2. Health risk assessment – systemic effects (1) REMINDER OF INHALED DOSE PG intake of 3.2 mg/day or 0.053 mg/kg bw/day for a 60-kg bw consumer Systemic absorption likely to be extensive. Ready metabolism to simple natural compounds Genotoxicity, carcinogenicity, reproductive/developmental toxicity, sensitisation data are reassuring Repeated dose toxicity is low – JECFA set an ADI of 25 mg/kg bw 2

3. A key study is the subchronic rat inhalation study by Suber et al. Ideally would assess study quality in terms of group sizes, extent of examination etc though such data not present in citing abstract Despite lack of detailed description, this study is deemed critical for the current assessment since it is subchronic in duration and involves the inhalation route of exposure Health risk assessment – systemic effects (2) 3

4. Systemic NOAEC = 160 mg/m 3, 6 hr/day, 5 days/wk for 90 days First, convert to a 13-wk NOAEL Use REACH default - a rat inhales 0.29 m 3 /kg bw over 6 hr Health risk assessment – systemic effects (2) 4

5. So in 6 hr, a rat inhales 46 mg/kg bw (160 x 0.29) = study NOAEL Next, a factor to account for the 5 days/wk exposure schedule NOAEL = 46 mg/kg bw/day x 5 days/7 days = 33 mg/kg bw/day Health risk assessment – systemic effects (2) 5

6. Health risk assessment – systemic effects (3) Next, convert to chronic value using REACH AF of 2 Lifetime NOAEL = 16.5 mg/kg bw/day Next convert to a human HCV by using traditional factors of 10 for interspecies and 10 for intraindividual differences Human HCV = 0.17 mg/kg bw/day The inhaled dose is 3.2 times lower than the derived HCV Conclusion: Based on this subchronic inhalation study, the exposure is tolerable 6

7. Health risk assessment – systemic effects (4) Useful also to consider Additional sources of exposure e.g. PG intake of 14.01 mg/kg bw/day as a food additive Humans are highly efficient at metabolising PG to innocuous products AFs of 10 may not be necessary for inter- and intra-individual differences 7

8. Health risk assessment – systemic effects (5) Use oral data for additional risk insights Absorption likely to be similar and extensive from both routes JECFA ADI of 25 mg/kg bw is almost 500 times higher than the exposure being assessed here (0.053 mg/kg bw/day) Typical dose from diet = 840 mg/day (for a 60 ‑ kg adult) 263 times more than e-liquid dose (3.2 mg/day) 8

9. Health risk assessment – systemic effects (6) Oral HCVs can be derived from the (sub)chronic rat feeding studies Oral TDI from 2-yr study. NOAEL = 1300 mg/kg bw/day AFs of 10 for interspecies and 10 for intraspecies variations Lifetime study so no duration AF needed Apply total AF = 100, yields an Oral TDI = 13 mg/kg bw/day This is 240-fold higher than the (inhaled) dose (0.053 mg/kg bw/day) 9

10. Health risk assessment – systemic effects (7) A long-term inhalation DNEL could also be derived from this study For absorption differences between routes, the default is to assume 100% inhalation and 50% oral, introducing an AF of 2 when deriving an inhalation DNEL from an oral study 10

11. Health risk assessment – systemic effects (7) This is considered unnecessary for PG – absorption likely to be 100% from both routes Health-precautionary to add an AF for first pass metabolism – perhaps 2 or 3 is adequate Leads to an inhalation DNEL of about 6.5 mg/kg bw/day (about 120-fold higher than the estimated dose) 11

12. Health risk assessment – systemic effects (8) Oral TDI from 140-day study. NOAEL = 7700 mg/kg bw/day AFs of 10 for interspecies and 10 for intraspecies variations and 2 for the use of a subchronic study Apply total AF = 200, yields an Oral TDI = 38.5 mg/kg bw/day This is 720-fold higher than the (inhaled) dose (0.053 mg/kg bw/day) 12

13. Health risk assessment – systemic effects (9) A long-term inhalation DNEL could also be derived from this study The default AF of 2 when deriving an inhalation DNEL from an oral study is considered unnecessary as absorption is likely to be 100% from both routes 13

14. Health risk assessment – systemic effects (9) Health-precautionary to add an AF for first pass metabolism – perhaps 2 or 3 is adequate Leads to an inhalation DNEL of about 19 mg/kg bw/day (about 360-fold higher than the estimated dose) 14

15. Use of OELs in setting systemic HCVs OELs range from 10-474 mg/m 3 Assume 10 m 3 air/work shift Inhaled doses of 100-4740 mg/day are acceptable Apply a factor of 5/7 to account for days/wk of exposure 15

16. Use of OELs in setting systemic HCVs Normal to apply a factor of 2 to account for higher susceptibility in general population [Possibly unnecessary for PG] Tolerable doses for consumers = 36-1690 mg/day 16

17. Systemic health risk overview table Summary table showing Margins of Exposure Conclusion: Highly unlikely to pose any systemic toxicity risks Benchmark Relevant route Benchmark (mg/kg bw/day) Exposure (mg/kg bw/day) MoE DietOral14.010.053263 REACH DNEL from unknown inhalation study Inhalation250.053469 Bibra DNEL from 13-wk inhalation study Inhalation0.170.0533.2 JECFA ADIOral250.053470 Bibra DNEL from 2- yr oral study Oral130.053243 Bibra DNEL from 2- yr oral study Inhalation6.50.053122 Bibra DNEL from 140-day oral study Oral38.50.053722 Bibra DNEL from 140-day oral study Inhalation190.053361 OELs 10-474 mg/m 3 36-1690 mg/day (10-474 mg/m 3 ) 3.2 (mg/day) 11-528 17

18. Health risk assessment – respiratory tract irritation (1) REMINDER OF INHALED CONCENTRATION PG intake of 3.2 mg/day 600 puffs/day Puff volume 55 mL Total puff volume = 0.033 m 3 Assumed even distribution in puffs Puff PG concentration = 97 mg/m 3 18

19. Health risk assessment – respiratory tract irritation (2) Key study 1 – Human data (Wieslander et al.) Volunteers exposed at mean conc of 309 mg/m 3 for 1 min - reduced tear film stability and sensations of eye irritation Likely to be whole-body exposure More detailed concentration data would be preferable Best estimate of LOAEC for acute exposure = 309 mg/m 3 19

20. Health risk assessment – respiratory tract irritation (2) No interspecies AF required as human data Factor of 3 for use of an LOAEC, not an NOAEC Debatable whether an intraspecies factor of 10 is required for a local effect, bibra prefers 3.2 (arguing that toxicokinetics are irrelevant) Generates a tolerable 1-min concentration of about 32 mg/m 3 20

21. Health risk assessment – respiratory tract irritation (3) For interest: Key study 2 – Rat data (Werley et al.) Rats exposed (nose-only) at 44.9 g/m 3 for 4 hr - mild respiratory tract irritation (localised bleeding around eyes/nose) seen on day 7 In the absence of further conc data in the Profile, LOAEC for acute exposure = 44.9 g/m 3 Further investigation reveals two lower concs (14.4 and 30.5 g/m 3 ) were tested and showed the same irritant effects More appropriate to use 14.4 g/m 3 as the LOAEC 21

22. Health risk assessment – respiratory tract irritation (3) Factor of 2.5 for interspecies toxicodynamics (toxicokinetic AF not required for local effects) Factor of 3 for use of an LOAEC, not an NOAEC More debatable whether an intraspecies factor of 10 is required for a local effect, bibra prefers 3.2 (arguing that toxicokinetics are again irrelevant) Generates a tolerable concentration of about 600 mg/m 3 (for human 4-hr exposure) 22

23. Health risk assessment – respiratory tract irritation (4) Consumer use - peak PG exposure at 97 mg/m 3 Such exposure will last for a few seconds, for 2/12 breaths during each minute Lasting maybe 10-40 minutes, repeated several times each day Such a concentration is roughly 3 times higher than the human derived NOAEC (32 mg/m 3 ) from human data but 6-fold lower than the human derived NOAEC (600 mg/m 3 ) from rat data The peak puff concentration is higher than the derived human “tolerable figure” (97 mg/m 3 cf 32 mg/m 3 ) 23

24. However, the e-cigarette exposure would be briefly (a 5-second puff) with rapid dilution from subsequent normal breaths The difference (12x) in the exposure duration for the human experiment (60 seconds) and the e-cigarette exposure (5 seconds) is greater than the exposure margin (3). Implies tolerability The rat LC50 study was not designed to assess irritation so a lower LOAEC may exist. However, not considered to be influential as human data exist Health risk assessment – respiratory tract irritation (5) 24

25. Health risk assessment – respiratory tract irritation (6) Consider the human exposure time in more detail Compare human exposure time (60 seconds) with the cumulative consumer exposure time during a similar 1-min period (5 sec/puff x 2 puffs/min = 10 secs) 25

26. Health risk assessment – respiratory tract irritation (6) Tolerable human exposure of 32 mg/m 3 based on volunteers exposed for a total of 60 seconds; consumers exposed at 97 mg/m 3 for 10 seconds Consumer exposure would be about 3 times higher but 6 times shorter duration Provides some evidence that respiratory tract irritation would not occur 26

27. Health risk assessment – respiratory tract irritation (7) Also useful to consider ratio of exposure to non-exposure parameters Take a single “intense” 40 ‑ minute session Ratio of e-cigarette exposure to non-exposure time is 1/5 (400 seconds/2000 seconds; assuming a 5-second puff exposure) 27

28. Health risk assessment – respiratory tract irritation (7) Ratio of exposure puffs to non ‑ exposure breaths is 1/5 (80/400) Ratio of exposure puff volume to non- exposure breath volume is 1/150 (0.0044 m 3 /0.6623 m 3 ) [worst-case] The 0.6623 m 3 figure is calculated by subtracting the total exposure puff volume in the 40 ‑ minute intense period from the total inhaled volume in 40 minutes (assuming a 1 m 3 per hour inhalation rate during the 16-hr day) 28

29. Health risk assessment – respiratory tract irritation (8) Take the full 16-hr daily use period Ratio of e-cigarette exposure to non- exposure time is 1/18 (3000 seconds/55,200 seconds) Ratio of exposure puffs to non ‑ exposure breaths is 1/18 (600/11,040) Ratio of exposure puff volume to non- exposure breath volume is 1/480 (0.033 m 3 /15.97 m 3 ) 29

30. Health risk assessment – respiratory tract irritation (9) Seems unlikely that exposure would be irritant 80/480 breaths (in a 40-min session) or 600/11,520 breaths (over a 16-hr use period) Exposure is limited to about 17% of the inhaled breaths in an intense session, so recovery time may be sufficient Critical studies involved whole-body exposure of humans and nose-only exposure of rats 30

31. Health risk assessment – respiratory tract irritation (9) e-Cigarette exposure is mouth-only Rodents are obligate nose-breathers and the nasal epithelium is a dry tissue compared with the mouth, where the production of about 1 L/day of saliva in humans keeps tissues moist In the human study the whole body exposure only induced irritant effects on the eyes (i.e. not as relevant here) 31

32. Health risk assessment – respiratory tract irritation (10) PG expected to dissolve in saliva; local tissues might experience an average concentration of around 0.0015% each minute Neat PG only mildly irritant to human eye and non-irritant to rabbit eye (presumably neat) Oral cavity tissues are probably less susceptible than nasal tissues E-cigarette exposure is mouth only; the effects in rats occurred in the (drier) nasal tissue 32

33. Health risk assessment – respiratory tract irritation (10) Conclusion: the intermittent nature of the brief exposures and the moist conditions in the mouth (compared to the rat nose) mean that respiratory tract irritation is unlikely [In 1 minute, 0.0107 mg of PG would be inhaled (3.2 mg/day x 2 puffs/min / 600 puffs/day) while 1 L/day of saliva is equivalent to 0.69 mL/min (1 x 1000 mL/1440 min). Therefore, if all the inhaled PG were to dissolve in the saliva, a concentration of about 0.0015% PG would result (0.0107 mg/0.69 mL = 0.015 mg/mL, equivalent to 0.0015 g in 100 mL i.e. 0.0015%)] 33