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. Collection of exposure data For systemic toxicity – daily intake in mg (or µg) Estimate daily intake from the e-liquid concentration (%) and liquid use (mL/day) For local toxicity – calculate puff concentration Based on total intake (mg/day), number of puffs and volume of puffs 3

4. Collect background exposure Used in cosmetics, pharmaceuticals, tobacco, food and drink Occurs naturally in a wide variety of foods A synthetic flavouring agent, intake: ~20-50 µg/kg bw/day or ~1200-3000 µg/day JECFA ADI of 0-1 mg/kg bw (1981) A fragrance ingredient Used as a tonic and masking agent in cosmetics 4

5. Exposure from e-liquid For systemic effects assessment Maltol intake stated to be 0.16 mg/day = 2.7 µg/kg bw/day for a 60-kg adult For assessment of respiratory tract irritation Puff maltol concentration = 0.16 mg/day in 0.033 mg/m 3 = 4.8 mg/m 3 5

6. Data source For this assessment, the primary data source was the 2015 JTI Toxicological profile 6

7. ADME data No oral or inhalation ADME data in Profile Based on low MWt and high water solubility (10,900 mg/L), absorption likely to be extensive following ingestion or inhalation Absorbed maltol “is predominantly metabolised to sulfate and glucuronic acid conjugates which are then eliminated in the urine” (JECFA, 2006) 7

8. ADME data Precautionary default is to assume 100% absorption when inhaled, 50% when ingested (as in REACH) Following iv injection into dogs, 57% of dose was excreted in the urine within 24 hr Most (88%) excretion occurred within 6 hr JECFA (2006) stated that maltol expected to be metabolized to innocuous products 8

9. Acute inhalation toxicity Humans – no data Laboratory animals – no useful data Mice exposed for 1 hr at an unspecified atmospheric concentration of maltol - Slight (14%) increase in activity If exposed after caffeine i.p. injection - a 50% decrease in motility Maltol was not detectable or was found only in trace amounts in serum 9

10. Repeated dose inhalation toxicity Humans – no data Laboratory animals – no data 10

11. Other toxicity data – Acute oral/dermal Human – no data Non-human Maltol is of moderate acute oral toxicity in lab animals Oral LD50 values: 1410 mg/kg bw in rats and guinea pigs 550 mg/kg bw in mice 1620 mg/kg bw in rabbits A single rabbit survived a skin application of 5000 mg/kg bw. Indicates a low acute dermal toxicity 11

12. Repeated oral dose studies (1) Human – no data Non-human Rats (50/sex/group, F 1 generation) Dietary administration 0, 100, 200 or 400 mg/kg bw/day for 2 yr Key study used by JECFA to derive an ADI 12

13. Repeated oral dose studies (1) Monitored survival, blood chemistry, haematology, and microscopic appearance of tissues and organs (range not specified in Profile). Presumably also clinical signs and body weight NOAEL = 100 mg/kg bw/day Blood biochemistry changes at 200 mg/kg bw/day (LOAEL) and above Note: JECFA took mg/kg to indicate mg/kg bw, not mg/kg diet. Original source should be consulted 13

14. Repeated oral dose studies (2) Other studies In diet to rats for 6 months NOAEL 200 mg/kg bw/day LOAEL 400 mg/kg bw/day (reduced body weight in females, increased cholesterol and creatinine levels and relative liver weight in males) 14

15. Repeated oral dose studies (3) In diet to mice for 18 months 0, 100, 200 and 400 mg/kg bw/day NOAEL was 200 mg/kg bw/day in males. LOAEL was 400 mg/kg bw/day – kidney and testes effects LOAEL in females was 100 mg/kg bw/day – all groups had a dose-related increase in serum alkaline phosphatase Note: uncertainty over whether the reported mg/kg figures are mg/kg bw or mg/kg diet 15

16. Repeated oral dose studies (4) Dogs (4/group, mixed sex) 0, 125, 250 and 500 mg/kg bw/day by capsule for 90 days LOAEL 125 mg/kg bw/day. A“moderate number” of Kupffer cells (in the liver) had haemosiderin deposits. More overt at 250 mg/kg bw/day Investigators set a ‘conservative’ no-effect level of 100 mg/kg bw/day 16

17. Repeated oral dose studies (5) JECFA consider the key NOAEL to be 100 mg/kg bw/day (in rats and dogs) LOAEL of 200 mg/kg bw/day in rats or 125 mg/kg bw/day in dogs (the latter based on minimal toxicity) Unclear why the mouse LOAEL was not selected 17

18. Repeated dermal dose studies Human – no data Non-human – no data 18

19. Genotoxicity overview (1) BACTERIAL MUTATION Mixed results in Ames bacterial reverse mutation tests Latest EFSA review (2015, not in Profile) says maltol is inactive in Ames Cites the new Ballantyne study. No mention of earlier positive results 19

20. Genotoxicity overview (2) MAMMALIAN CELLS IN VITRO Caused chromosome damage - micronuclei and chromosome aberrations Increased sister chromatid exchange in human and hamster cells 20

21. Genotoxicity overview (3) MAMMALS IN VIVO Intraperitoneal injection led to bone marrow chromosome damage in mouse bone marrow Gavage treatment of rats did not induce micronuclei in bone marrow cells Gavage treatment of rats did not cause liver DNA damage (Comet assay) 21

22. Genotoxicity overview (3) In 2015, EFSA accepted that maltol reached the bone marrow in the oral study and there was no concern for the genotoxicity of maltol in food EFSA report on maltol, dated 30 September 2015: http://www.efsa.europa.eu/sites/default /files/scientific_output/files/main_docu ments/4244.pdf 22

23. Genotoxicity overview (4) Higher quality and newer data generally indicate a lack of mutagenic potential Supported by two lifetime studies where maltol failed to induce cancer in rats or mice Most worrying result is the reported induction of micronuclei in mice following intraperitoneal injection in one 30-year-old in vivo study. 23

24. Genotoxicity overview (4) The situation is possibly akin to phenol, which is inactive following ingestion but mutagenic following injection Overall it seems highly unlikely that Expert Groups would approve, or regulators permit, maltol’s use as an added food flavour if it possessed significant genotoxic character Conclusion: Maltol is considered non- mutagenic in this assessment 24

25. Carcinogenicity (1) Human - no data Non-human - No inhalation cancer studies JECFA summarised two unpublished dietary studies Rats and mice (50/sex/species/group) 0, 100, 200 or 400 mg/kg bw/day for 2 years (rats) or 18 months (mice) Microscopic examination of “an extensive range of tissues” No evidence of carcinogenicity 25

26. Carcinogenicity (2) Profile includes (taken from CCRIS, 2006 ) “A number of studies have shown that maltol may induce carcinogenicity and toxicity but the mechanisms involved remain unknown. Therefore, we examined the ability of maltol to induce the cytochrome P450 1a1 (Cyp1a1), an enzyme known to play an important role in the chemical activation of xenobiotics to carcinogenic derivatives. ….. This is the first demonstration that … maltol … can directly induce Cyp1a1 gene expression in an AhR-dependent manner and represents a novel mechanism by which maltol promotes carcinogenicity and toxicity.” Highly unlikely that Expert Groups would support, or regulators permit, the direct addition of a mutagenic carcinogen to food Conclusion: Based on adequate rat and mouse studies, maltol is not considered carcinogenic 26

27. Reproductive and developmental toxicity Human – no data Non-human A 3-generation dietary study Rats given up to 400 mg/kg bw/day No adverse effects on reproduction or development 27

28. Other relevant data – skin irritation Human - No data for undiluted maltol No irritation when 10% in petrolatum was applied repeatedly (48-hr applications) in a maximization study (25 subjects) Used at 1% in petrolatum in patch-tests [presumably in 24-/48 ‑ hr covered contact]. Presumably non- irritating Non-human Maltol was moderately irritating to rabbit skin Probably applied undiluted for 24 hr, covered 28

29. Other relevant effects – sensitisation and intolerance No data on respiratory sensitisation Human Maltol (10% in petrolatum) did not induce skin sensitization in 25 volunteers given repeated dermal applications One report of an allergic reaction in a woman who developed eczema around the lips from use of a lip salve Patch tested with 1% maltol in petrolatum [presumably 24/48-hr covered contact] 29

30. Expert Group Health Criteria Values or Occupational Exposure Levels No inhalation HCVs or OELs were identified One oral HCV for long-term exposure JECFA set an ADI of up to 1 mg/kg bw/day (maintained in 2006) Based on an NOAEL of 100 mg/kg bw/day 30

31. Expert Group Health Criteria Values or Occupational Exposure Levels Evidently from the 2-yr rat dietary study JECFA presumably applied the traditional factors of 10 each for intra- and inter- species toxicity EFSA (2015) noted the JECFA reviews and stated that “…no EFSA consideration is required” 31