Regulatory Toxicology and Pharmacology 95 (2018) 102–114

Contents lists available at ScienceDirect

Regulatory Toxicology and Pharmacology

journal homepage: www.elsevier.com/locate/yrtph

Integrating chemical, toxicological and clinical research to assess the T

potential of reducing health risks associated with cigarette smoking through
reducing toxicant emissions
Kevin McAdam∗, James Murphy, Alison Eldridge, Clive Meredith, Christopher Proctor
Research and Development, British American Tobacco, Regents Park Road, Southampton, UK

A R T I C LE I N FO A B S T R A C T

Keywords: The concept of a risk continuum for tobacco and nicotine products has been proposed, which differentiates
Cigarettes products according to their propensity to reduce toxicant exposure and risk. Cigarettes are deemed the most
Prototype risky and medicinal nicotine the least. We assessed whether a Reduced-Toxicant Prototype (RTP) cigarette could
Public health sufficiently reduce exposure to toxicants versus conventional cigarettes to be considered a distinct category in
Tobacco
the risk continuum.
Toxicants
We present findings from both pre-clinical and clinical studies in order to examine the potential for reduced
Toxicology
Biomarkers of exposure smoke toxicant emissions to lower health risks associated with cigarette smoking. We conclude that current
Product regulation toxicant reducing technologies are unable to reduce toxicant emissions sufficiently to manifest beneficial dis-
Clinical studies ease-relevant changes in smokers.
These findings point to a minimum toxicant exposure standard that future potentially reduced risk products
would need to meet to be considered for full biological assessment.
The RTP met WHO TobReg proposed limits on cigarette toxicant emissions, however the absence of beneficial
disease relevant changes in smokers after six months reduced toxicant cigarette use, does not provide evidence
that these regulatory proposals will positively impact risks of smoking related diseases.
Greater toxicant reductions, such as those that can be achieved in next generation products e.g. tobacco
heating products and electronic cigarettes are likely to be necessary to clearly reduce risks compared with
conventional cigarettes.

1. Introduction
The risk continuum of tobacco and nicotine products developed by
McNeill and Munafò (McNeill and Munafò, 2013) hypothesises that
exposure to toxicants and health risks differ between types of tobacco
and nicotine products. Cigarettes and cigars are most harmful, and e-
cigarettes and medicinal nicotine products the least. Some product
categories, such as snus, have extensive epidemiological data on re-
lative health risks, whereas others, such as e-cigarettes are emerging
technologies with relatively few data. Additionally, some technologies,
such as those that heat rather than burn tobacco, are only very recently
coming into widespread consumer use in certain markets, despite being
developed and test marketed over three decades ago. In British Amer-
ican Tobacco (BAT) we recently presented a version of the product
continuum, ordered by levels of toxicants (Fig. 1) (British American
Tobacco, 2017). On account of emerging science on e-cigarettes
(Margham et al., 2016) and Tobacco Heating Products (Schaller et al.,
2016), along with previously published data on smokeless tobacco,
Snus (McAdam et al., 2013, 2015, 2016, 2017), we proposed that the
product continuum should place non-combustible tobacco and nicotine
products at the low end of toxicant exposure, with cigarettes at the
opposite extreme end of high toxicant exposure.
Cigarette smoking causes a range of serious diseases (Doll and Hill,
1950) in a dose-dependent manner and according to total duration of
smoking (Doll, 2004). Health risks diminish after cessation to a degree
dependent on the number of smoking-years. Therefore, after complete
cessation, risks can reduce and even return to never-smoker levels or
disease progression may be slowed (US Department of Health and
Human Services, 2014 and Stratton et al., 2001). Accordingly, the US
Institute of Medicine concluded that some of the harm caused by to-
bacco use might be reduced by the introduction of what it termed po-
tential reduced-exposure products (i.e., shown through scientific

∗
Corresponding author.
E-mail addresses: kevin_mcadam@bat.com (K. McAdam), james_murphy@bat.com (J. Murphy), alison_eldridge@bat.com (A. Eldridge), clive_meredith@bat.com (C. Meredith),
christopher_proctor@bat.com (C. Proctor).

https://doi.org/10.1016/j.yrtph.2018.03.005
Received 19 May 2016; Received in revised form 5 February 2018; Accepted 8 March 2018
Available online 09 March 2018
0273-2300/ © 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).
K. McAdam et al.
Abbreviations
BAT British American Tobacco
BoBE Biomarker of biological effect
BoE Biomarker of exposure
CC Commercial control (cigarette)
CO Carbon monoxide
EOS End of Study
FDA US Food and Drug Administration
HCI Health Canada intense smoking regime
HCN Hydrogen cyanide
ISO International Standardisation Organisation
studies to result in substantial reduction in exposure to one or more
tobacco toxicants, leading to reduced risk of disease and adverse ef-
fects) (Stratton et al., 2001). Which toxicants should be reduced or the
degree of reduction were not specified in the Institute of Medicine re-
port. Rather, clinical and other studies were expected to be used to
determine whether toxicant reductions could reasonably be expected to
result in reduced health risks.
The Institute of Medicine also noted the importance of regulatory
oversight for the assessment and marketing of potential reduced-ex-
posure products. In 2009, the US Food and Drug Administration (FDA)
began regulating tobacco after the introduction of the Family Smoking
Prevention and Tobacco Control Act. This legislation included the po-
tential for the FDA to accept claims that products were potential re-
duced-exposure products, termed in the regulation “modified risk to-
bacco products” (MRTPs), through demonstration of reduced toxicant
exposure and/or reduction in health risks and no adverse public health
effect on the population as a whole. Subsequently, the FDA issued draft
guidance on the types of studies needed (FDA, 2011), based on re-
commendations by the Institute of Medicine (2011).
The FDA formed the Tobacco Products Scientific Advisory
Committee (TPSAC) and amongst other issues asked it to identify
harmful or potentially harmful constituents (HPHCs) present in tobacco
and tobacco smoke. Lists of key toxicants in smoke have changed over
time as toxicological understanding and analytical chemical techniques
have improved. For instance, a list of 300 compounds in smoke was
collated by the UK Royal College of Physicians (1962), of which around
16 were thought to be carcinogenic. Hoffmann later defined a broader
list of 44 potential toxicants, which has been used by some regulators
including Health Canada (Hoffmann and Hecht, 1990; Health Canada
(2004)). In 2008, a WHO study group prioritised 9 smoke toxicants in
an assessment of possible regulatory approaches for reducing the harms
Fig. 1. Continuum of tobacco and nicotine products related to toxicant exposure.
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Regulatory Toxicology and Pharmacology 95 (2018) 102–114
MOA Mode of action
MOE Margin of exposure
RTP Reduced-toxicant prototype (cigarette)
TobReg WHO study group on tobacco product regulation
TPSAC FDA tobacco product science advisory committee
TRT Toxicant reducing technologies
TSNA Tobacco-specific nitrosamine
NFDPM Nicotine free dry particulate matter
NNK 4-[Methyl(nitroso)amino]-1-(3-pyridinyl)-1-butanone
NNN N-nitrosonornicotine, 3-(-1-Nitrosopyrrolidin-2-yl)pyr-
idine
WHO World Health Organization
associated with cigarette smoking (Burns et al., 2008). The FDA even-
tually identified a list of around 100 toxicants that were deemed in-
dividually to have toxic or potentially toxic properties (FDA, 2011).
Since the 1960s, many countries required manufacturers to print the
mainstream smoke yields on a per cigarette basis (Nicotine Free Dry
Particulate Matter, NFDPM ‘tar’; Nicotine and Carbon Monoxide, CO)
on the cigarette packaging, as measured under the ISO machine
smoking regimen (ISO, 2001). In the EU, a ceiling was applied to ci-
garettes permitting a maximum ISO yield of NFDPM = 10mg/cig; Ni-
cotine = 1.0mg/cig and CO = 10mg/cig (EU, 2014). Health Canada
and ANVISA (Brazil's Health Surveillance Agency) were the first reg-
ulators that required measurement and reporting of tobacco and smoke
toxicants. In Canada, values for a subset of these were printed on packs
of cigarettes until research showed that smokers did not understand the
information. Only a small number of other regulators, including those
in Venezuela, Nepal and Taiwan, have subsequently required reporting
of tobacco and smoke toxicants (Liu et al., 2011). The FDA now man-
dates measurement and disclosure of values for 18 harmful and po-
tentially harmful constituents, and is considering how to provide this
information so that it will be clearly understood by smokers.
Outside the USA, many countries have ratified the World Health
Organization (WHO) Framework Convention on Tobacco Control
(FCTC). The WHO also encourages measurement and disclosure of va-
lues for tobacco and smoke constituents and emissions. A network of
independent international analytical laboratories is working to estab-
lish standardised methods for assessment of various toxicants. A WHO
scientific advisory panel, the Study Group on Tobacco Product
Regulation (TobReg), has issued various reports, including a re-
commendation that 18 toxicants in tobacco smoke be monitored (Burns
et al., 2008), and the levels of nine be mandated for lowering (shown in
Fig. 2, together with FDA and Health Canada toxicant lists). These
K. McAdam et al. Regulatory Toxicology and Pharmacology 95 (2018) 102–114

Fig. 2. Graphical representation of Harmful and potentially harmful constituents of tobacco and tobacco smoke as defined by different regulatory groups.

toxicants were chosen because of their potential toxic effects and be- the RTP cigarette to reduce both exposure to toxicants and subsequent
cause some, such as nitrosamines, vary across tobacco blend styles. individual risk relative to commercial cigarettes (Shepperd et al.,
Key to Fig. 2: 2015). In this paper we also contextualise the findings of the RTP stu-
dies against regulatory proposals for reducing toxicant emissions from
cigarettes (Burns et al., 2008), and against recent studies of toxicant
Diagram segment Toxicant group (Liu et al., 2011) emissions from Next Generation Products (NGP) (Margham et al., 2016;
Poynton et al., 2017). Finally, we discuss here whether the scientific
Inner central circle: WHO Tobreg toxicants mandated for
data collected on the RTP cigarette are consistent with reduced toxicant
lowering as a ratio to nicotine
emission cigarettes forming a distinct category in the risk continuum.
Second circle: Toxicants recommended for annual
reporting by WHO TobReg
Third circle Additional toxicants identified for 2. Results
reporting by Health Canada reporting
list (together with those in inner and 2.1. Chemistry of tobacco-smoke toxicants
second circle)
Outer circle Compounds identified by FDA in their Tobacco smoke is a complex mixture of gases and volatile, semi-
list of HPHCs (additional to above three volatile, and involatile compounds that have been extensively char-
circles) acterised. Two tobacco-specific nitrosamines (TSNAs), 4-(methylni-
Parenthesised toxicants Toxicants identified by other authorities trosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine
not included on FDA HPHC list (NNN), are common to most lists of toxicants (US Department of Health
and Human Services, 2014). There are two principle sources of tox-
At BAT, we developed several toxicant reducing technologies and icants in smoke — transfer directly from the tobacco to the smoke, such
combined them using an optimal cigarette and blend design to produce as heavy metals (e.g., cadmium), and formation during combustion by
a prototype RTP cigarette (Fig. 3), aimed at reducing exposure to as pyrosynthesis or thermal breakdown e.g., carbon dioxide (CO) (Baker
many toxicants as feasibly possible while maintaining product accept- and Proctor, 1990). Some toxicants, such as TSNAs, are present in
ability from a sensorial, ritualistic and pharmacological perspective. smoke due to the operation of both mechanisms.
The RTP was evaluated in a series of pre-clinical and clinical studies. Toxicant precursor levels in tobacco blends can vary substantially
This publication summarises these studies describing the totality of the dependent on the varieties of tobacco, environmental and agrochemical
data package, including pre-clinical chemical studies (Dittrich et al., conditions during growing, and conditions during curing and storage
2014), and in vitro assessments focussing on both classical toxicological (Baker, 1999). The combustion conditions within a cigarette can also
(Crooks et al., 2015) and disease relevant (Oke et al., 2017) end points. vary dependent upon several factors, including its construction, how it
Furthermore, clinical studies were conducted to assess the potential of is smoked, and might affect individual compound yields and relative

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K. McAdam et al.
Fig. 3. Construction of the reduced toxicant prototype cigarette (Dittrich et al., 2014).
yields in smoke. Different filter materials (e.g., cellulose acetate fibres,
triacetin, and active carbon) affect smoke yields differently (Ashley
et al., 2014 and Branton et al., 2011a,b).
Commercial cigarettes have a wide range of toxicant yields
(Camacho et al., 2015). We have developed databases (McAdam et al.,
2012; Camacho et al., 2015) of those obtained under Health Canada
Intense (HCI) smoking conditions from three sources (Australian
Government, Department of Health and Ageing, 2002; Counts et al.,
2005 and Health Canada, 2004), although comparisons must be treated
with caution owing to limited standardisation between laboratories
(Counts et al., 2005; Gregg et al., 2004 and Intorp et al., 2009). We
removed data that were not available from all three sources (arsenic,
methyl ethyl ketone, nickel, and selenium yields) or were incomplete,
duplicated, or erroneous (e.g., in the Health Canada dataset two brands
seemed to have erroneously exchanged toluene and styrene yields; tar,
nicotine and CO yields were not provided for one brand; and multiple
instances of the same yield data were observed). Finally, we removed
data on reference products. The collated dataset had information on 39
toxicants in 120 cigarette brands from 16 countries or regions. Analysis
showed that most toxicants, particularly nitrogenous toxicants, such as
TSNAs and aromatic amines, were not normally distributed. Thus, al-
though unlikely to be fully representative of all cigarette products on
sale globally, with respect to either design features or brands, this da-
tabase does constitute a reasonable comparator set for toxicant yields
from reduced toxicant prototype cigarettes.
2.2. Reducing tobacco smoke toxicant yields: RTP design
Tobacco harm reduction is based on the premise that substituting
conventional cigarettes with a lower risk product will produce lower
levels of harm amongst smokers. Therefore, it is key that any potentially
reduced risk tobacco product would have reduced toxicity in compar-
ison to a conventional cigarette, while at the same time maintaining a
level of acceptability in terms of sensory, ritual and nicotine pharma-
cology. The design strategy for the RTP cigarette was to consider every
design feature to produce a product that could meet both of these goals.
Dittrich et al. (Dittrich et al., 2014) described the systematic study
that considered the effects of cigarette circumference and filter length
and concluded that a demi-slim cigarette design (c = 21.0 mm), to-
bacco rod and filter length of 56 mm and 37 mm respectively, produced
the lowest levels of toxicant emissions. The demi-slim cigarette format
was important as it enabled a total tobacco weight that was reduced
versus a kingsize cigarette, but was still sufficient to deliver an accep-
table sensory performance. The filter length was key as it enabled a
high filter additive loading with acceptable smoking mechanics (eg.
draw effort).
Several Toxicant Reducing Technologies (TRTs) were included in
the RTP cigarette, to further reduce key toxicants. The key technologies
were as follows:
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Regulatory Toxicology and Pharmacology 95 (2018) 102–114
(a) Tobacco Substitute Sheet (TSS), made from calcium carbonate,
bound with sodium alginate, loaded with glycerol (approximately
12.5%), and coloured with caramel, is added to a standard tobacco
blend. Upon combustion glycerol is released into smoke and re-
places some of the tobacco combustion products, diluting the yields
in the total NFDPM (McAdam et al., 2011).
(b) Blend Treated Tobacco (BTT), where tobacco is subjected to a
treatment process involving aqueous extraction and filtration to
remove a proportion of polyphenols and proteins, with insoluble
tobacco proteins removed by treatment with proteases, and sub-
sequent recombination of tobacco solids and aqueous extracts. This
blend treatment reduces yields of phenolics, aromatic amines, hy-
drogen cyanide, and other nitrogenous smoke constituents, al-
though increases in yields of formaldehyde and isoprene are ob-
served (Liu et al., 2011).
(c) Synthetic high-activity carbon (Blucher GmbH, Mettmanner,
Germany) replaces the carbon typically used in charcoal filters.
Spherical beads, combined with an optimised meso-/micro-pore
structure improves adsorption characteristics for various volatile
smoke toxicants (Branton et al., 2011a).
(d) Amine-functionalised resin, in bead form (CR20, Diaion, Mitsubishi
Chemical Corporation, Tokyo, Japan), is incorporated into filters,
which selectively reduced yields of HCN, volatile acids, and car-
bonyls (Branton et al., 2011b).
(e) Triethyl citrate is an alternative filter-plasticiser to triacetin, which
is used in many commercial cigarette brands, and enhances the
ability of the filter to reduce yields of phenols in mainstream smoke
(McAdam et al., 2012) without substantially altering the physical
quality of the filter.
(f) Split tipping is a novel filter ventilation technology that can be used
either as an alternative or in addition to conventional ventilation
methods to allow diffusion of vapours and gases in and out of the
filter when the cigarette is being smoked. With this design, con-
ventional tipping paper covers the join of the filter to the tobacco
rod and the mouth end of the filter, but between is an area of
naturally porous paper that allows diffusion and removal of volatile
toxicants (Dittrich et al., 2014).
We compared the RTP with a commercial comparator cigarette
(Dittrich et al., 2014), which was a high-volume BAT cigarette sold in
Germany in 2012, the market where the clinical study would be con-
ducted.
The target ISO tar and nicotine yields were, 7.0 and 0.7 mg/cig
respectively, in both products. The mainstream smoke yields were
matched for the cigarettes to ensure a fair comparison across the stu-
dies. Toxicant yields in mainstream smoke (Table 1) were lower with
the RTP cigarette than with the comparator product for all tested tox-
icants other than nicotine, fluorene, phenanthrene, and catechol, irre-
spective of machine smoking regime. Reductions of up to 25% were
K. McAdam et al. Regulatory Toxicology and Pharmacology 95 (2018) 102–114

Table 1
Mainstream smoke toxicant levels from the RTP and CC cigarettes (n = 5 replicates) (Dittrich et al., 2014).

Constituent CC RTP % change# p value CC RTP % change# p value

ISO machine smoking regime HCI machine smoking regime

Ammonia (μg/cig) 8.5 ± 0.4 4.1 ± 0.4 51.8 0.000 27.8 ± 1.9 11.6 ± 1.3 58.3 0.000
1-aminonaphthalene (ng/cig) 12.8 ± 0.8 6.5 ± 0.2 49.2 0.000 24.4 ± 0.8 12.9 ± 1.0 47.1 0.000
2-aminonaphthalene (ng/cig) 8.1 ± 0.9 4.5 ± 0.2 44.4 0.001 15.5 ± 0.7 8.7 ± 0.7 43.9 0.000
3-aminobiphenyl (ng/cig) 1.8 ± 0.2 1.0 ± 0.1 44.4 0.000 4.2 ± 0.3 2.2 ± 0.2 47.6 0.000
4-aminobiphenyl (ng/cig) 1.4 ± 0.1 0.7 ± 0.0 50.0 0.000 3.1 ± 0.2 1.6 ± 0.2 48.4 0.000
o-Toluidine (ng/cig) 49.5 ± 1.4 33.1 ± 0.4 33.1 0.000 101.3 ± 2.3 58.8 ± 4.3 42.0 0.000
Benzo[a]pyrene (ng/cig) 7.4 ± 0.7 5.9 ± 0.8 20.2 0.014 18.5 ± 1.8 13.6 ± 0.9 26.5 0.003
Formaldehyde (μg/cig) 21.5 ± 2.3 16.9 ± 1.7 21.4 0.009 64.4 ± 7.9 48.6 ± 4.3 24.5 0.008
Acetaldehyde (μg/cig) 393.2 ± 30.1 67.1 ± 12.5 82.9 0.000 1121.7 ± 39.5 576.1 ± 36.4 48.6 0.000
Acetone (μg/cig) 198.2 ± 13.1 10.6 ± 2.2 94.7 0.000 550.6 ± 16.2 256.4 ± 28.2 53.4 0.000
Acrolein (μg/cig) 43.6 ± 5.6 NQ 100.0 0.000 137.0 ± 5.3 61.6 ± 5.5 55.0 0.000
Propionaldehyde (μg/cig) 36.4 ± 3.2 NQ 100.0 0.000 102.8 ± 2.8 48.8 ± 4.2 52.5 0.000
Crotonaldehyde (μg/cig) 9.5 ± 1.3 BDL 100.0 0.000 46.9 ± 1.9 3.9 ± 1.4 91.7 0.000
Methyl ethyl ketone (μg/cig) 48.5 ± 3.8 NQ 100.0 0.000 141.0 ± 3.8 35.0 ± 7.0 75.2 0.000
Butyraldehyde (μg/cig) 26.3 ± 2.3 BDL 100.0 0.000 69.7 ± 2.8 16.9 ± 2.0 75.8 0.000
HCN (μg/cig) 84.8 ± 4.0 12.7 ± 1.1 85.0 0.000 316.3 ± 12.3 109.0 ± 2.7 65.5 0.000
Mercury (ng/cig) NQ BDL BDL NA 3.8 ± 0.4 NQ 100.0 0.000
Cadmium (ng/cig) 14.5 ± 0.8 3.2 ± 0.2 77.9 0.000 48.7 ± 1.2 9.7 ± 0.5 80.0 0.000
Lead (ng/cig) NQ BDL NA NA NQ NQ NA NA
Chromium (ng/cig) BDL BDL NA NA BDL BDL NA NA
Nickel (ng/cig) BDL BDL NA NA BDL BDL NA NA
Arsenic (ng/cig) NQ NQ NA NA NQ NQ NA NA
Selenium (ng/cig) BDL BDL NA NA BDL BDL NA NA
NO (μg/cig) 88.5 ± 4.7 31.3 ± 2.9 64.6 0.000 256.5 ± 32.6 87.4 ± 10.7 65.9 0.000
NNN (ng/cig) 69.5 ± 6.8 9.9 ± 1.0 85.8 0.000 171.4 ± 8.2 25.4 ± 2.0 65.2 0.000
NAT (ng/cig) 47.4 ± 2.1 21.5 ± 1.6 54.6 0.000 114.5 ± 2.7 55.9 ± 4.2 51.2 0.000
NAB (ng/cig) 8.9 ± 1.1 2.4 ± 0.3 73.0 0.000 18.2 ± 1.4 6.5 ± 0.3 64.3 0.000
NNK (ng/cig) 32.6 ± 3.7 NQ 100.0 0.000 79.9 ± 2.9 28.3 ± 2.8 64.6 0.000
Pyridine (μg/cig) 8.3 ± 0.3 NQ 100.0 0.000 36.7 ± 2.5 3.2 ± 0.9 91.3 0.000
Quinoline (μg/cig) 0.19 ± 0.01 0.08 ± 0.01 57.9 0.000 0.35 ± 0.02 0.12 ± 0.01 65.7 0.000
Styrene (μg/cig) 5.7 ± 0.3 0.7 ± 0.2 87.7 0.000 23.3 ± 1.4 2.3 ± 0.6 90.1 0.000
Hydroquinone (μg/cig) 43.8 ± 1.9 27.7 ± 1.6 36.8 0.000 113.1 ± 2.9 67.7 ± 5.9 40.3 0.000
Resorcinol (μg/cig) NQ BDL BDL NA 2.0 ± 0.2 1.7 ± 0.2 15.0 0.045
Catechol (μg/cig) 48.4 ± 2.4 49.4 ± 3.5 −2.0 0.415 99.5 ± 7.6 96.4 ± 8.8 3.1 0.570
Phenol (μg/cig) 9.5 ± 0.6 4.5 ± 0.9 52.6 0.000 14.6 ± 1.7 6.2 ± 0.7 57.5 0.000
m + p cresols (μg/cig) 6.7 ± 0.4 4.3 ± 0.5 35.8 0.000 11.5 ± 1.2 5.2 ± 0.4 54.8 0.000
o-cresol (μg/cig) 2.4 ± 0.2 1.6 ± 0.2 33.3 0.000 3.8 ± 0.4 1.8 ± 0.2 52.6 0.000
1,3-butadiene (μg/cig) 33.0 ± 1.1 4.3 ± 0.6 87.0 0.000 95.3 ± 3.5 52.5 ± 2.8 44.9 0.000
Isoprene (μg/cig) 283.8 ± 16.7 13.1 ± 2.5 95.4 0.000 817.0 ± 21.8 259.5 ± 18.3 68.2 0.000
Acrylonitrile (μg/cig) 5.6 ± 0.7 BDL 100.0 0.000 23.3 ± 3.5 4.7 ± 0.7 79.8 0.000
Benzene (μg/cig) 26.6 ± 2.8 BDL 100.0 0.000 68.5 ± 3.9 9.7 ± 1.1 85.8 0.000
Toluene (μg/cig) 44.1 ± 5.2 NQ 100.0 0.000 127.6 ± 9.1 NQ 100.0 0.000
Naphthalene (ng/cig) 257.6 ± 19.1 54.1 ± 3.6 79.0 0.000 1048.5 ± 38.1 142.0 ± 9.1 86.7 0.000
Fluorene (ng/cig) 141.4 ± 8.6 161.5 ± 4.6 −14.2 0.004 269.2 ± 8.6 238.8 ± 15.9 11.3 0.009
Phenanthrene (ng/cig) 99.7 ± 8.2 97.3 ± 2.7 2.4 0.568 182.4 ± 6.7 163.2 ± 10.1 105.0 0.012
Pyrene (ng/cig) 41.5 ± 2.0 37.2 ± 1.2 10.4 0.006 88.3 ± 2.7 70.9 ± 3.6 19.7 0.000
NFDPM (mg/cig) 7.1 ± 0.4 6.4 ± 0.3 9.9 0.017 26.3 ± 1.7 17.8 ± 1.2 32.3 0.000
Nicotine (mg/cig) 0.58 ± 0.04 0.64 ± 0.03 −10.3 0.031 1.59 ± 0.04 1.48 ± 0.06 6.9 0.014
CO (mg/cig) 7.4 ± 0.4 5.4 ± 0.3 27.0 0.000 22.7 ± 1.1 15.7 ± 0.8 30.8 0.000

#% change in mean yield of analyte in RTP smoke v analyte in CC smoke.

BDL: below detection limit; NQ: not quantified; NA: Not applicable.
Values for Arsenic, Chromium, Lead, Mercury, Nickel and Selenium were below quantification limits.

seen for polyaromatic hydrocarbons (including benzo[a]pyrene), for-
maldehyde, and resorcinol, up to 50% for CO, aromatic amines, 1,3-
butadiene, more than 50% for most TSNAs, carbonyls, NO, and am-
monia, and more than 80% for NNN and volatile species, such as HCN,
pyridine, acrylonitrile, 1,3-butadiene, benzene, styrene, naphthalene,
methyl ethyl ketone, and cadmium. The reductions were more balanced
across the range of measured toxicants than had been seen with earlier
prototype designs (McAdam et al., 2012), which was an important
design goal (Dittrich et al., 2014).
In sidestream smoke, measured under ISO smoking parameters
(Table 2), yields of all toxicants apart from some metals (whose yields
were below method reporting limits) and catechol (significance not
reached), were lower with the RTP than with the commercial com-
parator cigarette. The reductions ranged from approximately 10% for
formaldehyde to over 70% for NNN, with most being around 20–60%
(Dittrich et al., 2014).
We explored whether comparisons of multiple toxicant levels could
be simplified by creating a cumulative index (normalisation). Two ap-
proaches were examined (McAdam et al., 2012). The first method was
to sum the yields of the 39 toxicants for each cigarette to give a total
toxicant yield for each product. This approach, however, was of limited
utility because the total yield value is dominated by NFDPM, CO, and
nicotine. Even with exclusion of NFDPM, nicotine and CO, yields of
toxicants, such as acetaldehyde, isoprene and HCN are still orders of
magnitude greater than those of NNN, NNK, and benzo[a]pyrene and,
therefore, most constituents were not well represented. The second
method gave greater insight into the contribution of all toxicants. A
median value was calculated for each toxicant in the commercial

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K. McAdam et al. Regulatory Toxicology and Pharmacology 95 (2018) 102–114

Table 2
Sidestream smoke toxicant levels from the RTP and CC cigarettes measured using the ISO machine smoking regime (n = 5 replicates) (Dittrich et al., 2014).

Smoke constituent CC RTP

ISO smoking regime (mean ± SD) ISO smoking regime (mean ± SD)

Ammonia (μg/cig) 5901 2459

1-aminonaphthalene (ng/cig) 194.3 ± 7.9 92.0 ± 6.0
2-aminonaphthalene (ng/cig) 158.2 ± 4.2 76.7 ± 4.4
3-aminobiphenyl (ng/cig) 35.1 ± 1.3 14.9 ± 1.1
4-aminobiphenyl (ng/cig) 24.0 ± 1.5 10.1 ± 0.7
Benzo[a]pyrene (ng/cig) 118.9 ± 11.7 93.6 ± 5.4
Formaldehyde (μg/cig) 590 ± 92 524 ± 31
Acetaldehyde (μg/cig) 1535 ± 114 1120 ± 130
Acetone (μg/cig) 832 ± 60 586 ± 53
Acrolein (μg/cig) 339.3 ± 27.1 255.8 ± 32.6
Propionaldehyde (μg/cig) 158.5 ± 10.2 109.4 ± 8.8
Crotonaldehyde (μg/cig) 75.5 ± 5.7 57.0 ± 3.9
Methyl ethyl ketone (μg/cig) 200.3 ± 13.3 163.2 ± 13.7
Butyraldehyde (μg/cig) 97.0 ± 4.8 53.7 ± 4.2
HCN (μg/cig) 97.3 ± 10.1 53.7 ± 7.8
Mercury (ng/cig) NQ NQ
Cadmium (ng/cig) 207.6 ± 7.5 162.2 ± 10.0
Lead (ng/cig) BDL BDL
Chromium (ng/cig) NQ NQ
Nickel (ng/cig) NQ NQ
Arsenic (ng/cig) BDL BDL
Selenium (ng/cig) BDL BDL
NO (μg/cig) 2064 ± 62 883 ± 101
NNN (ng/cig) 89.9 ± 11.1 23.5 ± 1.3
NAT (ng/cig) 30.3 ± 3.7 15.1 ± 0.8
NAB (ng/cig) 9.8 ± 0.6 3.2 ± 0.3
NNK (ng/cig) 149.3 ± 8.9 73.3 ± 3.9
Pyridine (μg/cig) 269.9 ± 10.7 131.2 ± 7.7
Quinoline (μg/cig) 11.7 ± 0.4 5.8 ± 0.4
Styrene (μg/cig) 94.7 ± 6.4 48.7 ± 5.2
Hydroquinone (μg/cig) 93.5 ± 11.6 61.7 ± 2.6
Resorcinol (μg/cig) BDL BDL
Catechol (μg/cig) 71.1 ± 7.4 66.5 ± 4.4
Phenol (μg/cig) 225.6 ± 10.5 130.8 ± 6.2
m + p cresol (μg/cig) 73.6 ± 3.2 45.5 ± 3.0
o-cresol (μg/cig) 35.5 ± 1.5 23.5 ± 1.8
1,3-butadiene (μg/cig) 412 ± 40 261 ± 21
Isoprene (μg/cig) 3041 ± 248 1788 ± 116
Acrylonitrile (μg/cig) 119.7 ± 15.9 50.3 ± 8.0
Benzene (μg/cig) 281 ± 26 172 ± 19
Toluene (μg/cig) 558 ± 33 312 ± 28
NFDPM (mg/cig) 21.0 ± 1.4 16.5 ± 0.6
Nicotine (mg/cig) 4.01 ± 0.14 3.13 ± 0.14
CO (mg/cig) 40.0 ± 3.4 25.5 ± 2.9
CO2 (mg/cig) 305 ± 26 196 ± 11

dataset and was normalised to 100, against which the yields of toxicants
were scaled. The toxicants were each given an equal weighting for the
calculations using the Hoffmann lists as the basis for the toxicological
evaluation of each toxicant (Hoffmann and Hoffmann, 1997). The total
of the scaled values gave an overall normalised toxicant value per
product. Compared with all the brands in our commercial dataset, the
RTP had some of the lowest machine toxicant yields ever published for
a combustible cigarette for HCI smoke chemistry (McAdam et al.,
2012).
To establish the biological relevance of the reductions, we assessed
the RTP using in vitro tests focussed on classical toxicological and dis-
ease relevant endpoints and also performed clinical studies. For the
clinical study, the appearance of both the RTP and the comparator was
changed (from cork to white tipping) paper at the time of switching to
enable some level of blind-testing, though it should be noted the phy-
sical dimensions of the RTP and the length of filter were quite different
when compared to the commercial control product. The clinical study
would assess whether exposure and individual risk could be reduced
when smokers switched from a conventional cigarette to the RTP ci-
garette.
2.3. In vitro studies measuring toxicological endpoints
We conducted a battery of in vitro tests on particulate matter ob-
tained under the ISO and Health Canada Intense (HCI) machine
smoking regimes to assess the toxicity of the RTP in vitro relative to a
commercial cigarette (Crooks et al., 2015). The endpoints that were
assessed were bacterial mutagenicity (Ames test), mammalian cell
mutagenicity (Mouse Lymphoma Assay), genotoxicity (In Vitro Micro-
nucleus assay) and cytotoxicity (Neutral Red Uptake assay). In the
Ames test, the RTP particulate matter was less mutagenic in tester
strains TA98 and TA100, irrespective of smoking regime in comparison
to the commercial cigarette. The mouse lymphoma assay showed some
reductions in gene mutations and chromosome aberrations with the
RTP compared with control, but the findings were inconsistent.
Changes to chromosome structure and number were also reduced on
the in vitro micronucleus test irrespective of the smoking regime. Fi-
nally, the neutral red uptake assay identified some differences in cy-
totoxicity, but none reached significance. These data suggest that re-
ductions in toxicant yields are achievable without new additional
genotoxic hazards (Crooks et al., 2015).

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K. McAdam et al. Regulatory Toxicology and Pharmacology 95 (2018) 102–114

Fig. 4. Cell viability of H292 lung epithelial cells exposed to whole smoke from RTP and
CC cigarettes for 30 min (n = 9 replicates) (Oke et al., 2017).
Fig. 5. Intracellular Reactive Oxygen Species (ROS) production in NCI-H292 lung epi-
thelial cells exposed for 1 h to CSEaq from RTP and CC cigarettes, measured by the di-
The BTT used in the RTPs removes at least half of the tobacco chlorofluorescein (DCF) assay (n = 6 replicates) (Oke et al., 2017).
protein nitrogen, and more than 40% of the total polyphenols (Liu
et al., 2011). Reductions in protein nitrogen result in lower levels of
aromatic and heterocyclic amine protein products being generated on
smoking. These compounds are considered to be the main contribution
to cigarette smoke mutagenicity in the Ames assay (Mizusaki et al.,
1977).
Analysis of whole smoke, which included both particulate and va-
pour phases, rather than just particulate matter confirmed the reduced
cytotoxic profile with the RTPs (Fig. 4) (Crooks et al., 2015). Previous
studies (Horinouchi et al., 2016 and Noya et al., 2013) have shown that
volatile organic compounds (eg. aldehydes and ketones), are drivers of
cigarette smoke responses in cytotoxicity assays. As the RTP contains
filter additives that reduced levels of these compounds (≥70%) relative
to the commercial cigarette, the reduction in cytotoxicity is due mainly
to the carbon and ion-exchange technologies in the prototype filters.

2.4. In vitro studies measuring toxicological and disease relevant endpoints

Toxicology testing was also conducted using in vitro models of dis-

ease processes. Oxidative stress and inflammation are widely reported
to be driving factors in the development of atherosclerosis, chronic
obstructive pulmonary disease, and lung cancer. Cigarette smoking
increases oxidative stress and inflammation in the lungs and the vas-
cular system (Faux et al., 2009). Oke et al., therefore assessed in-
tracellular generation of reactive oxygen species, depletion of reduced
glutathione, and secretion of pro-inflammatory cytokines in lung epi-
thelial cells (Oke et al., 2017), in addition to an endothelial cell mi-
gration assay. Cells were exposed to particulate matter and whole
smoke to represent the particulate phase and combined particulate and
vapour phases. Cigarette smoke aqueous extract was used as a surrogate
for whole smoke with the reactive oxygen species and reduced glu-
tathione assays, which are not suitable for whole smoke exposure. All
extracts were generated using the ISO smoking regime.
All particulate matter experiments and whole-smoke pro-in-
flammatory cytokine analysis showed no significant differences be-
tween the RTP and conventional cigarettes. In contrast, cigarette smoke
aqueous extract from the RTPs induced significantly lower intracellular
generation of reactive oxygen species and reduced glutathione deple-
tion than the reference product (Figs. 5 and 6). Toxicological testing
suggested no increases in toxicological response, and importantly some
reductions, particularly for tests responsive to some of the irritants in
the vapour phase.
2.5. Computational toxicology
Computational toxicological tools were used to assess the potential
Fig. 6. Percentage levels of reduced glutathione relative to the untreated control cells in
NCI-H292 lung epithelial cells exposed for 1 h to CSEaq of RTP and CC cigarettes, mea-
sured by the GSH-Glo assay (n = 6 replicates) (Oke et al., 2017).
impact of reducing the toxicant yields to the extent achieved with the
RTP. Fowles and Dybing (Fowles and Dybing, 2003) prioritised the
hazards for 158 chemical constituents in tobacco smoke based on
published cancer potency factors and knowledge of typical yields in
smoke. They proposed that 1,3-butadiene, was the most influential
volatile compound in relation to cancer, and that four of the top five
cancer-related toxicants were aldehydes or small organic compounds,
contributing overall around 62.4% of the total cancer risk. They sug-
gested also that metals (e.g., arsenic and cadmium) contributed 18.2%
further risk and polyaromatic hydrocarbons only 0.8%. They associated
acrolein and acetaldehyde with respiratory disease and HCN and ar-
senic with cardiovascular disease. Fowles and Dybing noted limitations
in their estimates. For instance, the sum of the cancer risk indices they
had calculated was five times lower than that attributed to smoking in
the USA.
We have investigated various other possible paradigms to calculate
risks. For example, we have combined in silico and weight-of-evidence
approaches (margin of exposure (MOE), mode of action (MOA)), sup-
plemented with in vitro data. By this method, point of departure values
can be included in MOE calculations to support (or not) postulated
MOAs for specific toxicants (Cunningham et al., 2012). Comparison of

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K. McAdam et al. Regulatory Toxicology and Pharmacology 95 (2018) 102–114

Table 3
Margin of Exposure assessment of the emissions from the RTP cigarette versus the CC cigarette for the WHO list of toxicants mandated for lowering.

Analyte Unit CC cigarette yield Mean ( ± SD) CC MOE priority RTP cigarette yield Mean ( ± SD) RTP MOE priority

Acetaldehyde μg/cig 1121.7 ± 39.5 VERY HIGH 576.1 ± 36.4 HIGH

Acrolein μg/cig 137.0 ± 5.3 TOP 61.6 ± 5.5 TOP
B[a]P ng/cig 18.5 ± 1.8 LOW 13.6 ± 0.9 LOW
Benzene μg/cig 68.5 ± 3.9 HIGH 9.7 ± 1.1 MEDIUM
1,3-butadiene μg/cig 95.3 ± 3.5 HIGH 52.5 ± 2.8 HIGH
Carbon Monoxide mg/cig 22.7 ± 1.1 N/A 15.7 ± 0.8 N/A
Formaldehyde μg/cig 64.4 ± 7.9 TOP 48.6 ± 4.3 TOP
NNK ng/cig 79.9 ± 2.9 HIGH 28.3 ± 2.8 MEDIUM
NNN ng/cig 171.4 ± 8.2 MEDIUM 25.4 ± 2.0 LOW

these data with predicted target organ concentrations generated from
physiologically based pharmacokinetic modelling is a first step in
quantitative extrapolation from in vitro to in vivo.
An MOE is the ratio of a benchmark dose to the specific human
exposure. The European Food Safety Authority (EFSA, 2012) deems
MOEs greater than 10,000 to be low priority for risk management. We
have calculated MOE values from a wide range of different studies with
various disease endpoints to produce a series of values representative of
those in the literature. This enables assessment of the magnitude of the
potential risk associated with specific compounds. We defined the fol-
lowing priority bandings: ‘top’ (MOE 1–10), ‘very high priority’
(10–100), ‘high priority’ (100–1000), ‘medium priority’ (1000–10,000),
‘low priority’ (10,000–100,000), and ‘very low priority’ (> 1,000,000).
Use of these bandings shows (Table 3) that in all cases, the RTP had an
equal or lower priority MOE in comparison to the CC. Furthermore, the
MOEs suggest that toxicants in the RTPs, even those that are reduced
substantially, would potentially need to be reduced substantially fur-
ther to have beneficial health effects, perhaps beyond what might be
achievable in a cigarette.
With the exception of 1-OH pyrene, the BoE for the polycyclic
aromatic hydrocarbons, increased in the control group and RTP groups
(Table 4). The technologies used in the RTP, however, were not ex-
pected to substantially reduce these yields. Several BoEs increased in
control smokers after switching, presumably due to increases in con-
sumption. Urinary mutagenicity was similar for all smokers at baseline,
and differed substantially from that for non-smokers. At the end of the
study (EOS), mutagenicity in the RTP group was less than that in the
control smoker group, but remained substantially greater than that in
never smokers.
The BoE clearly showed the effect of increased consumption, and for
the control group, BoE levels typically increased between baseline and
the end of the study. However the reductions in the toxicant yield in the
RTP were such that reductions in BoE for some toxicants were still seen
in those who switched to this product. Good separation of control and
Table 4
Percentage changes in biomarker values for RTP cigarette and CC cigarette at the end of a
6-month switching study (Shepperd et al., 2015).

RTP Smokers Control Product Smokers

2.6. Clinical studies Biomarker ∼% Change at Biomarker ∼% Change at

the EOS the EOS
The RTP and comparator commercial cigarette were evaluated
further in a 6 month clinical study measuring both BoEs and BoBEs HMPMAa b
−75% 1-OHPa b −25%
NNNa b c −66% NNNc 0%
(Shepperd et al., 2013a,b). The study was reviewed by the Ärzte-
CEMAa b −59% o-tol 0%
kammer Hamburg, Hamburg, Germany (Processing Number PV3824) NABa b −44% Phenanthrene 2%
ethics board in Germany and registered with the US clinical database (‘total’)
the International Standard Randomised Controlled Trial Number NNALa b −40% MHBMA 4%
3-HPMAa b
−34% WBCs 4%
(ISRCTN) registry (ISRCTN81286286). Healthy volunteer regular
3-ABPa b −32% 3-ABP 9%
smokers smoked the control products for 2 weeks then either continued MHBMAa b
−31% NNALb 10%
or switched to an RTP for 6 months (Shepperd et al., 2015). An am- 1-OHPa b −30% Nicotine 11%
bulatory design was adopted except for several short-term residential Equivalentsb
clinic visits for biomarker sampling. Data from prospectively assessed NATa b −28% 2-ANb 12%
ECOa b −19% 8-iso-PGF2 type VIa b 13%
ex-smokers and never smokers provided background levels for bio-
4-ABPa b −17% HMPMAb 14%
markers. The study investigated whether BoBE could show differences 2-ANa b −10% 8-iso-PGF2 type III 18%
in smoking statuses and whether concentrations could return to never- 8-iso-PGF2 type VI −6% 3-HPMAb 19%
smoker levels after switching to the RTP (Haswell et al., 2014). o-tol −4% 4-ABPb 19%
We measured 23 urinary BoE (Shepperd et al., 2015), 5 urinary WBCs 0% Saliva Cotininea b 22%
8-iso-PGF2 type III 3% NATa b 25%
BoBE, and 22 blood BoBE (Haswell et al., 2014), as well as urine mu- Phenanthrene (‘total’) 19% NABa b 26%
tagenicity (Shepperd et al., 2015). An unexpected but significant and Nicotine Equivalentsa 26% CEMAa b 26%
b
substantial increase in cigarette consumption was seen during the
study, both in the arm that continued smoking the commercial control Saliva Cotininea b 28% Fluorene (‘total’) 36%
Naphthalene (‘total’) 55% ECOa b 37%
cigarette (an increase of 18.6% cigarettes per day) and the arm that
sICAM-1a b c 60% sICAM-1a b c 41%
switched to the RTP cigarette (an increase of 12.5% cigarettes per day) Fluorene (‘total’) 81% Napthalene (‘total’) 43%
(Shepperd et al., 2015). As this change was anticipated to affect the
outcomes of the study, it prompted an alert to the ethics committee, ‘total’ = sum of metabolites investigated.
a
formation of an independent data safety monitoring board, increased Denotes % changes with Baseline vs. end of study values statistically significant as
determined by evaluation of the simple effects in the statistical models that include only
monitoring of consumption in electronic diaries and monitoring of ci-
the smoking groups with cigarettes per day as a covariate.
garette consumption after the study. With these measures in place, both b
Denotes % changes with Baseline vs. end of study values statistically significant as
the ethics committee and data safety monitoring board approved con- determined by evaluation of the simple effects in the statistical models that include only
tinuation of the study. Additionally, we created a bespoke questionnaire the smoking groups without cigarettes per day as a covariate.
c
to assess the reasons behind increased consumption. Denotes statistical significance derived from analysis excluding extreme values.

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K. McAdam et al. Regulatory Toxicology and Pharmacology 95 (2018) 102–114

RTP was seen particularly for vapour phase toxicants such as acrolein. Table 5
The prime reason for increased consumption in control smokers was the Comparison of emissions from a conventional cigarette, RTP and two NGPs.
supply of free cigarettes, although there was also a perception that the
Analyte Units CC RTP Hybrid Electronic
white-tipped version of the control cigarette didn't last as long as the cigarettea cigaretteb Tobacco Cigaretted
cork-tipped version. For the RTP group, supply of free cigarettes was Mean Mean Productc Mean
also the main reason, but reduced satisfaction and a belief that the
Acetaldehyde μg/puff 112.62 54.35 0.084 0.106
study cigarettes might be less harmful than the smokers' usual brand
Acrolein μg/puff 13.76 5.81 0.057 0.070
were also given as possible reasons when the volunteers were surveyed. B[a]P ng/puff 1.86 1.28 BDL BDL
The BoBE data provided evidence for processes thought to be key (< 0.011) (< 0.005)
contributors to the development and progression of smoking-related Benzene μg/puff 6.88 0.92 BDL BDL
diseases, including oxidative stress and inflammation. Elevated levels of (< 0.002) (< 0.002)
1,3-butadiene μg/puff 9.57 4.95 BDL BDL
F2-isoprostanes are associated with oxidative stress (Milne et al., 2005;
(< 0.003) (< 0.003)
Rahman, 2005), and levels significantly reduce following smoking Carbon Monoxide mg/puff 2.28 1.48 0.055 0.057
cessation (Chehne et al., 2002; Flores et al., 2004; Morrow et al., 1995 Formaldehyde μg/puff 6.47 4.53 0.119 0.122
and Oguogho et al., 2000). In our study, isoprostane III levels differed NNK ng/puff 8.02 2.67 NQ NQ
(< 0.03) (< 0.02)
significantly between smokers and non-smokers, but not between
NNN ng/puff 17.21 2.40 0.017 0.054
smoker controls and RTP smokers. Raised white blood cell counts, an
indicator of inflammation, have been associated with smoking-related BDL = Below Detection Limit.
disease (Fröhlich et al., 2003 and Lao et al., 2009), but decrease after NQ = Not Quantified.
a
smoking cessation (Blann et al., 1997; Hammett et al., 2007 and Lao Comparator cigarette (Dittrich et al., 2014).
b
et al., 2009). White blood cell counts were significantly and sub- RTP cigarette (Dittrich et al., 2014).
c
Hybrid Tobacco Product (Poynton et al., 2017).
stantially higher in smokers than in non-smokers throughout the study d
Electronic Cigarette (Margham et al., 2016).
(Haswell et al., 2014). Values were higher in RTP than in control
smokers from baseline to the midpoint of the study and counts in-
2.7. Comparison of RTP toxicant emissions to those from next generation
creased in the smoker control group by the end of the study, but no
products
changes were significant. Similar patterns were seen for the neutrophil
and monocyte sub-fractions.
To contextualise emissions from next generation products against
11-dehydrothromboxane B2 (dTx), a metabolite of thromboxane
our study findings, published aerosol toxicant data was compiled
A2, is associated with platelet activation and smoking status (Frost-
against the list of toxicants proposed for mandated reduction by a WHO
Pineda et al., 2011; Lowe et al., 2009; Nowak et al., 1987 and
study group on Tobacco Product Regulation. (Burns et al., 2008). The
Wennmalm et al., 1991), which have been shown to decrease in smo-
next generation products selected for comparison were a novel tobacco
kers who quit smoking (Rångemark et al., 1993; Roethig et al., 2008
product (Poynton et al., 2017) and an e-cigarette (Margham et al.,
and Saareks et al., 2001). Throughout the study period, levels of dTx in
2016), both developed by BAT. To minimise uncertainties that could
smoking controls were significantly raised compared with those in non-
arise from potential differences in daily consumption of different ni-
smokers. In the RTP group, however, concentrations decreased sub-
cotine products the data is presented on a per-puff basis in Table 5.
stantially from baseline and were significantly lower than those in
These data show that the NGPs lead to substantial reductions in tox-
control smoking group at the end of the study (P = 0.002).
icant emissions in comparison to both the CC and RTP cigarettes, and at
An additional benefit of conducting a six month study was
a magnitude unlikely to be impacted by any differences in daily con-
that urinary levels of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol
sumption. These findings are consistent with published data showing
(NNAL), a metabolite of NNK, could be analysed over a significant time
per-puff toxicant emissions (as defined by WHO TobReg (Burns et al.,
period. The levels of NNAL were also significantly lower in the RTP
2008), FDA reporting list (FDA 2012) and Health Canada, 2004) from
group for the duration of the study.
these products to be 95–99% lower than from a reference cigarette
Recruitment of inflammatory cells from the vascular system is an
(Margham et al., 2016; Poynton et al., 2017). These findings suggest
essential part of the damage-repair process in endothelial dysfunction
that the NGPs should be considered as candidates for reduced risk
and tissue injury. Elevations in sICAM-1 and MCP-1 have implications
product assessment.
for the development of atherosclerosis (Deo et al., 2004; Gross et al.,
2012; Kusano et al., 2004 and Piemonti et al., 2009). Levels of MCP-1
differed significantly between smokers and non-smokers (Haswell et al., 2.8. Regulatory proposals on toxicants
2014). A significant reduction was observed in the control group,
whereas in the RTP group values did not differ significantly between TobReg proposed mandated ceilings for emissions of nine toxicants,
baseline and EOS. The implications of these findings are unclear, and Tables 5 and 6 (Burns et al., 2008; World Health Organisation, 2015).
whether changes have no effect or are detrimental needs to be assessed. Their proposals are precautionary, based on the substances being
By contrast, sICAM-1 levels did not differ between smokers and non- known to be harmful rather than there being a proven specific link
smokers at baseline, but became significantly higher in both smoking between reduced levels and lowered risk of human disease (World
groups (P < 0.001) and significantly higher in the RTP group than in Health Organization, 2008). The TobReg approach is complex, with
the smoker control group by the end of the study. three different models advanced. Two models focus upon the estab-
Modulation of serum lipids in smokers is related to the development lishment of ceiling values calculated as the ratios of toxicant emissions
and progression of cardiovascular disease, such as through decreased to nicotine when measured under the HCI smoking regime. With the
levels of high density lipoprotein cholesterol (Chelland-Campbell et al., first model, all tobacco products in a market must be assessed and
2008) and raised levels of other lipids and C-reactive protein. Beneficial emission ratios ranked from low to high, with the middle product be-
changes are seen in people who have quit smoking (Chelland-Campbell coming the reference for all others. For the two TSNAs, the ceiling value
et al., 2008; Hata and Nakajima, 2000 and Ohsawa et al., 2005) or would be the value of the median product, and for the other seven
switched to electronically heated tobacco products (Roethig et al., toxicants it would be 125% of the value for the median product. To be
2008). In our study, high density lipoprotein cholesterol levels were approved for sale, products (new and existing) would need to comply
significantly lower in smokers than in never-smokers and did not differ with all nine ceilings. These proposals, therefore, are likely to result in
between the RTP and smoker control groups. high non-compliance rates. For example, with NNN and NNK, 50% of

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K. McAdam et al. Regulatory Toxicology and Pharmacology 95 (2018) 102–114

products would automatically fail each TSNA limit.

Electronic Cigaretted In the second model TobReg defined two sets of fixed toxicant/ni-
cotine ratio ceilings—one for flue-cured product markets and one for

BDL (< 0.2)

BDL (< 0.1)
BDL (< 0.1)

NQ (< 0.8)
US-blended product markets—for those countries with limited labora-
tory capacity (Burns et al., 2008). These would be applied by regulators
Mean

3 without the need to identify the relevant values in their own market. A
2

2
4

2
more recent WHO TobReg report on smoke toxicants (World Health
Organisation, 2015) developed a third model wherein toxicant ceilings
would be set at a value three times the lowest toxicant emission of the
Hybrid Tobacco

products on the market.

BDL (< 0.4)
BDL (< 0.1)
BDL (< 0.1)

NQ (< 0.8)
We collected data for four countries (sampling 80–97% of the pro-
Productc

ducts within those markets) with distinct product attributes: pre-

Mean

dominately flue-cured cigarettes, predominately American-blended ci-

0.7
3
2

2
5

garettes, multiple charcoal filtered and reduced circumference

cigarettes, and mixed blend styles. Laboratory testing took a consider-
RTP cigaretteb

able time and was costly. Within-product variation over time was suf-
ficiently large to be a major determinant of whether a product passed or
Mean

failed to meet TobReg ceiling proposals (Eldridge et al., 2015, 2017).

389
42

35
11
32
19
17

Other key drivers for products failing to meet the proposed limits were
9
7

the TNSAs, NNN and NNK. Individually, failures to meet ceilings were
CC cigarettea

less extensive for the other seven toxicants, but the cumulative effect of
needing to be below all nine ceilings led to most products (typically
Mean

72–79%, around 600 products out of 781 tested) failing (Eldridge et al.,
705

108
86
12
43
60
14
41
50

2017). The impact of fixed ceiling values (model 2) was to produce

generally higher levels of non-compliance of between 70 and 100% of
TobReg fixed limit for

the products from these exemplar markets; similar conclusions were

Comparison of RTP and NGP emissions to WHO TobReg proposed ceiling values (model 1 and model 2) (Eldridge et al., 2017; Burns et al., 2008).

found for third model (Eldridge et al., 2017).

Canadian brands

The RTP we tested passed all individual toxicant ceilings under all
three models (Tables 6 and 7), with the RTP having emissions in the
range of 20–80% of the proposed ceilings. These findings raise concerns
15.4
670

as to the robustness of the WHO TobReg proposals; the failure of an

97
11
50
53

97
47
27

entirely compliant product to effect noticeable clinical benefits to its

consumers provides little evidence that these proposals would posi-
tively impact incidence of smoking related diseases. However, it is also
possible that longer time-scales may be needed for benefits to emerge.
TobReg fixed limits for
International brands

The NGPs also passed the TobReg limits, but by a considerably

greater margin than the RTP, reflecting the lower toxicant emissions
from the NGPs. These emissions were 1-3 orders of magnitude lower
than the TobReg ceilings. These NGPs may therefore reasonably be
Model 2

regarded as candidate reduced risk products, and therefore further in-

18.4
860

114
83
11
48
67

47
72

vestigation, including pre-clinical and clinical studies, is warranted.

3. Discussion
countries (Eldridge et al., 2017)
Range of ceiling values for 4

Evaluation of a reduced toxicant prototype cigarette in pre-clinical

and clinical studies demonstrated substantial reductions in broad
classes of laboratory measured toxicant emissions, lowered activities of
in vitro assays, as well as reduced in vivo exposure to toxicants.
However, the risk profile associated with its use (as monitored through
822–1001

BoBE) did not differ from that of a conventional cigarette. Overall, of

Model 1

16.2–19
82–106

For superscript letters a, b, c, d refer to Table 5 footnotes.

53–69
54–69

61–76
21–78
27–90

the suite of BoBEs examined, the RTP demonstrated potentially bene-

9–14

ficial effects only for levels of 11-dehydrothromboxane B2 but had

potentially adverse effects on sICAM-1 levels. Thus, while our data do
not challenge the basic premise that smoking-related diseases are
mg/mg nicotine
ng/mg nicotine

ng/mg nicotine
ng/mg nicotine
μg/mg nicotine
μg/mg nicotine

μg/mg nicotine
μg/mg nicotine

μg/mg nicotine

caused by prolonged and repeated exposure to toxicants or that risks

are dose related, they show little evidence that the modifications in-
troduced in the RTP modify risk.
Units

The reasons for these observations are unclear. Literature reports

indicate that changes in BoBE levels following smoking cessation occur
within the time frame of the RTP study (Shepperd et al., 2015). How-
ever, six months might not have been long enough to detect meaningful
Carbon Monoxide

BoBE changes in subjects who continue to smoke, albeit with a reduced

Formaldehyde
1,3-butadiene
Acetaldehyde

toxicant yield product. A substantially longer study may be required to

Acrolein

Benzene
Analyte

allow wider changes in BoBE to be observed. Also, it is possible that few

Table 6

B[a]P

NNN
NNK

or none of the toxicants reduced in the RTP have causative roles in

smoking-related diseases. Computational toxicological assessment to

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K. McAdam et al. Regulatory Toxicology and Pharmacology 95 (2018) 102–114

Table 7
Comparison of RTP and NGP emissions to WHO TobReg ceiling values calculated from 4 Countries using model 3 (World Health Organisation, 2015).

Analyte Units Range of WHO TobReg ceiling values from CC cigarettea RTP cigaretteb Hybrid Tobacco Electronic
four countries (Eldridge et al., 2017) Productc Cigaretted

Model 3 Mean Mean Mean/10 puffs Mean/10puffs

Acetaldehyde μg/cigarette 1780–2797 1122 576.1 0.84 1.06

Acrolein μg/cigarette 178–243 137 61.6 0.57 0.7
B[a]P ng/cigarette 17–28.2 18.5 13.6 BDL (< 0.11) BDL (< 0.05)
Benzene μg/cigarette 110–169 68.5 9.7 BDL (< 0.02) BDL (< 0.02)
1,3-butadiene μg/cigarette 103–202 95.3 52.5 BDL (< 0.03) BDL (< 0.03)
Carbon Monoxide mg/cigarette 33.3–55.5 22.7 15.7 0.55 0.57
Formaldehyde μg/cigarette 95.2–181 64.4 48.6 1.19 1.22
NNK ng/cigarette 37.9–110.1 79.9 28.3 NQ (< 0.3) NQ (< 0.25)
NNN ng/cigarette 32.3–118.1 171.4 25.4 0.17 0.54

For superscript letters a, b, c, d refer to Table 5 footnotes.

prioritise toxicants as drivers of disease also suggests that levels of some
respiratory irritant precursors (e.g., acrolein and the aldehydes) might
need to be reduced more than achieved with the RTP to have any effects
on BoBE.
Ambulatory clinical trials of the kind performed with the RTP are
largely blind to subject-product compliance, and the degree to which
the subjects use the test product exclusively outside of the clinical
setting. Significantly increasing cigarette consumption, or frequently
smoking other cigarette brands, between clinical visits could diminish
the potential of the RTP to reduce overall toxicant exposure and any
consequential potential health benefits. Most BoE have shorter half-life
times than BoBEs, and significant non-compliance of this kind could
minimise changes in BoBE while registering significant BoE reductions.
Compliance checking is challenging when one cigarette product is
switched to another in a trial, as the presence or absence of any com-
bustion markers cannot be used to monitor compliance, unlike with
cessation studies or when a cigarette is switched to a non-combustible
product. In the RTP trial a number of steps were taken to monitor
compliance. These included:
i) use of electronic diaries to monitor cigarette consumption,
ii) comparison of numbers of collected study cigarettes with diary
entries,
iii) interrogation of changes in long-lived Biomarkers of Effective Dose
(BoED - such as DNA and protein adducts of 4-aminobiphenyl and
acrylonitrile) in comparison to their urinary BoE,
iv) comparison of changes in certain slow changing Biomarkers of
Exposure (such as NNAL the metabolite of NNK), and their ratios to
nicotine (to account for consumption changes) in comparison to
laboratory smoke yield reductions.
Significant increases in cigarette consumption were observed in the
ambulatory periods of the RTP study in comparison to those measured
in the clinical stages. Reductions in BoED were slightly smaller than
with their corresponding BoE; similar findings were observed with
changes in NNAL/TNeq in comparison to smoke yield reductions, but in
both cases alignment between the two comparators was good. From
these observations, we can conclude that some degree of non-com-
pliance may have existed during ambulatory study arms, but without a
substantial impact on study findings.
Given the overall lack of response in BoBE with RTP users, these
results point to a minimum toxicant exposure level above which im-
provements in risk profile are unlikely to be found. This is a vital
finding as future candidate reduced risk products would need to de-
monstrate the likelihood of toxicant exposures lower than those mea-
sured for the RTP. Comparison of toxicant emissions from Next
Generation Products to those from the RTP and control cigarette sug-
gest that NGPs meet this criterion, and therefore have the potential to
reduce the risks associated with smoking. However, although toxicant
reductions from NGPs were 95–99% lower than from a reference ci-
garette, the reductions were not complete, and therefore some level of
risk associated with their use may exist.
4. Conclusions
This paper illustrates that it is possible to develop prototype cigar-
ette products that can reduce toxicant emissions and exposure com-
pared with conventional cigarettes. However, the reductions in emis-
sions (despite meeting toxicant emission ceilings identified by a WHO
study group) and subsequent exposure did not manifest beneficial dis-
ease relevant health outcomes in a long term clinical study, with little
change in BoBE after 6 months' use.
Thus, while we cannot say with certainty that there would be no
public health benefit associated with the observed toxicant reductions,
as proposed by WHO TobReg, it is likely that more substantial reduc-
tions in exposure will be needed to produce measurable reductions in
health risks. We conclude, therefore, that there is insufficient evidence
to support cigarettes with reduced toxicant emissions as a distinct ca-
tegory in a risk continuum.
These results also point to a minimum toxicant exposure baseline,
that candidate RRPs would need to overcome before consideration for
testing via full pre-clinical and clinical assessment. A comparison of the
emissions from Next Generation Products show that they have much
lower toxicant levels than both conventional and RTP cigarettes. Next
generation products therefore represent a highly promising approach
for reducing the harm associated with cigarette smoking.
Acknowledgements
The authors appreciate the contributions of David Rushforth, David
Dittrich, Richard Fiebelkorn, Ivan Flores, Christopher Shepperd, Nik
Newland, Fiona Cunningham, Stacy Fiebelkorn, Ian Crooks,
Oluwatobiloba Oke, David Azzopardi, Mike McEwan, Frazer Lowe,
Alison Bushby and Rebecca Such.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.
doi.org/10.1016/j.yrtph.2018.03.005.
Transparency document
Transparency document related to this article can be found online at
http://dx.doi.org/10.1016/j.yrtph.2018.03.005.
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