CLP CLASSIFICATION AND LABELLING REQUIREMENTS OF NICOTINE CONTAINING E-LIQUIDS

Prepared by

Dr Dilip Arvind Bhuva Document created: January, 2015 1st update: August, 2015 2nd update: October, 2015

This document is a property of Xyfil Ltd.

CLP Classification of e-liquids Dr Dilip Arvind Bhuva, Xyfil Ltd. This document has been made public by Xyfil Ltd. as many manufacturers of the eliquids include hazard pictograms and statements that are – at most times – irrelevant and therefore supplying misleading information about the CLP classification of these products. This document is intended for sharing information on the CLP classification of e-liquids and is intended for our customers, e-cigarette users and other manufacturers and businesses that operate in the e-cigarette industry. This document can be cited as:

Bhuva, D.A. 2015. CLP classification and labelling requirements of nicotine containing eliquids. Xyfil Ltd. Available from: [insert web link to the document]

Legal Disclaimer The information provided in this document is accurate and represents the best information available to us – and believed to be reliable – at the time of publication. This information is given in good faith without any representation or warranty, expressed or implied, regarding its accuracy or correctness and we assume no liability – legal, personal or otherwise – resulting from its use. Although the information given in this document is used for designing the products manufactured by Xyfil Ltd., any manufacturer, supplier, or distributor must satisfy themselves that the information on their product and their labels are correct and comply with all the relevant legislations. In no event shall Xyfil Ltd. be liable for any claims, losses, or damages to any third party or for lost profits or any special, indirect, incidental, consequential or exemplary damages, howsoever arising, even if Xyfil Ltd. has been advised of the possibility of such damages. We advise that the information provided in this document should be used along with all the relevant legislation and/or literature.

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CLP Classification of e-liquids Dr Dilip Arvind Bhuva, Xyfil Ltd.

The CLP classification and selection of labelling information for our products is based mainly on the assessment carried out according to: a) “Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures”1 (this legislation is referred to as ‘CLP regulation’ in this document); b) Any subsequent “Adaptations to Technical Progress (ATP)” made to the CLP regulations. As of August 2015, six amendments have been made to the CLP regulations2. c) “Guidance on the Application of the CLP Criteria” version 4.1, last updated on June, 20153; d) “Guidance on Labelling and Packaging in accordance with Regulation (EC) No 1272/2008”4. e) The European Agreement concerning the International Carriage of Dangerous Goods by Road (ADR). ECE/TRANS/242, Vol. I and II and corrigenda5 (this legislation is referred to as ‘ADR-2015’ in this document). f) Inland transport of dangerous goods (by air, road and inland waterway) Directive 2008/68/EC6. These can be downloaded from links below in the footnotes.

http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2008:353:0001:1355:EN:PDF http://ec.europa.eu/enterprise/sectors/chemicals/documents/classification/index_en.htm#h2-4 3 http://echa.europa.eu/documents/10162/13562/clp_en.pdf 4 https://echa.europa.eu/documents/10162/13562/clp_labelling_en.pdf 5 http://www.unece.org/trans/danger/publi/adr/adr2015/15contentse.html 6 http://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:02008L0068-20130515&from=EN 1 2

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CLP Classification of e-liquids Dr Dilip Arvind Bhuva, Xyfil Ltd.

List of Abbreviations ATE

Acute Toxicity Estimates

CLP

Classification, labelling and packaging of substances and mixtures

EC50

Half maximal effective concentration

ECITA

Electronic Cigarette Industry Trade Association

ErC50

The concentration at which a 50% inhibition of growth rate is observed

KOW

Octanol-Water partition coefficient

LC50

The concentration (in air or water) at which 50% of the test population died. i.e. The median lethal concentration.

LD50

The dose (amount of test material) at which 50% of the test population died. i.e. The median lethal dose

LDLO

The lowest lethal dose

mg/kg.bw

milligram per kilo-body weight

NOEC

No Observed Effect Concentration

pH

Potential of Hydrogen

ppb

Parts per billion

ppm

Parts per million

TPD

Tobacco Product Directive

TWA

Time-Weighted Average

Notes: The phrase “CLP regulation” and Regulation “(EC) No 1272/2008” are used interchangeably throughout this document and they both refer to the, same, European Union CLP regulation that has regulation identity: “REGULATION (EC) No 1272/2008 of the European parliament and of the council of 16 December 2008 on classification, labelling and packaging of substances and mixtures” and came into force on 16th December 2008 whilst amending and repealing ‘Directives 67/548/EEC’ and ‘Directives 1999/45/EC’, and amending regulation (EC) No 1907/2006.

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CLP Classification of e-liquids Dr Dilip Arvind Bhuva, Xyfil Ltd.

Contents Legal Disclaimer ............................................................................................................................................. 2 List of Abbreviations..................................................................................................................................... 4 1. INTRODUCTION ....................................................................................................................................... 7 2. NICOTINE .................................................................................................................................................... 9 2.1 Animal Toxicity Data for Nicotine .............................................................................................. 10 2.2 Major/relevant Human Toxicity data for Nicotine: ............................................................ 11 2.3 Carcinogenicity, Genotoxicity & Reproductive Toxicity .................................................... 12 2.4 Specific Risk Groups ........................................................................................................................ 12 2.5 Eco-Toxicity Data ............................................................................................................................. 13 2.6 Environmental Fate ......................................................................................................................... 14 2.7 Additional Hazards .......................................................................................................................... 14 3. PROPYLENE GLYCOL ........................................................................................................................... 15 3.1 Animal Toxicity Data for Propylene Glycol ............................................................................ 16 3.3 Carcinogenicity, Genotoxicity & Reproductive Toxicity .................................................... 18 3.4 Specific Risk Groups ........................................................................................................................ 18 3.5 Eco-toxicity Data for Propylene Glycol .................................................................................... 19 3.6 Environmental Fate ......................................................................................................................... 20 3.7 Additional Hazards .......................................................................................................................... 20 4. GLYCEROL (VEGETABLE GLYCERINE) ........................................................................................ 21 4.1 Animal Toxicity Data for Glycerol .............................................................................................. 22 4.2 Major Human Toxicity Data for Glycerol ................................................................................. 23 4.3 Carcinogenicity, Genotoxicity & Reproductive Toxicity .................................................... 23 4.4 Specific Risk Groups ........................................................................................................................ 24 4.5 Eco-toxicity Data for Glycerol ...................................................................................................... 25 4.6 Environmental Fate ......................................................................................................................... 25 4.7 Additional Hazards .......................................................................................................................... 26 5. 7.2% (W/V) NICOTINE IN PROPYLENE GLYCOL ................................................................... 27 5.1 Physical Hazards ............................................................................................................................... 27 5.2 Health Hazard Classification ........................................................................................................ 27 5.3 Environmental Hazards ................................................................................................................. 29 5.4 Conclusion ........................................................................................................................................... 31 Page 5 of 54

CLP Classification of e-liquids Dr Dilip Arvind Bhuva, Xyfil Ltd. 6. SUMMARY TABLES FOR CLP CLASSIFICATION OF NICOTINE SOLUTIONS. ............. 32 7. TRANSPORT LABELS ........................................................................................................................... 38 7.1 Hazard class of nicotine solutions for transport purposes .............................................. 38 7.2 Do you require a transport label? .............................................................................................. 40 8. CONCLUSIONS ......................................................................................................................................... 42 APPENDIX A .................................................................................................................................................. 43 APPENDIX B .................................................................................................................................................. 48 B.1 Boundaries of classification for acute toxicity hazards ..................................................... 48 B.2 Contents of the label ....................................................................................................................... 50 B.3 General rules for the application of labels.............................................................................. 51 B.4 Location of information on the label ........................................................................................ 51 B.5 Dimensions and make-up of the label elements .................................................................. 52 B.6 Allowed (relevant) exceptions .................................................................................................... 52 B.7 Interplay between Transport and CLP labelling system .................................................. 54

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CLP Classification of e-liquids Dr Dilip Arvind Bhuva, Xyfil Ltd.

1. INTRODUCTION There is a lot of debate among e-cigarette (here after referred to as e-cigs) industry stakeholders on the CLP classification of nicotine containing solutions that are intended to be used with e-cigs (referred to as e-liquid(s) here after). Some stake holders argue that since e-liquids (or e-cigs) have nicotine in them and since nicotine has legally binding harmonised classification in the EU, all e-liquids should carry the same classification as that of pure nicotine along with all the hazard pictograms and warning statements7,8. Whilst others argue that EU CLP experts have got the classification of pure nicotine wrong (i.e. wrongly classified as toxic) and thus any product containing nicotine should not be classified under CLP regulations in the first place9,10. Such misconception is also evident among trading standards with some trading standard agencies going to the extent of declaring that those products (containing nicotine ≤2.5% w/w) that do not carry GHS06 and/or GHS09 and relevant risk phrases are liable to be seized for non-compliance with the CLP regulations11,12,13. To the best of our knowledge, to date only ‘Bibra proposal’ has come close to correctly classifying the e-liquids according to the CLP classification (classification carried out by a chemical company named ‘Bibra Ltd.’ on behalf of ECITA)14. The ‘Bibra proposal’ has taken – in general – a correct approach of using the acute toxicity estimate (ATE) values (as described for classification of mixtures) for assessing the CLP classification of e-liquids; and their approach for selection of the LD50 values is appropriate (for oral and dermal toxicity). However, their assessment completely ignored the other toxicity categories such as aquatic toxicity (even though nicotine has harmonised classification in this category), the implications of weight/weight and weight/volume solutions, and transport label considerations. Thus in order to address these differences of opinion and to provide complete assessment of e-liquids for labelling purposes, Xyfil Ltd. has undertaken CLP classification of e-liquids and findings are presented in this document.

http://hibiscus-plc.co.uk/blog/e-cigarettes-clp-labelling-and-tactile-warning-labels/ http://www.totallywicked-eliquid.co.uk/blog/2101/e-liquid-is-it-really-all-that-toxic/ 9 http://ecigintelligence.com/e-liquids-not-as-dangerous-as-regulators-think/ 10 http://escape-ecigarettes.co.uk/blog/EU-to-remove-toxic-warning-labels-from-e-cigarettes 7 8

11

https://www.google.co.uk/url?sa=t&rct=j&q=&esrc=s&source=web&cd=3&cad=rja&uact=8&ved=0CDE QFjACahUKEwjz88S02eDIAhVJuBQKHRO3C1M&url=https%3A%2F%2Fwww.sheffield.gov.uk%2Fdms% 2Fscc%2Fmanagement%2Fcorporate-communications%2Fdocuments%2Fbusinessindustry%2Ftradingstandards%2FElectronic_Cigarettes_Guidance.pdf&usg=AFQjCNGblUDQrJuZAIQIadIvqbO3FweApA&bvm =bv.105841590,d.bGQ http://www.darlington.gov.uk/media/540498/E-Cigarette_-_Trader_Guidance.pdf http://www3.hants.gov.uk/tradingstandards/tradingstandards-business/ts-business-safety/tsguideelectronic-cigarettes.htm 14 http://www.ecita.org.uk/ise/07-qs-chapter-4-classification-labelling-and-packaging-clp-regulations2010 12 13

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CLP Classification of e-liquids Dr Dilip Arvind Bhuva, Xyfil Ltd. We manufacture e-liquids that are intended to be used in electronic cigarette (e-cigs) devices. An e-liquid contains specific concentrations of nicotine, propylene glycol, glycerol and other flavour ingredients that impart taste to the e-liquid and subsequent smoke generated from its use in e-cig devices (commonly referred to as “vapour” or “vaping” in the industry). The classification should be performed for each type of eliquid product as the mixtures may have different toxicity properties – and thus different CLP classification; however, TPD2 regulations may not allow use of flavour ingredients/additives that would pose a (higher) risk to human health when compared to that of nicotine15. Thus it is likely that the finalised e-liquid products that are placed on the market post TPD2 may have the same CLP classification as the base nicotine solution (i.e. without any additives such as flavourings). Thus here the CLP classification is based on the nicotine, propylene glycol and glycerol. However, the CLP classification will be revisited for each and every product during the notification process of the e-liquids under TPD2 even though they are likely to carry the same classification as that of these ‘base nicotine solutions’.

Clause 36.7 of the Draft statutory instrument: The Tobacco and Related Products Regulations 2016. https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/440989/SI_tobacco_p roducts_acc.pdf 15

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CLP Classification of e-liquids Dr Dilip Arvind Bhuva, Xyfil Ltd.

PART A: Gathering and Analysing Relevant Information. 2. NICOTINE General Name IUPAC Name EC No CAS No Molecular Weight Molecular Formula XLogP3 Boiling Point Melting Point Flash Point Auto Ignition Vapour Density (air=1) Vapour Pressure Log Kow pH TWA (mg/m3) Harmonised Classification Index No (ISO) Harmonised Classification21 (ISO) Harmonised Labelling10 (ISO)

Nicotine 3-(N-methyl-2-pyrrolidinyl)pyridine 200-193-3 54-11-5 162.23156 g/mol C10H14N2 1.2 247 oC at atmospheric pressure16 -79 oC at atmospheric pressure16 111 oC17 244 oC 5.6118 0.038 mm Hg at 25 oC19 (8.83 Pa) 1.1720 10.2 (for 0.05 Molar solution) 0.5 (skin) 614-001-00-4 Acute Tox. 1; H310 (dermal/skin) Acute Tox. 3 *; H301 (Oral) Aquatic Chronic 2; H411 GHS06; H310 GHS09; H301 Dgr; H411

* This is the minimum classification (for pure nicotine) given by the Harmonised classification under CLP ((EC) No 1727/2008). If the evaluator has more data that classify the substance in a more hazardous class, then the higher toxicity class needs to be chosen.

Although the toxicity classification of nicotine has been harmonised under the CLP regulation, the classification is for the pure form. However in the electronic cigarettes and e-liquids, the nicotine used is highly diluted with either propylene glycol or glycerol

Lide, D.R. CRC Handbook of Chemistry and Physics 88TH Edition 2007-2008. CRC Press, Taylor & Francis, Boca Raton, FL 2007, p. 3-386 17 Experimental determination according to EU method A.9 (Flash-Point). 2014. 18 Clayton, G. D. and F. E. Clayton (eds.). Patty's Industrial Hygiene and Toxicology: Volume 2A, 2B, 2C: Toxicology. 3rd ed. New York: John Wiley Sons, 1981-1982., p. 2741 19 Boublik, T., Fried, V., and Hala, E., The Vapour Pressures of Pure Substances. Second Revised Edition. Amsterdam: Elsevier, 1984. 20 Hansch, C., Leo, A., D. Hoekman. Exploring QSAR - Hydrophobic, Electronic, and Steric Constants. Washington, DC: American Chemical Society., 1995., p. 76 21 Table 3.1 List of harmonised classification and labelling of hazardous substances to Annex VI of CLP Regulation (EC) No 1272/2008. 16

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CLP Classification of e-liquids Dr Dilip Arvind Bhuva, Xyfil Ltd. or both (along with flavour ingredients). Thus the CLP classification of the nicotine containing mixtures (e-liquid) need to be evaluated by using the classification criteria for mixtures as laid down in the CLP regulation ((EC) No 1272/2008).

2.1 Animal Toxicity Data for Nicotine Model Rat Rat Rat Rat

Route Skin Oral subcutaneous Intraperitoneal

Rat

intramuscular

Rat Rat

Intravenous parenteral

Rabbit

Skin

Rabbit Rabbit

Intraperitoneal Intramuscular

Rabbit

Intravenous

Dose 140mg/kg.bw 50 mg/kg.bw 25mg/kg.bw 14.56 mg/kg.bw 15mg/kg.bw

Comments LD50 LD50 LD50 LD50

Reference 22 23 24 25

LDLO. Behavioural: convulsions or effect on seizure threshold; ataxia; Peripheral nerve and sensation: flaccid paralysis without anaesthesia (usually neuromuscular blockage) 2.8 mg/kg.bw LD50 34mg/kg.bw LDLO. Lungs, thorax, or respiration: other changes 50 mg/kg.bw LD50. Respiratory depression; convulsions or effect on seizure threshold 14mg/kg.bw LD50 30mg/kg.bw LDLO. Behavioural: convulsions or effect on seizure threshold; ataxia; Peripheral nerve and sensation: flaccid paralysis without anaesthesia (usually neuromuscular blockage) 6.25mg/kg.bw LD50

26

27 28

29

30 31

32

World Review of Pest Control. Vol. 9, Pg. 119, 1970. Sine C (1993). Nicotine. In: Farm chemicals handbook '93, p. C245. 24 Farmakologiya i Toksikologiya Vol. 47(5), Pg. 85, 1984. 25 Journal of Pharmacology and Experimental Therapeutics. Vol. 124, Pg. 350, 1958. 26 Science. Vol. 127, Pg. 1054, 1958. 27 Drugs in Japan Vol. -, Pg. 916, 1995. 28 Journal of Pharmacology and Experimental Therapeutics. Vol. 48, Pg. 317, 1933. 29 Trochimowicz HJ, Kennedy GL Jr and Krivanek ND. Heterocyclic and miscellaneous nitrogen compounds. 1994. 30 Proceedings of the Society for Experimental Biology and Medicine. Vol. 58, Pg. 231, 1945. 31 Science. Vol. 127, Pg. 1054, 1958. 32 Proceedings of the Society for Experimental Biology and Medicine. Vol. 58, Pg. 231, 1945. 22 23

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CLP Classification of e-liquids Dr Dilip Arvind Bhuva, Xyfil Ltd.

2.2 Major/relevant Human Toxicity data for Nicotine: Type Dose Comments References Effect on 4 mg nicotine Gum increased significantly maternal heart rate, 33 Human foetus chewing gum systolic and diastolic blood pressure. There was no influence on foetal heart rate or foetal blood flow. Oral ingestion 5 gm of 17 year old smoker; committed and collapsed, 34 concentrated pulseless. The patient was hospitalised and died nicotine 64 hr after ingestion pesticide solution (concentration not specified) Dermal Nicotine patch Two patients developed vasculitis after 3 days 35 use of nicotine patches. They recovered fully after these were stopped. On challenge, the symptoms reappeared confirming the casual link between nicotine patches use and development of vasculitis Epidemiology Nicotine patch Meta-analysis/review. In total the study 36 study included data from 5501 patients using nicotine patches and 3752 patients receiving placebo. Several minor effects were elevated among nicotine patch users, these included sleep disturbances, nausea or vomiting, localised skin irritation and respiratory symptoms. However, the background rate and risk ratios varied widely among different studies Case study Pure nicotine Splash of pure nicotine base in the eye caused 37 base severe pain, much conjunctival reaction & corneal infiltration. Eventually, eye healed with partial opacification of cornea.

Lindblad A, Marsal K; J Perinat Med 15 (1): 13-9 (1987) Krieger, R. (ed.). Handbook of Pesticide Toxicology. Volume 1, 2nd ed. 2001. Academic Press, San Diego, California., p. 120 35 Van der Klauw MM et al; Br J Dermatol 134 (2): 361-4 (1996) 36 Greenland S et al; Drug Saf. 1998 Apr;18(4):297-308 37 Grant, W. M. Toxicology of the Eye. 2nd ed. Springfield, Illinois: Charles C. Thomas, 1974., p. 747 33 34

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CLP Classification of e-liquids Dr Dilip Arvind Bhuva, Xyfil Ltd.

2.3 Carcinogenicity, Genotoxicity & Reproductive Toxicity Nicotine can rapidly cross placenta and enter the foetus. In some reports, nicotine in higher doses interfered with osteogenesis in mice and chick embryos. Chronic nicotine treatments of pregnant rats throughout the gestation period produced subtle neurological changes that exhibited as behavioural or electrophysiological alterations. Although smoking (traditional cigarettes) is generally associated with impaired growth and other congenital defects in a foetus, nicotine itself is not thought to be involved in these processes38. Furthermore, literature reports indicate that nicotine is neither an initiator nor a promoter of tumours in mice39. In the Ames and mammalian cell cytogenic assays, nicotine did not exhibit any significant genotoxic activity. In one report, nicotine has been shown to be teratogenic in mice treated cutaneously with a dose of 25 mg/kg.bw27. Thus, nicotine – especially in the concentrations used in e-liquid – may not exhibit any carcinogenicity, teratogenicity, genotoxicity or reproductive toxicity.

2.4 Specific Risk Groups Nicotine has been associated with a decrease in breathing movement if the mother is exposed to nicotine in the last trimester. Nicotine also passes freely into the breast milk but the concentrations would generally be clinically non-significant (average: 91 ppb in one study). However, heavy smoking (20-30 cigarettes per day) may alter the supply of milk and cause nausea and vomiting in the infant. Thus pregnant and breast feeding women can be regarded as a specific risk group that needs to exercise caution when using e-cigs.

US Department of Health and Human Services (1988). A Report of the Surgeon General, The Health consequences of smoking. Nicotine addiction. US Department of Health and Human Services, Public Health Service. Office of the Assistant Secretary for Health, on Smoking and Health 32-33: 601-602 39 PDR (1987) Physician's desk reference. 41st ed. Barnhart E. 1070-72 38

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CLP Classification of e-liquids Dr Dilip Arvind Bhuva, Xyfil Ltd.

2.5 Eco-Toxicity Data Model Xenopus laevis Daphnia magna (age: 98% flow through, 10-11 oC, pH purity 8.2, hardness 140 mg/L CaCO3, conductivity 260 μmhos/cm, dissolved oxygen 90% (70-100%). 0.938 mM for 24 LC50. Conditions: freshwater, 45 hr; 100% purity static, 18-20 oC (50,000-75,000) LC50. Conditions: freshwater, 46 μg/L for 10 days renewal

Dawson DA et al; Teratog Carcinog Mutagen 8 (6): 329-38 (1988) Lilius H et al; Aquat Toxicol 30: 47-60 (1994) 42 Lilius H et al; Environ Toxicol Chem 14 (12): 2085-8 (1995) 43 Perry and Smith. Bull. Environ. Contam. Toxicol., 41, 604-608 (1998) 44 Passino-Reader DR et al; J Gt Lakes Res 21 (3): 373-83 (1995) 45 Corbet SA et al; Entomol Exp Appl 94 (3): 295-307 (2000) 46 Best JB, Morita M; Hydrobiologia 227: 375-83 (1991) 40 41

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CLP Classification of e-liquids Dr Dilip Arvind Bhuva, Xyfil Ltd.

2.6 Environmental Fate An estimated Koc value of 100, determined from a log Kow of 1.17 and a regressionderived equation47, indicates that nicotine is expected to have high mobility in soil and is not expected to adsorb to suspended solids and sediments. However, the pKb1 and pKb2 values of 6.16 and 10.9648 indicate that this compound will exist - almost entirely in the cation form. The cations generally adsorb more strongly to soils containing organic carbon and clay than their neutral counterparts49. Volatilization of nicotine from moist soil surfaces and/or water is not expected to be an important fate process given an estimated Henry's Law constant of 3.0x10-9 atm-cu m/mole, determined using a fragment constant estimation method50. Similarly, nicotine is not expected to volatilize from dry soil surfaces based upon a vapour pressure of 0.038 mm of Hg at 25 oC. If released to air, a vapour pressure of 0.038 mm of Hg at 25 oC indicates nicotine will exist solely as a vapour in the atmosphere. Vapour-phase nicotine will be degraded in the atmosphere by reaction with photochemically-produced hydroxyl radicals; the halflife for this reaction in air is estimated to be 4 hours. Nicotine does not contain chromophores that absorb at wavelengths >290 nm and therefore is not expected to be susceptible to direct photolysis by sunlight51. An estimated BCF score of 3 was calculated in fish, using a log Kow of 1.17 and a regression-derived equation, which suggests that the potential for bio-concentration in aquatic organisms is low. Lastly, it has been experimentally determined ‒ using OECD Guideline 301 B ‒ that nicotine has ready biodegradability under aerobic conditions; and the degradation at the end of 10day period was 72%.

2.7 Additional Hazards No additional hazards to report.

Lyman WJ et al; Handbook of Chemical Property Estimation Methods. Washington, DC: Amer Chem Soc pp. 4-9 (1990) 48 Tomlin C; The Pesticide Manual. 10th Ed, Surrey,UK: Crop Protection Pub, p. 735 (1994) 49 Doucette WJ; pp. 141-188 in Handbook of Property Estimation Methods for Chemicals. Boethling RS, Mackay D, eds. Boca Raton, FL: Lewis Publ (2000) 50 Meylan WM et al; Environ Toxicol Chem 18: 664-72 (1999) (7) Maeda S et al; Agric Biol Chem 42: 145560 (1978) 51 Lyman WJ et al; Handbook of Chemical Property Estimation Methods. Washington, DC: Amer Chem Soc pp. 8-12 (1990) 47

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CLP Classification of e-liquids Dr Dilip Arvind Bhuva, Xyfil Ltd.

3. PROPYLENE GLYCOL General Name IUPAC Name EC No CAS No Molecular Weight Molecular Formula XLogP3 Boiling Point Melting Point Flash Point Auto Ignition Vapour Density (air=1) Vapour Pressure Log Kow pH TWA (mg/m3) Harmonised Classification Index No Harmonised Classification Harmonised Labelling

Propylene Glycol Propane-1,2-diol 200-338-0 57-55-6 76.09442 g/mol C3H8O2 -0.9 187.6 oC at atmospheric pressure52 -60 oC at atmospheric pressure52 104 oC53 371 oC 2.6 0.13 mm Hg at 25 oC (Extrapolated) -0.92 Unknown Not Established Not classified Not classified Not classified

Lide, D.R. CRC Handbook of Chemistry and Physics 88TH Edition 2007-2008. CRC Press, Taylor & Francis, Boca Raton, FL 2007, p. 3-444 53 Martin AE, Murphy FH; Glycols, Propylene Glycols. Kirk-Othmer Encyclopedia of Chemical Technology (1994). John Wiley & Sons, Inc. Online Posting Date: December 4, 2000 52

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CLP Classification of e-liquids Dr Dilip Arvind Bhuva, Xyfil Ltd.

3.1 Animal Toxicity Data for Propylene Glycol Model Rat Rat Rat Rat Rat Rabbit Rabbit Rabbit

Route Oral Subcutaneous Intraperitoneal Intramuscular Intravenous Skin Oral Intramuscular

Dose 22000mg/kg.bw 22500mg/kg.bw 6660mg/kg.bw 14000mg/kg.bw 6423mg/kg.bw 20800mg/kg.bw 18000mg/kg.bw 6300mg/ml

Rabbit

Intravenous

3099mg/kg.bw

Comments Reference 54 LD50 55 LD50 56 LD50 57 LD50 58 LD50 59 LD50 60 LD50 LDLo. Coma, respiratory 61 stimulation, somnolence. 62 LD50

3.2 Major Human Toxicity Data for Propylene Glycol Type Patch testing

Dose

Pads containing propylene glycol (100%) attached to forearm for 2 hr. Patch testing Semi-occlusive or occlusive epicutaneous application. (Amount not specified; 100% purity) Topical (skin) Cream containing

Comments

Refere nces

Observation duration: 7 days; n = 6 humans. No irritation of the skin observed/reported.

63

n > 300 humans. The study demonstrates that it is not irritating to skin or eye and does not cause skin sensitisation by skin contact.

64

n = 204 humans. Conclusion: Not sensitising or

65

Organization for Economic Cooperation and Development; Screening Information Data Set for 1,2Dihydroxypropane (57-55-6) p.6 (2001). 55 Interagency Collaborative Group on Environmental Carcinogenesis, National Cancer Institute, Memorandum, June 17, 1974Vol. 17JUN1974. 56 Lewis, R.J. Sr. (ed) Sax's Dangerous Properties of Industrial Materials. 11th Edition. Wiley-Interscience, Wiley & Sons, Inc. Hoboken, NJ. 2004., p. 3061. 57 Interagency Collaborative Group on Environmental Carcinogenesis, National Cancer Institute, Memorandum, June 17, 1974Vol. 17JUN1974. 58 Lewis, R.J. Sr. (ed) Sax's Dangerous Properties of Industrial Materials. 11th Edition. Wiley-Interscience, Wiley & Sons, Inc. Hoboken, NJ. 2004., p. 3061. 59 European Commission, ESIS; IUCLID Dataset, Propane-1,2-diol (57-55-6) p.45 60 Amdur, M.O., J. Doull, C.D. Klaasen (eds). Casarett and Doull's Toxicology. 4th ed. New York, NY: Pergamon Press, 1991., p. 705. 61 Journal of Pharmacology and Experimental Therapeutics. Vol. 44, Pg. 109, 1932. 62 European Commission, ESIS; IUCLID Dataset, Propane-1,2-diol (57-55-6) p.53 63 Organization for Economic Cooperation and Development; Screening Information Data Set for 1,2Dihydroxypropane (57-55-6) p.56 (2001). 64 Organization for Economic Cooperation and Development; Screening Information Data Set for 1,2Dihydroxypropane (57-55-6) p.17 (2001). 54

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CLP Classification of e-liquids Dr Dilip Arvind Bhuva, Xyfil Ltd. application Inhalation (mist)

12% propylene glycol. Geometric mean of 309 mg/cu m (range 176-851 mg/cu m). Exposure for 1 minute

Patch testing

100% glycol

Propylene

PC12 cells

0.2 to 20% v/v

Metaanalysis/revi ew

---

Inhalation (smoke from theatrical fog generators)

Surveillance study conducted over 2 years on population frequently exposed to such glycol smoke

irritating to the skin. n = 27 humans, non-asthmatic. Exposed during realistic training conditions. The included an estimate of tear film stability break up time, nasal patency by acoustic rhinometry, dynamic spirometry, and a doctor's administered questionnaire on symptoms. Conclusion: short exposure to PG mist may cause acute ocular and upper airway irritation in non-asthmatic subjects. A few may also react with cough and slight airway obstruction. N = 1556 humans. 194 subjects were observed with positive reactions with 4 patients identified as having “true allergy”. These patients were later tested with lower concentrations (3.2, 10 & 32%) of propylene glycol and only 9, 12 and 20 patients showed positive reactions respectively. Concentrations above 1% produced significant increase in dopamine release. Ocular exposure causes mild ocular irritation with hyperaemia. Gastrointestinal disturbances, nausea and vomiting have been observed after ingestion. (No dose specified). 439 actors from 16 musicals were studied for irritant effects to the respiratory tract and eyes by employing a baseline questionnaire, daily checklists, and medical evaluation. No clinically significant adverse impact on pulmonary function or in rates of asthma associated with exposure to propylene glycol was noted. However, there was an increased reporting of respiratory, throat, and nasal symptoms, and vocal folds inflammation in cases of elevated localized air concentrations following release of glycol smoke. It was recommended that exposures to propylene glycol by actors not exceed peak or ceiling concentrations of 40 mg/cu m.

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69

70

Organization for Economic Cooperation and Development; Screening Information Data Set for 1,2Dihydroxypropane (57-55-6) p.60 (2001). 66 Wieslander G et al; Occup Environ Med 58 (10): 649-55 (2001) 67 Cosmetic Ingredient Review Expert Panel; J Am Coll Toxicol 13 (6): 437-91 (1994) 68 Hattori T et al; Res Commun Mol Pathol Pharmacol 107 (3-4): 323-9 (2000) 69 Poisons Information Monograph 443: Propylene glycol (May 1994). 70 DHHS/NTP-CERHR; NTP-CERHR Monograph on the Potential Human Reproductive and Developmental Effects of Propylene Glycol (March 2004) NIH Pub No. 04-4482 p.II-29 . 65

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CLP Classification of e-liquids Dr Dilip Arvind Bhuva, Xyfil Ltd.

3.3 Carcinogenicity, Genotoxicity & Reproductive Toxicity Propylene Glycol has been used in many consumer products worldwide for decades and no cancer risk has been associated with exposure to propylene glycol. Many extensive studies have been conducted in the laboratory – including lifetime studies – on a variety of species and none reported an associated increase in tumour rates. There is a general consensus that there is no cancer risk associated with exposure to propylene glycol and this is in agreement with the American Chemistry Council’s report71.

3.4 Specific Risk Groups 

 





Hyperosmolarity and lactic acidosis, both of which occur most frequently in patients (specifically Infant and neonatal age) with consumption of large quantities of propylene glycol. Symptoms were also observed on administration of propylene glycol to children under age of 4, pregnant women, and patients with hepatic or renal failure.72 Adverse effects from propylene glycol may occur in patients treated with disulfiram or metronidazole.73 Propylene glycol is metabolized in the liver by the alcohol and aldehyde dehydrogenase enzyme pathway, and the possibility exists that young infants, patients with renal or hepatic impairment, and certain patient groups (females, Asians, Native Alaskans, Native Americans) may be at increased risk of propylene glycol-associated adverse effects if they receive amprenavir oral solution because of diminished ability to metabolize propylene glycol (due to alcohol dehydrogenase polymorphism). Estimated dose: ~1650 74 mg/kg.bw/day. In a patient with renal failure who was unable to excrete propylene glycol in the urine, such retention caused severe central nervous system depression. In addition, lactic acidosis was a prominent feature in this patient; with a large anion gap and a lactic acid level of 80 mEq/l.75 Patients with impaired liver or kidney function would be at increased risk for developing propylene glycol toxicity. In patients with renal insufficiency, high propylene glycol levels have been associated with lactic acidosis

71http://msdssearch.dow.com/PublishedLiteratureDOWCOM/dh_0046/0901b80380046c80.pdf?filepath

=propyleneglycol/pdfs/noreg/117-01656.pdf&fromPage=GetDoc 72 Rowe, R.C., Sheskey, P.J., Quinn, M.E.; (Eds.), Handbook of Pharmaceutical Excipients 6th edition Pharmaceutical Press, London, England 2009, p. 593. 73 Rowe, R.C., Sheskey, P.J., Quinn, M.E.; (Eds.), Handbook of Pharmaceutical Excipients 6th edition Pharmaceutical Press, London, England 2009, p. 593. 74 McEvoy, G.K. (ed.). American Hospital Formulary Service - Drug Information 2003. Bethesda, MD: American Society of Health-System Pharmacists, Inc. 2003 (Plus Supplements)., p. 626. 75 Haddad, L.M., Clinical Management of Poisoning and Drug Overdose. 2nd ed. Philadelphia, PA: W.B. Saunders Co., 1990., p. 700.

Page 18 of 54

CLP Classification of e-liquids Dr Dilip Arvind Bhuva, Xyfil Ltd. (hyperlactemia). Propylene glycol has been found in the blood of alcoholics with cirrhosis of the liver without detectable measurable blood alcohol levels.76

3.5 Eco-toxicity Data for Propylene Glycol Model Selenastrum capricornutum Daphnia magna (age 6-24hr) Skeletonema costatum

Carassius auratus Cyprinodon variegatus Daphnia magna Mysidopsis bahia Oncorhynchus mykiss Oryzias latipes Pimephales promelas ≤7 days)

Dose 19000 mg/l for 96 hr >10000 mg/l for 24, 48 hr ErC50=19300 mg/l for 72 hr. 5300, 12000, 25000, 51000 and 97000 mg/l.

Comments EC50. 14 day growth rate

Reference 77

EC50. Conditions: fresh water, static, 20 oC, pH≥7.0 ErC50. 14 day NOEC=5000 mg/l for LC50. Conditions: static 24 hr 48000 ppm for LC50. 96 hr 43500 mg/l for LC50. Conditions: static 48 hr 18800 mg/l for LC50. Conditions: static 96 hr 51600 mg/l for LC50. Conditions: static 96 hr >1000 mg/l for LC50. Conditions: freshwater, 24 & 48 hr static, 10, 20, 30 oC (age: 29485-39339 LC50. Conditions: freshwater, mg/L for 96 hrs renewal, 20 oC

78

79

80

81

82

83

84

85

86

Monograph on the Potential Human Reproductive and Developmental Effects of Propylene Glycol (March 2004) NIH Pub No. 04-4482 p.II-44. 77 Organization for Economic Cooperation and Development; Screening Information Data Set for 1,2Dihydroxypropane (57-55-6) p.6 (2001). 78 Kuhn R et al; Water Res 23 (4): 495-9 (1989) as cited in the ECOTOX database. 79 Organization for Economic Cooperation and Development; Screening Information Data Set for 1,2Dihydroxypropane (57-55-6) p.6 (2001). 80 European Commission, ESIS; IUCLID Dataset, Propane-1,2-diol (57-55-6) p.36 (2000 CD-ROM edition). 81 USEPA/Office of Pesticide Programs; Reregistration Eligibility Decision Document - Propylene glycol and Dipropylene glycol p.13 EPA-739-R-06-002 (September 2006). 82 Organization for Economic Cooperation and Development; Screening Information Data Set for 1,2Dihydroxypropane (57-55-6) p.5 (2001). 83 Organization for Economic Cooperation and Development; Screening Information Data Set for 1,2Dihydroxypropane (57-55-6) p.6 (2001). 84 Organization for Economic Cooperation and Development; Screening Information Data Set for 1,2Dihydroxypropane (57-55-6) p.5 (2001). 85 Tsuji S et al; J Hyg Chem (Eisei Kagaku) 32 (1): 46-53 (1986) as cited in the ECOTOX database. 86 Cornell JS et al; Environ Toxicol Chem 19 (6): 1465-72 (2000) as cited in the ECOTOX database. 76

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3.6 Environmental Fate 



 







Propylene glycol does not contain chromophores that absorb at wavelengths >290 nm and therefore is not expected to be susceptible to direct photolysis by sunlight. If released to soil, propylene glycol is expected to have very high mobility based on an estimated Koc of 1, determined from a log Kow of -0.92 and a regressionderived equation.87 If released into water, propylene glycol is not expected to adsorb to suspended solids and sediment based on the estimated Koc of 1. Volatilization from moist soil or water surfaces is not expected to be an important fate process based on an estimated Henry's Law constant of 1.3x10-8 atm-cu m/mole, derived from its vapour pressure of 0.13 mm of Hg, and assigned value for water solubility of 1x106 mg/L (miscible).88 Vapour-phase propylene glycol is degraded in the atmosphere by reaction with photochemically-produced hydroxyl radicals; the half-life for this reaction in air is estimated to be 32 hours, calculated from its rate constant of 1.2x10-11 cu cm/molecule-sec at 25 oC89 Propylene glycol was mineralized (73-78%) in laboratory studies conducted using an agricultural soil over a 51 day incubation period, suggesting biodegradation will be an important environmental fate process in soil. An estimated BCF of 3, calculated from log Kow of -0.92 and a regression-derived equation, suggests the potential for bio concentration in aquatic organisms is low.

3.7 Additional Hazards 

None to report

Estimation Program Interface (EPI) Suite. Ver. 4.11. Nov, 2012. Yalkowsky SH, He Y; Handbook of Aqueous Solubility Data: An Extensive Compilation of Aqueous Solubility Data for Organic Compounds Extracted from the AQUASOL dATAbASE. Boca Raton, FL: CRC Press LLC, (2003). 89 Atkinson R; J Phys Chem Ref Data Monograph 1 (1989) (4) Lyman WJ et al; Handbook of Chemical Property Estimation Methods. Washington, DC: Amer Chem Soc pp. 8-12 (1990). 87 88

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CLP Classification of e-liquids Dr Dilip Arvind Bhuva, Xyfil Ltd.

4. GLYCEROL (VEGETABLE GLYCERINE) General Name

Glycerol (Glycerine/Glycerin)

IUPAC Name

propane-1,2,3-triol

EC No

200-289-5

CAS No

56-81-5

Molecular Weight

92.09382 g/mol

Molecular Formula

C3H8O3

XLogP3

-1.8

Boiling Point

290 oC at atmospheric pressure (decomposes)90

Melting Point

18.1 oC at atmospheric pressure91

Flash Point

177 oC (open cup)92

Auto Ignition

393 oC 93

Vapour Density (air=1)

3.1794

Vapour Pressure

1.68x10-4 mm Hg at 25 oC95

Log Kow

-1.7696

pH

Neutral to litmus test97

TWA (mg/m3) Harmonised Classification Index No

5 mg/m3 (respirable fraction) & 15 mg/m3 (total dust) Not classified

Harmonised Classification

Not classified

Harmonised Labelling

Not classified

O'Neil, M.J. (ed.). The Merck Index - An Encyclopedia of Chemicals, Drugs, and Biologicals. Whitehouse Station, NJ: Merck and Co., Inc., 2006., p. 774. 91 Lide, D.R. CRC Handbook of Chemistry and Physics 88TH Edition 2007-2008. CRC Press, Taylor & Francis, Boca Raton, FL 2007, p. 3-268. 92 International Labour Office. Encyclopedia of Occupational Health and Safety. Vols. I&II. Geneva, Switzerland: International Labour Office, 1983., p. 971. 93 Lewis, R.J., Sr (Ed.). Hawley's Condensed Chemical Dictionary. 12th ed. New York, NY: Van Nostrand Rheinhold Co., 1993, p. 566 94 Sax, N.I. Dangerous Properties of Industrial Materials. 6th ed. New York, NY: Van Nostrand Reinhold, 1984., p. 1479. 95 Daubert, T.E., R.P. Danner. Physical and Thermodynamic Properties of Pure Chemicals Data Compilation. Washington, D.C.: Taylor and Francis, 1989.. 96 Hansch, C., Leo, A., D. Hoekman. Exploring QSAR - Hydrophobic, Electronic, and Steric Constants. Washington, DC: American Chemical Society., 1995., p. 7. 97 O'Neil, M.J. (ed.). The Merck Index - An Encyclopedia of Chemicals, Drugs, and Biologicals. Whitehouse Station, NJ: Merck and Co., Inc., 2006., p. 774. 90

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CLP Classification of e-liquids Dr Dilip Arvind Bhuva, Xyfil Ltd.

4.1 Animal Toxicity Data for Glycerol Model Rat

Route Oral

Rat Rat

Subcutaneous Intraperitoneal

Rat Rat

Intravenous Inhalation

Rat (SpragueDawleyderived (Cr1:CD))

Inhalation

Mouse Mouse Mouse

Oral Subcutaneous Intraperitoneal

Mouse Rabbit Rabbit Guinea Pig

Intravenous Skin Oral Oral

Dose 12.6 gm/kg.bw

Comments Reference LD50. Behavioural: general 98 anaesthetic, muscle weakness. Liver: other changes 99 0.1 gm/kg.bw LD50 4.42 gm/kg.bw LD50. Cardiac: Other changes; 100 behavioural: toxic psychosis; kidney, ureter, & bladder: other changes 101 5.57 gm/kg.bw LD50 102 >570 mg/cu m/1 LC50 hr Mean aerosol Nose only inhalation. For 2 103 concentrations weeks (n=10 rats), all three of 0, 1.93, 3.91 or concentrations of glycerol 7.0 mg exhibited minimal to mild glycerol/L of squamous metaplasia of the filtered room air epithelium lining the base of for 6 hr/day for the epiglottis. For 13 weeks 2 weeks. AND, (n=75 rats), results were 0.033, 0.167, or same as those for 2 week 0.662 mg exposure period glycerol/L of filtered room air for 6 hr/day for 13 weeks. 104 4.1 gm/kg.bw LD50 105 0.091 gm/kg.bw LD50 8.70 gm/kg.bw LD50. Behavioural: altered 106 sleep time (including change in righting reflex) 107 4.25 gm.kg.bw LD50 108 >10 gm/kg.bw LD50 109 27 gm/kg.bw LD50 110 7.75 gm/kg.bw LD50

Federation Proceedings, Federation of American Societies for Experimental Biology. Vol. 4, Pg. 142, 1945. 99 Drugs in Japan Vol. 6, Pg. 215, 1982. 100 Research Communications in Chemical Pathology and Pharmacology. Vol. 56, Pg. 125, 1987. 101 Arzneimittel-Forschung. Drug Research. Vol. 26, Pg. 1581, 1976. 102 BIOFAX Industrial Bio-Test Laboratories, Inc., Data Sheets. Vol. 9-4/1970. 103 Renne et al., 2-week and 13-week inhalation studies of aerosolized glycerol in rats. Inhalation Toxicology: International Forum for Respiratory Research, Vol. 4, 2, 1992. 104 Farmatsevtichnii Zhurnal Vol. (6), Pg. 56, 1977. 105 Drugs in Japan Vol. 6, Pg. 215, 1982. 106 Arzneimittel-Forschung. Drug Research. Vol. 28, Pg. 1579, 1978. 107 Journal of the American Pharmaceutical Association, Scientific Edition. Vol. 39, Pg. 583, 1950. 108 BIOFAX Industrial Bio-Test Laboratories, Inc., Data Sheets. Vol. 9-4/1970. 109 Delaware State Medical Journal. Vol. 31, Pg. 276, 1959. 98

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CLP Classification of e-liquids Dr Dilip Arvind Bhuva, Xyfil Ltd.

4.2 Major Human Toxicity Data for Glycerol Type

Dose

Comments

Refere nces

Oral

1.428 gm/kg.bw

Behavioural: headache. Gastrointestinal: nausea or vomiting No significant differences were observed between the test and control (63 workers) groups indicating that glycerol may not cause decrease sperm quality Slightly irritating in case of 0.05ml/closed patch test. Furthermore, the participants assigned a maximum irritation score of 4 (on a scale of 9) at 14th day during the 21 days test period. Increased serum glyceride concentration was observed with males showing significantly higher increase

111

Fertility (sperm quality)

64 male employees of glycerol manufacturer Closed skin 0.05 ml or 10% patch test solution for 21 consecutive days. Oral

Acute or chronic (42 days). Exact concentration/qu antity not specified Oral 30ml of 95% 14 volunteers (10 men, 4 women). No obvious glycerol (mixed signs of toxicity or effect on food consumption. with orange juice) Eye exposure 100% glycerol Specular microscopy showed extensive changes (drops) in the appearance of the endothelium most of which disappeared within 90 minutes after the exposure ended. Oral Average daily 10 men & 4 women. No toxic effects reported intake of 24 with a slight tendency towards an increase in gm/kg.bw per day, body weight. for 50 days

112

113

4.3 Carcinogenicity, Genotoxicity & Reproductive Toxicity Glycerol has been tested for its carcinogenic or genotoxicity effects in few studies and no studies have reported a link between glycerol exposure and carcinogenicity. A standard mutation test, Ames test, showed no induced mutations in the bacteria (with or without metabolic activation). In other tests that involved mammalian cell gene mutation test and chromosomal aberration tests (using Chinese hamster ovary and rat bone marrow), glycerol did not induce any statistically significant mutation of chromosomal aberrations and these incidences were not concentration dependent

Journal of Industrial Hygiene and Toxicology. Vol. 23, Pg. 259, 1941. "Toxicology of Drugs and Chemicals," Deichmann, W.B., New York, Academic Press, Inc., 1969Vol. -, Pg. 288, 1969. 112 European Chemicals Bureau; IUCLID Dataset, Glycerol (56-81-5) (2000 CD-ROM edition). 113 WHO/FAO: Expert Committee on Food Additives. Summary of Toxicological Data of Certain Food Additives Series 48: Aliphatic acyclic diols, triols, and related substances (56-81-5) (2002). 110 111

Page 23 of 54

CLP Classification of e-liquids Dr Dilip Arvind Bhuva, Xyfil Ltd. indicating that the results are of no biological relevance 114. Thus glycerol is not considered to induce any carcinogenicity, genotoxicity or reproductive toxicity.

4.4 Specific Risk Groups  







Chances of dehydration are increased in patients with geriatric, senile, or already dehydrated patients115. Moreover, caution needs to be exercised when geriatric patients are given glycerol as these patients may have age-related renal function impairment116. However, no such relationship of age to the adverse effects of glycerol has been established. When metabolised, glycerol may cause slight hyperglycaemia and glycosuria and thus caution must be observed during oral administration in diabetic patients117. Risk-benefit should be considered when Glycerol is administered to patients with severe dehydration, cardiac, renal or hepatic disease, since shift in body water may aggravate these conditions and may lead to pulmonary oedema and/or congestive heart failure104, 105. No specific, age-related effects of glycerol are expected in paediatric population.

United Nations Environment Programme: Screening Information Data Sheets on Glycerol (56-81-5) (March 2002) Available from, as of July 14, 2009. 115 McEvoy, G.K. (ed.). American Hospital Formulary Service - Drug Information 93. Bethesda, MD: American Society of Hospital Pharmacists, Inc., 1993 (Plus Supplements, 1993)., p. 1774. 116 US Pharmacopeial Convention; US Pharmacopeia Dispensing Information (USP DI); Drug Information for the Health Care Professional 12th ed, V.I p.1456 (1992). 117 McEvoy, G.K. (ed.). American Hospital Formulary Service - Drug Information 93. Bethesda, MD: American Society of Hospital Pharmacists, Inc., 1993 (Plus Supplements, 1993)., p. 1774. 114

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CLP Classification of e-liquids Dr Dilip Arvind Bhuva, Xyfil Ltd.

4.5 Eco-toxicity Data for Glycerol Model Microcystis aeruginosa Entosiphon sulcatum Carassius auratus Daphnia magna Scenedesmus quadricauda

Dose 2900 mg/l

Comments Reference Toxicity threshold (cell 118 multiplication inhibition test) 3200 mg/l Toxicity threshold (cell multiplication inhibition test) >5000 mg/L for LC50 24 hr >10000 mg/L for LC50. Conditions: freshwater, 119 24 hr static, 20-22 deg C EC3 (3% Conditions: Static, freshwater, 120 effective pH=7.0 before adding test concentration) = material. >10,000 mg/L (for 8 days)

4.6 Environmental Fate 

  



Glycerol does not contain chromophores that absorb (sun) light at wavelengths >290 nm and therefore it is not expected to be susceptible to direct photolysis by sunlight121. If released to soil, glycerol is expected to have very high mobility based upon an estimated Koc of 1, determined from a structure estimation method122. If released into water, glycerol is not expected to adsorb to suspended solids and sediment based upon the estimated Koc of 1. Volatilization from moist soil or water surfaces is not expected to be an important fate process based upon an estimated Henry's Law constant of 1.7x108 atm-cu m/mole123. According to a model of gas/particle partitioning of semi-volatile organic compounds in the atmosphere124, glycerol is expected to exist in both, vapour and particulate, phases given that it has vapour pressure of 1.68x10-4 mm Hg at 25 oC125.

Verschueren, K. Handbook of Environmental Data of Organic Chemicals. 2nd ed. New York, NY: Van Nostrand Reinhold Co., 1983., p. 695. 119 Bringmann G, Kuhn R; Z Wasser-Abwasser-Forsch 10 (5): 161-1 (1977) as cited in the ECOTOX database. Available from, as of July 7, 2011. 120 Bringmann and Kuehn. Mitt. Internat. Verein. Limnol. 21: 275-284 (1980) 121 Lyman WJ et al; Handbook of Chemical Property Estimation Methods. Washington, DC: Amer Chem Soc pp. 8-12 (1990). 122 Meylan WM et al; Environ Sci Technol 26: 1560-67 (1992) (3) Hine J, Mookerjee PK; J Org Chem 40: 292-8 (1975). 123 Hine J, Mookerjee PK; J Org Chem 40: 292-8 (1975). 124 Bidleman TF; Environ Sci Technol 22: 361-367 (1988). 125 Daubert TE, Danner RP; Physical and Thermodynamic Properties of Pure Chemicals Data Compilation. Washington, DC: Taylor and Francis (1989). 118

Page 25 of 54

CLP Classification of e-liquids Dr Dilip Arvind Bhuva, Xyfil Ltd. 





Vapour-phase glycerol is degraded in the atmosphere by reaction with photochemically-produced hydroxyl radicals; the half-life for this reaction in air is estimated to be 7 hours, calculated from its rate constant of 1.9x10-11 cu cm/molecule-sec at 25 oC126. Glycerol, present at 100 mg/L, reached 63% of its theoretical BOD in 2 weeks using an activated sludge inoculum at 30 mg/L in the Japanese MITI test 127. Aerobic biodegradation rate constants of 0.258/day and 0.200/day in respirometric test systems employing activated sludge have also been reported, corresponding to 68% and 78% degradation, respectively128. This suggests that biodegradation is an important and major fate of glycerol in the environment. An estimated BCF of 3, calculated from log Kow of -1.76129 and a regressionderived equation130, suggests the potential for bio concentration in aquatic organisms is low.

4.7 Additional Hazards 

None to report

Meylan WM, Howard PH; Chemosphere 26: 2293-99 (1993). NITE; Chemical Risk Information Platform (CHRIP). Biodegradation and Bioconcentration. Tokyo, Japan: Natl Inst Tech Eval. 128 Reuschenbach P et al; Water Res 37: 1571-1582 (2003). 129 Hansch C et al; Exploring QSAR. Hydrophobic, Electronic, and Steric Constants. ACS Prof Ref Book. Heller SR, consult. ed., Washington, DC: Amer Chem Soc p. 7 (1995). 130 US EPA; Estimation Program Interface (EPI) Suite. Ver. 4.1. Jan, 2010. 126 127

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CLP Classification of e-liquids Dr Dilip Arvind Bhuva, Xyfil Ltd.

PART B: CLP CLASSIFICATION OF PRODUCTS 5. 7.2% (W/V) NICOTINE IN PROPYLENE GLYCOL 5.1 Physical Hazards From all the available physical information, the mixtures under classification do not pose any physical hazards and does not require classification (or cannot be classified) under this category. The only important and relevant note is that the given nicotine mixtures are believed to be combustible and thus care should be taken to avoid exposure to open flames.

5.2 Health Hazard Classification According to CLP regulations (EC No 1272/2008), if the data is not available on the mixture itself or on similar mixtures, then the hazard in this category can be calculated from the summation formula as given in the Annex I: 3.1.3.6.1 of CLP regulation (EC 1272/2008), provided the toxicity values are available for all the ingredients. The Acute Toxicity Estimate (ATE) of the mixture is determined by calculation from the ATE values (or LD50) for all relevant ingredients according to the following formula for Oral, Dermal or Inhalation Toxicity: 100 C𝑖 =∑ ATE𝑚𝑖𝑥 ATE𝑖 𝑛

............. Equation 1 where: Ci = concentration of ingredient i ( % w/w or % v/v) i = the individual ingredient from 1 to n n = the number of ingredients ATEi = Acute Toxicity Estimate of ingredient i.

In order to apply this to the 7.2% (w/v) nicotine mixtures, the w/v percentage will first need to be converted to w/w percentage and most appropriate acute animal toxicity doses need to be selected for solving the summation equation. 𝑤 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑠𝑢𝑏𝑠𝑡𝑎𝑛𝑐𝑒 𝑖𝑛 𝑔𝑚 𝑝𝑒𝑟 𝑚𝑙 %= × 100 𝑤 𝑆𝑝𝑒𝑐𝑖𝑓𝑖𝑐 𝑔𝑟𝑎𝑣𝑖𝑡𝑦

...............Equation 2 Given that the specific gravity of the mixture/solution is 1.038 (determined experimentally) and substituting all the values in equation 2 we get, 𝑤 7.2 × 10−2 𝑔𝑚 %= × 100 = 6.94% 𝑤 1.038 Page 27 of 54

CLP Classification of e-liquids Dr Dilip Arvind Bhuva, Xyfil Ltd. Therefore, 7.2% (w/v) nicotine in propylene glycol is equivalent to 6.94% (w/w) nicotine in propylene glycol solution. Similarly, 7.2% (w/v) nicotine in glycerol is equivalent to 6.01% (w/w) nicotine in glycerol as the specific gravity for this solution is 1.198 (determined experimentally; calculations not shown). Furthermore, the CLP regulations states that “The preferred test species for evaluation of acute toxicity by the oral and inhalation routes is the rat, while the rat or rabbit are preferred for evaluation of acute dermal toxicity”. Also, if more than one reliable toxicity end points are available, a conservative approach should be used in selecting the ATE values. The list of selected values for calculation is given below in the Table 5.1. Table 5.1: Selected ATE for health hazard calculations Chemical

Oral (LD50; Rat)

Dermal (LD50; Rabbit) 50 mg/kg.bw 20,800 mg/kg.bw >10,000 mg/kg.bw

Inhalation (LC50; Rat) Nicotine 50 mg/kg.bw Not available Propylene Glycol 22,000 mg/kg.bw Not available Glycerol 12,600 mg/kg.bw No reliable data available. The LD50 values for Propylene glycol and Glycerol will be ignored during the calculation (i.e. solving ............. Equation 1) as these values are above 2,000 mg/kg.bw.

Acute Oral Toxicity Solving ............. Equation 1 we get, 100 C𝑖 =∑ ATE𝑚𝑖𝑥 ATE𝑖 𝑛

𝑤 100 (% 𝑤 ) ATE𝑚𝑖𝑥

ATE𝑚𝑖𝑥 =

𝑤 6.94 (% 𝑤 ) = 𝑚𝑔 50 ( ) 𝑘𝑔. 𝑏𝑤

50 × 100 𝑚𝑔 = 720.46 6.94 𝑘𝑔. 𝑏𝑤

Thus, the estimated ATE for 7.2% (w/v) nicotine in Propylene glycol or glycerol is 720.46 mg/kg.bw, which indicates that the given mixture is classified into category 4 for this toxicity category, based on Table 3.1.1 of CLP regulation (see Appendix A). Similarly, calculations can be done - by using values given in table 1 and solving equation 1 – for 7.2% nicotine in Glycerol which would classify the mixture in category 4 for oral toxicity since the derived ATE value is 831.95 mg/kg.bw. Page 28 of 54

CLP Classification of e-liquids Dr Dilip Arvind Bhuva, Xyfil Ltd. Acute Dermal Toxicity Solving ............. Equation 1 for 7.2% (w/v) nicotine in glycerol we get, 100 C𝑖 =∑ ATE𝑚𝑖𝑥 ATE𝑖 𝑛

𝑤 100 (% 𝑤 ) ATE𝑚𝑖𝑥

ATE𝑚𝑖𝑥 =

𝑤 6.01 (% 𝑤 ) = 𝑚𝑔 50 ( ) 𝑘𝑔. 𝑏𝑤

50 × 100 𝑚𝑔 = 831.95 6.01 𝑘𝑔. 𝑏𝑤

Thus, the derived ATE value for acute dermal toxicity of 7.2% (w/v) nicotine in glycerol is 831.95 mg/kg.bw. Similarly, the derived ATE value for 7.2% (w/v) nicotine in propylene glycol would be 720.46 mg/kg.bw. These derived ATE values place both the products in category 3 for dermal toxicity (see table 3.1.3 of CLP regulation). Acute Inhalation Toxicity Classification under this toxicity category is not possible due to lack of sufficient and reliable evidence.

5.3 Environmental Hazards For environmental hazards, the assessment of a mixture – which itself hasn’t been tested for environmental toxicity - can be based on ‘bridging principles’ or ‘summation method’. Since there are no environmental data available on similar mixtures, bridging principles are not applicable (according to Annex I, 4.1.3.4 of CLP regulation) and the summation method should be used (Annex I, 4.1.3.5 of CLP regulation). Furthermore, since nicotine (pure form) is classified under EU harmonised system for its aquatic toxicity (Chronic category 2), the additivity formula given in Annex I, 4.1.3.5.2 cannot be used and summation method given in Annex I, 4.1.3.5.5 of CLP regulations can be directly applied in this case. In the mixtures under classification, the propylene glycol and glycerol are considered as not having any substantial acute or chronic aquatic toxicity (as evident from ATE values in Table 5.2; calculations not shown) and thus these components are ignored while employing the summation method.

Page 29 of 54

CLP Classification of e-liquids Dr Dilip Arvind Bhuva, Xyfil Ltd. Table 5.2: Selected ATE for Environmental calculations Chemical

ErC50 (acute)

NOEC

Dose

Model

Dose

Nicotine

37 mg/L

3.2 mg/L

Propylene Glycol

19300 mg/L

Glycerol

2900 mg/L

Desmodesmus subspicatus (algae); 72 hrs Skeletonema costatum (algae); 72 hrs Microcystis aeruginosa (Cyanobacteria); not specified

Model

Desmodesmus subspicatus (algae); 72 hrs ≤5300 mg/L Skeletonema costatum (algae); 72 hrs EC3 = ~10,000 Scenedesmus mg/L quadricauda (algae); 8 days

Acute Aquatic Toxicity Nicotine is neither classified as having an acute aquatic toxicity under harmonised classification nor does the aquatic toxicity data suggest that nicotine has a potential of acute aquatic toxicity. Moreover, nicotine is determined to be rapidly degraded in the environment and has very low potential for bioaccumulation (Kow=1.17; >70% degradation in 28 days; however, NOEC is ≥1 mg/mL and therefore chronic implications) further supporting the notion that nicotine may not pose any significant acute toxicity threat to the aquatic ecosystem. Thus the mixtures under consideration are not classified under this toxicity category. Chronic Aquatic Toxicity Pure nicotine is classified as having chronic aquatic toxicity category 2 under harmonised classification and labelling of hazardous substances (Annex VI, table 3.1 of regulation (EC) No 1272/2008); and this is a legally binding classification – for this particular toxicity category – to all the manufacturers, importers and all downstream users (see clause 17 of the introductory notes of CLP regulation). Using this classification and applying summation method given in Annex I, 4.1.3.5.5 and table 4.1.2 (see Appendix A) we get following classification:

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CLP Classification of e-liquids Dr Dilip Arvind Bhuva, Xyfil Ltd. Mixture

Percentage of Multiplication components from factor chronic category 2

7.2% (w/v) 6.93% (w/w) nicotine propylene glycol 7.2% (w/v) 6.01% (w/w) nicotine Glycerol

Adjusted sum of Results for all toxic classification components with aquatic toxicity

None

6.93%

10

69.3%

None

6.01%

10

60.1%

Cannot classify under category 2 as the sum is