A Comparative In Vitro Toxicological Screening of a Closed-End Heated Tobacco Product *
, , , , , , , , , , , , , , and | Dec 16, 2023
About this article
Published Online: Dec 16, 2023
Page range: 146 - 156
Received: Jun 05, 2023
Accepted: Nov 23, 2023
DOI: https://doi.org/10.2478/cttr-2023-0018
© 2023 Zhenyu Niu et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.
Figure 1.
Figure 2.
Salmonella typhimurium strains characteristics, source and positive controls.
| Strains | Mutation | Antibiotic resistance | Positive controls | |
|---|---|---|---|---|
| Without S9 (μg/plate) | With S9 (μg/plate) | |||
| TA98 | His D3052 | Ampicillin | 2-Nitrofluorene (4 μg/plate) | 2-Amlinofluorene (10 μg/plate) |
| TA100 | His G46 | Ampicillin | 4-Nitroquinoline |
2-Amlinofluorene (10 μg/plate) |
In vitro mammalian cell micronucleus test.
| Positive controls | Ultimate density |
|---|---|
| Cyclophosphamide A (short-term + S9) | 0.2 μg/mL |
| Mitomycin C (short-term - S9) | 1.0 μg/mL |
| Mitomycin C (long-term - S9) | 0.5 μg/mL |
Revertant colonies of Salmonella typhimurium obtained following exposure to different doses of TPM from HTP-A and HTP-B.
| Dose (μg/plate) | TA98 | TA100 | |||
|---|---|---|---|---|---|
| HTP-A | HTP-B | HTP-A | HTP-B | ||
| −S9 | |||||
| NC | 38.0 ± 2.4 | 38.0 ± 2.4 | 115.3 ± 3.8 | 115.3 ± 3.8 | |
| 0 | 25.7 ± 4.0 | 25.7 ± 4.0 | 121.7 ± 6.0 | 121.7 ± 6.0 | |
| 50 | 24.0 ± 3.7 | 30.3 ± 4.0 | 124.0 ± 4.1 | 108.0 ± 9.9 | |
| 500 | 35.0 ± 3.7 | 28.7 ± 4.1 | 120.3 ± 8.7 | 105.0 ± 8.3 | |
| 1000 | 30.7 ± 2.4 | 33.3 ± 5.7 | 114.3 ± 6.2 | 129.3 ± 5.3 | |
| 2000 | 30.7 ± 3.8 | 32.7 ± 4.5 | 120.7 ± 7.9 | 125.3 ± 3.1 | |
| PC | 899.0 ± 24.0 | 899.0 ± 24.0 | 2719.7 ± 55.5 | 2719.7 ± 55.5 | |
| +S9 | |||||
| NC | 60.0 ± 5.9 | 60.0 ± 5.9 | 156.3 ± 2.9 | 156.3 ± 2.9 | |
| 0 | 51.0 ± 5.1 | 51.0 ± 5.1 | 107.0 ± 6.4 | 107.0 ± 6.4 | |
| 50 | 48.3 ± 7.6 | 48.3 ± 3.1 | 116.0 ± 19.0 | 100.0 ± 4.3 | |
| 500 | 48.7 ± 7.4 | 56.0 ± 4.3 | 112.0 ± 10.7 | 100.7 ± 5.4 | |
| 1000 | 49.0 ± 7.1 | 40.3 ± 5.2 | 113.7 ± 12.4 | 100.7 ± 9.2 | |
| 2000 | 51.0 ± 2.8 | 41.7 ± 7.0 | 94.0 ± 16.8 | 105.3 ± 1.2 | |
| PC | 2437.3 ± 106.0 | 2437.3 ± 106.0 | 781.7 ± 95.7 | 781.7 ± 95.7 | |
Selected toxicant yields from two HTPs and 3R4F.
| Analyte | Unit | HTP-A | HTP-B | 3R4F |
|---|---|---|---|---|
| Nicotine | mg/stick | 0.47 ± 0.01 | 0.98 | 1.05 |
| Total particulate matter | mg/stick | 29.06 ± 1.36 | 30.15 | 24.23 |
| mg/mg nicotine | 61.83 ± 2.90 | 30.77 | 23.08 | |
| Carbon monoxide | mg/stick | 0.19 ± 0.01 | 0.25 | 24.69 |
| mg/mg nicotine | 0.40 ± 0.02 | 0.26 | 23.52 | |
| Formaldehyde | μg/stick | 1.08 ± 0.18 | 4.46 ± 0.56 | 26.60 ± 2.14 |
| μg/mg nicotine | 2.30 ± 0.38 | 4.55 ± 0.57 | 25.33 ± 2.04 | |
| Acetaldehyde | μg/stick | 123.5 ± 19.53 | 172.42 ± 6.10 | 722.47 ± 36.11 |
| μg/mg nicotine | 262.77 ± 41.55 | 175.94 ± 6.22 | 688.07 ± 34.39 | |
| Acrolein | μg/stick | 4.78 ± 0.91 | 10.90 ± 0.18 | 71.92 ± 2.84 |
| μg/mg nicotine | 1.02 ± 1.94 | 11.12 ± 0.18 | 68.50 ± 2.70 | |
| 1,3-Butadiene | μg/stick | NQ | NQ | 8.13 ± 0.17 |
| μg/mg nicotine | NQ | NQ | 7.74 ± 0.16 | |
| Benzene | μg/stick | 0.17 ± 0.00 | 0.36 ± 0.05 | 49.95 ± 1.54 |
| μg/mg nicotine | 0.36 ± 0.00 | 0.37± 0.05 | 47.57 ± 1.47 | |
| Benzo( |
ng/stick | NQ | NQ | 24.00 ± 0.64 |
| ng/mg nicotine | NQ | NQ | 22.86 ± 0.61 | |
| NNK | ng/stick | 6.61 ± 0.86 | 5.32 ± 0.89 | 281.00 ± 16 |
| ng/mg nicotine | 14.06 ± 0.83 | 5.43 ± 0.91 | 267.62 ± 15 | |
| NNN | ng/stick | 1.79 ± 0.26 | 8.37 ± 0.55 | 163.43 ± 11.19 |
| ng/mg nicotine | 3.81 ± 0.55 | 8.54 ± 0.56 | 155.65 ± 10.66 |
Revertant colonies of Salmonella typhimurium obtained following exposure to different doses of TPM from 3R4F.
| Dose (μg/plate) | TA98 | TA100 | ||
|---|---|---|---|---|
| −S9 | +S9 | −S9 | +S9 | |
| NC | 38.0 ± 2.4 | 60.0 ± 5.9 | 115.3 ± 3.8 | 156.3 ± 2.9 |
| 0 | 25.7 ± 4.0 | 51.0 ± 5.1 | 121.7 ± 6.0 | 107.0 ± 6.4 |
| 50 | 38.3 ± 8.2 | 87.7 ± 5.2 | 125.7 ± 8.5 | 105.0 ± 3.7 |
| 100 | 28.3 ± 7.5 | 124.3 ± 5.4* | 127.3 ± 7.6 | 115.3 ± 7.5 |
| 250 | 32.7 ± 6.1 | 237.7 ± 11.8* | 143.3 ± 8.3 | 133.0 ± 4.1 |
| 500 | 34.7 ± 2.1 | 233.7 ± 33.0* | 137.7 ± 11.1 | 171.7 ± 13.0 |
| PC | 899.0 ± 24.0 | 2437.3 ± 106.0 | 2719.7 ± 55.5 | 781.7 ± 95.7 |
Analytical procedures used for HTP aerosol and cigarette smoke chemical analyses.
| Analyte | Brief description | Reference |
|---|---|---|
| Nicotine | By gas chromatography with flame ionization detection | ISO 10315 ( |
| CO | By nondispersive infrared photometry | ISO 8454 ( |
| Polycyclic aromatic hydrocarbons | Extracted by hexane and analyzed by GC-MS | CRM 91 ( |
| Extracted by acetic acid and analyzed by HPLC-MS/MS | CRM 75 ( |
|
| Volatile organic compounds | By GC-MS using simultaneous trapping of adsorbent cartridges in a Cambridge filter without cryogenic impinger | WHO SOP 09 ( |
| Carbonyl compounds | By HPLC, using simultaneous trapping of adsorbent cartridges in a Cambridge filter without cryogenic impinge | WHO SOP 08 ( |
Cell genotoxicity of different TPM fractions of HTP-A and HTP-B, and 3R4F.
| Sample | Concentration (μg/mL) | MN frequencies (%) | ||
|---|---|---|---|---|
| 3h + 21h -S9 | 3h + 21h +S9 | 24h + 0h -S9 | ||
| Vehicle | 11 | 1.49 ± 0.23 | 1.52 ± 0.54 | 1.08 ± 0.08 |
| HTP-A | ||||
| 75 | 1.37 ± 0.33 | 1.28 ± 0.04 | 1.29 ± 0.08 | |
| 150 | 1.08 ± 0.37 | 1.20 ± 0.07 | 1.19 ± 0.04 | |
| 300 | 1.05 ± 0.07 | 1.21 ± 0.01 | 1.40 ± 0.08 | |
| 600 | 1.15 ± 0.17 | 1.10 ± 0.10 | 1.30 ± 0.01 | |
| HTP-B | ||||
| 75 | 1.11 ± 0.02 | 1.20 ± 0.14 | 1.26 ± 0.16 | |
| 150 | 1.33 ± 0.22 | 1.07 ± 0.18 | 1.28 ± 0.11 | |
| 300 | 1.36 ± 0.30 | 1.28 ± 0.01 | 1.33 ± 0.13 | |
| 600 | 1.69 ± 0.30 | 1.29 ± 0.21 | 1.32 ± 0.11 | |
| 3R4F | ||||
| 14.3 | 2.03 ± 0.21 | 2.16 ± 0.92 | 2.53 ± 0.05 | |
| 28.5 | 3.21 ± 1.02 | 2.46 ± 0.98 | 2.85 ± 0.20 | |
| 57.0 | 3.93 ± 1.34 | 2.31 ± 1.29 | 2.65 ± 0.05 | |
| 114.0 | 4.49 ± 1.58 | 2.67 ± 1.49 | 3.03 ± 0.30 | |
| PC | - | 5.68 ± 0.29 | 5.08 ± 0.55 | 6.09 ± 0.37 |
| NC | - | 1.40 ± 0.30 | 1.32 ± 0.30 | 1.20 ± 0.11 |
Overview of test product specifications. The heating device of HTP-A is branded as Mr. Yeah and the tobacco sticks carry the brand FARSTAR. HTP-B consists of an IQOS™ branded heating device and TEREA branded tobacco sticks.
| Parameter | HTP-A | HTP-B | Combustible cigarette |
|---|---|---|---|
| Commercial product (name) | Mr. Yeah | IQOS™ | 3R4F |
| Puffing air flow design | Closed-end | Open-end | Open |
| Heater design | External heating | Central heating | Combustion |
| Maximum heating temp. (°C) | 260 | 350 | / |
| Puff n° under HCI regime | 8 | 12 | 8 |
| Tobacco stick-length (mm) | 45 | 45 | 84 |
| Tobacco stick-weight (mg) | 721.70 | 664.30 | 1035.00 |
| Tobacco stick-diameter (mm) | 7.36 | 7.18 | 7.98 |