1. bookVolume 30 (2021): Issue 4 (November 2021)
Journal Details
License
Format
Journal
eISSN
2719-9509
First Published
01 Jan 1992
Publication timeframe
4 times per year
Languages
English
access type Open Access

A Laboratory Method to Measure Skin Surface Staining by Cigarette Smoke, Tobacco Heating Products and E-Cigarettes

Published Online: 02 Jan 2022
Page range: 158 - 166
Received: 16 Mar 2021
Accepted: 20 Aug 2021
Journal Details
License
Format
Journal
eISSN
2719-9509
First Published
01 Jan 1992
Publication timeframe
4 times per year
Languages
English
Summary

Exhaled or side-stream cigarette smoke (CS) may visually stain a consumer's skin over time. Tobacco heating products (THPs) and e-cigarettes (ECs) have reduced staining potential because they do not produce side-stream aerosols and their exhaled aerosols have significantly reduced levels of toxicants, particles and odour. Here we assess discolouration of porcine skin in vitro after exposure to particulate matter (PM) or aerosols from CS (3R4F), two THPs (glo and glo sens) and an EC (iSwitch Maxx). PM was prepared by capturing aerosols on Cambridge filter pads and eluting with dimethyl sulfoxide (DMSO). Abattoir-obtained porcine skin samples were incubated with PM or DMSO control at 37 °C between 0 and 6.0 h. For aerosol assessment, porcine skin samples were exposed to between 50 and 400 puffs of the products, or air control, using a smoking machine. Colour profiles and staining levels of each skin sample were measured at different timepoints and puff thresholds using a spectrophotometer. Staining increased with time and dose, the greatest changes being observed following exposure to aerosols and PM from CS. THP, EC and control values were significantly different from CS after 0.5 h exposure to PM or 50 puffs of aerosols. The minimal staining induced by THPs and EC was comparable to controls. These data suggest that THPs and ECs could offer hygiene benefits to consumers who switch from smoking cigarettes. Further studies are required to assess the longer-term effects of THPs and ECs on skin discoloration. [Contrib. Tob. Nicotine Res. 30 (2021) 158–166]

Keywords

INTRODUCTION

Electronic cigarettes (ECs) have become widely available and have increased in popularity since their introduction to the market. More recently, tobacco heating products (THPs) have become commercially available, but so far are used less widely as availability varies by country and regulation. ECs are relatively simple devices that heat an e-liquid, creating an inhalable aerosol. Most e-liquids consist of 1,2-propylene glycol, vegetable glycerol, water and flavours, and can be purchased with or without nicotine. THPs heat tobacco rods (often known as consumables or sticks) to temperatures of 200–350 °C, which vaporise nicotine and other volatile compounds without the combustion/pyrolysis of the tobacco (1, 2). Both classes of device s release the aerosol only when inhaled by the consumer. By contrast, conventional cigarettes burn at temperatures up to 950 °C, producing mainstream smoke, but also smoulder between puffs, producing side-stream smoke. Overall, cigarette smoke (CS) releases more than 7,000 chemicals, including many known toxicants (3, 4) and can contribute to room odour and staining (5, 6) as well as staining of consumers’ teeth (7, 8, 9) and skin (10).

THP and EC aerosols contain significantly fewer toxicants than CS (1, 2, 11, 12, 13), and there is growing consensus that THPs and ECs hold the potential to reduce the health risks associated with smoking (14, 15, 16, 17, 18). In addition to risk reduction, THPs and ECs might have hygiene or social benefits for smokers who switch. Laboratory methods have been developed to quantify tooth enamel sample and surface staining following exposure to CS or THP and EC aerosols (6, 9). Consistently, CS stains samples, whereas changes with THP and EC aerosols are minimal and close to controls. As far as we are aware, no comparison of skin staining by CS, THP and EC aerosols has been performed. In the current study, a laboratory method was developed to assess the exposure of porcine skin samples to emissions from CS, THP and EC aerosols.

MATERIALS AND METHODS
Chemicals and reagents

All chemicals and reagents were purchased from Sigma-Aldrich (Gillingham, UK, or St Louis, MO, USA) unless otherwise stated.

Test articles

Four test products were used in this study: 3R4F Kentucky reference cigarettes, iSwitch Maxx e-cigarettes (British American Tobacco, Southampton, UK) and the glo and glo sens THP (British American Tobacco; Table 1). Prior to use, the 3R4F cigarettes were conditioned for a minimum of 48 h and a maximum of 10 days and the glo THP tobacco rods (Neostiks) were conditioned for 48 h to 5 days by storing at 22 ± 1 °C and 60 ± 3% relative humidity, according to the ISO 3402:1999 standard (19). The EC e-liquid cartridges and glo sens THP tobacco pods were stored at room temperature. All devices were fully charged before use.

Products assessed for skin staining.

Product Source Consumable E-liquid nicotine (mg/mL) Puffs per product/cartridge Puffs per tobacco pod
3R4F reference cigarette University of Kentucky N/A N/A 10 N/A
iSwitch Maxx e-cigarette British American Tobacco Virginia tobacco 5 80 N/A
glo THP British American Tobacco Bright tobacco Neostiks N/A 8 N/A
glo sens THP British American Tobacco Mixed fruit 0 150 50
Porcine sample preparation

Ex vivo pig abdominal skin was retrieved in an abattoir and immediately placed on ice. In the laboratory, the skin was prepared by clipping the surface hair and removing excess subcutaneous fat. Slices of 500–750 μm were cut with a dermatome, from which cylindrical biopsy punches of 5 mm diameter were obtained. Prepared skin punch samples were stored at −20 ± 5 ºC until required. On the day of analysis, skin punches were removed from the freezer, brought to room temperature and examined for obvious defects, such as tears, fissures or scratches. Prior to use, skin punches were incubated for 10 min at standard culture conditions (5 ± 1% CO2 and 37 ± 1 °C) in phenol-free Hanks’ Balanced Salt Solution, containing calcium and magnesium (Thermo Fisher Scientific, Grand Island, NY, USA, 14025-092), and then washed in calcium- and magnesium-free Dulbecco's Phosphate Buffered Saline (PBS) (Thermo Fisher Scientific, 14190-144).

Particulate matter preparation

3R4F CS and aerosols from glo, glo sens or iSwitch Maxx were generated using LM20X or LM20E linear smoking machines (Borgwaldt, Hamburg, Germany). The iSwitch Maxx was tested at the highest power level. Specific puffing regimes were used for each product (Table 2). The particulate fraction of CS or aerosol from each product was collected on 44-mm Cambridge filter pads (CFPs, Whatman, Maidstone, UK) and the particulate matter (PM) was eluted with dimethylsulfoxide (DMSO) as described previously (20, 21). Briefly, CFPs were weighed before and after aerosol collection to determine the weight of the captured aerosol. CFPs were placed in 100-mL glass bottles and DMSO added to achieve a concentration of 24 mg/mL. Bottles were covered with foil and placed, at room temperature, on an orbital shaker set at 150 rpm for 25 min. The PM was then extracted from each CFP under vacuum, 1-mL aliquots were prepared and stored in glass vials at −80 °C until required.

Product puffing regimes.

Product Regime Puff volume (mL) Puff duration (s) Puff interval/frequency (s) Vent blocking Puff profile
3R4F reference cigarette HCI 55 2 30 100% Bell
iSwitch Maxx e-cigarette CRM81 55 3 30 None Square
glo THP HCIm 55 2 30 None Bell
glo sens THP CRM81 55 3 30 None Square

Abbreviations:
CRM81

CORESTA recommended method no. 81 (30)

HCI

Health Canada intense smoking regime (29)

HCIm

Health Canada intense smoking regime modified with no vent blocking

N/A

Not Applicable

THP

Tobacco Heating Product

PM exposure

Cylindrical punch biopsy skin samples were placed dermis down into 500 μL of product PM or DMSO and incubated under standard culture conditions (5 ± 1% CO2 and 37 ± 1 °C). Samples were removed at 0.25, 0.5, 1.0, 3.0 and 6.0 h (three skin samples per timepoint) and colorimetric readings were taken. Each experiment was repeated at least three times.

Aerosol exposure

Cylindrical biopsy skin punches were placed into 12-well hanging inserts (Transwell®, Corning, Lowell, MA, USA) and then into a VITROCELL® 12/6 CF module (Waldkirch, Germany) with Hanks’ Buffered Salt Solution at ambient room temperature. Using product-specific puffing regimes (Table 2) and a VITROCELL® VC1® engine, samples were exposed to 50, 100, 200 or 400 puffs of 3R4F CS, glo sens, iSwitch Maxx or air control (three skin samples per dose). Three or more independent experiments were performed for each product or control, glo was not assessed as an aerosol.

Colour measurements

Prior to exposure, the colour profile of each pig skin punch sample was determined using a CM-700d spectrophotometer (Konica Minolta Business Solutions, Greenville, SC, USA) with 5-mm aperture that was calibrated using the manufacturer-supplied white tile before use. Four measurements per skin samples were taken and the sample was rotated 90° between each measurement. Throughout the colorimetric analysis, the operator maintained a uniform specimen measuring port-to-tissue surface distance and ambient lighting to minimize variability and bias in measurements. Colour readings were captured, stored in the CM-700d using the SpectraMagic NX software (Konica Minolta Business Solutions) and the results were exported to a Microsoft Excel document. Samples exposed to PM or DMSO were rinsed in 500 μL phosphate-buffered saline before analysis. Those exposed to aerosol or air were removed from the exposure chamber and placed directly on to the CM-700d spectrophotometer aperture.

Colour profiles and staining levels were calculated at baseline and at every timepoint or puff number using the “Commission Internationale de L’éclairage L*a*b* method”. L* is a measure of lightness and a* and b* are measures of green-red and blue-yellow colour components, respectively (6, 9, 22). Changes in values from baseline and between treatments were determined in Excel by calculating ΔL*, Δa*, Δb* and ΔE (total difference) with the following equation:

ΔE=((ΔL*)2+(Δa*)2+(Δb*)2) \Delta E = \sqrt {\left({{{\left({\Delta L*} \right)}^2} + {{\left({\Delta a*} \right)}^2} + {{\left({\Delta b*} \right)}^2}} \right)}
Statistical methods

The data analysis for this paper was generated using SAS software, Version 9.4 of the SAS System for Windows (Copyright © 2021 SAS Institute Inc., SAS and all other SAS Institute Inc. product or service names are registered trademarks or trademarks of SAS Institute Inc., Cary, NC, USA.) Generalised linear models were used to assess the differences in ΔL*, Δa*, Δb* and ΔE values between the products and reference cigarettes. The significance threshold for difference (α) was set at p = 0.05. Post-hoc Tukey adjustment for pairwise comparisons was also used.

RESULTS
Particulate matter skin sample exposure

Exposure to 3R4F PM resulted in darkening and discoloration of the skin samples, with effects increasing over time. After 0.25 h, 3R4F ΔL* values were significantly lower than glo sens and DMSO values (p < 0.05, Table 3) indicating darkening of the skin samples. After 0.5 h, 3R4F ΔL* values were also significantly lower than glo and iSwitch Maxx values (p < 0.05). After 0.25 h exposure, 3R4F Δa* values (green to red), were significantly higher than glo and DMSO (p < 0.0001) demonstrating reddening of the skin following the exposure. At 0.5 h, all products and DMSO Δa* values were significantly lower (p < 0.0001) than 3R4F. The Δb* values (blue to yellow) following 3R4F exposure were significantly higher than glo, glo sens and DMSO control from 0.25 h demonstrating that the skin yellows with exposure. From 0.5 h, iSwitch Maxx and all other products Δb* values were significantly lower than 3R4F (p < 0.0001). From 0.25 h, total colour changes, shown by the ΔE value (Figure 1, a), were significantly higher for 3R4F than DMSO and all products except iSwitch. At 0.5 h, glo, glo sens, iSwitch Maxx and DMSO control ΔE values were significantly lower than the 3R4F value (p < 0.0001). All THP and EC values were similar to those for the DMSO control throughout the timepoints assessed (Figure 1, a and b).

Mean ΔL*, Δa*, Δb* and ΔE and standard deviation values following the exposure of skin samples for 0.25, 0.5, 1.0, 2.0, 4.0 and 6.0 h to particulate matter generated from 3R4F cigarettes, glo and glo sens THP, iSwitch Maxx EC or DMSO as a control.

Hours 3R4F glo glo sens iSwitch Maxx DMSO

Mean SD Mean SD Mean SD Mean SD Mean SD
ΔL* (lightness)
0.25 −9.00 2.19 −7.76 2.45 −6.98 b 2.86 −8.41 2.60 −7.30 b 2.80
0.5 −10.76 2.60 −9.02 b 2.27 −8.35 b 3.16 −8.61 b 2.40 −8.64 a 3.27
1.0 −12.87 2.50 −10.26 b 2.63 −8.76 a 5.29 −9.70 b 3.65 −10.01 a 3.69
2.0 −14.19 2.48 −9.45 a 2.21 −8.77 a 4.50 −9.33 a 3.58 −9.20 a 2.79
4.0 −15.62 2.28 −8.74 a 2.45 −7.63 a 4.48 −8.22 a 3.05 −8.68 a 2.70
6.0 −16.76 2.54 −8.13 a 2.88 −6.89 a 4.13 −7.75 a 2.91 −8.29 a 3.11
Δa* (green-red)
0.25 0.49 0.83 −0.29 a 0.34 0.25 0.55 0.52 0.48 −0.22 a 0.75
0.5 1.34 0.82 0.52 a 0.85 0.33 a 0.54 0.45 a 0.66 0.35 a 0.98
1.0 1.78 0.81 0.16 a 1.10 0.24 a 0.95 0.13 a 0.56 0.35 a 0.98
2.0 2.72 0.80 −0.28 a 1.30 −0.03 a 0.63 0.08 a 0.53 0.11 a 1.01
4.0 4.07 0.75 −0.07 a 1.35 0.03 a 0.60 −0.02 a 0.47 0.18 a 0.81
6.0 4.98 0.79 0.12 a 1.27 0.21 a 0.49 0.29 a 0.58 0.41 a 0.95
Δb* (blue-yellow)
0.25 2.66 3.49 −0.70 a 2.02 1.21 b 1.26 1.41 1.35 0.13 a 2.39
0.5 5.86 2.17 0.70 a 2.27 1.05 a 1.11 1.17 a 1.91 0.84 a 2.71
1.0 7.90 1.84 0.70 a 2.37 0.05 a 1.51 1.61 a 0.86 0.49 a 2.24
2.0 9.35 2.59 0.64 a 2.06 0.38 a 0.74 1.49 a 1.02 0.34 a 2.61
4.0 10.20 3.21 −0.10 a 1.70 −0.31 a 0.78 0.72 a 1.28 −0.36 a 1.79
6.0 9.64 3.32 −0.69 a 1.63 −0.45 a 0.45 −0.38 a 2.00 −1.06 a 2.55
ΔE (total difference)
0.25 10.11 1.87 8.03 b 2.53 7.34 a 2.52 8.66 2.59 7.70 a 2.85
0.5 12.62 2.19 9.39 a 2.19 8.56 a 3.04 8.96 a 2.28 9.25 a 2.97
1.0 15.41 1.94 10.64 a 2.48 9.02 a 5.15 9.99 a 3.35 10.39 a 3.47
2.0 17.47 2.03 9.78 a 2.17 8.87 a 4.42 9.62 a 3.28 9.63 a 2.74
4.0 19.40 2.05 8.99 a 2.48 7.73 a 4.42 8.36 a 3.06 8.88 a 2.76
6.0 20.22 2.77 8.38 a 2.97 6.99 a 4.04 8.14 a 2.59 8.84 a 2.96

Figure 1

Changes in porcine skin sample colour following exposure to particulate matter or aerosol from cigarettes, tobacco heating products or e-cigarettes. Values are means and standard deviations.

Aerosol skin exposure

Exposure to 3R4F CS aerosol resulted in darkening and discoloration of punch skin samples, with dose-dependent changes observed for ΔL*, Δa* and ΔE* values (Table 4). After 50 puffs, 3R4F CS ΔL* values were significantly lower than those for glo sens, iSwitch Maxx and air control (all p < 0.0001). Skin reddening was seen with 3R4F CS exposure compared with the other products and control, with the difference in Δa* values becoming significant from 50 puffs (all doses p < 0.0001). Skin yellowing, represented by Δb* values, increased following 3R4F exposure, differing significantly from glo sens, iSwitch Maxx and air control values at all puff numbers (all p < 0.0001). However, 3R4F Δb* values reached a plateau at 200 puffs and decreased at 400 puffs. The ΔE values (Figure 1 c), indicating overall colour change, were significantly higher for 3R4F CS than for glo sens, iSwitch Maxx and air control at 50–400 puffs (all p < 0.0001). As for exposure to PM, all THP and EC values were comparable to air control at all doses (Figure 1, c and d).

Mean ΔL*, Δa*, Δb* and ΔE and standard deviation values following the exposure of skin samples to 50–400 puffs of 3R4F cigarettes, glo sens THP, iSwitch Maxx EC or air as a control.

Puffs 3R4F glo sens iSwitch Maxx Air

Mean SD Mean SD Mean SD Mean SD
ΔL* (lightness)
50 −9.11 1.88 1.22a 1.69 −0.15 a 1.79 1.36 a 2.42
100 −8.90 2.86 0.72 a 1.53 −0.04 a 1.96 1.52 a 1.39
200 −15.55 2.84 0.28 a 1.24 −0.86 a 2.07 1.75 a 1.67
400 −22.73 3.05 0.69 a 1.35 −2.11 a 2.74 2.11 a 2.66
Δa* (green-red)
50 5.39 1.24 −0.15 a 0.49 −0.16 a 0.18 −0.15 a 0.29
100 4.48 1.69 −0.03 a 0.25 −0.04 a 0.20 −0.07 a 0.35
200 8.93 1.72 −0.09 a 0.27 −0.20 a 0.29 −0.14 a 0.35
400 11.50 1.39 −0.21 a 0.32 −0.49 a 0.36 −0.21 a 0.39
Δb* (blue-yellow)
50 18.35 2.68 −0.65 a 0.68 −1.10 a 0.77 −0.66 a 0.96
100 17.90 3.18 −0.24 a 0.45 −0.15 a 0.76 −0.44 a 0.84
200 19.68 2.73 −0.88 a 1.11 −0.89 a 1.24 −0.93 a 0.95
400 14.53 3.92 −1.35 a 0.69 −1.64 a 1.20 −1.33 a 1.20
ΔE (total difference)
50 18.04 1.50 1.89 a 1.37 1.94 a 1.11 2.32 a 2.03
100 21.57 3.01 1.38 a 1.11 1.77 a 1.12 2.19 a 1.93
200 26.86 2.44 1.62 a 1.02 2.26 a 1.51 2.62 a 1.90
400 29.68 2.31 1.99 a 0.87 3.44 a 2.13 3.23 a 1.79

= Significantly different from 3R4F p < 0.0001

= Significantly different from 3R4F p < 0.05

DISCUSSION

In this study, significant differences were noticed for skin darkening and discoloration after exposure to CS versus aerosol from THP and EC. By contrast, changes with THP and EC exposure remained similar to those seen with DMSO and air controls by time and dose. Value changes indicated darkening, reddening and yellowing of skin after CS exposure.

Consensus is growing that THPs and ECs hold great potential for reducing the health risk associated with cigarette smoking (14, 15, 16, 17, 18). The aerosols produced by THPs and ECs differ greatly from CS, and studies have confirmed they contain significantly less toxicants (1, 2, 4, 11, 12, 13). In addition to risk reductions, there could be hygiene and/or social consideration benefits for smokers who switch to THPs and ECs, which seem to be of importance to consumers. A recent survey of Japanese THP consumers highlighted social consideration and hygiene as motivations for switching from smoking to using THPs. Consumers also believed that THPs are less harmful to people around them and have reduced odour (23).

Numerous countries now restrict smoking indoors. Before these bans, the impact of CS could be easily visualised as yellow or brown staining on surfaces and a characteristic odour left on hair, clothing and furnishing fabrics. Staining and odour are due to exhaled and side-stream CS produced as a cigarette burns between puffs. CS is composed of two phases, the particulate, also known as “tar”, and the vapor phase (3, 4). The particulate colour is thought to come from the burning of the tobacco in the cigarette, which then deposit on surfaces resulting in yellowing or brown staining (5, 6, 7, 8, 9, 10). Unlike a burning cigarette, THPs and ECs release an aerosol only when consumers inhale on the product, this lack of side-stream aerosol might reduce staining of surfaces such as furnishing fabric and wallpaper (6) and also the staining of consumers’ hands. THP and EC reduced staining levels are also possibly due to the fact that THP devices heat rather than burn the tobacco contained in the consumable and that the majority of EC e-liquids do not contain tobacco.

In this study, the accelerated staining methods developed for enamel, wallpaper and cotton samples (6, 9) were adapted to enable the exposure of porcine skin samples. Porcine skin was selected, as samples are routinely used for in vitro testing due to structural and functional similarities to human skin (24, 25). Two exposure methods were used – submerging in PM extracts and exposure to aerosol. The capture of the particulate fraction of CS is widely used to assess tobacco products in vitro and also THP and EC products (20, 21, 26). In this study, the contributions of CS to skin darkening and discoloration was confirmed by PM exposure and indicated a time-related effect. Likewise, aerosol studies, which are more aligned to consumer exposure, showed dose-related increases in skin sample darkening and discoloration with CS. Limited staining or discoloration was observed following exposure to the THP and EC PM or aerosol. In the current aerosol study, a dose response was not observed for 3R4F Δb* values, but was observed for 3R4F ΔL*, Δa* and ΔE values. Increasing Δb* values with 3R4F dose was observed for the PM study and also in previous aerosol studies (6, 9), differences could be due to the surface of the skin which is not as uniform as enamel, wallpaper or cotton.

A limitation of this study is that the experimental method delivers mainstream, but not side-stream CS and the ECs were operated at the highest power during aerosol collection, which might have over-represented THP and EC exposure and under-represented CS exposure. Nevertheless, clear and significant differences seen with mainstream CS suggest that staining levels would also differ with side-stream CS.

The data produced in this study support published findings that detail yellowing of the skin, fingernails and facial hair by CS (10). Although we assessed short-term exposures, studies looking at long-term CS exposure have proposed that prominent wrinkles, gauntness and a grey colour to the facial skin are due to CS. Twin studies in which one twin is a smoker and the other a non-smoker also highlight CS-induced changes to the skin (10, 27, 28). Long-term switching studies would enable a further understanding of long-term effects of THP and EC on skin structure and allow investigation of whether the effects of CS exposure are reversible.

CONCLUSIONS

We describe a novel method developed to assess skin sample staining by CS, THP or EC aerosols. CS exposure significantly increased the level of skin sample staining in a dose-dependent manner, whereas the THP and EC aerosol exposure resulted in minimal staining. These data suggest that THPs and ECs may have hygiene benefits for consumers who switch to exclusive use of these products. Further studies are required to assess the long-term impact on skin of consumers who switch from smoking to using ECs or THPs.

Figure 1

Changes in porcine skin sample colour following exposure to particulate matter or aerosol from cigarettes, tobacco heating products or e-cigarettes. Values are means and standard deviations.
Changes in porcine skin sample colour following exposure to particulate matter or aerosol from cigarettes, tobacco heating products or e-cigarettes. Values are means and standard deviations.

Products assessed for skin staining.

Product Source Consumable E-liquid nicotine (mg/mL) Puffs per product/cartridge Puffs per tobacco pod
3R4F reference cigarette University of Kentucky N/A N/A 10 N/A
iSwitch Maxx e-cigarette British American Tobacco Virginia tobacco 5 80 N/A
glo THP British American Tobacco Bright tobacco Neostiks N/A 8 N/A
glo sens THP British American Tobacco Mixed fruit 0 150 50

Mean ΔL*, Δa*, Δb* and ΔE and standard deviation values following the exposure of skin samples to 50–400 puffs of 3R4F cigarettes, glo sens THP, iSwitch Maxx EC or air as a control.

Puffs 3R4F glo sens iSwitch Maxx Air

Mean SD Mean SD Mean SD Mean SD
ΔL* (lightness)
50 −9.11 1.88 1.22a 1.69 −0.15 a 1.79 1.36 a 2.42
100 −8.90 2.86 0.72 a 1.53 −0.04 a 1.96 1.52 a 1.39
200 −15.55 2.84 0.28 a 1.24 −0.86 a 2.07 1.75 a 1.67
400 −22.73 3.05 0.69 a 1.35 −2.11 a 2.74 2.11 a 2.66
Δa* (green-red)
50 5.39 1.24 −0.15 a 0.49 −0.16 a 0.18 −0.15 a 0.29
100 4.48 1.69 −0.03 a 0.25 −0.04 a 0.20 −0.07 a 0.35
200 8.93 1.72 −0.09 a 0.27 −0.20 a 0.29 −0.14 a 0.35
400 11.50 1.39 −0.21 a 0.32 −0.49 a 0.36 −0.21 a 0.39
Δb* (blue-yellow)
50 18.35 2.68 −0.65 a 0.68 −1.10 a 0.77 −0.66 a 0.96
100 17.90 3.18 −0.24 a 0.45 −0.15 a 0.76 −0.44 a 0.84
200 19.68 2.73 −0.88 a 1.11 −0.89 a 1.24 −0.93 a 0.95
400 14.53 3.92 −1.35 a 0.69 −1.64 a 1.20 −1.33 a 1.20
ΔE (total difference)
50 18.04 1.50 1.89 a 1.37 1.94 a 1.11 2.32 a 2.03
100 21.57 3.01 1.38 a 1.11 1.77 a 1.12 2.19 a 1.93
200 26.86 2.44 1.62 a 1.02 2.26 a 1.51 2.62 a 1.90
400 29.68 2.31 1.99 a 0.87 3.44 a 2.13 3.23 a 1.79

Product puffing regimes.

Product Regime Puff volume (mL) Puff duration (s) Puff interval/frequency (s) Vent blocking Puff profile
3R4F reference cigarette HCI 55 2 30 100% Bell
iSwitch Maxx e-cigarette CRM81 55 3 30 None Square
glo THP HCIm 55 2 30 None Bell
glo sens THP CRM81 55 3 30 None Square

Mean ΔL*, Δa*, Δb* and ΔE and standard deviation values following the exposure of skin samples for 0.25, 0.5, 1.0, 2.0, 4.0 and 6.0 h to particulate matter generated from 3R4F cigarettes, glo and glo sens THP, iSwitch Maxx EC or DMSO as a control.

Hours 3R4F glo glo sens iSwitch Maxx DMSO

Mean SD Mean SD Mean SD Mean SD Mean SD
ΔL* (lightness)
0.25 −9.00 2.19 −7.76 2.45 −6.98 b 2.86 −8.41 2.60 −7.30 b 2.80
0.5 −10.76 2.60 −9.02 b 2.27 −8.35 b 3.16 −8.61 b 2.40 −8.64 a 3.27
1.0 −12.87 2.50 −10.26 b 2.63 −8.76 a 5.29 −9.70 b 3.65 −10.01 a 3.69
2.0 −14.19 2.48 −9.45 a 2.21 −8.77 a 4.50 −9.33 a 3.58 −9.20 a 2.79
4.0 −15.62 2.28 −8.74 a 2.45 −7.63 a 4.48 −8.22 a 3.05 −8.68 a 2.70
6.0 −16.76 2.54 −8.13 a 2.88 −6.89 a 4.13 −7.75 a 2.91 −8.29 a 3.11
Δa* (green-red)
0.25 0.49 0.83 −0.29 a 0.34 0.25 0.55 0.52 0.48 −0.22 a 0.75
0.5 1.34 0.82 0.52 a 0.85 0.33 a 0.54 0.45 a 0.66 0.35 a 0.98
1.0 1.78 0.81 0.16 a 1.10 0.24 a 0.95 0.13 a 0.56 0.35 a 0.98
2.0 2.72 0.80 −0.28 a 1.30 −0.03 a 0.63 0.08 a 0.53 0.11 a 1.01
4.0 4.07 0.75 −0.07 a 1.35 0.03 a 0.60 −0.02 a 0.47 0.18 a 0.81
6.0 4.98 0.79 0.12 a 1.27 0.21 a 0.49 0.29 a 0.58 0.41 a 0.95
Δb* (blue-yellow)
0.25 2.66 3.49 −0.70 a 2.02 1.21 b 1.26 1.41 1.35 0.13 a 2.39
0.5 5.86 2.17 0.70 a 2.27 1.05 a 1.11 1.17 a 1.91 0.84 a 2.71
1.0 7.90 1.84 0.70 a 2.37 0.05 a 1.51 1.61 a 0.86 0.49 a 2.24
2.0 9.35 2.59 0.64 a 2.06 0.38 a 0.74 1.49 a 1.02 0.34 a 2.61
4.0 10.20 3.21 −0.10 a 1.70 −0.31 a 0.78 0.72 a 1.28 −0.36 a 1.79
6.0 9.64 3.32 −0.69 a 1.63 −0.45 a 0.45 −0.38 a 2.00 −1.06 a 2.55
ΔE (total difference)
0.25 10.11 1.87 8.03 b 2.53 7.34 a 2.52 8.66 2.59 7.70 a 2.85
0.5 12.62 2.19 9.39 a 2.19 8.56 a 3.04 8.96 a 2.28 9.25 a 2.97
1.0 15.41 1.94 10.64 a 2.48 9.02 a 5.15 9.99 a 3.35 10.39 a 3.47
2.0 17.47 2.03 9.78 a 2.17 8.87 a 4.42 9.62 a 3.28 9.63 a 2.74
4.0 19.40 2.05 8.99 a 2.48 7.73 a 4.42 8.36 a 3.06 8.88 a 2.76
6.0 20.22 2.77 8.38 a 2.97 6.99 a 4.04 8.14 a 2.59 8.84 a 2.96

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