GroupMG Companies Murat Gkay

Division: ANASYN Biotechnology & Research

Technologiezentrum
Vor dem Kreuzberg 17
72070 Tbingen
Germany
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Characterization and Chemical Safety Assessment
of the Vapor of Electronic Cigarette Liquid
(e-Liquid)



prepared for


Hellenic Commercial Union of
Electronic Cigarettes and Vaping (ESHTA) Greece

Greece









GroupMG Companies Murat Gkay
Division: ANASYN Biotechnology & Research
Technologiezentrum
Vor dem Kreuzberg 17
72070 Tbingen
Germany
www.groupmg.com
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24 August 2012

Client: Hellenic Commercial Union of Electronic
Cigarettes and Vaping (ESHTA) Greece

Requestor: Vice President Mr. Tasos Tsakirakis

Study: Characterization and Chemical Safety Assessment of the Vapor of
Electronic Cigarette Liquid (e-Liquid)

Sample no.: LIQUA Bright TOBACCO Italian Flavours LOT B161

Safety report no.: 082012ECLGR02

1. Summary
The vapor of the liquid sample, used in electronic cigarettes, was analyzed by headspace gas
chromatography- mass spectrometry (HSGC-MS) to identify the ingredients in the vapor
phase and their relative concentrations.


2. Experimental
The vapor was generated by heating the liquid sample to the temperature of 65 C in a sealed
vial for 5 minutes. Afterwards 10L of the vapor were injected directly into the GC-MS
instrument.
The sample was characterized on a Hewlett Packard 6830-6890 GC-MS instrument equipped
with a DB-5MS 25m x 0,25mm, df= 0,25m capillary column. Observed peaks were
identified based on spectral matches with commercial MS libraries. Relative concentrations
were estimated from the peak areas.


GroupMG Companies Murat Gkay
Division: ANASYN Biotechnology & Research
Technologiezentrum
Vor dem Kreuzberg 17
72070 Tbingen
Germany
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3. Results & Discussion
A Total Ion Chromatogram (TIC) of 10 L of the vapor phase of the LIQUA Bright
TOBACCO Italian Flavours e-cigarette liquid is shown in Figure 1. Spectral library matches
identified two ingredients propylene glycol and nicotine with retention times of about 3.4
minutes and 17.7 minutes respectively. The signal intergral value of Propylene glycol (the
first peak) was significantly higher than the other ingredient. Table 1 summarizes the
estimated relative % of each ingredient in the vapor phase, generated at 65 C, of the smoke
liquid. This analysis assumed that all components, being volatile at this temperature, were
observed in the headspace GC-MS analysis and that the sensitivity of the detector to each
compound was similar.
Chemical safety assessment was performed by considering general hazard and toxicity data
from the safety data sheets obtained from Sigma Aldrich and the safety and toxicity data
published at the online resources www.hazard.com and http://toxnet.nlm.nih.gov/index.html.


















GroupMG Companies Murat Gkay
Division: ANASYN Biotechnology & Research
Technologiezentrum
Vor dem Kreuzberg 17
72070 Tbingen
Germany
www.groupmg.com
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Figure 1: TIC of the e-Liquid vapor of the sample LIQUA Bright TOBACCO Italian
Flavours
Signal assignments: propylene glycol (1), nicotine (2).
1
2
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Division: ANASYN Biotechnology & Research
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Signal no. Name % in Vapors
(Peak Area %)
CAS No. Comments
1 Propylene
glycol
88.4 57-55-6 Not a hazardous substance or
mixture according to Regulation
(EC) No. 1272/2008.
This substance is not classified as
dangerous according to Directive
67/548/EEC.
May be harmful if inhaled.
May cause respiratory tract
irritation.
May be harmful if swallowed.
May be harmful if absorbed through
skin.
May cause skin irritation.
Causes eye irritation.
2 Nicotine 11.6 54-11-5


H301 Toxic if swallowed.
H310 Fatal in contact with skin.
H411 Toxic to aquatic life with
long lasting effects.
May be harmful if inhaled. May
cause respiratory tract irritation.
May cause eye irritation.

Table 1: TIC Peak Report and toxicological aspects for sample LIQUA Bright TOBACCO
Italian Flavours



4. Chemical Safety Assessment of Each Identified Component

4.1 Propylene Glycol

CAS-No. : 57-55-6. For this product a material safety data sheet is available. This product is
used as a laboratory chemical and for manufacturing substances.
It is not a hazardous substance or mixture according to Regulation (EC) No. 1272/2008.
This substance is not classified as dangerous according to Directive 67/548/EEC.

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Division: ANASYN Biotechnology & Research
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The product does not need to be labeled in accordance with EC directives or respective
national laws.
Formula : C
3
H
8
O
2
Molecular Weight : 76,09 g/mol
Appearance Form: liquid, clear, viscous
Colour: colourless
Melting point/freezing point-60 C
Initial boiling point and boiling range 185 - 189 C
Flash point 103 C - closed cup
Flammability (solid, gas) no data available
Upper explosion limit: 12,5 %(V)
Lower explosion limit: 2,6 %(V)
Vapour pressure 0,11 hPa at 20 C
Vapour density 2,63 - (Air = 1.0)
Relative density 1,036 g/cm3

Toxicity:
LD50 Oral - rat - 20.000 mg/kg
LD50 Dermal - rabbit - 20.800 mg/kg
LD50 Intramuscular - rat - 14 g/kg
LD50 Intravenous - dog - 26 g/kg
LD50 Intraperitoneal - rat - 6.660 mg/kg
LD50 Subcutaneous - rat - 22.500 mg/kg
LD50 Intravenous - rat - 6.423 mg/kg
LD50 Intraperitoneal - mouse - 9.718 mg/kg
Remarks: Lungs, Thorax, or Respiration:Chronic pulmonary edema. Kidney, Ureter,
Bladder:Changes in both tubules and glomeruli. Blood:Changes in spleen.
LD50 Subcutaneous - mouse - 17.370 mg/kg
Remarks: Behavioral:Change in motor activity (specific assay). Behavioral:Muscle
contraction or spasticity.Cyanosis
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LD50 Intravenous - mouse - 6.630 mg/kg
LD50 Intravenous - rabbit - 6.500 mg/kg
Skin - Human - Mild skin irritation - 7 d
Serious eye damage/eye irritation
Eyes - rabbit - Mild eye irritation
IARC: No component of this product present at levels greater than or equal to 0.1% is
identified as probable, possible or confirmed human carcinogen by IARC.
May be harmful if inhaled. May cause respiratory tract irritation. May be harmful if
swallowed. May be harmful if absorbed through skin. May cause skin irritation. Causes eye
irritation.

Signs and Symptoms of Exposure:
Gastrointestinal disturbance, Nausea, Headache, Vomiting, Central nervous system
depression.

Ecological Information:
Toxicity to fish: mortality NOEC- Pimephales promelas (fathead minnow) - 52.930 mg/l -96h
Toxicity to daphnia and other aquatic invertebrates: mortality NOEC - Daphnia - 13.020 mg/l
- 48 h
EC50 - Daphnia magna (Water flea) - > 10.000 mg/l - 48 h

Final Toxicolgical Assessment
Propylene glycol is used as a solvent in many pharmaceuticals, including oral, injectable
and topical formulations (notably, diazepam, which is insoluble in water, uses propylene glycol
as its solvent in its clinical, injectable form). It is also used as a humectant food additive, labeled
as E number E1520.
Other fields of application of propylene glycol are cosmetics (as a moisturizer), food,
toothpaste, shampoo, mouth wash, hair care and tobacco products.


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Division: ANASYN Biotechnology & Research
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Because of its low chronic oral toxicity, propylene glycol was classified by the U. S. Food and
Drug Administration as "generally recognized as safe" (GRAS) for use as a direct food additive.



4.2 Nicotine

CAS-No. : 54-11-5. For this product a material safety data sheet is available. This product is
used as a laboratory chemical and for manufacturing substances.
Classification according to Regulation (EC) No 1272/2008 [EU-GHS/CLP]
Acute toxicity; Dermal (Category 1)
Acute toxicity, Oral (Category 3)
Chronic aquatic toxicity (Category 2)
Classification according to EU Directives 67/548/EEC or 1999/45/EC
Very toxic in contact with skin. Toxic if swallowed. Toxic to aquatic organisms. May cause
long-term adverse effects in the aquatic environment.
Labeling according Regulation (EC) No 1272/2008 [CLP]

Pictogram :


Signal word: Danger

H301 Toxic if swallowed.
H310 Fatal in contact with skin.
H411 Toxic to aquatic life with long lasting effects.
According to European Directive 67/548/EEC as amended.
R25 Toxic if swallowed.
R27 Very toxic in contact with skin.
R51/53 Toxic to aquatic organisms, may cause long-term adverse effects in the
aquatic environment.
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Division: ANASYN Biotechnology & Research
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Hazard Symbols:

Formula : C
10
H
14
N
2
Molecular Weight : 162,23 g/mol
Appearance Form: clear, liquid
Colour: dark brown
pH 10,2
Melting point/range: -79 C
Initial boiling point and boiling range: 247 C at 993 hPa
Flash point: 101 C - closed cup
Vapour pressure: 0,051 hPa at 25 C
Vapour density: 5,6 - (Air = 1.0)
Relative density: 1,010 g/cm
3
at 20 C
completely miscible with water
Partition coefficient: n-octanol/water log Pow: 1,17
Solubility in other solvents: Ethanol 50 g/l

Toxicity:
LD50 Oral - rat - 50 mg/kg
IARC: No component of this product present at levels greater than or equal to 0.1% is
identified as probable, possible or confirmed human carcinogen by IARC.
Possible risk of congenital malformation in the fetus.
May be harmful if inhaled. May cause respiratory tract irritation. May be fatal if swallowed.
May be fatal if absorbed through skin. May cause skin irritation. May cause eye irritation.

Signs and Symptoms of Exposure prolonged or repeated exposure:
Can cause Vomiting, Diarrhoea, Convulsions, To the best of our knowledge, the chemical,
physical, and toxicological properties have not been thoroughly investigated.

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Division: ANASYN Biotechnology & Research
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Ecological Information:
Toxicity to fish: LC50 - Oncorhynchus mykiss (rainbow trout) - 4 mg/l - 96,0 h
Toxicity to daphnia and other aquatic invertebrates: EC50 - Daphnia magna (Water flea) -
0,24 mg/l - 48 h.
Very toxic to aquatic life.

Final Toxicolgical Assessment
The LD50 of nicotine is 50 mg/kg for rats and 3 mg/kg for mice. 3060 mg (0.51.0 mg/kg)
can be a lethal dosage for adult humans [1, 2]. It is unlikely that a person would overdose on
nicotine through smoking alone, although overdose can occur through combined use of
nicotine patches or nicotine gum and cigarettes at the same time [3]. In low doses (an
average cigarette yields about 1 mg of absorbed nicotine), the substance acts as
a stimulant in mammals, while high amounts (3060 mg) can be fatal [1, 3]. This stimulant
effect is the main factor responsible for the dependence-forming properties of tobacco smoking.
Due to the low concentration of nicotine in the vapor phase of the cigarette liquid, compared
to the amount of nicotine in tobacco smoke (0.8 3.0 mg per cigarette), the inhaled
concentration can be assessed as less hazardous to health than the inhaled nicotine
concentrations in tobacco (cigarette) smoke.

References:
[1] http://www.inchem.org/documents/pims/chemical/nicotine.htm#PartTitle:7.%20TOXICOLOGY
[2] Okamoto M, Kita T, Okuda H, Tanaka T, Nakashima T (Jul 1994). "Effects of aging on acute
toxicity of nicotine in rats". Pharmacol Toxicol. 75 (1): 16
[3] http://learn.genetics.utah.edu/content/addiction/drugs/overdose.html


Assessor: Dr. rer. nat. Ozan Gkay
(Chief of the Division ANASYN)