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. Identification and background data CAS RN: 8006-90-4 Used in cosmetics, pharmaceuticals, tobacco, food and drink, home products and pesticides Used as a flavouring in foods and drinks Estimated intake from flavouring use is 1.18 mg/kg bw/day (about 71 mg day for a 60-kg person) Fragrance ingredient Cosmetics - masking, perfuming, refreshing and tonic ingredient 3

4. Exposure Peppermint oil (PO) intake = 7.2 mg/day (0.12 mg/kg bw/day for a 60-kg adult) [for systemic effects assessment] 600 puffs/day Puff volume 55 mL Total puff volume = 0.033 m 3 Puff PO concentration = 218.2 mg/m 3 [for local effects assessment] 4

5. ADME data Oral – rapidly absorbed Inhaled – absorbed through the lungs (variable extent) and rapidly eliminated As an oil, probably water insoluble 5

6. Acute inhalation toxicity Human – no useful data No adverse effects at undisclosed concentration (Study of peppermint aromatherapy for relief of post ‑ operative nausea) 6

7. Acute inhalation toxicity Menthol at up to 60% in peppermint oil Menthol data relevant Transient euphoria, involuntary eye movement, double vision and unsteadiness in a 13-yr-old who inhaled menthol (estimated 4 mg/kg bw) in a volatile oil preparation 7

8. Repeated dose inhalation toxicity Humans No data on peppermint oil alone No adverse effects in 14 individuals who repeatedly inhaled an unknown amount of essential oil mix (peppermint, basil and helichrysum) for 1 week, through a personal inhaler 8

9. Repeated dose inhalation toxicity Menthol data Workers at a factory producing menthol Exposed to menthol at 12-380 mg/m 3 plus a range of fragrance oils Evidence of liver and kidney damage 9

10. Repeated dose inhalation toxicity Woman smoked 80 menthol cigs/day for 3 months About 0.8 mg/kg bw/day CNS effects - digestive disorders, vomiting, irritability, unsteadiness, insomnia, speech disorders, tremors, confusion, mental depression and slowed heart rate Symptoms disappeared 3 weeks after she changed to a non-mentholated brand 10

11. Repeated dose inhalation toxicity Laboratory animals - menthol Rats (4/group) Inhaled l-menthol (6.75 hr/day, 5 days/wk) – for up to 52 exposures No effects at up to 0.9 mg/m 3 At 1.6 mg/m 3 - no overt toxicity; detailed tissue examination found only lung congestion and inflammation 11

12. Repeated dose inhalation toxicity Laboratory animals - menthol Mice, exposed at 100 mg/m 3 for 3 months [no other details] “Regression changes” in liver and kidney Monkeys (four), inhaled 40 mg/kg bw/day as an aerosol, 8 hr/day for 14 days No overt signs of toxicity 12

13. Other toxicity data - Acute oral/dermal Human PO directly into stomach or colon, or single ingestion at 0.1-0.24 ml – reduced spasms of the smooth muscles of the gastrointestinal tract Oral PO at 182 mg by capsule reduced intra-gastric pressure, contractility and appetite 13

14. Other toxicity data - Acute oral/dermal Human A man who swallowed a “peppermint capsule” [quantity not specified] had a severe burning sensation in the anus [probably a local effect] 0.1 ml PO (in 20 ml saline) instilled into the bowel of five volunteers resulted in abdominal cramps/pain (4 cases) and nausea (1 case) 14

15. Other toxicity data - Acute oral/dermal Non-human PO oral LD50s: >2400 mg/kg bw in rats and mice In rats, oral PO at ~750 mg/kg bw led to a 70% increase in bile flow (not seen at 7.5 mg/kg bw) No change in liver enzyme activity or in liver histology. “Doses 2-3 orders of magnitude greater than recommended for human use “ No acute dermal toxicity on PO identified 15

16. Repeated oral dose studies Human Various clinical trials where PO was given in capsule form for the treatment of irritable bowel syndrome Doses: 90-1080 mg/day [or 1.5-18 mg/kg bw/day for a 60-kg adult] Effects included: burning sensations, neurological disturbances and reduced spasms of the GI tract smooth muscles 16

17. Repeated oral dose studies Irregular or reduced heart beat reported from chronic consumption of ‘large quantities’ [not further defined] of peppermint candy No adverse effects in 47 pregnant women given PO [dose not specified], twice a day for 2 weeks 17

18. Repeated oral dose studies Non-human Rats (12 males/group) PO (1-2% pulegone) by oral gavage [0], 20, 150 or 500 mg/kg bw/day for 5 wk No overt toxicity, effects on body weight, blood biochemistry or haematology, and no changes at post mortem NOAEL 500 mg/kg bw/day 18

19. Repeated oral dose studies Rats (14/sex/group) PO (1.1% pulegone) by oral gavage 0, 10, 40 or 100 mg/kg bw/day for 90 days No overt toxicity, effects on body weight, haematology or blood biochemistry. Nephropathy in high-dose males was irrelevant to humans (male rat specific). ‘Cyst-like spaces’ in the cerebellum at top dose; no other signs NOEL was established as 40 mg/kg bw/day 19

20. Repeated oral dose studies Further investigation: JECFA noted that cyst-like spaces were also seen in one, but not another, 28-day rat study with pulegone at 160 mg/kg bw/day. May have been artefacts arising from inadequate tissue preparation Rats given oral doses of PO at about 75 mg/kg bw/day for 28 days A 45% increase in alkaline phosphatase activity. No effect on bile flow or liver histology “Doses 2-3 orders of magnitude greater than those recommended for human use” 20

21. Repeated oral dose studies Dogs (3/sex/group) Oral capsules PO (containing 1-2% pulegone) [0], 25 or 125 mg kg bw/day for 5 wk No overt toxicity, effects on body weight, blood biochemistry or haematology, and no tissue changes at post mortem (including the brain) NOAEL 125 mg/kg bw/day 21

22. Repeated oral dose studies Also high-quality dietary 2-yr studies on dl-menthol In rats and mice (50/sex/species/group) Rats 0, 190 and 375 mg/kg bw/day Mice 0, 300 and 600 mg/kg bw/day Comprehensive tissue examination TERA reports NOAELs of 188 (rats) and 667 (mice) mg/kg bw/day [Decreased weight in top-dose rats] 22

23. Repeated dermal dose studies No data in humans or laboratory animals Data gap not critical for current assessment 23

24. Genotoxicity Overview: PO inactive in several Ames tests (though oil toxicity may have limited the test value) l- and dl-menthol inactive in Ames tests Mammalian cell assays for chromosome aberration were generally reassuring 24

25. Genotoxicity Mammalian cell assays for sister chromatid exchange - inactive or equivocal Mammalian cell assay for mutation - inactive In vivo chromosome aberration - no data on PO but l- and dl-menthol inactive Conclusion: Peppermint oil probably lacks significant genotoxic character 25

26. Carcinogenicity Human – no data Non-human – 1 PO study (limited) Mice (52 males/group, 260 control (toothpaste base) 0, 4 or 16 mg/kg bw/day by gavage, 6 days/wk for 80 wk Examination (presumably microscopic) of brain, liver, lung and kidney, and assessed for malignant lymphomas No evidence of carcinogenic activity 26

27. Carcinogenicity Limited assay Current guidelines generally require 50/sex/group, with microscopic examination of a comprehensive range of tissues and organs, and treatment of at least three dose groups (in addition to the controls). This study was not designed specifically to assess PO 27

28. Carcinogenicity High-quality dietary studies on dl-menthol Rats 0, 190, 375 mg/kg bw/day Mice 0, 300 and 600 mg/kg bw/day 50/sex/group for 2 years Comprehensive tissue examination No clear evidence of carcinogenicity 28

29. Reproductive and developmental toxicity Human PO - used to induce menstruation; avoid during pregnancy Non-human Female rats (10/groups) given PO 0, 150, 750 or 1500 mg/kg bw/day from 7 days prior to mating, through mating, gestation and delivery to postnatal day 4 NOAEL 150 mg/kg bw/day LOAEL 750 mg/kg bw/day - maternal toxicity, increased numbers of stillborn pups and decreased pup viability and pup body weight 29

30. Other relevant data – skin irritation Human No skin reactions were reported in a woman who repeatedly applied neat PO (containing 10% menthol) to the skin PO is not generally considered a skin irritant in humans Non-human PO is irritant to rabbit skin [no details provided] 30

31. Other relevant data – eye irritation Human Neat PO splash - loss of corneal epithelium, corneal infiltration, pigment release into the anterior chamber with deposits on the back of the cornea; recovery in 16 days Eye irritation was reported in workers at a factory producing menthol (which may be present at up to 60% in peppermint oil) and a range of fragrance oils; atmospheric levels of menthol were 12 ‑ 380 mg/m 3 Non-human No data 31

32. Other relevant data – respiratory tract irritation No PO data Humans Workers producing menthol (12-380 mg/m 3 found in air) and a range of fragrance oils reported respiratory tract irritation Non-human Rabbits exposed by inhalation (once per day) to an aerosol of a 1% menthol solution for 9 months [dose unspecified] showed damage to the nasal passages and sinuses 32

33. Other relevant data – respiratory tract irritation TERA (2014/2015) summarised several relevant menthol studies Mice - 100 mg/m 3 for 15 mins – mild irritation – changes in respiratory function Mice – no irritation at 115 mg/m 3 for 30 mins, periocular wetness and deaths at 140 mg/m 3 Mice – RD50 – 288 mg/m 3 for 30 mins 33

34. Other relevant effects – gastrointestinal irritation Human PO administered directly into stomach or colon) at 0.1-0.24 mL Substantial anti-spasmodic effect on the smooth muscles of the gastrointestinal tract; apparently menthol altered calcium movement across cell membranes 34

35. Other relevant effects – gastrointestinal irritation Direct colonic instillation of PO can cause both muscle relaxation and stimulation e.g. at 200 mL of a mixture of 8 mL PO and 0.2 mL of Tween 80 per 1 L water 35

36. Other relevant effects – gastrointestinal irritation Human Oral PO is used to treat irritable bowel syndrome; little dose information; one study reports 0.2 mL PO in a capsule and 3-6 capsules/day [about 600-1200 mg/day] Non-human PO exhibits spasmolytic activity on smooth muscles of experimental animals 36

37. Other relevant effects – sensitisation and intolerance Human Asthmatic responses have been reported from the use of PO in toothpastes, mouthwashes and foods Sensitisation reactions around the mouth have followed use of lip balm, toothpaste, mouthwash, food, other cosmetics and medicaments containing PO 37

38. Other relevant effects – sensitisation and intolerance Anaphylactic reaction after ingestion of a peppermint sweet. He previously had nasal congestion, cough and wheeze from cultivating mint plants in his back yard, as well and itchy mouth and mild tongue/lip swelling after use of peppermint toothpaste Avoidance prevented recurrence of the symptoms A near-fatal reaction to the ingestion of PO has also been described 38

39. Other relevant effects – sensitisation and intolerance Another man had an asthma attack after ingesting a menthol-flavoured sweet. Immediate reduction in lung function when brushing teeth with menthol toothpaste; a 30- second mouth-rinse with peppermint oil (8 mm 3 diluted in 2 ml alcohol) produced an adverse change in lung function Wheezing from chewing gum containing “peppermint” was reported in a US patient 39

40. Expert Group Health Criteria Values or Occupational Exposure Levels PO - no inhalation HCVs/OELs or HCVs for other routes of exposure Menthol - recently assessed - TERA OARS Set a menthol WEEL of 6.4 mg/m 3 to avoid respiratory tract irritation in 8-hr exposure Set a 15-mins menthol STEL of 19.2 mg/m 3 40