Methyl salicylate was the only flavorant compound detected in the ‘‘wintergreen” products. Also given in Table 3 are the levels of methyl salicylate and ethyl salicylate in the two SLT products labeled as ‘‘straight”; no other flavorant compounds were detected in the ‘‘straight” products. 3.2. Confectionery products The results for levels of menthol, methyl salicylate and total flavorant compounds in selected confectioner products are provided in Table 4. Menthol was found to be the primary flavorant compound for the ‘‘mint group” of flavored confectionery products considered here (‘‘peppermint”, ‘‘spearmint”, ‘‘Winterfrost”, ‘‘Coolmint”, ‘‘Cryst O Mint”, ‘‘Sweet Mint”, ‘‘Bold Mint”, and ‘‘Freshmints”). In addition to menthol, the three ‘‘spearmint” sub-brands were found to contain relatively high levels of carvone. Of the two enantiomeric forms of carvone, the R-( ) form is associated with the spearmint fragrance. By way of comparison, in work from 10 years ago, Gelal et al. (1999) report 5.3 mg/g for menthol in an unnamed sub-brand of ‘‘Altoids candy”.

4. Discussion Fig. 1a provides a comparison of the levels (mg/g) of menthol in the ‘‘mint group” of SLT products considered here vs. the ‘‘mint group” of confectionery products. On an overall average basis, the SLT levels were found to be about 50% higher than the confectionery levels. For the top five SLT products, the average was found to be 4.34 mg/g, as compared to 3.48 mg/g for the top five confectionery products. Fig. 1b illustrates that the levels (mg/g) of methyl salicylate in the ‘‘wintergreen group” of SLT products are much higher than those in the ‘‘wintergreen group” of confectionery products. For the top five SLT products, the average was found to be 23.8 mg/g, which is eight times higher than the average of 3.01 mg/g for the top five ‘‘wintergreen group” confectionery products. Fig. 1c provides a comparison of the total levels (mg/g) of flavorant compounds for the SLT products vs. the confectionery products; the highest levels of flavorant compounds found in the SLT products greatly exceed the highest levels found in the confectionery products. As part of their GRAS program, the Flavor and Extract Manufacturers Association has tabulated average ‘‘usual use” and average ‘‘maximum use” levels for important flavorant compounds (e.g., see Burdock, 2009). Table 5 and Figs. 2 and 3 summarize these averages for menthol and methyl salicylate and compare them with the corresponding top five averages obtained here for SLT and for the selected confectionery products. It is obvious from Figs. 2 and 3 that some SLT products utilize menthol and methyl salicylate at levels that are much elevated relative to those found in candies and gum. Fig. 4 provides comparisons of the levels of flavorant compounds on a mg/serving basis. For SLT, a 2 g serving is assumed based on available information for regular users (Henningfield et al., 1997). For confectionery products, serving size information is often provided on the packaging; the values utilized here are provided in Table 6. For many of the SLT products in the ‘‘mint group”, the mg/serving values are larger than the mg/serving values for the products in the ‘‘mint group” of confectionery products.

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0

Tic Tac Wintergreen

LifeSavers Wint O Green Sugar Free

Tic Tac Bold Mint

Icebreakers Coolmint

Altoids Spearmint

avg. ± sd for all

5 ←2.8

Longhorn Long Cut Wintergreen

10

Husky Long Cut Wintergreen

←14.3

Skoal Pouches Wintergreen

Grizzly Long Cut Wintergreen

mg/g

Altoids Wintergreen

Icebreaker Wintergreen

Tic Tac Wintergreen

20 15

avg. ± sd “winterg. group”

0

confectionery

25

Hawken Wintergreen

avg. ± sd “mint group”

Longhorn Long Cut Wintergr.

5

Husky Long Cut Wintergreen

10

Skoal Pouches Wintergreen

15

Grizzly Long Cut Wintergreen

mg/g

Altoids Peppermint

←22.0

20

SLT

30

avg. ± sd “winterg. group”

Hawken Wintergreen

←2.1

c. Tot. Flav. Compounds (mg/g) 35

confectionery

25 Eclipse Winterfrost

Icebreaker Coolmint

1

Skoal Bandit Pouches Mint

2

30

3.2 → Skoal Pouches Mint

3

Husky Long Cut Mint

4

Timberwolf Packs Mint Pouches

mg/g

5

Longhorn Long Cut Mint

avg. ± sd “mint group”

Tic Tac Bold Mint

6

SLT

LifeSavers Wint O Green

confectionery Altoids Spearmint

SLT

b. Methyl Salicylate (mg/g) 35

LifeSavers Wint O Green Sugar Free

a . M e nt h ol (m g /g ) 7

←3.2

avg. ± sd for all

0

Fig. 1. Levels (mg/g) of menthol, methyl salicylate and total flavorant compounds in smokeless tobacco (SLT) and confectionery products. Values for the products with the top five highest levels in each category are given with error bars giving ±1 sd for three replicates. Upper and lower edges of the dashed boxes give the mean ± 1 standard deviation (sd), respectively, for all products examined.

Table 5 Top five levels of menthol and methyl salicylate from among ‘‘mint” flavored SLT products, ‘‘wintergreen” flavored SLT products, ‘‘mint” flavored confectionery products, and ‘‘wintergreen” flavored confectionery products. Also given are average ‘‘usual” use levels and average ‘‘maximum” use levels as tabulated by Burduck (2009) according to 1994 FEMA (Flavor and Extract Manufacturers Association) data. FEMA-1994a

This work SLT top five (mg/g)

Menthol Methyl 0.6 a

4.3

Confectionery top five (mg/g)

3.5 salicylate

Chewing gum

Hard candy

Soft candy

Ave. of usual values (mg/g)

Ave. of maximal values (mg/g)

Ave. of usual values (mg/g)

Ave. of maximal values (mg/g)

Ave. of usual values (mg/g)

Ave. of maximal values (mg/g)

0.46 23.8

2.3 3.0

1.4 2.1

2.1 7.0

0.13 1.9

0.18 2.0

0.8

Values obtained from Burdock (2009).

5

Menthol in “Mint” Types of Smokeless Tobacco, Candy, and Gum

Methyl Salicylate in “Wintergreen” Types of 25

4

confectionery (candies)

milligrams 3 of flavorant per gram 2

chewing gum

20 milligrams 15 of flavorant per gram 10

hard candy

1 0

soft candy 5 5 top top this work

l ua x us ma FEMA

Smokeless Tobacco, Candy, and Gum smokeless tobacco

smokeless tobacco

l ua x us ma FEMA

l ua x us ma FEMA

5 0

chewing gum confectionery (candies)

5 5 top top this work

l ua x us ma FEMA

hard candy l ua x us ma FEMA

soft candy

l ua x us ma FEMA

Fig. 2. Comparison for menthol of the average for the top five smokeless tobacco (SLT) products and the top five confectionery products investigated in this work with levels reported by Burdock (2009) citing 1994 FEMA (Flavor and Extract Manufacturers Association) data for average ‘‘usual use” and average ‘‘maximum use”.

Fig. 3. Comparison for methyl salicylate of the average for the top five smokeless tobacco (SLT) products and the top five confectionery products investigated in this work with levels reported by Burdock (2009) citing 1994 FEMA (Flavor and Extract Manufacturers Association) data for average ‘‘usual use” and average ‘‘maximum use”.

In Fig. 4a, the average for the ‘‘mint group” of SLT products (7.1 mg/ portion) is 2.5 times the average for the ‘‘mint group” of confectionery products (2.8 mg/serving). Fig. 4b provides an analogous

comparison of the two ‘‘wintergreen groups”, in which case the average for the SLT products (44 mg/serving) was found to be 7 times the average for the confectionery products (6.1 mg/serving).

C. Chen et al. / Food and Chemical Toxicology 48 (2010) 755–763

confectionery

confectionery

20 10

←6.1

avg. ± sd “winterg. group”

0

Eclipse Spearmint

Altoids Peppermint

LifeSavers Wint O Green

8.7

Altoids Spearmint

avg. ± sd for all ←2

LifeSavers Wint O Green Sugar Free

30

Skoal Pouches Wintergreen

40 Longhorn Long Cut Wintergreen

mg/serving

Tic Tac Wintergreen

Altoids Wintergreen

50

Grizzly Long Cut Wintergreen

0

Longhorn Long Cut Wintergreen

10

Husky Long Cut Wintergreen

←2.8

Skoal Pouches Wintergreen

30

Grizzly Long Cut Wintergreen

40

Breath Savers Wintergreen

←43.9

LifeSavers Wint O Green

50

SLT

60 LifeSavers Wint O Green Sugar Free

avg. ± sd “winterg. group”

mg/serving

Tic Tac Bold Mint

Eclipse Spearmint

c. Tot. Flav. Compds. (mg/serv.) 70

60

20

avg. ± sd “mint group”

0

SLT

Hawken Wintergreen

Skoal Pouches Mint

2

Longhorn Long Cut Mint

4

6.4 → Skoal Bandit Pouches Mint

6

Timberwolf Packs Mint Pouches

mg/serving

8

Husky Long Cut Mint

avg. ± sd “mint group”

10

Eclipse Winterfrost

12

Altoids Spearmint

confectionery

SLT

Altoids Peppermint

14

b. Methyl Salicylate (mg/serv.) 70

Husky Long Cut Wintergreen

a. Menthol (mg/serv.)

Hawken Wintergreen

760

←5.2

avg. ± sd for all

Fig. 4. Levels (mg/serving) of menthol, methyl salicylate and total flavorant compounds in smokeless tobacco (SLT) and confectionery products. Values for the products with the top five highest levels in each category are given with error bars giving ±1 sd for three replicates. Upper and lower edges of the dashed boxes give the mean ± 1 standard deviation (sd), respectively, for all products examined. Assumed serving size for SLT is 2 g; assumed serving sizes for confectionery products are given in Table 6.

Table 6 Serving size information for confectionary products.

‘‘Mint group” Altoids peppermint Altoids spearmint Eclipse spearmint Eclipse winterfrost Icebreakers coolmint LifeSavers mints Cryst O Mint LifeSavers sweet mint Tic Tac bold mint Tic Tac freshmints Tic Tac spearmint ‘‘Wintergreen group” Altoids wintergreen Breathsavers wintergreen Icebreaker wintergreen LifeSavers Wint O Green LifeSavers Wint O Green sugar free Tic Tac wintergreen a b c

Number of pieces per serving assumed in this work

Mass per serving (g)

3a 3a 3a 3a 1a 1b

2a 2a 2a 2a 0.8a 2.29b

1c 1a 1a 1a

3.75c 0.49a 0.49a 0.49a

3a 4a

2a 1.8a

1a 1c

0.8a 4c

1c

3.5c

1a

0.49a

Values taken from packaging. No serving size information on packaging; assumed serving size equals 1 piece. Packaging information indicates serving size equals 4 pieces.

For total flavorant compounds (Fig. 4c), the overall average for the SLT products (2 g/serving) was found to be 5.5 times that for the confectionery products. If a specific SLT consumption rate and body weight are assumed, the data obtained in this study can also be examined in terms of the resulting daily intake (DI, mg/kg-day) values for the flavorant compounds (Fig. 5). DI values are used routinely in evaluations of chronic oral intake of chemicals of possible concern (Lu, 1995), including food additives (Burdock, 2002). The FAO/WHO Joint Expert Committee on Food Additives (JECFA) has established maximum acceptable daily intake (ADI) values that set limits on the amounts of food additives that ‘‘can be ingested daily over a lifetime without appreciable health risk” (IPCS, 1987). For menthol and methyl salicylate, the ADI values are 4 and 0.5 mg/kg-day,

respectively (JECFA, 2001, 2004). These values have been obtained by consideration of a safety factor of 1/100 together with the ‘‘no observed adverse effects level” (NOAEL) values of 375 and 50 mg/ kg-day for chronic oral delivery of menthol and methyl salicylate in rats (e.g., NCI, 1978; Belsito et al., 2007). Calculations here initially assume an adult body weight of 60 kg and 100% absorption of the flavorant compounds from both the SLT products and the confectionery products. In a study involving users of Copenhagen SLT, Kotlyar et al. (2007) report a use range of 1.5– 3.5 tins/week; at 34 g/tin, this corresponds to 7–17 g/day. For regular SLT users Benowitz et al. (1989) assumed 15 g/day, which we also assume for the calculations that follow. For the confectionery products, two possibilities are considered: (a) 3 servings/day; and (b) 7 g/day, the latter value being the average rate of ‘‘candy” consumption obtained from two surveys conducted by US Department of Agriculture (Wilson et al., 1997; USDA, 1997). For menthol, most the SLT products in the ‘‘mint group” considered in this work give computed DI values for 100% absorption that would be of the same order of magnitude, though less than the ADI for that compound: the overall average DI for the ‘‘mint group” is 0.20ADI, the average for the top five is 0.27ADI. For the confectionery products, assuming 7 g/day, the overall average DI for menthol for the ‘‘mint group” is 0.062ADI; the average for the top five is 0.10ADI. Thus, the ‘‘mint group” of SLT products at 15 g/day and 100% absorption would on average yield menthol at rates that are several times larger than for the confectionery products at 7 g/day: 0.20/0.062 = 3 based on overall averages; 0.27/0.10 = 3 for the top five (see Table 7). Similar results are obtained when consumption of the confectionery products is assumed to be 3 servings/day (Table 7). For methyl salicylate, assuming 100% absorption, all the SLT products in the ‘‘wintergreen group” would give computed DI values that are much larger than the ADI for that compound, the overall average being 11ADI; the average for the top five is 12ADI. By comparison, for methyl salicylate in the ‘‘wintergreen group” of confectionery products and the assumption of 7 g/day, the overall average DI is 0.65ADI; the average for the top five is 0.70ADI. Thus, not only would use of the ‘‘wintergreen group” of SLT products at 15 g/day and 100% absorption yield methyl salicylate at a significantly higher rate than the ADI, but the rate would be many times larger than for the confectionery products: 11/0.65 = 17 based on overall averages; 12/0.7 = 17 for the top five (see Table 6). Similar

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C. Chen et al. / Food and Chemical Toxicology 48 (2010) 755–763

b. Methyl Salicylate – 100% Absorption, 60 kg BW

a. Menthol – 100% Absorption, 60 kg BW SLT (15 g/day)

1 0

Altoids Wintergreen

Icebreaker Wintergreen

Tic Tac Wintergreen

LifeSavers Wint O Green

confectionery (3 serv./day) LifeSavers Wint O Green Sugar Free

2

Altoids Wintergreen Breath Savers Wintergreen Tic Tac Wintergreen

3

LifeSavers Wint O Green

4

LifeSavers Wint O Green Sugar Free

mg/kg-day

Eclipse Spearmint Tic Tac Bold Mint

5

Longhorn Long Cut Wintergreen

0.0

6 Grizzly Long Cut Wintergreen Husky Long Cut Wintergreen Skoal Pouches Wintergreen

0.2

7

Hawken Wintergreen

0.4

confectionery (7 g/day)

8

confectionery (3 serv./day) Altoids Spearmint Altoids Peppermint Eclipse Winterfrost

0.6

Skoal Pouches Mint

0.8

Timberwolf Packs Mint Pouches Husky Long Cut Mint Longhorn Long Cut Mint Skoal Bandit Pouches Mint

mg/kg-day

1.2

Altoids Peppermint Eclipse Winterfrost

Tic Tac Bold Mint Altoids Spearmint Icebreakers Coolmint

1.4

1.0

SLT (15 g/day)

9

confectionery (7 g/day)

1.6

ADI (FAO/WHO)

Fig. 5. Daily intake values for menthol and methyl salicylate in top five SLT and top five confectionery products. Error bars give ±1 standard deviation (sd) for three replicates. Maximum acceptable daily intake (ADI) values as recommended by the World Health Organization (WHO): 4 and 0.5 mg/kg-day for menthol (JECFA, 2004) and methyl salicylate (JECFA, 2001), respectively.

Table 7 Acceptable daily intake (ADI) values for menthol and methyl salicylate (mg/kg-day) as established by the FAO/WHO Joint Expert Committed on Food Additives (JECFA, 2001, 2004), and the daily intakes (DI) of those compounds normalized by their respective ADI values for the ‘‘mint group” of confectionary products, the ‘‘mint group” of smokeless tobacco products, the ‘‘wintergreen group” of confectionary products, and the ‘‘wintergreen group” of smokeless tobacco products. Mint flavorant

ADI (mg/kg-day)

Menthol Menthol

4 4

Wintergreen flavorant

ADI (mg/kg-day)

Methyl salicylate Methyl salicylate

0.5 0.5

‘‘Mint group” SLT 15 g SLT/day

Top five average: Overall average:

DI ADI

DI ADI

7 g/day

100% absorption

10% absorption

100% absorption

100% absorption

0.27 0.20

0.027 0.020

0.056 0.035

0.10 0.062

‘‘Wintergreen group” SLT 15 g SLT/day

Top five average: Overall average:

‘‘Mint group” confectionary products 3 servings/day

DI ADI

DI ‘‘Wintergreen group” confectionary products ADI 3 servings/day 7 g/day

100% absorption

10% absorption

100% absorption

100% absorption

12 11

1.2 1.1

0.69 0.61

0.70 0.65

results are obtained when consumption of the confectionery products is assumed to be 3 servings/day (Table 7). As with nicotine, the actual percentages of flavorant compounds absorbed by SLT users will always be at least somewhat less than 100% of the amounts that are chemically extractable from the SLT. For the flavorant compounds, rough guidance on actual percentages under typical use conditions can be obtained by what is known about nicotine uptake from SLT products, though nicotine uptake can be reduced by pH effects related to the alkaloid nature of nicotine (Tomar and Henningfield, 1997). For example, a clinical study by Lunell and Lunell (2005) indicated that about 50% of the chemically extractable amounts of nicotine found in four brands of Swedish snus was absorbed at steady state use. Since the log10 octanol/water partition coefficient (log10 Kow) values of menthol (3.4; Griffin et al., 1999) and methyl salicylate (2.6; Simonich et al., 2000) are larger than that of nicotine (1.2; Sangster, 1997), the bioavailability of menthol and methyl salicylate from SLT may be lower than that of nicotine. Thus, although assuming 100% absorption is the most conservative toxicological approach, Table 7 also includes DI calculations assuming 10% absorption. At 10% absorption, both compounds would be delivered at rates comparable to those from the confectionary products considered here, but still for methyl salicylate the DI is 1.1ADI (Table 7). When administered orally, both methyl salicylate and aspirin are hydrolyzed relatively quickly to salicylic acid (Davison et al., 1961; Higgs et al., 1987). Similarly, after percutaneous administration of methyl salicylate by means of whole-body bathing in water

amended with methyl salicylate, Pratzel (1987) reported finding only salicylic acid and salicyluric acid in humans plasma samples, and no methyl salicylate ester. These results suggest that it is toxicologically relevant to compare DI values for methyl salicylate as salicylic acid to DI values for aspirin as salicylic acid. The FDA (2009) maximum recommended therapeutic dose (MRTD) for aspirin is 66.7 mg/kg-day, or 51.1 mg/kg-day as salicylic acid. For a 60 kg body weight, this is about 12 aspirin tablets per day at 325 mg each. The average for the top five SLT methyl salicylate values was found to be 23.8 mg/g, or 21.6 mg/g as salicylic acid. Assuming an SLT use rate of 15 g/day, a 60 kg body weight, and 100% absorption, the result is 5.4 mg/kg-day, or more than 10% of the value resulting from the MRTD for aspirin. This is a matter of additional concern given that like aspirin, both methyl salicylate and salicylic acid affect platelet function and alter blood coagulation (CIR, 2003). Indeed, in a study involving 3874 participants in six randomized clinical trials, use of aspirin in combination with other oral anticoagulants has been associated with more than double the frequency of intracranial hemorrhage (Hart et al., 1999). Even more specifically, topical application of a gel containing methyl salicylate has been implicated in a problematic intensification (‘‘potentiation”) of the drug warfarin in a previously stable 22year old patient receiving anticoagulant therapy (Joss and LeBlond, 2000). In summary, for the ‘‘mint type” SLT products, menthol: (1) was found to be present at levels (in mg/g) that average 50% higher than in the ‘‘mint type” confectionery products; (2) if 100%

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absorbed, would be delivered on average at 0.2ADI for an SLT use of 15 g/day; and (3) if 100% absorbed, would be delivered on average at 3 times the rate from the mint-type confectionery products assuming a typical rate of 7 g/day of confectionery products. For wintergreen-type SLT products, methyl salicylate: (1) was found to be present at levels that averaged eight times the level in wintergreen-type confectionery products; (2) if 100% absorbed, would be delivered on average at 11ADI for methyl salicylate for an SLT use of 15 g/day; and (3) if 100% absorbed, would be delivered on average at 17 times the rate from the wintergreen-type confectionery products assuming a typical rate of 7 g/day of confectionery products. Moreover, habitual use of wintergreen-flavored SLT products may be contraindicated in patients receiving anticoagulant therapy. Conflict of Interest The authors declare that there are no conflict of interest. Acknowledgments This work was supported by the Cooley Family Fund for Critical Research of the Oregon Community Foundation, and through the support of Regina M. Dowd, Michael J. Dowd, Patrick J. Coughlin, Keith F. Park, and Steven T. Huff. We gratefully acknowledge Cambridge Isotope Laboratories for donating the nicotine-d3 internal standard material used in this research. References Alpert, H.R., Koh, H., Connolly, G.N., 2008. Free nicotine content and strategic marketing of moist snuff tobacco products in the United States: 2000–2006. Tob. Control 17, 332–338. Aungst, B.J., Rogers, N.J., 1988. Site dependence of absorption-promoting actions of laureth-9, Na salicylate, Na2EDTA, and aprotinin on rectal, nasal, and buccal insulin delivery. Pharm. Res. 5, 305–308. Belsito, D., Bickers, D., Bruze, M., Calow, P., Greim, H., Hanifin, J.H., Rogers, A.E., Saurat, J.H., Sipes, I.G., Tagami, H., 2007. A toxicologic and dermatologic assessment of salicylates when used as fragrance ingredients. Food Chem. Toxicol. 45, S318–S361. Benowitz, N.L., Jacob III, P., Yu, L., 1989. Daily use of smokeless tobacco: systemic effects. Ann. Inter. Med. 111, 112–116. Bhatia, S.P., McGinty, D., Letizia, C.S., Api, A.M., 2008. Fragrance material review on menthol. Food Chem. Toxicol. 46, S209–S214. Burdock, G.A., 2002. Regulation of flavor ingredients. In: Kotsonis, F.A., Mackey, M. (Eds.), Nutritional Toxicology, second ed. Taylor & Francis, New York, NY, pp. 316–339. Burdock, G.A. (Ed.), 2009. Fenaroli’s Handbook of Flavor Ingredients, sixth ed., CRC Press, Boca Raton, FL. B&W (Brown and Williamson Tobacco Corporation), 1978. RJR low tar flavoring practices. Bates Number 570389459/9484. Available at: (accessed on 27.09.09). B&W (Brown and Williamson Tobacco Corporation), 1986. Summary data on menthol. Bates Numbers 503226730/6749. Available at: (accessed on 27.09.09). B&W Tobacco Co., Byfield Snuff Co., Conwood Co., L.P., Helme Tobacco Co., House of Windsor Inc., National Tobacco Co., The Pinkerton Tobacco Co., RC Owen Co., Fred Stoker & Sons Inc., United States Tobacco Co., 1994. Smokeless tobacco ingredient list as of 940404. Bates Number 566415479/5524. Available at: (accessed on 27.09.09). Carpenter, C.M., Wayne, G.F., Pauly, J.L., Koh, H.K., Connolly, G.N., 2005. New cigarette brands with flavors that appeal to youth: tobacco marketing strategies. Health Affairs 24, 1601–1610. CDC (Centers for Disease Control and Prevention), 2008. 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. Federal Register 73[121], pp. 35395–35402. Celebucki, C.C., Ferris Wayne, G., Connolly, G.N., Pankow, J.F., Chang, E.I., 2005. Characterization of measured menthol in 48 US sub-brands. Nicotine Tob. Res. 7, 523–531. CIR (Cosmetic Ingredient Review Panel, Cosmetic, Toiletry, Fragrance Association), 2003. Safety assessment of salicylic acid, butyloctyl salicylate, calcium salicylate, C12–15 alkyl salicylate, capryloyl salicylic acid, hexyldodecyl salicylate, isocetyl salicylate, isodecyl salicylate, magnesium salicylate, MEAsalicylate, ethylhexyl salicylate, potassium salicylate, methyl salicylate, myristyl salicylate, sodium salicylate, TEA-salicylate, and tridecyl salicylate. Int. J. Toxicol. 22, 1–108.

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