TEST REPORT. Tobacco smoke vs. flavoured e-liquid vapour of CULAMI Toxic effects on cultured human lung cells after long-term exposure

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1 Page 1 (6) Dartsch Scientific GmbH Oskar-von-Miller-Str. 10 D Schongau CULAMI GmbH & Co. KG c/o Herrn Michael Opel Kurt-Schumacher-Platz 11 D Bochum Oskar-von-Miller-Straße 10 D Schongau, Germany Fon Diessen: Fon Schongau: Fax: Web: TEST REPORT September 2, 2015 Tobacco smoke vs. flavoured e-liquid vapour of CULAMI Toxic effects on cultured human lung cells after long-term exposure Tobacco cigarette and e-liquids The investigations were done by using a common cigarette brand of medium strength with 10 mg tar, 0.8 mg nicotine und 10 mg carbon monoxide. For comparison, the following e-liquids from CULAMI GmbH & Co. KG, D Bochum, Germany, were examined as pooled samples mixed from 2 ml of each liquid with 18 mg/ml nicotine: Pool 1 (all liquids with 18 mg/ml nicotine) (1) Fresh Green, Charge , best before 05/2017; (2) Orient Apfeltabak, Charge , best before 05/2017; (3) Hypnotic, Charge , best before 05/2017. Pool 2 (all liquids with 18 mg/ml nicotine) (1) RY4, Charge , best before 07/2017; (2) Erdbeer Eisbonbon, Charge , best before 05/2017; (3) Bourbon Vanille, Charge , best before 05/2017. Simulation of smoking and vaping to obtain the primary extract In order to simulate the real smoking or vaping conditions, a special apparatus was used which allows to vary the frequency, length and the depths of the puffs (Figure 1). For smoking of three cigarettes, 3 x 10 puffs with a duration of 3 seconds and a pause of 15 seconds between two puffs was presumed. See Vansickel AR et al. (2010): A clinical laboratory model for evaluating the acute effects of electronic cigarettes : Nicotine delivery profile and cardiovascular and subjective effects. Cancer Epidemiology, Biomarkers, and Prevention 19:

2 Page 2 (6) For the e-cigarette (egrip OLED from Joyetech with vaporiser 1.7 Ω and 3.3 V = 6.5 W electrical power) 30 puffs of 4 to 5 seconds and a pause of 15 seconds between two puffs were conducted. The smoke of the cigarettes and the vapour of the liquids were aspirated by a suction pump and passed into 20 ml of cell culture medium at 37 C buffered with 10 mm HEPES. The resulting primary extracts had a neutral ph value of 7.4 ± 0.3. The extract was brownish-yellow for cigarette smoke and colourless for e-liquid vapour. Both primary extracts were filtrated sterile by pressing them through a sterile porous membrane (porous size 0.45 µm) and added to the lung cell cultures as described in the next chapter. or 10 puffs of 3-5 seconds with 30 seconds resting p eriod Gas-washing bottle # 1 Gas-washing bottle # 2 T T Pinchcock as flow regulator to suction pump Prim ary extract for cell culture experim ents Water for gas cleaning to avoid pump damage Fig. 1: Experimental setup for simulation of smoking or vaping. The suction pump on the right generates an adjustable underpressure which aspirates the smoke or vapour and passes it into the culture medium in the left gas-washing bottle # 1. This yields the primary extract which is used either undiluted or diluted stepwise for further cell culture experiments. The right gas-washing bottle # 2 is only for gas cleaning to avoid pump damage. Experimental setup The investigations were done with human lung carcinoma cells (cell line A549; ECACC, Salisbury, UK) which are widely used in current scientific research all over the world. See Cervellati F et al.( 2014): Comparative effects between electronic and cigarette smoke in human keratinocytes and epithelial lung cells. Toxicology in Vitro 28: Cells were routinely cultured as mass cultures in a Binder CO 2 incubator at 37 C with a moist atmosphere of 5 % CO 2 and 95 % air. Culture medium was DMEM/Ham s F12 (1:1)

3 Page 3 (6) supplemented with 10 % fetal bovine serum and 100 Units/ml of penicillin & 100 µg/ml of streptomycin. All cell culture reagents were from GE Healthcare Life Sciences, D Cölbe, Germany. For the experiments, cells were taken from 80 to 90 % confluent mass cultures and were seeded in 5 ml culture medium into cell culture flasks (55 cm 2 of growth area) at a density of 1,000 cells/flask to yield a single cell distribution. On the second day after seeding cells were completely attached and spread to the bottom of the flasks. Then, fresh culture medium and the appropriate amounts of primary extract were added to yield the following e-liquid test concentrations: vol%. For cigarette smoke, only 5 vol% of primary extract was added. Pure extract medium in the same dilution served as reagent control (= 0 vol%). On days 4 and 8 of incubation, the evaporated liquid in the culture dishes was balanced by the addition of 0.7 ml of deionised sterile water to keep the osmolarity of the culture medium in the dishes as constant as possible. The lung cells were cultured for a total of 14 days after seeding, i.e. 12 days under the influence of the primary extracts. During this time period the single lung cells were mitotically active with at least five cell divisons so that cell clusters (clones) were finally obtained from the single cells. The number of cell divisions are adequate to a time period of several years in vivo. Because of the long-term exposure, any toxic effect becomes more pronounced than in short-term cultures with an exposure period of only one day (see test report on acute toxicity). After 14 days, culture medium was discarded and the cells were fixed with methanol for 2 minutes and stained with Coomassie-Giemsa solution according to Romanowsky. By this dye combination, cell nuclei are stained red and cytoplasm is stained blue. The stained cultures were air-dried and photographed with a Nikon D300 digital SLR and a macro lens at a magnification of 1:2. The photos were processed by a digital image analysis system (Wimasis Image Analysis, ibidi GmbH, München). The results of the number of cell clones in the flasks are presented in tabular form. The investigations were done in triplicate. Results & conclusions The primary extract of cigarette smoke did not yield any clones or viable single cells at a concentration of 5 vol% after 12 days of continuous exposure. In contrast, human lung cells which were exposed to the both extracts of e-liquid vapour did not show any morphological signs of toxicity or a visually reduced number of clones at all concentrations tested (Fig. 2). Quantitative examination of the number of cell clones showed a cloning efficiency which did not differ from the appropriate controls (Tab. 1). Thus, a toxic effect of CULAMI e-liquid vapour after long-term exposure was not observed. This demonstrates that vaping of the e-liquids tested here is an alternative to tobacco smoking with much lower health risks.

4 Page 4 (6) Annotation: The study was conducted by using pooled e-liquids, but the results are also valid for the single liquids. Moreover, for this present study e-liquids with the highest nicotine concentration of 18 mg/ml were used. The results are also valid for liquids with the same ingredients, but lower nicotine concentrations. Investigator and responsible for the correctness of the presented experiments and results. Schongau September 2, Prof. Dr. Peter C. Dartsch Diplom-Biochemiker

5 Page 5 (6) A: Primary extract = control D: Primary extract = control B: Pool 1 10 vol% E: Pool 2 10 vol% C: Pool vol% F: Pool vol% Fig. 2: Representative macroscopic photographs of the lung cell cultures after 12 days of exposure to undiluted elution medium (control; A, D), 10 vol% of primary extract (B, E) and undiluted primary extract (100 vol%; C, F) of CULAMI liquid pool 1 (B, C) and CULAMI liquid pool 2 (E, F).

6 Page 6 (6) Tab. 1: Tabular presentation of the results for both CULAMI liquid pools. Data represent mean value ± standard deviation of three experiments. Pool 1 Pool 2 Sample M.W. ± S.D. M.W. ± S.D. Control (= 0 vol%) ± ± 4.84 Primary extract 10 vol% ± ± 3.82 Primary extract 25 vol% ± ± 3.22 Primary extract 50 vol% ± ± 3.47 Primary extract 100 vol% (= undiluted) ± ± 3.53 Clon.eff. = Cloning efficiency für clones > px M.W. = Mean value; S.D. = Standard deviation

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