In Vitro Cytotoxicity and Genotoxicity of Representative E-liquid Flavor Mixtures WWW.ALTRIA.COM/ALCS-SCIENCE 2019 CORESTA Smoke Science & Product Technology Conference (SSPT2019), Hamburg, Germany, 6-10 October 2019 Utkarsh Doshi, Jingjie Zhang, Ashutosh Kumar & K. Monica Lee Altria Client Services LLC, Richmond, VA 23219 INTRODUCTION ▶ Flavor compounds that are added to food and orally consumed fall within “generally recognized as safe (GRAS)” category. ▶ Limited safety data exists for inhalation route of exposure for flavor compounds used in inhalable e-vapor products. ▶ Flavor compounds in e-vapor products are commonly available as mixtures which makes their hazard characterization resource and time-demanding. ▶ The Approach: – Group flavors based on their structural similarities. – Identify representative flavors from each group based on available toxicological data. – Divide the flavors into subgroup mixtures (called pre-blends) based on their solubility and chemical reactvity. – Mix the pre-blends in an e-liquid carrier (PG/VG) to prepare test mixtures with or without nicotine. – Subject representative mixtures to preclinical toxicity (cytotoxicity and genotoxicity) testing. ▶ To identify drivers of toxicological response, subject pre-blends to additional testing. Toxicological Evaluation of Flavor Mixtures Test Articles Carrier (PG/VG/Nicotine) Test Formulation Test Formulation + Nicotine Mutagenicity Negative Negative Negative Ames Assay s PG VG Nicotine 38 Flavors In Vitro Assays Cytotoxicity Genotoxicity Test Formulations Aldehydes Acetals Ketones Phenols Terpenes Acids Pyridines Esters 38 Flavor Compounds E-Vapor Industry 5000+ Flavor Compounds 200-300 ▶ Test Articles: – E-liquids ▪ Carrier (Propylene Glycol : Vegetable Glycerol (80:20) + 2% Nicotine v/v) ▪ Test Formulation (18.6% flavor) ▪ Test Formulation (18.6% flavor) + 2% Nicotine – Pre-blends ▪ Pre-blend I A , I B , I C , II, III & IV. ▶ Salmonella Mutagenicity (Ames) Assay: E-liquids were tested in five Salmonella typhimurium strains: TA1537, TA98, TA100, TA1535 and TA102 according to OECD 471 1 . The maximum concentration tested up to 100 μL/plate. ▶ Neutral Red Uptake (NRU) Assay: BALB/c 3T3 cells were incubated in presence of the vehicle control (DMSO)/positive control (sodium lauryl sulfate)/the test e-liquids or pre-blends for ~48 h according to OECD 129 2 . The maximum concentration tested was up to 0.5% (v/v). ▶ In Vitro Micronucleus (MNvit) Assay Using TK6 Cells: The e-liquids were evaluated for micronucleus induction according to OECD 487 3 in TK6 cells during short (4 h) incubations with and without S9, and long (27 h) incubations without S9 followed by an extended recovery of 40 h. Cytotoxicity was checked to set the testing concentration, with the maximum concentration tested up to 1.5% (v/v). METHODS AND MATERIALS Flavor compounds “generally recognized as safe” for oral consumption are commonly used in inhalable e-vapor products for which insufficient safety data may exist. The hazard characterization of each flavor and various flavor mixtures is resource and time-demanding. Here we explored a pragmatic approach, where we selected representative flavors and tested for in vitro cytotoxicity and genotoxicity. From a group of commonly used individual flavors (>200) in e-vapor products, 38 flavors were selected using structural grouping and available toxicological data. These flavors were mixed in a carrier (PG/VG/water) to prepare a test mixture (prototype flavor mixture with up to 18 % flavor load), with and without nicotine, and were subjected to a standard CORESTA battery of in vitro cytotoxicity (Neutral Red Uptake [NRU]) and genotoxicity (Ames and micronucleus [MN]) assays. Test mixtures (with and without nicotine) were negative in the Ames mutagenicity assay but showed cytotoxicity in all three assays including NRU assay. In the MN genotoxicity assay, the test mixture with nicotine was negative but the test mixture without nicotine provided equivocal results. To further identify the potentially responsible flavor(s) for the cytotoxicity response in the NRU assay of the test mixtures, we divided the 38 flavors into five subgroup mixtures according to their solubility and chemical reactivity, and tested them using NRU cytotoxicity assay. Results suggested that subgroup mixtures containing certain flavors – for example, ethyl maltol, furaneol and isopulegol – were more cytotoxic, consistent to literature findings as in vitro cytotoxicant/irritant. The results align with the overall systematic toxicity evaluation approach, deconstructing mixtures into subsets of flavors, ultimately in support of flavor read-across assessment. ABSTRACT RESULTS REFERENCES CONCLUSIONS ▶ Representative flavor mixtures did not show mutagenicity and genotoxicity in the in vitro assays ▶ Representative flavor mixtures showed cytotoxicity in the in vitro assay, however the cytotoxicity was driven by few selected flavors or flavor groups ▶ Use of a read across approach in combination with systematic toxicity evaluation (deconstructing mixtures into subsets of flavors) can reduce the list of compounds for thorough toxicological evaluation 1. OECD. (1997). OECD Guideline for Testing Chemicals Test Guideline 471, Bacterial Reverse Mutation Test. 2. OECD. (2010). OECD Guidance Document on Using Cytotoxicity Tests to Estimate Starting Doses for Acute Oral Systemic Toxicity Tests 3. OECD. (2016). OECD Guideline for Testing Chemicals Test Guideline 487, In vitro Mammalian Cell Micronucleus Test. In vitro studies (cytotoxicity & genotoxicity) were conducted at Bioreliance (Millipore Sigma), Rockville, Maryland USA NRU Assay Pre-blends IA, IB and II were the major contributors to toxicity. 0 20 40 60 80 100 120 140 0.0001 0.001 0.01 0.1 1 Percent Viability (Relative to Vehicle Control) Concentration of E-liquid (% (v/v)) Neutral Red Uptake Cytotoxicity Assay Pre-blend IA Pre-blend IB Pre-blend IC Pre-blend II Pre-blend III Pre-blend IV 0 10 20 30 40 50 60 Pre-blend IA Pre-blend IB Pre-blend IC Pre-blend II Pre-blend III Pre-blend IV 1/EC50 (%v/v) Cytotoxicity Potency of Pre-blends Genotoxicity NRU Assay 0 25 50 75 100 125 150 0.0001 0.001 0.01 0.1 1 Percent Viability (Relative to Solvent Control) Concentration of E-liquid (% (v/v)) Neutral Red Uptake Cytotoxicity Assay Carrier (PG/VG/Nicotine) Test Formulation Test Formulation + Nicotine 50% Viability Cytotoxicity Evaluation of Pre-blends (IA, IB, IC, II, III, IV) Micronucleus Assay Cytotoxicity 0 20 40 60 80 100 120 0.00 0.10 0.20 0.30 0.40 % Viability (Relative Population Doubling) Concentration of E-liquid (%v/v) Cytotoxicity in TK6 cells Test Formulation + Nic 4h - S9 4h + S9 27h - S9 0 20 40 60 80 100 120 0.00 0.50 1.00 1.50 2.00 % Viability (Relative Population Doubling) Concentration of E-liquid (%v/v) Cytotoxicity in TK6 cells PG/VG/Nic 4h - S9 4h + S9 27h - S9 0 20 40 60 80 100 120 0.00 0.10 0.20 0.30 0.40 % Viability (Relative Population Doubling) Concentration of E-liquid (%v/v) Cytotoxicity in TK6 cells Test Formulation 4h - S9 4h + S9 27h - S9 Test Articles Genotoxicity Carrier (PG/VG/Nicotine) Negative Test Formulation Equivocal Test Formulation + Nicotine Negative 0 20 40 60 80 100 0.0 0.2 0.4 0.6 0.8 1.0 1.2 DMSO 0.04 0.08 0.14 Cytotoxicity (%) % Micronuclei Concentration (%v/v)) In Vitro Micronucleus Assay (Test Formulation-Nicotine/4h+S9) % Micronuclei Cytotoxicity * Upper limit of vehicle historical control Criteria For Positive Genotoxicity Call All 3 criteria have to be met: • Statistical Significance (p≤0.05, Fisher exact) • Outside of vehicle historical control • Significant for trend * p≤0.05, Fisher exact test 2019_STPOST38_Doshi.pdf SSPT2019 - Document not peer-reviewed by CORESTA
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In Vitro Cytotoxicity and Genotoxicity of Representative E-liquid Flavor Mixtures
WWW.ALTRIA.COM/ALCS-SCIENCE
2019 CORESTA Smoke Science & Product Technology Conference (SSPT2019),
Hamburg, Germany, 6-10 October 2019
Utkarsh Doshi, Jingjie Zhang, Ashutosh Kumar & K. Monica Lee
Altria Client Services LLC, Richmond, VA 23219
INTRODUCTION▶ Flavor compounds that are added to food and orally consumed fall within “generally recognized
as safe (GRAS)” category.▶ Limited safety data exists for inhalation route of exposure for flavor compounds used in inhalable
e-vapor products.▶ Flavor compounds in e-vapor products are commonly available as mixtures which makes their
hazard characterization resource and time-demanding.▶ The Approach:
– Group flavors based on their structural similarities.– Identify representative flavors from each group based on available toxicological data.– Divide the flavors into subgroup mixtures (called pre-blends) based on their solubility and
chemical reactvity.– Mix the pre-blends in an e-liquid carrier (PG/VG) to prepare test mixtures with or without nicotine.– Subject representative mixtures to preclinical toxicity (cytotoxicity and genotoxicity) testing.
▶ To identify drivers of toxicological response, subject pre-blends to additional testing.
Toxicological Evaluation of Flavor Mixtures
Test Articles Toxicity Mutagenicity
Carrier (PG/VG/Nicotine) None (Max: 100µL/plate) Negative
Test Formulation >0.75µL/plate (-S9); >5µL/plate (+S9) Negative
Test Formulation + Nicotine >1.56µL/plate (-S9); >3µL/plate (+S9) Negative
Test Articles Toxicity Mutagenicity
Carrier (PG/VG/Nicotine) None (Max: 100µL/plate) Negative
Test Formulation >0.75µL/plate (-S9); >5µL/plate (+S9) Negative
Test Formulation + Nicotine >1.56µL/plate (-S9); >3µL/plate (+S9) Negative
Ames Assay
>200 Compounds
CommonlyUsed Flavors
PGVG
Nicotine38 Flavors
In Vitro AssaysCytotoxicity GenotoxicityTest Formulations
Aldehydes
Acetals
Ketones
Phenols
Terpenes Acids
PyridinesEsters
38 Flavor Compounds
E-Vapor Industry
5000+ Flavor Compounds 200-300
▶ Test Articles:– E-liquids
▪ Carrier (Propylene Glycol : Vegetable Glycerol (80:20) + 2% Nicotine v/v)▪ Test Formulation (18.6% flavor)▪ Test Formulation (18.6% flavor) + 2% Nicotine
– Pre-blends▪ Pre-blend IA, IB, IC, II, III & IV.
▶ Salmonella Mutagenicity (Ames) Assay: E-liquids were tested in five Salmonella typhimurium strains: TA1537, TA98, TA100, TA1535 and TA102 according to OECD 4711. The maximum concentration tested up to 100 μL/plate.
▶ Neutral Red Uptake (NRU) Assay: BALB/c 3T3 cells were incubated in presence of the vehicle control(DMSO)/positive control (sodium lauryl sulfate)/the test e-liquids or pre-blends for ~48 h according toOECD 1292. The maximum concentration tested was up to 0.5% (v/v).
▶ In Vitro Micronucleus (MNvit) Assay Using TK6 Cells: The e-liquids were evaluated for micronucleusinduction according to OECD 4873 in TK6 cells during short (4 h) incubations with and without S9, andlong (27 h) incubations without S9 followed by an extended recovery of 40 h. Cytotoxicity was checkedto set the testing concentration, with the maximum concentration tested up to 1.5% (v/v).
METHODS AND MATERIALS
Flavor compounds “generally recognized as safe” for oral consumption are commonly used in inhalable e-vapor products for which insufficient safety data may exist. The hazard characterization of each flavor andvarious flavor mixtures is resource and time-demanding. Here we explored a pragmatic approach, where weselected representative flavors and tested for in vitro cytotoxicity and genotoxicity. From a group of commonly usedindividual flavors (>200) in e-vapor products, 38 flavors were selected using structural grouping and availabletoxicological data. These flavors were mixed in a carrier (PG/VG/water) to prepare a test mixture (prototype flavormixture with up to 18 % flavor load), with and without nicotine, and were subjected to a standard CORESTAbattery of in vitro cytotoxicity (Neutral Red Uptake [NRU]) and genotoxicity (Ames and micronucleus [MN])assays. Test mixtures (with and without nicotine) were negative in the Ames mutagenicity assay but showedcytotoxicity in all three assays including NRU assay. In the MN genotoxicity assay, the test mixture with nicotinewas negative but the test mixture without nicotine provided equivocal results. To further identify the potentially responsible flavor(s) for the cytotoxicity response in the NRU assay of the test mixtures, we divided the 38 flavors into five subgroup mixtures according to their solubility and chemical reactivity, and tested them using NRU cytotoxicity assay. Results suggested that subgroup mixtures containing certain flavors – for example, ethyl maltol, furaneol and isopulegol – were more cytotoxic, consistent to literature findings as in vitro cytotoxicant/irritant. The results align with the overall systematic toxicity evaluation approach, deconstructing mixtures into subsets of flavors, ultimately in support of flavor read-across assessment.
ABSTRACT RESULTS
REFERENCES
CONCLUSIONS▶ Representative flavor mixtures did not show mutagenicity and genotoxicity in the in vitro assays▶ Representative flavor mixtures showed cytotoxicity in the in vitro assay, however the cytotoxicity was
driven by few selected flavors or flavor groups▶ Use of a read across approach in combination with systematic toxicity evaluation (deconstructing
mixtures into subsets of flavors) can reduce the list of compounds for thorough toxicological evaluation
1. OECD. (1997). OECD Guideline for Testing Chemicals Test Guideline 471, Bacterial Reverse Mutation Test.2. OECD. (2010). OECD Guidance Document on Using Cytotoxicity Tests to Estimate Starting Doses for Acute Oral Systemic Toxicity Tests3. OECD. (2016). OECD Guideline for Testing Chemicals Test Guideline 487, In vitro Mammalian Cell Micronucleus Test.In vitro studies (cytotoxicity & genotoxicity) were conducted at Bioreliance (Millipore Sigma), Rockville, Maryland USA
NRU Assay
Pre-blends IA, IB and II were the major contributors to toxicity.
0
20
40
60
80
100
120
140
0.0001 0.001 0.01 0.1 1
Perce
nt Vi
abilit
y(R
elativ
e to
Vehic
le Co
ntrol)
Concentration of E-liquid (% (v/v))
Neutral Red Uptake Cytotoxicity Assay
Pre-blend IA Pre-blend IB Pre-blend ICPre-blend II Pre-blend III Pre-blend IV
Cytotoxicity Assessment of Pre-blends
Pre-blends IA, IB and II were the major contributors to toxicity. Examples of flavors reported to be in vitro cytotoxic/irritant:
- IA (isopulegol)- II (furaneol, ethyl maltol)
Utkarsh Doshi l Regulatory Affairs l Altria Client Services l TSRC Sept 17, 2019 l Final 4
0
10
20
30
40
50
60
Pre-blendIA
Pre-blendIB
Pre-blendIC
Pre-blendII
Pre-blendIII
Pre-blendIV
1/EC5
0 (%
v/v)
Cytotoxicity Potency of Pre-blends
Genotoxicity
NRU Assay
Utkarsh Doshi l Regulatory Affairs l Altria Client Services l TSRC Sept 17, 2019 l Final 3
0
25
50
75
100
125
150
0.0001 0.001 0.01 0.1 1
Perc
ent V
iabilit
y(R
elativ
e to
Solve
nt C
ontro
l)
Concentration of E-liquid (% (v/v))
Neutral Red Uptake Cytotoxicity Assay
Carrier (PG/VG/Nicotine)Test FormulationTest Formulation + Nicotine
50% Viability
Cytotoxicity Evaluation of Pre-blends
(IA, IB, IC, II, III, IV)
Micronucleus Assay Cytotoxicity
Utkarsh Doshi l Regulatory Affairs l Altria Client Services l TSRC Sept 17, 2019 l Final 1
0
20
40
60
80
100
120
0.00 0.10 0.20 0.30 0.40
% V
iabilit
y (R
elativ
e Pop
ulat
ion
Doub
ling)
Concentration of E-liquid (%v/v)
Cytotoxicity in TK6 cellsTest Formulation + Nic
4h - S9
4h + S9
27h - S9
0
20
40
60
80
100
120
0.00 0.50 1.00 1.50 2.00
% V
iabilit
y (R
elativ
e Pop
ulat
ion
Doub
ling)
Concentration of E-liquid (%v/v)
Cytotoxicity in TK6 cellsPG/VG/Nic
4h - S9
4h + S9
27h - S9
0
20
40
60
80
100
120
0.00 0.10 0.20 0.30 0.40
% V
iabilit
y (R
elativ
e Pop
ulat
ion
Doub
ling)
Concentration of E-liquid (%v/v)
Cytotoxicity in TK6 cellsTest Formulation
4h - S9
4h + S9
27h - S9
Test Articles Genotoxicity
Carrier (PG/VG/Nicotine) Negative
Test Formulation Equivocal
Test Formulation + Nicotine Negative
Utkarsh Doshi l Regulatory Affairs l Altria Client Services l TSRC Sept 17, 2019 l Final 2
0
20
40
60
80
100
0.0
0.2
0.4
0.6
0.8
1.0
1.2
DMSO 0.04 0.08 0.14
Cyto
toxic
ity (%
)
% M
icron
uclei
Concentration (%v/v))
In Vitro Micronucleus Assay(Test Formulation-Nicotine/4h+S9)
% Micronuclei
Cytotoxicity
*Upper limit of vehicle historical control
Criteria For Positive Genotoxicity CallAll 3 criteria have to be met:• Statistical Significance (p≤0.05, Fisher exact)• Outside of vehicle historical control• Significant for trend
Utkarsh Doshi l Regulatory Affairs l Altria Client Services l TSRC Sept 17, 2019 l Final 2
0
20
40
60
80
100
0.0
0.2
0.4
0.6
0.8
1.0
1.2
DMSO 0.04 0.08 0.14
Cyto
toxic
ity (%
)
% M
icron
uclei
Concentration (%v/v))
In Vitro Micronucleus Assay(Test Formulation-Nicotine/4h+S9)
% Micronuclei
Cytotoxicity
*Upper limit of vehicle historicalcontrol
Criteria For Positive Genotoxicity CallAll 3 criteria have to be met:• Statistical Significance (p≤0.05, Fisher exact)• Outside of vehicle historical control• Significant for trend
* p≤0.05, Fisher exact test
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