2. 1.Collection of relevant data – toxicity and exposure 2.Selection of critical studies and/or HCVs 3.Health risk assessment – systemic 4.Health risk assessment – respiratory tract irritation PRESENTATION OVERVIEW

3. For systemic toxicity – daily intake in mg (or µg) Obtain from concentration in e-liquid (%) and liquid use (ml/day) For local toxicity – calculate puff concentration Based on total intake (mg/day), number of puffs and volume of puffs COLLECTION OF EXPOSURE DATA 3

4. Used as a flavouring in food and drink, pharmaceuticals, tobacco, and cosmetics No measured environmental or occupational air concentration data Food flavouring intakes estimated at about 43-135 and 5.4 mg/person/day in US and Europe respectively COLLECT BACKGROUND EXPOSURE DATA 4

5. For this assessment, the primary data source was the 2015 JTI Toxicological profile The REACH dossier (as publically disseminated) was consulted for the inclusion of DNELs and to ensure all key data was captured COLLECTION OF TOXICITY DATA 5

6. Ethyl vanillin intake = 0.28 mg/day [for systemic effects assessment] Puff volume 55 mL 600 puffs/day Total puff volume = 0.033 m 3 Puff ethyl vanillin concentration = 8.5 mg/m 3 [for local effects assessment] EXPOSURE EXAMPLE 6

7. ORAL ADME DATA Rapidly and extensively absorbed Metabolised primarily to ethyl vanillic acid (@60%), but also ethyl vanillyl alcohol (@17%) Excreted primarily in the urine within 24 hr 7

8. Inhaled – no in vivo data Log Kow (@1.6), high water-solubility (2822 mg/L) and low MWt (166) indicate extensive and rapid absorption, and wide distribution Precautionary default is to assume 100% absorption (as in REACH) INHALATION ADME DATA 8

9. If available, start with most appropriate key studies – acute and repeated inhalation Other route studies give additional insights into toxicity potential – oral, dermal Mutagenicity, carcinogenicity, reproductive/developmental, sensitisation, etc May use read-across or QSAR data to fill gaps and/or enrich data set TOXICITY INTRO 9

10. Humans - No data Laboratory animals - No data ACUTE AND REPEATED INHALATION TOXICITY 10

11. Human No data identified Non-human Oral LD 50 = 1590-4470 mg/kg bw in rats and rabbits Oral LDLo = 3000 mg/kg bw in rabbits CNS effects at high oral dose. Dermal LD 50 = >7940 mg/kg bw in rabbits >2000 mg/kg bw in rats (from REACH ) OTHER TOXICITY DATA: ACUTE ORAL/DERMAL 11

12. Human No data identified Non-human Studies 1 & 2 [Quality unknown – but longest duration] Study 1: Rats (5 male/group) fed diets containing 0, 2 or 5% for 1 yr (about 0, 1000 or 2500 mg/kg bw/day) Study 2: Rats (12/sex/group) fed diets containing 0, 0.5, 1 or 2% for 2 yr (about 0, 250, 500 or 1000 mg/kg bw/day) Periodic blood examinations. Histopathology of major tissues. No adverse effects; NOAEL 2.5 and 1 g/kg bw/day REPEATED ORAL DOSE STUDIES 12

13. Study 3. [FDA guideline; Hooks et al., 1992] Rats (20/sex/group) In diet at 0, 500, 1000 or 2000 mg/kg bw/day for 90 days. Comprehensive examination. NOAEL 500 mg/kg bw/day; LOAEL 1000 mg/kg bw/day REPEATED ORAL DOSE STUDIES 13

14. Critical effects: ↓ bodyweight gain and impaired food efficiency in high dose group Biochemical changes (e.g. ALP and cholesterol) in high dose group ↑ liver and spleen weights (mid and high dose groups) Enlarged cervical lymph nodes (mid and high dose groups) Dose-related ↑ in liver inflammation and bile duct hyperplasia (mid and high dose groups) Histopathological changes in the spleen and lymphoid tissue (mid and high dose groups) REPEATED ORAL DOSE STUDIES 14

15. Study 4. [Deichmann & Kitzmuller, 1940 – quality?] Rats (unspecified number) Gavage, 64 mg/kg bw/day, for 10 wk Extent of examination unclear ↓growth rate, and damage to heart, kidney, liver, lung, spleen and stomach REPEATED ORAL DOSE STUDIES 15

16. Mutation Negative in several Ames bacterial reverse mutation tests Positive in mammalian cell assays for mutation Negative for mutagenic activity in vivo (Drosophila) Conclusion: Mutagenic potential cannot be ruled out on this basis. GENOTOXICITY 16

17. Clastogenicity Negative in mammalian cell assays for chromosome aberration (although polyploidy seen in one study) Equivocal in mammalian cell assays for sister chromatid exchange Negative for in vivo chromosome aberrations in vivo Conclusion: Inactive for chromosome aberrations. SCE and polyploidy seen. GENOTOXICITY 17

18. Human data None identified. Non-human Study 1: Rats (5 male/group) fed diets containing 0, 2 or 5% for 1 yr (about 0, 1000 or 2500 mg/kg bw/day) Study 2: Rats (12/sex/group) fed diets containing 0, 0.5, 1 or 2% for 2 yr (about 0, 250, 500 or 1000 mg/kg bw/day) Periodic blood examinations. Histopathology of major tissues. No increase in tumour incidence [Limited pre-guideline assays. Generally require 50/sex/group, microscopic examination of a comprehensive range of tissues and organs, and treatment of at least 3 dose groups daily for 2 yr] CARCINOGENICITY 18

19. Human No data identified. Non-human No adverse effects on reproductive organs (e.g. weight and histopathology of ovaries and testes) in 90-day study. Fertility effects not adequately assessed (e.g. 1-, 2- or multi-gen studies). Notably males. REPRODUCTIVE AND DEVELOPMENTAL TOXICITY 19

20. Non-human cont. Female rats (10/group) Gavaged at 0, 200, 1000 or 2000 mg/kg bw/day ≥ 1 wk pre-mating to 4 days after birth. Maternal toxicity at all doses (e.g. growth) No adverse effects observed on the offspring. No effects on ‘female reproductive performance’ or developmental effects REPRODUCTIVE AND DEVELOPMENTAL TOXICITY 20

21. Human 2% produced mild skin irritation (48-hr closed patch) in 25 subjects. 5% was not irritating (probably 24/48-hr covered patch) in 200 patients. ‘Aldehydes’ are irritating to the eyes and mucous membranes of the respiratory tract. [Also -ve in human cornea in vitro tests (from REACH)] Non-human Ethyl vanillin did not irritate rabbit skin (500 mg, 24 hr, covered) or rabbit eyes (100 mg, 24 hr) (from JTI profile) Was slightly irritating to rabbit skin and eyes (from REACH) OTHER RELEVANT DATA: IRRITATION 21

22. No data on respiratory sensitisation Human No skin sensitisation reactions in maximisation test with 25 subjects at 2%. Case of allergic occupational contact dermatitis to cutting fluid and several ingredients (including ethyl vanillin) Non-human Not sensitising in LLNA [not further described in JTI profile – more detail in REACH dossier]. Equivocal in guinea pigs (from REACH dossier). No convincing immunotoxicity effect in orally exposed mice. OTHER RELEVANT EFFECTS: SENSITISATION AND IMMUNOTOXICITY 22

23. No Expert Group inhalation HCVs identified. No worker OELs. No worker or gen. pop. inhalation DNELs determined. EXPERT GROUP INHALATION HCVs AND OELS 23

24. Other HCVs HCV (mg/kg bw/day) BasisReference Oral acceptable daily intake (ADI) 0-3Not applicable JECFA, 1996; 2002 OTHER KEY HCVs Also considered as GRAS (US FDA) 24