Tobacco Smoking Behaviour Sub-Group · 2016. 4. 26. · ISO 3534-2:Statistics — Vocabulary and...

Tobacco Smoking Behaviour Sub-Group

Technical Report

Results of the 2014 CORESTA

Part-Filter Method Ring Trial

and Comparison with the 2012 Ring Trial

June 2015

Authors:

Kathryn Nother and Peter Clayton

British American Tobacco, R&D Southampton, U.K.

Statistical Analysis:

Thomas Verron and Rémi Julien

Imperial Tobacco Group – SEITA, France

Study Coordinator:

Peter Clayton


Sub-Group Coordinator:

Krishna Prasad


Table of Contents

1. Summary ............................................................................................................................. 3

2. Recommendation ................................................................................................................. 3

3. Introduction ......................................................................................................................... 4

4. 2012 Part Filter Method Ring Trial ..................................................................................... 6

4.1 Results ................................................................................................................................ 7

4.2 Learnings and Conclusions ............................................................................................... 8

4.3 Possible Sources of Variation ........................................................................................... 8

4.4 Recommendations ............................................................................................................. 9

5. 2014 Part Filter Method Ring Trial ................................................................................... 10

5.1 Key differences between 2012 and 2014 Ring Trials ................................................... 11

5.2 The results of the 2014 Ring Trial .................................................................................. 11

6. Conclusion......................................................................................................................... 22

7. References ......................................................................................................................... 23

Appendix 1 ............................................................................................................................... 24

Appendix 2 ............................................................................................................................... 32

Appendix 3 ............................................................................................................................... 38

CTR-TSB 2012-2014 Part Filter Method Ring Trial – June 2015 3/41

1. Summary

In 2011, under the auspices of CORESTA organisation, a Ring Trial of the Part-Filter

Method was completed and subsequently reported in 2012 and published in 2013. Soon

afterwards, also in 2013, a further Ring Trial of the method was completed and reported in

2014. In this report the results of the 2014 Ring Trial are communicated and compared with

the 2012 Ring Trial. The results and conclusions of both Ring Trials are reported

concurrently to allow a degree of comparison to be made with the aim of assessing whether

the method should be recognised by the CORESTA organisation as a recommended method.

The aim of two Ring Trials conducted in 2011 (termed the 2012 Ring Trial) and in 2013

(termed the 2014 Ring Trial) was to assess the standardised method agreed by the CORESTA

Smoking Behaviour Sub-Group. Specifically the output of the Ring Trial was to gain a

measure of the repeatability and reproducibility of estimated nicotine and tar yields across

the participating laboratories. The repeatability (r) is defined as variation of results within a

laboratory, and the reproducibility (R) is defined as variation of results within and between

laboratories.

It needs to be highlighted that direct comparison between the two Ring Trial outcomes should

be avoided because the test regimes used between the two Ring Trial differed; the 2014 test

regimes were somewhat more intense and, in our judgment, more human-like. Additionally

differences in the cohort of participating laboratories and their number also changed.

Conclusions reached from the 2014 Ring Trial:

CV for repeatability (within lab variation, termed CV r (%), for estimated nicotine and NFDPM is satisfactory. This value was also satisfactory in the previous Ring Trial study

CV for reproducibility (within and between lab variation, termed CV R(%), for estimated nicotine were in the range 5 to 13%, compared to 13 to 25% in 2012, a considerable

improvement. This indicates the method is now under control.

CV for reproducibility for estimated NFDPM by UV were in the range 20 to 34%, compared to 6 to 48% previously, this indicates this measurement requires further

attention CV for reproducibility for estimated NFDPM by tip solanesol were in the range

9 to 13%, compared to 6 to 58% in the 2012 Ring Trial.

Estimated NFDPM by tip solanesol is similar to estimated NFDPM by tip UV absorbance. However only 5 laboratories submitted data for NFDPM by tip solanesol; these data are

reported in appendix 3.

2. Recommendation

It was the opinion of the Ring Trial co-ordinator that the protocol (as detailed in Appendix 2)

for estimated nicotine yield from tip nicotine and estimated NFDPM yield from tip solanesol

should be considered by the CORESTA Scientific Commission and Board as the basis of a

Recommended Method. This opinion was concurred with, at a gathering of the Smoking

Behaviour sub-group convened on 4th

October 2013 where these results were presented and

discussed.


Note on the comparability of CV r(%) and CV R(%) between the 2014 and 2012 Ring

Trials

In the 2014 Ring Trial the coefficients of variation of repeatability ( CV(sr) ) and

Reproducibility ( CV(sR) ) are calculated by following the formula mentioned in the standard

ISO 3534-2:Statistics — Vocabulary and symbols — Part 2: Applied statistics:

𝐶𝑉(𝑠𝑟) =𝑆𝐷(𝑟)

𝑀𝑒𝑎𝑛 𝑎𝑛𝑑 𝐶𝑉(𝑠𝑅) =

𝑆𝐷(𝑅)

𝑀𝑒𝑎𝑛

In the 2012 report the coefficients of variation named CVr and CVR have been calculated

from r and R and therefore,

𝐶𝑉𝑟 = 2.828𝑥𝐶𝑉(𝑠𝑟) 𝑎𝑛𝑑 𝐶𝑉𝑅 = 2.828𝑥𝐶𝑉(𝑠𝑅)

In order to facilitate comparison with the results of 2012 study, the table below lists the Mean

and the Coefficients of Variation of repeatability and Reproducibility obtained in the two last

studies (2012 and 2014). It has to be noted that the number of participants varies throughout

the studies.

3. Introduction

This report sets out the results of two Ring Trials conducted on the Part-Filter Method

reported in 2012 and 2014. The reason this document reports both Ring Trial concurrently is

to allow a measure of comparison to be made between them to aid a decision to be made

regarding whether to advance the methodology towards becoming a CORESTA

Recommended Method. It should be emphasized however that direct comparison between

the two Ring Trial outcomes needs to be avoided because of differences in the cohort of

participating laboratories and their number. Nevertheless there is indicative value in the

Tables displayed throughout the report in showing that the variation associated with the

methodology is under control when it utilised across laboratories worldwide.

Outline of the Part Filter Method for the Estimation of Mouth Level Exposure

A non-invasive method of assessing the mouth level exposure (MLE) obtained by the

individual smokers is to use the part filter method (PFM). The technique has gained a

measure of popularity and is used by a number of laboratories within and beyond the tobacco

industry.1-8

The theory and practice of the PFM is described elsewhere1,6

and the technique

will not be reviewed in this report. The basic principle is that the amount of ‘tar’ and nicotine

that emerges from the filter is proportional to the amount of ‘tar’ and nicotine deposited in a

section of the smoked filter. The part filter method utilises smoked cigarette filters discarded

by smokers as a means of estimating the mouth level exposure obtained by the individual

smoking the cigarette. This is accomplished by comparing nicotine, UV absorbing species

and solanesol extracted from a portion of the smoked filter. Values of these extracted

components from human smoked filters are compared with machine smoked filter extracts

where the yield of nicotine and NFDPM are known. A variety of smoking machine regimes,

designed to encompass the range of typical human smoking yields are used to define the

relationship between smoking yields and the tip extracted components. This then allows for

human smoked tip extracts to be correlated to the yields of nicotine and NFDPM pertaining

to the individual smoker which should be viewed as an estimate rather than a direct

measurement. The correlation, referred to as calibration smoking, is completed on the same

batch of cigarette consumed by the smoker. The technique is referred to as the part-filter

method (PFM) because, filters are cut to 5 - 10mm lengths from mouth-end ensuring neither

ventilation holes nor carbon components are included in the cut filter section.7


To date there is no agreed methodological standard for the PFM. The aim of two Ring Trials

was to conduct an initial Ring Trial on an agreed methodology to determine intra and inter

laboratory variation, and then incorporate the learning from this into a revised methodology.

A second Ring Trial would assess laboratory variation on this revised methodology. If the

results of the Ring Trial offered encouragement, showing that variation was under control,

the methodology could be potentially advanced as a CORESTA Recommended Method for

the Part-Filter Method.

Human smoke yields may be estimated by comparison of the nicotine content of filter

extracts from human smoked cigarettes with those from machine smoked cigarettes for which

the smoke yield is known. Similarly, the UV absorbance (at 310 nm) of the filter extracts are

related to NFDPM collected on the smoking machine Cambridge Filter Pad (CFP). For each

cigarette product a range of regimes are smoked which cover typical human smoking

behaviour parameters in terms of puff volumes, durations and intervals. Machine smoke

yields (CFP nicotine and CFP NFDPM) and extracts from part filters (tip nicotine, tip UV

absorbance) from these calibrations are used to produce linear regression equations. These

regressions, together with tip data from human smoked cigarettes can be used to estimate the

MLE for humans for nicotine and tar. An additional alternative estimation of the human

MLE for tar can also be made based on the regression of CFP NFDPM and solanesol

extracted from part filters. Solanesol is a component of tobacco which is transferred

unaltered into smoke and a proportion of which is deposited in the filter; tip solanesol is co-

extracted from the part filter along with nicotine and UV absorbing species.

Overview of the initial CORESTA Part Filter Method Ring Trial reported in 2006

The leading features of this study, Co-ordinated by Dr Paul Nelson of RJ Reynolds Tobacco

Company were:

Each laboratory applied their own methods and procedures

Three different cigarette products were smoked and test filters were produced at two intensities

The co-ordinating laboratory sent out the machine-smoked test filter tips to participating laboratories together with cigarettes of the same batch for laboratories to conduct

calibration smoking

Following calibration smoking and analysis of tips and pads, laboratories estimated yields of NFDPM and nicotine for each product. Nine laboratories participated, seven returned

data

The main conclusions reached by the 2006 Ring Trial study were:

Due to overlapping of methods and few labs performing identical methods, impact of individual method differences (solvent, internal standard, extraction etc.) could not be

determined

No difference was observed between linear and rotary smoking machine calibration

A method with defined smoked calibration regimes was considered to be the best choice for standardization of the method

The smoked calibration regimes should be as simple as possible; some laboratories may struggle to achieve very intense puffing regimes.


4. 2012 Part Filter Method Ring Trial

The proposal to set up a Ring Trial was made by delegates attending a meeting of the

CORESTA Smoking Behaviour Sub-Group (SB SG) on 8th

July 2010. Following this a draft

protocol of the proposed Ring Trial was circulated to all laboratories which had expressed an

interest in taking part. An update meeting of the Sub-Group occurred in September 2010 at

Edinburgh where the protocol details were ratified and the decision was endorsed to proceed

with the Ring Trial. The subsequent timetable of the Ring Trial was as shown below:

Ring Trial Timetable

Set-up meeting, SB SG meeting July 2010

Smoked tip generation August

Update meeting to CORESTA SB SG September

Tips and cigarettes despatched to laboratories September

Laboratories report data January 2011

Presentation made to CORESTA SB SG April

Update made to CORESTA SB SG October

Presentation to CORESTA Joint Study Groups October

Draft report presented to CORESTA SB SG April 2012

Draft report submitted to CORESTA Scientific Commission June 2012

Report issued December 2012

should complete a single defined set of calibration smoking regimes. To this end it was

agreed the calibration smoking regimes to be used were defined in Ring Trial protocol.

Test tips were produced from two cigarette products using two smoking regimes:

65mL puff volume /1.5s puff duration / 50s puff frequency – High Regime Smoking

40mL puff volume / 2.0s puff duration / 60s puff frequency - Low Regime Smoking

The test products analysed in the Ring Trial consisted of 4 levels, detailed in Table 1

Table 1. Description of four levels of smoked tips used in the 2012 Ring Trial

1mg ISO tar product 1 Low Regime Smoking Appendix 1 Figures 1

and 5 Dunhill White

1mg ISO tar product 1 High Regime Smoking Appendix 1 Figures 2

and 6 Dunhill White

10mg ISO tar product 2 Low Regime Smoking Appendix 1 Figures 3

and 7 Marlboro Red

10mg ISO tar product 2 High Regime Smoking Appendix 1 Figures 4

and 8 Marlboro Red


Participants of the 2012 Ring Trial

Twelve laboratories agreed to take part in the Ring Trial – all twelve laboratories returned

data; the participating laboratories are shown below:

Participants

Imperial Tobacco Limited Montreal, Canada

Zhengzhou Tobacco Research Institute of CNTC Zhengzhou, PRC

PHILIP MORRIS Research Laboratories GmbH Köln, Germany

BAT (Germany) GmbH Bayreuth, Germany

FILTRONA Jarrow, UK

Japan Tobacco Inc Tokyo, Japan

KT&G Central Research Institute Daejeon, ROK

LABSTAT Kitchener, Canada

BAT Souza Cruz Porte Allegra, Brazil

Reemtsma Cigarettenfabriken GmbH Hamburg, Germany

BAT GR&D Southampton, UK *

Imperial Tobacco Group Fleury-les-Aubrais, France **

* Study Coordination; ** Statistical Support

4.1 Results

Table 2. 2012 Ring Trial: Coefficient of variation, estimated nicotine yields

Estimated Nicotine yield from tip nicotine

Coefficient of variation repeatability (%)

Coefficient of variation reproducibility (%)

1mg ISO ‘tar’, low smoking regime 16.3 23.5

1mg ISO ‘tar’, high smoking regime 3.9 24.5



Table 3. 2012 Ring Trial: Coefficient of variation, estimated NFDPM yields

Estimated NFDPM yield from tip UV absorbance








Data sets were received from all twelve laboratories.

Twelve labs reported estimated nicotine yields from tip nicotine from 3 replicates for each of the 4 test tips. The results are reported in Table 4 showing the grand mean for

each level, the calculated repeatability and reproducibility and the associated coefficient

of variation.

Nine labs additionally reported estimated NFDPM yield from tip UV absorbance from 3 replicates for each of the 4 test tips. The results are reported in Table 5 showing the

grand mean for each level, the calculated repeatability and reproducibility and the

associated coefficient of variation.

Six labs further reported estimated NFDPM yield from tip solanesol from 3 replicates for each of the 4 test tips. In view of the limited number of laboratories who completed this

aspect of the Ring Trial the results are presented in Appendix 3, Table 2, showing the

coefficients of variation at each level.

Estimated NFDPM from tip solanesol is very similar to estimated NFDPM from Tip UV absorbance, however significantly fewer laboratories performed the former measurement

than the latter meaning that direct comparisons should not be made.

The collated statistical data were evaluated according to ISO Standard 5725-2:1994. Outliers were determined in accordance with the protocol using Cochran’s and Grubb’s

tests.

Single value graphs are depicted in Appendix 1 Figures 1 – 8 where data points for each

replicate are shown as black cross symbols, laboratory means as green dots. The green line

represents the line of the grand mean, the blue and red lines are the lines of the 95% and 99%

confidence interval limits respectively between a reference value (the grand mean of all the

laboratories) based on ISO 5725-6.

4.2 Learnings and Conclusions

Both low and high regime smoked test tips should be smoked more intensively to better reflect human smoking behaviour

Pads must be weighed outside the holder – as residual water in holder is not characterised in NFDPM calculation

Replicate calibration smoking must be completed on separate days - independent smoking of replicates

The effects of regime O (Appendix 2, Table 1) were considered - non-smoked blank regime (cut filters extracted to determine residual levels present in unsmoked tips). The

inclusion of regime O as part of the smoking calibration regimes was significant; regime

O should constitute one of the calibration regimes as stated in the protocol. Many

laboratories omitted to include these data points; this may be because amounts detected

fell below the laboratory’s reporting limits. Subsequent comment: Despatched tips in the

2014 all participating laboratories determined regime O values.

4.3 Possible Sources of Variation

Calibration Smoking

Smoking Regime F (70mL / 1.5s / 20s) may be difficult for a number of laboratories to

undertake and may account for variation in the estimates.


Measurements of Tip Extract Solution

Tip extraction solvent - methanol is preferred. The use of ethanol or IPA will result in incomplete extraction of tips

Measurement of low / high levels nicotine and UV absorbance in tip extract solution may be constrained by laboratory reporting limits

The use of an Internal Standard added to tip extraction solvent is recommended

Measurements of Pad Extract Solution

Weighing of Cambridge Filter Pad – pads should be weighed OUTSIDE the holder. This was stressed in the protocol; the weighing of the pad whilst it is within the pad holder will

result in some of the water present remaining within the pad holder assembly, and hence

not being characterised in the water assay. The effect will be most apparent with high

intensity regimes, especially the vent blocked regime which showed considerable

variation.

4.4 Recommendations

A further Ring Trial incorporating the learnings from the 2012 RT was recommended by the

coordinator and discussed and endorsed by the SB SG (meeting in September 2011).

Table 4. 2012 Ring Trial Repeatability (r) and Reproducibility (R) of Estimated Nicotine

from tip nicotine

Estimated Nicotine yield from tip nicotine

Number of Labs

included in statistical evaluation

Grand Mean

(mg/cig)

r (mg/cig)

R (mg/cig)

CV r (%)

CV R (%)

1mg ISO ‘tar’ low regime 9 of 12 0.11 0.048 0.070 16.3 23.5

1mg ISO ‘tar’ high regime 12 of 12 0.35 0.038 0.242 3.9 24.5



Table 5. 2012 Ring Trial: Repeatability (r) and Reproducibility (R) of Estimated NFDPM

from tip UV absorbance

Estimated NFDPM from tip UV absorbance

Number of Labs


Grand Mean

(mg/cig)

r (mg/cig)

R (mg/cig)

CV r (%)

CV R (%)






5. 2014 Part Filter Method Ring Trial

Following the completion of the previous Ring Trial in 2011 and reporting to the CORESTA

joint study group meeting in October in October 2011, a meeting of the SB SG made the

decision to carry out a further Ring Trial on the PFM. The objective was to capitalise on the

momentum of the previous Ring Trial and to incorporate the learning of the completed RT

into an updated method protocol. It was anticipated variability of the methodology could be

reduced by the incorporated changes.

2014 Ring Trial Timetable

Set up meeting at SB SG following report of 2012 RT October 2011

Draft protocol circulated April 2012

Invitation email sent to laboratories June

Smoked tip generation September

Tip and cigarette despatched October

Analysis at laboratories Nov- March

Reporting of data Jan - Apr 2013

Data analysis May - August

Update report to CORESTA SB SG April

Ring Trial results presented to SB SG October

Report & findings submitted to SC February 2014

Report & Findings Presentation made to CORESTA Congress September 2014

Initial Ring Trial Participants

+ Lorillard Tobacco Company Greensboro, USA

# Zhengzhou Tobacco Research Institute of CNTC Zhengzhou, PRC

Philip Morris International Neuchatel, Switzerland

+ BAT (Germany) GmbH Bayreuth, Germany

+ FILTRONA Jarrow, UK

+ Japan Tobacco Inc Tokyo, Japan

+ KT&G Central Research Institute Daejeon, ROK

+ LABSTAT Kitchener, Canada

+ BAT Souza Cruz Porte Allegra, Brazil

+ Arista Laboratories Inc Richmond, USA

+ BAT GR&D Southampton, UK *

+ Imperial Tobacco Group, Fleury-les-Aubrais, France**

+Returned data # customs prevented receipt of samples

*Study Coordination **Statistical Support

Nine laboratories included in statistical evaluation, one excluded as high outlier

Learnings from 2012 were incorporated into the smoking calibrations used in the 2014 Ring

Trial which are described in the Ring Trial protocol (Appendix 2). The test products analysed

in the Ring Trial consisted of 4 levels as detailed in Table 6.


Table 6. Description of four levels of smoked tips used in Ring Trial

1mg ISO tar product 1 Low Regime Smoking Figures 1 and 5 Dunhill White

1mg ISO tar product 1 High Regime Smoking Figures 2 and 6 Dunhill White

10mg ISO tar product 2 Low Regime Smoking Figures 3 and 7 Marlboro Red

10mg ISO tar product 2 High Regime Smoking Figures 4 and 8 Marlboro Red

5.1 Key differences between 2012 and 2014 Ring Trials

Calibration regimes simplified

Introduction of a 4 puff regime

Elimination of the vents blocked regime

Different test tip regimes used: test tip regimes

- Low Regime Smoking: 40mL/2.0s/60s → 55mL/2.0s/60s - High Regime Smoking: 65mL/1.5s/50s → 65mL/2.0s/30s

Test tip regimes were made more intense to make more ‘human-like’

- Pad and tip extraction procedure described in detail

There were changes in the laboratory cohort participating in the Ring Trial

5.2 The results of the 2014 Ring Trial

Table 7. 2014 Ring Trial: Coefficient of variation, estimated nicotine yields

Estimated nicotine yield from tip nicotine







Table 8. 2014 Ring Trial: Coefficient of variation, estimated NFDPM (nicotine free dry

particulate matter, commonly known as ‘tar’) yields

Estimated NFDPM yield from tip UV absorbance








Data sets used in the statistical evaluation were received from ten laboratories; of these two

were excluded for reasons of non-compliance to the protocol. Data from eight labs were used

in the statistical evaluation of estimated nicotine yields from tip nicotine from 3 replicates for

each of the 4 test tip levels. Data from seven labs were used in the statistical evaluation of

estimated NFDPM yield from tip UV absorbance from 3 replicates for each of the 4 test tip

levels. According to ASTM criteria9 this number of participating laboratories is sufficient for

a valid inter-laboratory comparison.

The tabulated results for estimated nicotine yields are detailed in Table 9 showing the grand

mean for each level, the calculated repeatability and reproducibility and the associated

coefficient of variation. CV for repeatability for estimated nicotine and NFDPM remains

satisfactory between the two Ring Trials. CV for reproducibility were in the range 5 to 13%

(mean = 9.6%), compared to 13 to 25% in 2012 (mean = 19.0%) – a considerable

improvement. According to AIAG10

a general rule for the variation introduced by the

measurement system is acceptable if the error is under 10%. According to this criterion the

results of the 2014 Ring Trial, the method is under control.

The tabulated results for estimated NFDPM from tip UV are detailed in Table 10 showing the

grand mean for each level, the calculated repeatability and reproducibility and the associated

coefficient of variation. CV for reproducibility for estimated NFDPM by UV were in the

range 20 to 34% (mean = 26.2%), compared to 6 to 48% (mean = 20.3%) previously.

According to AIAG10

if the variation introduced by the measurement system is between 10

and 30%, the system may be acceptable depending on the importance of application, cost of

measurement device, cost of repair, and other factors. However this level of variation

suggests that the method requires further attention.

Five labs further reported estimated NFDPM yield from tip solanesol from 3 replicates for

each of the 4 test tip levels. The results are reported in Appendix 3 Table 1 showing the grand

mean for each level, the calculated repeatability and reproducibility and the associated

coefficient of variation. (These data are reported in an Appendix because the number of

participating laboratories is below the minimum stipulated by ASTM which states that the

precision of a method should not be based on an assessment of fewer than six laboratories).9

Estimated NFDPM from tip solanesol is very similar to estimated NFDPM from tip UV

absorbance, however, as mentioned above, significantly fewer laboratories performed the

former measurement than the latter meaning that caution should be exercised in comparing

the two results. Nevertheless Appendix 3 Figure 5 shows that there is excellent correlation

between the two data sets. In the plot of NFDPM by tip solanesol against NFDPM by tip UV

the gradient of the line (through the origin) equals 0.9454, suggesting that tip solanesol

method gives approximately 5% higher NFDPM yield. At the meeting of the CORESTA SB

SG (October 2013) which considered the results of the Ring Trial, it was decided that a

CORESTA Recommended Method should incorporate the estimation of NFDPM from tip

solanesol in addition to nicotine yield from tip nicotine.

Single value graphs for the 2014 Ring Trial are depicted in Figures 1 - 8 where data points for

each replicate are shown as black cross symbols, laboratory means as green dots. The green

line represents the line of the grand mean, the blue and red lines are the lines of the 95% and

99% confidence interval limits respectively between a reference value (the grand mean of all

the laboratories) based on ISO 5725-6. Graphical representation of the equivalent data from

the 2012 Ring Trial is similarly presented in Appendix 1 Figures 1 – 8.

The collated statistical data were evaluated according to ISO Standard 5725-2:1994. Outliers

were determined in accordance with the protocol using Cochran’s and Grubb’s tests.


Table 9. 2014 Ring Trial Repeatability (r) and Reproducibility (R) of Estimated Nicotine

from tip nicotine

Estimated Nicotine yield from Tip Nicotine

Number of Labs


Grand Mean

(mg/cig)

r (mg/cig)

R (mg/cig)

CV r (%)

CV R (%)





Table 10. 2014 Ring Trial: Repeatability (r) and Reproducibility (R) of Estimated NFDPM

from Tip UV Absorbance

Estimated NFDPM from Tip UV absorbance

Number of Labs


Grand Mean

(mg/cig)

r (mg/cig)

R (mg/cig)

CV r (%)

CV R (%)






Figure 1. 2014 Ring Trial: Estimated Nicotine Yield from tip nicotine - Product 1, low smoking

Eight laboratories included in statistical evaluation, none excluded as outlier


Figure 2. 2014 Ring Trial: Estimated Nicotine Yield from tip nicotine - Product 1, high smoking




Eight laboratories included in statistical evaluation, one excluded as high outlier


Figure 5. 2014 Ring Trial: Estimated NFDPM Yield from tip UV absorbance - Product 1, low smoking

Seven laboratories included in statistical evaluation, none excluded as outlier


Figure 6. 2014 Ring Trial: Estimated NFDPM Yield from tip UV absorbance - Product 1, high smoking



Figure 8. 2014 Ring Trial: Estimated NFDPM Yield from tip UV Absorbance - Product 2, high smoking



6. Conclusion

The decision to carry out two Ring Trials in swift secession allowed the learnings from the

first to be incorporated into the second. The revised PFM protocol was tested across a range

of laboratories worldwide.

The resulting assessment of intra and inter laboratory variation of the PFM showed that estimated nicotine yields from tip nicotine was well controlled (data was interpreted from

eight laboratories).

Estimated NFDPM from tip UV absorbance was shown to be less well controlled (data was interpreted from seven laboratories) suggesting further work is required to reduce

method variability. It is the viewpoint of the study coordinator that the heterogeneous

nature of (UV absorbing) smoke components absorbed onto the filter material, and its

extracted liquid, is intrinsically chemically unstable; small differences in the treatment of

tips will exaggerate variability in the method. Therefore further effort is not justified in

attempts to reduce the variability associated with this aspect of the PFM.

The estimation of NFDPM from tip solanesol confers the advantage that solanesol is a single chemical species, non-volatile, stable and is relatively abundant in extracted

solution. Solanesol extracted from part filters has been used to estimate NFDPM yields of

smokers outside the tobacco industry but suffered from reduce numbers of participating

laboratories (data from five laboratories). The estimation of NFDPM from tip solanesol

was similar to the estimation of NFDPM from tip UV absorbance - in this Ring Trial the

latter method produced approximately 5% greater estimates of NFDPM.

It is recommended that the PFM involving the estimation of nicotine and NFDPM MLE using

nicotine and solanesol retained in the part filter respectively are advanced as a CORESTA

Recommended Method.


7. References

1. St.Charles, F.K., Ashley, M., Shepperd, C.J., Clayton, P., and Errington Beiträge zur Tabakforschung International / Contributions to Tobacco Research; 23:232-243 (2009) A Robust Method for Estimating Human Smoked Cigarette Yields from Filter Analysis Data

2. J.L. Pauly, RJ O'Connor, G.M. Paszkiewicz, K.M. Cummings, M.V. Djordjevic and P.G.

Shields Cancer Epidemiol Biomarkers Prev; 18(12):3321-3333 (2009) Cigarette filter-based assays as proxies for toxicant exposure and smoking behaviour--a literature review

3. P.M. Clayton, A. Cunningham and J.D.H. van Heemst Analytical Methods; 2(8):1085-1094 (2010) Quantification of four tobacco-specific nitrosamines in cigarette filter tips using liquid chromatography-tandem mass spectrometry

4. D.L. Ashley, R.J. O'Connor, J.T. Bernert, C.H. Watson, G.M. Polzin, R.B. Jain, D. Hammond, D.K. Hatsukami, G.A. Giovino, K.M. Cummings, A. McNeill, L. Shahab, B. King, G.T. Fong, L. Zhang, Y. Xia, X. Yan and J.M. McCraw Cancer Epidemiol Biomarkers Prev; 19:1389-1398 (2010) Effect of differing levels of tobacco-specific nitrosamines in cigarette smoke on the levels of biomarkers in smokers

5. Y.S. Ding, T. Chou, S. Abdul-Salaam, B. Hearn and C.H. Watson Cancer Epidemiol Biomarkers Prev; 21:39-44 (2012) Development of a method to estimate mouth-level benzo[a]pyrene intake by filter analysis

6. Regulatory Toxicology and Pharmacol; 61(3): Supplement 1 (2011) Six supplement articles

7. C.J. Shepperd, FK St. Charles, M. Lien and M. Dixon Beiträge zur Tabakforschung International / Contributions to Tobacco Research; 22:176-184 (2006) Validation of methods for determining consumer smoked cigarette yields from cigarette filter analysis

8. T. Hyodo, K. Minagawa, T. Inoue, J. Fujimoto, N. Minami, R. Bito, and A. Mikita Regulatory Toxicology and Pharmacology; 63:486-498 (2013) Estimation of mouth level exposure to smoke constituents of cigarettes with different tar levels using filter analysis

9. ASTM E691-09 Standard Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method

10. Automotive Industry Action Group (AIAG) (2010) Measurement Systems Analysis, 4th edition. Chrysler Group LLC, Ford Motor Company and General Motors Supplier Quality Requirements Task Force

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Pauly%20JL%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Djordjevic%20MV%22%5BAuthor%5D


Appendix 1


Twelve laboratories included in statistical evaluation, one excluded as high outlier one excluded as low outlier

+

++

+

+

+

+

++

++

+

+++

++

+++

+ +++

+

++

+++

+++

+++

Brand DW_L

Labs

Estim

ate

d N

ico

tin

e

1 2 3 4 5 6 7 8 9 10 11 12

-0.05

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

+

++

+

+

+++

+

++

+++

+ +++

+

++

+++

+++

G_out_h

G2_out_l G2_out_l

Lab Number

Est

imat

ed N

ico

tin

e (

mg

/cig

)

0.165

0.046

165

0.110

4816



Twelve laboratories included in statistical evaluation, none excluded as outlier

0.60

++++++

+++

+++

+++

+

++ ++++++

+++

+

+

+

+++

+++

Brand DW_H

Labs

Estim

ate

d N

ico

tin

e

1 2 3 4 5 6 7 8 9 10 11 12

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

0.65

++++++

+++

+++

+++

+

++ ++++++

+++

+

+

+

+++

+++

Lab Number

Est

imat

ed N

ico

tin

e (

mg

/cig

)

0.09

0.35


Figure 3. 2012 Ring Trial: Estimated Nicotine Yield from tip nicotine – Product 2, low smoking


+++++

+

+++

+

++

++

+

+

++

+

+

+

+++

++

+++

+

+++

+++

Brand MR_L

Labs

Estim

ate

d N

ico

tin

e

1 2 3 4 5 6 7 8 9 10 11 12

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

+++++

+

+++

+

++

++

+

+

++

+

+

+

+++

++

+++

+

+++

+++

Lab Number

Est

imat

ed N

ico

tin

e (m

g/c

ig)

0.88

1.25

0.50




++

+

+

++

+++

+++

+++

++

+

+

+

+

+

++

+

++

+

+

+

+++

+++

Brand MR_H

Labs

Estim

ate

d N

ico

tin

e

1 2 3 4 5 6 7 8 9 10 11 12

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

++

+

+

++

+++

+++

+++

++

+

+

+

+

+

++

+

++

+

+

+

+++

+++

Lab Number

Est

imat

ed N

ico

tin

e (

mg

/cig

)

1.08

0.66

1.50



Nine laboratories included in statistical evaluation, none excluded as outlier

0.38

Lab Number

Est

imat

ed N

FD

PM

(m

g/c

ig)

+++

++

+

++

+

++

+

+

+

+

+

++

+++

+++

++

+

Brand DW_L

Labs

Estim

ate

d N

FD

PM

1 2 3 4 5* 6 7* 8 9 10 11* 12

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

+++

++

+

++

+

++

+

+

+

+

+

++

+++

+++

++

+

2.35

0.98



Nine laboratories included in statistical evaluation, none excluded as outlier

Lab Number

Est

imat

ed N

FD

PM

(m

g/c

ig)

+++

+++

+

+

+

+++

+

+

+

+

+

+ +++

+

+

+

+

+

+

Brand DW_H

Labs

Estim

ate

d N

FD

PM

1 2 3 4 5* 6 7* 8 9 10 11* 12

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

+++

+++

+

+

+

+++

+

+

+

+

+

+ +++

+

+

+

+

+

+

3.43

5.17

1.69




Est

imat

ed N

FD

PM

(m

g/c

ig)

Lab Number

+++

+++

++

+

+

+

+ +

+

+ ++

+

+

++

++

+

++

+

Brand MR_L

Labs

Estim

ate

d N

FD

PM

1 2 3 4 5* 6 7* 8 9 10 11* 12

8

9

10

11

12

13

14

15

16

+++

+++

++

+

+

+

+ +

+

+ ++

+

+

++

++

+

G_out_h

12.7

1

9.12

10.9

1




Lab Number

Est

imat

ed N

FD

PM

(m

g/c

ig)

++

+

+

++

+

+

+

+++

+++++

+

+

++

++

+

+

++

Brand MR_H

Labs

Estim

ate

d N

FD

PM

1 2 3 4 5* 6 7* 8 9 10 11* 12

9

10

11

12

13

14

15

16

17

18

19

20

21

++

+

+

++

+

+

+

+++

+++++

+

+

++ +

++

G_out_h

16.60

10.08

13.35

6


Appendix 2

Protocol for 2014 CORESTA Smoking Behaviour Sub-Group

Part Filter Method Ring Trial

Pre-amble: Below is the protocol of the PFM Ring Trial reported in 2014

1. Objective

Following the CORESTA Smoking Behaviour Sub-Group (SBSG) Filter Analysis Ring Trial

completed in 2011 and reported in 2012, a proposal was made in April 2012 to the meeting

Sub-Group for a further Ring Trial. The objective of the further Ring Trial was to maintain

and improve the repeatability of the previous Ring Trial by measuring the estimated nicotine

yields across participating laboratories using part filter method as described in this protocol

which incorporated learning from the previous Ring Trial.

2. Introduction

Each participating laboratory will conduct machine smoking to produce calibration data for

each product. Test tips, produced at the Study Co-ordination Centre (BAT Southampton),

will be sent to each laboratory where they will be analysed for tip nicotine. Participating

laboratories are additionally encouraged to make measurements of tip UV absorbance and tip

solanesol using the same methanol tip extract solution.

Regression equations obtained from smoking calibration data will be applied to the tip data to

produce estimated nicotine and tar/NFDPM yields for the test tips, and these estimates will be

returned to the Study Co-ordination Centre in Southampton on a standard form.

The key aspects of the method that must be followed are outlined in the protocol. Each

laboratory will use their own dedicated analytical methods for measurement of water and

nicotine yield, and the measurement of tip nicotine (and tip UV absorbance and tip solanesol).

However, analytical recommendations based on the learning from the previous Ring Trial

were incorporated into this protocol.

3. Procedure

3.1. Cigarettes

Product 1 1mg ISO tar Dunhill White (UK market)

Product 2 10mg ISO tar Marlboro Red (UK market)

Four hundred of each product will be supplied by the Study Co-ordination Centre to each

participating laboratory for calibration smoking. The cigarettes must not be conditioned

before calibration smoking, it is recommended that cigarettes are taken from freshly opened

packs prior to smoking. However, it is permissible to store unopened packets of cigarette in a

conditioned environment.


3.2. Calibration Smoking

Participating laboratories will construct calibration lines according to part filter analysis

methodology using the smoking regimes detailed in Table 1. On each occasion five cigarettes

will be smoked (3 cigarettes only for regimes C, D and F), and the smoke condensate will be

collected onto a 44mm Cambridge filter pad (CFP). Following the determination of TPM,

the resultant pads are extracted in 20mL extracting solvent for the determination of smoke

yield. Measurements are made for pad water and pad nicotine facilitating the calculation of

NFDPM.

Table 1: Calibration Smoking Regimes

Regime Volume

(mL) Duration

(s) Interval

(s) CFP Size

(mm) Smoked

Length/Puffs Number of

Cigs

A 35 2 60 44 OT + 3mm 5

B 60 2 30 44 4 puffs 5

C 60 2 30 44 OT + 3mm 3

D 70 2 30 44 OT + 3mm 3

E 40 2 30 44 OT + 3mm 5

F 70 1.5 20 44 OT + 3mm 3

O Blank 5

NOTE: It is especially important that calibration smoking CFPs are weighed outside the pad

holder. This is substantially different to the procedure outlined in ISO 3308. The weighing of

pads in the holder will result in some moisture not being characterised by the pad water

measurement - for instance some water is deposited in inaccessible parts of the pad holder.

The time interval between the removal of the pad from the holder, the weighing of the pad

and placing the pad in a sealed vessel should be reduced as far as possible – this is to

minimise evaporative losses. CFPs must be extracted and analysed for nicotine and water

immediately following smoking.

For regime C, D and F, only three cigarettes are smoked onto the CFP. An adjustment must

be made in the calculations to account for the reduced number of cigarettes used for these two

regimes. Regime B, D, E and F differs from previous draft protocol. Please only

include Regime F if your smoking machine allows this regime - laboratories are

requested to use their judgement.

Either rotary or linear smoke engines may be used to achieve these smoke regimes.

Repeat the regimes in Table 1 on different days to account for daily variation. Determine

smoke yields for nicotine (in mg/cig) at each of the smoke regimes; each calibration smoke

regime will be performed twice on different days.

The measurements of water and nicotine yields are subtracted from Total Particulate Matter

(TPM) yields to calculate smoke yields for tar/NFDPM (in mg/cig) at each of the smoke

regimes. For regime O nicotine and tar/NFDPM yields are set to zero.


3.3. Treatment of Tips

Smoked cigarette tips obtained from calibration smoking are cut to 10mm mouth-end portions

and extracted in methanol containing an internal standard; the recommended internal standard

is n-heptadecane. Five tips (three in the case of regimes C, D and F) should be extracted in

20mL of methanol containing a suitable internal standard such n-heptadacane (50g/mL). It

is important to ensure the extraction procedure is able to extract nicotine (and UV absorbing

species and solanesol) deposited in cellulose acetate filter tips with high and consistent

efficiency. An example of one way to ensure this is to extract 5 tips in 20mL of extraction

solvent using a 150mL round-bottom flask, the extraction is completed on an orbital shaker

operating at 150rpm for a minimum of 30 minutes. The resultant extract solution is then

vialed and analysed within 12 hours for UV absorbance, or within 4 days (stored at +4C) for

nicotine or solanesol. Further specific details are available on request. Please record any

deviation from the method described above and report to the Study Co-ordination Centre.

Smoked calibration tips are analysed together in the same batch as test tips forwarded from

the Study Co-ordination Centre as detailed below.

4. Test and Calibration Filter Tip Analysis

Test tips for each product will be produced at the Study Co-ordination Centre using two

smoking regimes (High and Low), giving a total of four levels. The tips will be cut to 10mm

lengths and immediately frozen by the Study Co-ordination Centre until dispatch. Test tips

will be defrosted just before dispatch and will be sent to laboratories in an unfrozen (ambient)

state together with cigarette products. Receiving laboratories are requested to freeze on

arrival test tips but not the cigarette products.

Laboratories will each receive three tins each containing 15 tips for each level, giving a total

of twelve tins*. Each tin will be labelled with a code to be used for reporting the results. The

test tips must be kept frozen until analysis. Table 2 gives details of the smoking used to

produce the test tips provided to laboratories. Each cigarette product is analysed in three

independent replicate measurements (both high and low test tips are analysed jointly).

* The dimensions of the tin are 40mm diameter, 80mm height.

Table 2: Numbers Test Tips per Laboratory

HIGH test tips: 65mL/2s/30s LOW test tips: 55mL/2s/60s

Tin 1 Tin 2 Tin 3 Tin 1 Tin 2 Tin 3

1 mg Product 1 15 15 15 15 15 15

10 mg Product 2 15 15 15 15 15 15

The test tips will be analysed in three stages as described in sections 4.1, 4.2 and 4.3.

Regime O provides blank tips to be analysed for tip nicotine (and tip UV absorbance / tip

solanesol if conducted The quantities measured in these tips will be very low and often will

be below laboratory reporting limits. The measured values must be included to construct the

calibration graphs.


4.1. Tin 1: Replicate 1 of Product 1

The following outline procedure refers only to Product 1, however, it is recommended that

Products 1 and 2 are smoked and analysed together on the same days.

On Day 1 move all tins labelled Product 1, Tin 1 (two tins) into a fridge. (+4C).

Complete calibration smoking for product 1 over two days (Day 1 and Day 2) as described in

Section 3.2. CFP samples are extracted and analysed on the day of smoking, tips are cut to

10mm lengths and stored in the dark at +4C.

At the end of Day 2 there should be two tins containing test tips (each containing 15 tips), ten

or twelve tins containing smoked calibration tips (each containing 5 or 3 tips), and two tins

containing unsmoked blank tips (each containing 5 tips)

On Day 3 remove all tins from fridge. Randomly divide the contents of each test tip tin of 15

test tips into three samples; each containing five test tips. All test and calibration filter tips

lengths are measured to nearest 0.01mm allowing the calculation of the mean tip length (2

decimal places) of each extraction. Extract tips in 20 mL tip extract solution as described in

Section 3.3. Analyse tip extracts for nicotine [and tip UV absorbance and tip solanesol] using

the methods routinely used in your laboratory.

Subsequently determine tip nicotine normalised to 10mm tip length (mg/10mm tip) [tip UV

absorbance normalised to 10mm tip length (arbitrary units) / tip solanesol normalised to

10mm tip length (mg/10mm tip)] of the tip extract.

In Summary

Day 1 Smoking of first set of calibration smoking regimes, CFP analysis, calibration tips

stored at 4C. Test tips Product 1, Tin 1 (2 tins – High and Low) placed in 4C storage.

Day 2 Smoking of second set of calibration smoking regimes, CFP analysis, calibration tips

stored at 4C.

Day 3 Remove all tins from fridge. Measurement of lengths of test and calibration tips.

Extraction of tips. Analysis of tip extract solution.

Calibrations lines are produced by plotting the 10 or 12 smoked calibration points and 2

unsmoked blanks for the cigarette product to obtain unweighted linear regression equations.

Table 3: Calibration Graphs to Construct

Graph Graph Y axis X axis

G1 Nicotine Yield calibration Nicotine Yield (mg/cig) Normalised tip nicotine

(mg/10mm tip)

G2 NFDPM-UV calibration NFDPM Yield (mg/cig) Normalised tip UV

(arbitrary units/10mm tip)

G3 NFDPM-sol calibration NFDPM Yield (mg/cig) Normalised tip solanesol

(mg/10mm tip)

The calibration lines (slope and intercept data) are used to estimate the nicotine [NFDPM]

yields of the test tips using tip nicotine [tip UV absorbance and tip solanesol data (mg/tip)].

For each product, use the linear regression equations described in Table 3 and the test tip data

from the same product to estimate the yields of the test tips.

Estimated Nicotine Yield (mg/cig) = slope(G1) * tip nicotine (mg/tip) + intercept (G1)


Estimated NFDPM Yield (mg/cig) = slope(G2) * tip UV absorbance (units/tip) + intercept (G2)

Estimated NFDPM Yield (mg/cig) = slope(G3) * tip solanesol (mg/tip) + intercept (G3)


On Day 4 move all tins labelled Product 1, Tin 2 (two tins) into a fridge. (+4C).





or twelve tins containing smoked calibration tips (each containing 5 or 3 tips) and two tins

containing unsmoked blank tips (each containing 5 tips).





Section 3.3. Analyse tip extract for nicotine [and tip UV absorbance and tip solanesol].

In Summary:




stored at 4C.

Day 6 Remove all tins from the fridge. Measurement of lengths of test and calibration tips.



On Day 7 move all tins labelled Product 1, Tin 3 (two tins) into a fridge (+4C).





or twelve tins containing smoked calibration tips (each containing 5 or 3 tips) and two tins

containing unsmoked blank tips (each containing 5 tips).





Section 3.3. Analyse tip extract for nicotine [and tip UV absorbance and tip solanesol].

In Summary:




stored at 4C.

Day 9 Remove all tins from the fridge. Measurement of lengths of test and calibration tips.


Using this procedure it is possible to smoke and analyse all three replicates of both cigarette

products in nine days.


5. Reporting Results

5.1 Report the raw data and individual test results for the calibration and tip data in the spreadsheets provided. Nicotine yield results rounded to 3 decimal places.

5.2 Comments from the operators on any deviation from the documented analytical procedure should be reported in the comments column of the spreadsheet.

5.3 Information regarding any irregularities or disturbances during the measurement, including change of operator, together with a statement as to which measurements were

performed by which operator, and the reasons for any missing results.

5.4 The date when the samples were received and storage details and when analysed.

5.5 Information regarding the equipment used.

5.6 The completed spreadsheets shall be returned to Peter Clayton and Kate Nother at R&D Centre, Regents Park Road, Southampton SO15 8TL, [email protected] and

[email protected]

5.7 The Statistical plan on the received data will be in accordance with ISO 5725-2.

6. Timings

There will be an update meeting of the CORESTA Smoking Behaviour Sub-Group Filter

Analysis Ring Trial at the CORESTA Congress in Sapporo in September.

Smoked tips together with cigarette products will be forwarded to nominated technical

specialists of each laboratory in October 2012.

A provisional timetable is detailed below:

RT2 Reported, SBSG agrees for further Ring Trial (RT3) September 2011

Set-up meeting, draft protocol circulated April 2012

Invitation for RT3 emails sent out June

Revised protocol circulated July

Draft protocol sent to laboratories July/August

Smoked tip generation September

Update to CORESTA SB SG meeting September

Protocol agreed September

Agreed Protocol sent to laboratories October

Tips and cigarettes despatched to laboratories October

Analysis at laboratories Oct. – Jan.

Reporting of data from Laboratories February 2013

Data analysis March

Presentation to CORESTA SB SG April

Update to CORESTA SB SG October

Presentation to CORESTA Joint Study Groups October


Appendix 3

Table 1. 2014 Ring Trial Repeatability (r) and Reproducibility (R) of Estimated NFDPM

by tip solanesol

Estimated NFDPM yield

from Tip Solanesol

Number of Labs included in statistical evaluation

Grand Mean

(mg/cig)

r (mg/cig) R

(mg/cig) CV r (%)

CV R (%)

1mg ISO ‘tar’ low regime

5 of 5 3.24 0.637 1.068 6.9 11.7

1mg ISO ‘tar’ high regime

4 of 5 7.30 0.499 1.773 2.4 8.6

10mg ISO ‘tar’ low regime

5 of 5 13.87 3.206 4.900 8.2 12.5

10mg ISO ‘tar’ high regime

5 of 5 25.15 3.066 6.168 4.3 8.7

Figure 1. 2014 Ring Trial: Estimated NFDPM from tip solanesol - Product 1, low smoking


Figure 2. 2014 Ring Trial: Estimated NFDPM from tip solanesol - Product 1, high smoking

Figure 3. 2014 Ring Trial: Estimated NFDPM from tip solanesol - Product 2, low smoking


Figure 4. 2014 Ring Trial: Estimated NFDPM from tip solanesol - Product 2, high smoking

Figure 5. 2014 Ring Trial: Values of Estimated NFDPM from tip solanesol against

Estimated NFDPM from tip UV


Table 2. 2012 Ring Trial: Coefficients of variation associated with estimated NFDPM yields

Estimated NFDPM yield from tip solanesol



1mg ISO ‘tar’, low smoking regime

16.2 57.8

1mg ISO ‘tar’, high smoking regime

4.2 15.5

10mg ISO ‘tar’, low smoking regime

5.6 5.6

10mg ISO ‘tar’, high smoking regime

6.2 12.8

1. Summary2. Recommendation3. Introduction4. 2012 Part Filter Method Ring Trial4.1 Results4.2 Learnings and Conclusions4.3 Possible Sources of Variation4.4 Recommendations

5. 2014 Part Filter Method Ring Trial5.1 Key differences between 2012 and 2014 Ring Trials5.2 The results of the 2014 Ring Trial

6. Conclusion7. ReferencesAppendix 1Appendix 21. Objective2. Introduction3. Procedure4. Test and Calibration Filter Tip Analysis5. Reporting Results6. Timings

Appendix 3

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