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In Vitro Toxicity Testing of Tobacco Smoke Sub-Group
Technical Report
Neutral Red Uptake Assay
Proficiency Study
November 2015
Author:
Betsy Bombick
RJ Reynolds, U.S.A.
Co-Author:
Alexander Hauleithner
JTI R&D, OEKOLAB, Austria
Trial Coordinator:
Betsy Bombick
RJ Reynolds, U.S.A.
Sub-Group Coordinator:
Kei Yoshino
Japan Tobacco Inc., Japan
Table of Contents
1. Summary ............................................................................................................................. 3
2. Introduction ......................................................................................................................... 3
3. Participants .......................................................................................................................... 4
4. Study Protocol ..................................................................................................................... 4
5. Results – Raw Data ............................................................................................................. 5
6. Statistical Analysis and Interpretations ............................................................................... 6
6.1 Reported concentration ranges .................................................................................. 6
6.2 Determination of EC50 values .................................................................................... 7
6.3 Statistical Power – Minimum Detectable Difference (MDD) δ .............................. 11
6.4 Repeatability and Reproducibility analysis (r&R) .................................................. 14
6.5 Comparison of Samples – Relative Potency Plots ................................................... 18
6.6 Summary of findings ............................................................................................... 20
7. Discussion and Results ..................................................................................................... 21
7.1 Data Audit ................................................................................................................ 21
7.2 Audit Findings ......................................................................................................... 21
8. Conclusions ....................................................................................................................... 21
9. References ......................................................................................................................... 22
Appendix A: Summary Data for Each Laboratory ................................................................... 23
IVT–CTR NRU Proficiency Study – November 2015 3/30
1. Summary
A Neutral Red Uptake (NRU) assay proficiency study was conducted by member laboratories
of the CORESTA In Vitro Toxicity Testing Task Force (now aSub-Group since January
2015). Given that different procedures (internal or regulatory) are followed by the
participating laboratories, the purpose was not to develop a CORESTA Recommended
Method but to evaluate the cytotoxicity of a mainstream smoke extract from three cigarettes
using a common study protocol. This enabled laboratories to evaluate method proficiency,
compare results with those of other laboratories, and obtain an external audit of
documentation procedures that might identify potential areas for improvement. Each
laboratory therefore has been able to benefit both technically (scientific methods) and
administratively (documentation procedures) from their participation.
Eleven laboratories participated in the study. The protocol was approved by each laboratory
manager and allowed each laboratory to determine which cell line(s) to use. Neutral Red
Uptake (NRU) cytotoxicity assays were conducted on condensates (particulate matter
collected on Cambridge filter pads and extracts prepared in dimethylsulfoxide) from three
cigarettes where the cytotoxicity had previously been shown to be significantly different.
The cigarettes were coded as follows:
• A: 3R4F Kentucky Reference Cigarette
• B: Base Web (100% reconstituted tobacco sheet after water extraction)
• C: Recon (100% reconstituted tobacco sheet)
Smoke condensates were prepared using the ISO smoking regime at one laboratory, these
were frozen at approximately -70°C, and shipped on dry ice to each laboratory. NRU assays
were conducted following the protocol. Four cell lines were used (CHO, Balb/c 3T3, HEP-
G2, A549). Raw data, worksheets and summary conclusions were submitted to the Study
Coordinator. Data and worksheets were forwarded by the Coordinator to the Sub-Group
auditor for quality assurance audits. A common statistical analysis was conducted on coded
raw data by the Study Statistician.
Participants generally followed the prescribed protocol. Deviations were minor and were
considered unlikely to have a significant impact on the study outcome. Various cell lines
were employed by the laboratories and differences were found in the ability of the cell lines to
discriminate between the three samples.
2. Introduction
At the October 2009 meeting of the CORESTA In Vitro Toxicity Testing Task Force (Aix-en-
Provence, 2009), the Task Force confirmed its intention to conduct a neutral red uptake
proficiency study with the following purpose:
To conduct a proficiency testing program to evaluate the cytotoxicity of cigarette smoke
particulate matter of different products using the Neutral Red Uptake cytotoxicity assay.
Each participating laboratory will use their preferred cell line(s) to evaluate the test
samples. Experimental design will be harmonized as much as possible.
The objective of this study was to evaluate the NRU cytotoxicity assay ability to discriminate
the cytotoxic potential of mainstream smoke particulate matter extracts from three cigarette
samples (cigarette smoke condensate, CSC). The NRU assay is a rapid, reliable and
economical method for screening compounds for potential cytotoxicity. The NRU assay is
based on the ability of viable cells to incorporate and bind neutral red dye following exposure
IVT–CTR NRU Proficiency Study – November 2015 4/30
of the cell culture to the test agent. If the test agent is toxic to the cells and injures the plasma
and/or lysosomal membrane, the dye will not be retained in the cells. The dye is extracted
from the lysosomes, quantified spectrophotometrically and compared to a control cell culture
(Borenfreund & Puerner, 1985). Cytotoxicity is expressed as a concentration-dependent
reduction in NRU, providing an integrated signal of cell integrity and growth inhibition
(NIH/ICCVAM).
3. Participants
Table 1 - List of Participating Laboratories
Participants Study Contribution
Arista Laboratories Collecting and providing condensates
Covance Laboratories Ltd, UK
Imperial Tobacco Canada Limited
Japan Tobacco Inc.
KT&G Central Research Institute
Labstat International ULC
Lorillard Tobacco Company
Ökolab/JTI Statistical Analysis
Philip Morris Research Laboratories
ITG/Reemtsma Providing test cigarettes
Zhengzhou Tobacco Research Institute of CNTC
BAT provided Audit / QA Review but did not participate in the study.
4. Study Protocol
The protocol, summarized below, was reviewed and approved by a Study Manager from each
participating laboratory. The approved Study Plan is available upon request.
1. Samples:
3R4F University of Kentucky Reference Cigarette
Base Web: an unventilated cigarette made from 100% reconstituted tobacco
sheet from water-extracted tobacco, without addition of the extract (NOTE:
“Base web” is the name ITG/Reemtsma used for this particular test cigarette).
Recon: an unventilated cigarette made from 100% reconstituted tobacco sheet
from water-extracted tobacco, with addition of the extract
The cigarettes were provided by Imperial Tobacco Group/REEMTSMA. Statistically
significant differences in cytotoxicity had already been demonstrated between the Base
Web and Recon cigarettes in the Imperial Tobacco Group/REEMTSMA laboratory
using HEP-G2 cells.
2. Cigarettes were conditioned and smoked under ISO conditions (ISO 3308:2000/Amd
1:2009) using a RM-20 smoking machine. Twenty cigarettes were smoked at a time
and TPM collected on 92-mm pads. Pads were stored frozen at -70C.
3. Step 2 was repeated in order to generate sufficient CSC for use by all of the study
participants.
IVT–CTR NRU Proficiency Study – November 2015 5/30
4. Three independent smoking sessions were performed on each sample on different days.
Smoke chemistry results are presented in the following table (mean ± 1 standard
deviation):
Table 2 - Smoke chemistry results (mean ± 1 standard deviation)
Code Sample
ID Cigs
smoked TPM/Pad
(mg) TPM
(mg/cig) Water
(mg/cig) Nicotine (mg/cig)
Tar (mg/cig)
A1 3R4F 220 2420.7 11 1.17 ± 0.15 0.35 ± 0.01 8.67 ± 0.16
A2 3R4F 220 2409.2 10.95 1.39 ± 0.20 0.37 ± 0.02 8.93 ± 0.21
A3 3R4F 220 2396 10.89 1.18 ± 0.15 0.35 ± 0.02 8.11 ± 0.17
B1 Base Web 180 2626.6 14.59 1.82 ± 0.45 NQ 12.22 ± 0.45
B2 Base Web 180 2619.6 14.55 1.62 ± 0.16 NQ 12.65 ± 0.17
B3 Base Web 180 2550.4 14.17 1.9 ± 0.23 NQ 12.62 ± 0.23
C1 Recon 280 2805 10.02 0.73 ± 0.09 0.73 ± 0.04 9.62 ± 0.13
C2 Recon 280 2798.8 10 1.10 ± 0.16 0.69 ± 0.03 8.97 ± 0.21
C3 Recon 280 2799.4 10 0.98 ± 0.01 0.70 ± 0.02 9.14 ± 0.02
(NQ: not quantified)
5. Pads of each replicate were combined and extracted to provide 20 mg TPM/ml in
DMSO.
6. Each participant received 4 aliquots of each condensate extract (0.5 ml each), for a total
of 36 vials (4 aliquots x triplicate smokings x 3 cigarettes).
7. Smoke condensates were coded as follows:
A1: Cigarette A Replicate 1
A2: Cigarette A Replicate 2
A3: Cigarette A Replicate 3
B1: Cigarette B Replicate 1
B2: Cigarette B Replicate 2
B3: Cigarette B Replicate 3
C1: Cigarette C Replicate 1
C2: Cigarette C Replicate 2
C3: Cigarette C Replicate 3
8. Each participating laboratory determined which cell line(s) to use.
9. Work sheets were sent to each participating laboratory for recording experimental
information and data.
5. Results – Raw Data
Laboratories provided experimental information, raw data, and summary data using the
provided worksheets. Summary results as provided by each individual laboratory are reported
in Appendix A. Raw data are available upon request.
The following table summarizes the cell lines used by different laboratories. Nine
laboratories used CHO cells, 3 used Balb/c 3T3 cells, 2 used HEP-G2 and 2 used A549 cells.
IVT–CTR NRU Proficiency Study – November 2015 6/30
Table 3 - LAB Codes and Tested Cell Lines
Lab Code Cell Lines
Lab A CHO-K1
Lab B CHO-K1 Balb/c
Lab C Balb/c 3T3
Lab D CHO Balb/c 3T3
Lab E CHO-K1
Lab F CHO HEP-G2
Lab G CHO-K1 HEP-G2
Lab H CHO-K1
Lab I CHO A549
Lab J CHO-WBL
Lab K A549
Total 9 3 2 2
6. Statistical Analysis and Interpretations
Data analysis was performed with consideration to the following aspects of the study data:
• Reported concentration ranges
• Determination of EC50 values
• Statistical Power – Minimum Detectable Difference (MDD) δ
• Repeatability and Reproducibility analysis (r&R)---for CHO cells only (9 laboratories)
• Comparison of Samples – Relative Potency Plots
Further details are available upon request.
6.1 Reported concentration ranges
Participating laboratories reported the following concentration ranges for testing.
Table 4 - Reported concentration ranges
Lab Code Cell Lines Concentration Range Reported (μg TPM/mL)
Lab A CHO-K1 <25, 50, 75, 100, 125, 150, 175, 200>
Lab B CHO-K1 <10, 20, 30, 50, 75, 100, 125, 150>
Balb/c <10, 20, 30, 50, 75, 100, 125, 150>
Lab C Balb/c 3T3 <160, 140, 120, 100, 80, 60, 40, 20>
Lab D CHO <200, 175, 150, 125, 100, 75, 50, 25>
Balb/c 3T3 <200, 175, 150, 125, 100, 75, 50, 25>
Lab E CHO-K1 <200, 160, 140, 120, 100, 75, 50, 10>
Lab F CHO <2.5, 10, 24, 38, 52, 66, 80, 100>
HEP-G2 <1, 4, 9.6, 15.2, 20.8, 26.4, 32, 40>
Lab G CHO-K1 <300, 200, 100, 75, 50, 25, 12.5, 5>
IVT–CTR NRU Proficiency Study – November 2015 7/30
Lab Code Cell Lines Concentration Range Reported (μg TPM/mL)
HEP-G2 <300, 200, 100, 75, 50, 25, 12.5, 5>
Lab H CHO-K1 <80, 70, 60, 50, 40, 30, 20, 10>
Lab I CHO <10, 25, 35, 50, 65, 80, 100, 125>
A549 <25, 50, 100, 150, 200, 250, 300, 400>
Lab J CHO-WBL <20, 40, 50, 60, 80, 100, 125, 150>
Lab K A549 <200, 181.14, 164.07, 148.6, 134.59, 121.9, 110.41, 100>
6.2 Determination of EC50 values
The EC50 is defined as the concentration that causes a response halfway between minimum
and maximum responses.
The following approach was used to determine the EC50 values.
- Fit a sigmoidal dose-response curve and determine the best fit values for the log EC50, the
Hill slope and the bottom and top plateaus“, of a 4 parameter nonlinear regression model.
- Done with Built-in function “DoseResp“,
- Software ORIGIN 8.0 SR4, OriginLab Corporation
There is evidence that absolute absorbance results of edge wells are influenced by diffuse
light. Therefore, those measurements are not included in the calculations. Data points
remaining for the estimation of regression parameters are the mean of 6 values per
concentration and plate.
Please note, when evaluating the results of the nonlinear regression fit, it is important to check
afterwards whether the parameter values make sense.
The following plots show the EC50-values ± 0.95 CI for all samples and cell lines, as they
were reported by the laboratories and determined with the formula and software described
above.
Overall, reported and determined EC50-values do not diverge too much. Observed differences,
especially those in width of the confidential interval of the mean EC50 may result from
different data selection, outlier exclusion or different model assumptions (e.g. fixing upper
and lower plateau within the nonlinear regression) in the respective laboratories. Nevertheless,
the absolute level of EC50 was reproducible in almost all cases, indicating a high standard of
data evaluation of NRU data within tobacco industry.
IVT–CTR NRU Proficiency Study – November 2015 8/30
Figure 1 - Reported vs. determined EC50. Sample A. CHO cells
Figure 2 - Reported vs. determined EC50. Sample B. CHO cells
IVT–CTR NRU Proficiency Study – November 2015 9/30
Figure 3: Reported vs. determined EC50. Sample C. CHO cells
Figure 4: Reported vs. determined EC50. Sample A-C. BALB/c-cells
B C D B C D B C D
0
25
50
75
100
125
150
Sample CSample B
BALB/c
me
an
EC
50
µg
/mL
(+
/- 0
.95
CI)
LAB Code
EC50 reported by LAB
EC50 determined
Sample A
IVT–CTR NRU Proficiency Study – November 2015 10/30
Figure 5 - Reported vs. determined EC50. Sample A-C. HEP-G2 and A549 cells.
The following two plots show the mean EC50 values for Samples B and C in relation to the
respective EC50 of Sample A.
For CHO cells all laboratories found Sample B and Sample C to be more cytotoxic than
Sample A. Eight out of nine laboratories found a cytotoxicity ranking of B > C > A at the
EC50 level.
Figure 6 - Comparison of Samples for CHO cells, normalized with Sample A
Samples B and C were found to be more cytotoxic than Sample A in Balb/c cells. A contrary
effect is found in HEP-G2 cells. In A549 cells the difference between all samples at the EC50
level is least distinct.
F G F G F G I K I K I K
0
25
50
75
100
125
150
175
200
225
A B C
A549HEP G2
me
an
EC
50
µg
/mL
(+
/- 0
.95
CI)
LAB Code
EC50 reported by LAB
EC50 determined
A B C
A B -- D E F G H I J --
0
20
40
60
80
100
Comparison of Samples CHO cells
EC
50 (
µg
/mL)
perc
ent of S
am
ple
A
LAB Code Sample A (100%)
Sample B
Sample C
IVT–CTR NRU Proficiency Study – November 2015 11/30
Figure 7 - Comparison of Samples for Balb/c, HEP G2 and A549 cells, normalized with Sample A.
6.3 Statistical Power – Minimum Detectable Difference (MDD) δ
Using a two-tailed t-test the question can be answered how different the mean EC50 values of
two samples must be before a difference would be detected. The sample size is three
replicates (n=3), the significance level is set to α=0.05 and we want to have a 90% chance
(power) of detecting a difference, if one exists.
[1] νβ(1),να,
2
ptt
n
2sδ
In above formula [1] the t parameters are derived from the t-distribution table and the
variances (s2) are pooled variance across samples.
The example given in Figure 8 can be interpreted as follows. If two Samples’ EC50 values
differ at least in 15 units (ABS(EC50A- EC50B)≥15) (y-axis) than the LAB would need at least
3 replicates (x-axis) to detect this difference with a t-test. To detect a difference between two
samples of 10, the LAB would need at least 4 replicates.
B C D F G I K
0
20
40
60
80
100
120
A549HEP-G2
Comparison of Samples Balb/c, HEP G2 and A549 cells
EC
50
(µ
g/m
L)
pe
rce
nt o
f S
am
ple
A
LAB Code
Sample A (100%)
Sample B
Sample C
Balb/c
IVT–CTR NRU Proficiency Study – November 2015 12/30
Figure 8 - Example of Minimum Detectable Difference (MDD) plot.
In the following MDD plots are displayed for all four cell lines.
Figure 9 - Minimum Detectable Difference (MDD) per LAB for CHO cells.
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
0
5
10
15
20
25
30
LAB X
Min
imu
m D
ecte
cta
ble
Diffe
ren
ce
(M
DD
)
be
twe
en
EC
50
va
lue
s o
f tw
o s
am
ple
s
number of replicate tests
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
0
10
20
30
40
50
60
Min
imum
Decte
cta
ble
Diff
ere
nce (
MD
D)
be
twee
n E
C50
valu
es o
f tw
o s
am
ple
s
number of replicate tests
LAB A LAB G
LAB B LAB H
LAB D LAB I
LAB E LAB J
LAB F
CHO Cells
IVT–CTR NRU Proficiency Study – November 2015 13/30
Figure 10 - Minimum Detectable Difference (MDD) per LAB for BALB/c cells
Figure 11 - Minimum Detectable Difference (MDD) per LAB for HEP G2 cells
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
0
10
20
30
40
50
60
BALB/c Cells
Min
imum
Decte
cta
ble
Diffe
ren
ce (
MD
D)
be
twee
n E
C50
valu
es o
f tw
o s
am
ple
s
number of replicate tests
LAB B
LAB C
LAB D
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
0
10
20
30
40
50
60
HEP G2 Cells
Min
imum
Decte
cta
ble
Diffe
ren
ce (
MD
D)
be
twee
n E
C50
valu
es o
f tw
o s
am
ple
s
number of replicate tests
LAB F
LAB G
IVT–CTR NRU Proficiency Study – November 2015 14/30
Figure 12 - Minimum Detectable Difference (MDD) per LAB for A549 cells
As one would expect, increasing the number of replicates decreases the detectable difference
between samples. Nevertheless, the effect flattens and three replicates is a reasonable number
to detect a difference between samples of 20%.
e.g. with CHO cells the average EC50 values are between ~40-60µg/mL for the test samples.
The majority of laboratories would then need three replicates to detect a difference of
10µg/mL (approx. 20%) in EC50.
6.4 Repeatability and Reproducibility analysis (r&R)
Per request of Study participants, r&R analysis of the CORESTA in vitro TF NRU
Collaborative Study was performed for CHO cells; r&R analyses could not be conducted for
the other cell lines since the number of LAB EC50 results for other cell lines is ≤ 3. r&R
analysis was conducted following the statistical model provided by ISO 5725-2 (1994) “basic
method for the determination of repeatability and reproducibility of a standard measurement
method”. ISO 5725-2 (1994) provides methods to calculate mean values and standard
deviations for repeatability (sr) and reproducibility (sR), with outliers removed. First step is
the determination of straggling (0.95) and outlying (0.99) LAB results using graphical and
numerical outlier detection techniques. Final r&R values are then derived using outlier
removed data.
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
0
10
20
30
40
50
60
70
A549 Cells
Min
imum
Decte
cta
ble
Diffe
ren
ce (
MD
D)
be
twee
n E
C50
valu
es o
f tw
o s
am
ple
s
number of replicate tests
LAB I
LAB K
IVT–CTR NRU Proficiency Study – November 2015 15/30
Figure 13 - MANDEL’s h. Graphical outlier detection techniques.
MANDEL’s h is a “between-laboratory consistency” technique, indicating a deviation of the
laboratory mean when compared with the general mean. LAB D delivered outlying (0.99)
EC50 values for Samples A and C, and one straggling (0.95) result for Sample B.
Figure 14 - MANDEL’s k. Graphical outlier detection techniques.
MANDEL’s k-statistic is a “within-laboratory consistency statistic” which indicates a
measurement deviation in one laboratory when compared with the pooled laboratory standard
deviation. LAB J delivered straggling (0.95) within laboratory standard deviations for
Samples A-C.
In ISO 5725-2 (1994), COCHRAN’s test is recommended for examining the within-
laboratory consistency, and GRUBBS’ test for examining the between-laboratory consistency,
numerically. COCHRAN’s test failed to detect any straggler or outlier. GRUBBS’ test
identified LAB D Sample C as an outlier.
33,02
37,60
60,86
84,12
88,69
18,25
22,93
46,77
70,60
75,28
19,98
24,79
49,23
73,68
78,49
LAB A LAB B -- LAB D LAB E LAB F LAB G LAB H LAB I LAB J --
30
40
50
60
70
80
90
Sample B
me
an
EC
50 (
µg
/mL
)
MANDEL's h CHO cells LAB mean EC50
(µg/mL)
LAB A LAB B -- LAB D LAB E LAB F LAB G LAB H LAB I LAB J --
20
30
40
50
60
70
80
me
an
EC
50 (
µg
/mL
)
Sample A
LAB A LAB B -- LAB D LAB E LAB F LAB G LAB H LAB I LAB J --
20
30
40
50
60
70
80
LAB mean
overall mean
0.95 straggling limit
0.99 outlying limit
Sample C
me
an
EC
50 (
µg
/mL
)
16,65
14,06
10,54
8,90
12,52
10,57
LAB A LAB B -- LAB D LAB E LAB F LAB G LAB H LAB I LAB J --
0,0
2,5
5,0
7,5
10,0
12,5
15,0
17,5
20,0Sample B
LA
B S
TD
EV
MANDEL's k CHO cells LAB STDEV of EC50
(µg/mL)
LAB A LAB B -- LAB D LAB E LAB F LAB G LAB H LAB I LAB J --
0,0
2,5
5,0
7,5
10,0
12,5
LA
B S
TD
EV
Sample A
LAB A LAB B -- LAB D LAB E LAB F LAB G LAB H LAB I LAB J --
0,0
2,5
5,0
7,5
10,0
12,5
15,0
LAB STDEV
0.95 straggling limit
0.99 outlying limit
Sample C
LA
B S
TD
EV
IVT–CTR NRU Proficiency Study – November 2015 16/30
All results of graphical and numerical outlier detection techniques are summarized in the
following table. Both graphical outlier detection techniques are more likely to detect an
outlier than their corresponding numerical technique. To avoid excessive data exclusion, only
those outliers detected by GRUBBS’ or COCHRAN’s test were removed.
Table 5 - Summary of outlying and straggling results
LAB mean (EC50) LAB STDEV (EC50)
MANDEL's h GRUBBS' MANDEL's k COCHRAN's
Sample A outlier (0.99) LAB D
straggler (0.95) LAB J
Sample B outlier (0.99)
straggler (0.95) LAB D LAB J
Sample C outlier (0.99) LAB D* LAB D*
straggler (0.95) LAB J
* discarded
After removal of outlying data (LAB D, Sample C), the mean, repeatability standard deviation
sr, repeatability r, reproducibility standard deviation sR and reproducibility R were calculated
from the remaining data following the statistical model given in ISO 5725-2.
The final r&R values are listed in the following table. RSDr and RSDR were calculated as r
and R in percent of the mean.
Table 6 - Final r&R values for Samples A-C. Cell line: CHO
SAMPLE MEAN EC50
ug/ML sr sR r R RSDr RSDR N
Sample A 60.9 8.4 14.7 23.4 41.3 38 68 9
Sample B 46.8 5.3 14.1 14.8 39.4 32 84 9
Sample C 45.0 5.8 7.5 16.1 21.1 36 47 8
Please note that the exclusion of Sample C of LAB D leads to a reverse cytotoxicity ranking
in terms of mean EC50 values (Sample C more cytotoxic than Sample B). This reverse
ranking should not be overestimated; both samples are close to each other, but clearly
distinguished from Sample A. Additionally, the exclusion of Sample C, LAB D values leads
to smaller R values, when compared with the R values of Sample A and Sample B.
The following graphs show LAB mean EC50 ± r&R for Samples A-C.
IVT–CTR NRU Proficiency Study – November 2015 17/30
Figure 15 - EC50 (µg/mL) ± r&R for Sample A.
Figure 16 - EC50 (µg/mL) ± r&R for Sample B.
LAB A LAB B -- LAB D LAB E LAB F LAB GLAB H LAB I LAB J --
0
25
50
75
100
125
150 LAB EC50 (µg/mL)
repeatability r
reproducibility R
EC
50
(µ
g/m
L)
± r
&R
SAMPLE A
LAB A LAB B -- LAB D LAB E LAB F LAB GLAB H LAB I LAB J --
0
25
50
75
100
125
150 LAB EC50 (µg/mL)
repeatability r
reproducibility R
EC
50
(µ
g/m
L)
± r
&R
SAMPLE B
IVT–CTR NRU Proficiency Study – November 2015 18/30
Figure 17 - EC50 (µg/mL) ± r&R for Sample C.
6.5 Comparison of Samples – Relative Potency Plots
To address the question whether a particular parameter (log(EC50)) differs between samples,
several approaches can be used. T-tests between log(EC50) values are a common approach.
The power of this approach for the present data is addressed in a previous part.
The results of an alternative approach are presented in the following. Global nonlinear
regression (sharing fit parameters across datasets) is used to check whether the EC50 values
(or any other EC level) of two samples are the same. The idea is to turn the question of
comparing curves (or EC values) into questions of model comparison. (H. J. Motulsky, et al.,
2003)
Figure 18 - Example of a Relative Potency Plot.
LAB A LAB B -- LAB D LAB E LAB F LAB GLAB H LAB I LAB J --
0
25
50
75
100
125
150
LAB EC50 (µg/mL)
repeatability r
reproducibility R
EC
50
(µ
g/m
L)
± r
&R
SAMPLE C
LAB D outlier
(excluded)
10 25 50 75 90
0,0
0,5
1,0
1,5
2,0
Sample A
Sample is less cytotoxic than Sample A
rela
tive
po
ten
cy
EC level
Sample is more cytotoxic than Sample A
Interpretation of "Relative Potency" plots
IVT–CTR NRU Proficiency Study – November 2015 19/30
The interpretation of relative potency plots is as follows: in relative potency plots, the
reference sample A (K3R4F) is set to 1.0, equaling 100%. The relative potency of test
samples is checked at various EC-levels, ranging from EC5 to EC95 (step 5, x-axis). This
results in a relative potency line for the test sample of interest, compared with the reference
sample. Relative Potency is displayed in percentages, and is therefore easy to interpret.
If the line is in the upper part of the plot, the test sample is more cytotoxic than the reference
cigarette. If it is in the lower part, the test sample is less cytotoxic. In the above example, the
test sample is about 10-20% more cytotoxic than Sample A, depending on the EC-level
observed.
One major advantage of this approach is that the (relative) cytotoxic potency of test samples is
not limited to one single EC-level (i.e. EC50) but can be checked at various levels, and thus
concentrations. For example, the relative potency line of test samples may cross the line of
the reference sample, meaning that the test samples are less cytotoxic in low concentrations
and more cytotoxic in high concentrations, relative to the reference sample (see results for
A549 cells, Figure 24 below). This may provide a deeper insight in underlying biological
processes.
Figure 19 - Relative Potency Plot for CHO cells.
The relative potency plots for CHO cells indicate that both, Base Web cigarettes (Sample B)
and Reconstituted cigarettes (Sample C), have a higher cytotoxic potency than the reference
sample K3R4F.
Sample A
Sample A
Sample A
10 25 50 75 90
0,0
0,5
1,0
1,5
2,0
rela
tive
po
ten
cy
EC Level
LAB A10 25 50 75 90
0,0
0,5
1,0
1,5
2,0
LAB B
rela
tive
po
ten
cy
EC Level
10 25 50 75 90
0,0
0,5
1,0
1,5
2,0
LAB D
rela
tive
po
ten
cy
EC Level
10 25 50 75 90
0,0
0,5
1,0
1,5
2,0
LAB E
rela
tive
po
ten
cy
EC Level
C>B>A
10 25 50 75 90
0,0
0,5
1,0
1,5
2,0
LAB F
rela
tive
po
ten
cy
EC Level
10 25 50 75 90
0,0
0,5
1,0
1,5
2,0
LAB G
rela
tive
po
ten
cy
EC Level
10 25 50 75 90
0,0
0,5
1,0
1,5
2,0
LAB H
rela
tive
po
ten
cy
EC Level
Relative Potency plots for CHO cells
10 25 50 75 90
0,0
0,5
1,0
1,5
2,0
LAB I
rela
tive
po
ten
cy
EC Level
10 25 50 75 90
0,0
0,5
1,0
1,5
2,0
Sample A Sample B Sample C
LAB J
rela
tive
po
ten
cy
EC Level
IVT–CTR NRU Proficiency Study – November 2015 20/30
Figure 20 - Relative Potency Plot for BALB/c, HEP-G2 and A549 cells.
In Balb/c cells, both test samples are more cytotoxic than the K3R4F. It seems that HEP-G2
cells react differently, with the reference sample being most cytotoxic. Finally, in A549 cells
the relative potency of test samples in clearly dependent on the respective EC-level. At the
EC50-level all three test samples are equal in cytotoxic potency.
6.6 Summary of findings
CHO cells: 9 laboratories
• All 9 laboratories could differentiate between Sample A and Sample B
• All 9 laboratories could differentiate between Sample A and Sample C
• 6 out of 9 laboratories (66.7%) could differentiate between Sample B and Sample C
• 8 out of 9 laboratories (88.9%) found a Cytotoxicity Ranking of B>C>A
• 1 laboratory found Sample C to be more cytotoxic than Sample B
Balb/c cells: 3 laboratories
• All 3 laboratories could differentiate between all Samples
• 2 laboratories found a Cytotoxicity Ranking of B>C>A
• 1 laboratory found a Cytotoxicity Ranking of C>B>A
HEP-G2 cells: 2 laboratories
• Both laboratories found Sample A to be more cytotoxic than the other samples
A549 cells: 2 laboratories
• Cytotoxic Potency was dependent on the respective EC-level. Samples A-C almost equal
at EC50-level.
Sample A
Sample A
Sample A
10 25 50 75 90
0,0
0,5
1,0
1,5
2,0
LAB B
rela
tive
po
ten
cy
EC Level
10 25 50 75 90
0,0
0,5
1,0
1,5
2,0
LAB C
rela
tive
po
ten
cy
EC Level
10 25 50 75 90
0,0
0,5
1,0
1,5
2,0
LAB D
rela
tive
po
ten
cy
EC Level
10 25 50 75 90
0,0
0,5
1,0
1,5
2,0
LAB F
rela
tive
po
ten
cy
EC Level
10 25 50 75 90
0,0
0,5
1,0
1,5
2,0
LAB G
rela
tive
po
ten
cy
EC Level
10 25 50 75 90
0,0
0,5
1,0
1,5
2,0
Relative Potency plots for Balb/c, HEP-G2 and A549 cells
LAB I
rela
tive
po
ten
cy
EC Level
10 25 50 75 90
0,0
0,5
1,0
1,5
2,0
LAB K
Sample A Sample B Sample C
A549
HEP-G2
rela
tive
po
ten
cy
EC Level
Balb/c
IVT–CTR NRU Proficiency Study – November 2015 21/30
Cell lines # of laboratories Cytotoxic Potency
(based on EC50 results)*
CHO 9 All: B > A; C > A
8 laboratories: B > C > A
6 laboratories: B > C
Balb/c 3T3 3 2 labs: B > C > A
1 lab: C > B > A
HEP-G2 2 A > B and C
A549 2 No significant differences
* Note: samples are ranked in terms of cytotoxic potential, not actual EC50 value. For
example, using CHO cells, B is more cytotoxic than A. The actual EC50 value for B is
lower than A---the 50% point was achieved at a lower concentration of B than with A,
indicating greater cytotoxicity of B.
7. Discussion and Results
7.1 Data Audit
• Data sets were reviewed against the Study protocol to determine if the assays had been
carried out as agreed.
• Data sampling procedure used was based on British Standard 6001-4:2005.
• Information related to the generation and distribution of test items was not included in the
audit (conditioning, smoking, nicotine + water analysis, storage and distribution)
7.2 Audit Findings
Some differences in methodology were observed. These differences included technical
details (i.e., plate washing, preparation of desorb solution, time of sample preparation, plate
layout) and analytical details (i.e., number of blank wells, calculation of relative absorbency,
outlier identification). The specific impact of these differences is unknown, but the majority
of the audit findings were minor and considered unlikely to impact the final results overall.
Further details of the audit findings are available upon request.
8. Conclusions
Participants generally followed the prescribed study protocol. Deviations were minor and
were considered unlikely to have a significant impact on the study outcome. Various cell
lines were employed by the laboratories. With CHO cells in particular, the data showed good
reproducibility both within and between the laboratories.
When applying the NRU assay, various cell lines can be employed for the testing of CSC
cytotoxicity, and participating laboratories used a variety of cell lines. Differences were
found in the ability of specific cell lines to discriminate between samples. Laboratories may
choose specific cell lines for a variety of reasons, including the following:
• metabolic capability
• growth characteristics
IVT–CTR NRU Proficiency Study – November 2015 22/30
• multi-functional utility
• regulatory guidelines
For the specific purpose of the proficiency trial, using the same cell line should lead to less
variability between laboratories. However given the individual needs of a particular
laboratory, this may not always be possible. The Sub-Group did not determine whether a
specific cell line is advantageous or generally recommended.
Results of the study indicate that the assay can be efficiently and successfully performed. The
NRU assay is applicable to assess the cytotoxicity of cigarette smoke condensate, and can be
used as a tool for comparing cytotoxicity.
9. References
1. Borenfreund, E. & Puerner, J. A. (1985). A simple quantitative procedure using
monolayer cultures for cytotoxicity assays (HTD/NR-90). Journal of Tissue Culture
Methods, 9, 7-9.
2. Test Method Protocol for the BALB/c 3T3 Neutral Red Uptake Cytotoxicity Test, a Test
for Basal Cytotoxicity for an In Vitro Validation Study, Phase III. Based on Standard
Operating Procedure Recommendation from an International Workshop Organized by the
Interagency Coordinating Committee on the Validation of Alternative Methods
(ICCVAM) http://iccvam.niehs.nih.gov/methods/acutetox/acutetox.htm
3. Minimum Detectable Difference for Two-Sample t-Test for Mean, Equation adapted
from Zar (1984). Biostatistical analysis. Prentice-Hall, Inc. Englewood N.J.
4. H. J. Motulsky, et al. (2003). Fitting models to biological data using linear and nonlinear
regression. A practical guide to curve fitting., GraphPad Software Inc., San Diego
IVT–CTR NRU Proficiency Study – November 2015 23/30
Appendix A: Summary Data for Each Laboratory
LCI (lower confidence interval) and UCI (upper confidence interval) were collected and
provided as optional data and the CIs were not used for the comparison between samples.
Regarding the comparison result, please see original conclusion.
A-1. Lab A (CHO-K1)
Sample Replicate EC50 Mean STD CV% LCI UCI
A
1 61.89
63.69 2.25 3.53 58.1 69.3 2 62.97
3 66.21
B
1 38.78
38.76 1.93 4.98 34 43.5 2 36.82
3 40.67
C
1 41.35
43.15 5.26 12.18 30.1 56.2 2 39.04
3 49.08
Ratio A/B Ratio A/C Ratio B/C
Replicate 1
1.60 1.50 0.94
Replicate 2
1.71 1.61 0.94
Replicate 3
1.63 1.35 0.83
A-2.1. Lab B (CHO-K1)
Sample Replicate EC50 Mean STD CV% LCI UCI
A
1 - 48.91 6.86 14.03 - -
2 - 53.36 11.45 21.46 - -
3 - 46.22 2.62 5.66 - -
B
1 - 34.94 2.45 7.03 - -
2 - 32.52 3.52 10.81 - -
3 - 28.35 2.21 7.78 - -
C
1 - 38.67 1.34 3.47 - -
2 - 46.7 2.86 6.12 - -
3 - 35.24 0 0 - -
Ratio A/B Ratio A/C Ratio B/C
Replicate 1
1.40 1.26 0.90
Replicate 2
1.64 1.14 0.70
Replicate 3
1.63 1.31 0.80
IVT–CTR NRU Proficiency Study – November 2015 24/30
A-2.2. Lab B (Balb/c)
Sample Replicate EC50 Mean STD CV% LCI UCI
A
1 - 117.25 5.63 4.8 - -
2 - 91.31 5.59 6.13 - -
3 - 96.41 0 0 - -
B
1 - 80.71 3 3.72 - -
2 - 59.83 2.89 4.83 - -
3 - 61.52 3.96 6.43 - -
C
1 - 101.38 5.24 5.17 - -
2 - 70.25 3.56 5.07 - -
3 - 76.72 4.77 6.22 - -
Ratio A/B Ratio A/C Ratio B/C
Replicate 1
1.45 1.16 0.80
Replicate 2
1.53 1.30 0.85
Replicate 3
1.57 1.26 0.80
A-3. Lab C (Balb/c 3T3)
Sample Replicate EC50 Mean STD CV% LCI UCI
A
1 0.076
0.076 0.002 2.019 0.072 0.079 2 0.077
3 0.074
B
1 0.059
0.056 0.004 6.438 0.047 0.065 2 0.057
3 0.052
C
1 0.063
0.064 0.002 3.27 0.059 0.069 2 0.066
3 0.062
Ratio A/B Ratio A/C Ratio B/C
Replicate 1
1.29 1.21 0.94
Replicate 2
1.35 1.17 0.86
Replicate 3
1.42 1.19 0.84
IVT–CTR NRU Proficiency Study – November 2015 25/30
A-4.1. Lab D (CHO)
Sample Replicate EC50 Mean STD CV% LCI UCI
A
1 73.47
89.38 13.8 15.44 - - 2 96.55
3 98.11
B
1 65.32
75.25 9.98 13.27 - - 2 75.14
3 85.28
C
1 72.97
82.11 8.41 10.24 - - 2 83.85
3 89.51
Ratio A/B Ratio A/C Ratio B/C
Replicate 1
1.12 1.01 0.90
Replicate 2
1.28 1.15 0.90
Replicate 3
1.15 1.10 0.95
A-4.2. Lab D (Balb/c)
Sample Replicate EC50 Mean STD CV% LCI UCI
A
1 61.35
70.12 16.73 23.86 - - 2 59.61
3 89.41
B
1 60.22
61.77 13.69 22.17 - - 2 48.92
3 76.17
C
1 42.55
53.48 11.23 21 - - 2 52.89
3 64.99
Ratio A/B Ratio A/C Ratio B/C
Replicate 1
1.02 1.44 1.42
Replicate 2
1.22 1.13 0.92
Replicate 3
1.17 1.38 1.17
IVT–CTR NRU Proficiency Study – November 2015 26/30
A-5. Lab E (CHO-K1)
Sample Replicate EC50 Mean STD CV% LCI UCI
A
1 - 42.36 3.73 8.8
- - 2 - 42.53 1.93 4.54
3 - 40.85 2.65 6.49
B
1 - 51.38 4.11 7.99
- - 2 - 41.52 7.33 17.64
3 - 38.29 3.24 8.47
C
1 - 31.63 0.96 3.05
- - 2 - 27.9 3.07 11.01
3 - 28.07 1.17 4.16
Ratio A/B Ratio A/C Ratio B/C
Replicate 1
0.82 1.34 1.62
Replicate 2
1.02 1.52 1.49
Replicate 3
1.07 1.46 1.36
A-6.1. Lab F (CHO)
Sample Replicate EC50 Mean STD CV% LCI UCI
A
1 39.81
42.67 2.917 6.835
38.85 40.79
2 42.56 41.57 43.58
3 45.64 44.57 46.74
B
1 35.01
38.477 3.01 7.822
34.1 35.95
2 40 39.16 40.86
3 40.42 39.43 41.44
C
1 34.07
38.99 4.313 11.062
33.29 34.87
2 40.78 39.9 41.68
3 42.12 41.24 43.01
Ratio A/B Ratio A/C Ratio B/C
Replicate 1
1.14 1.17 1.03
Replicate 2
1.06 1.04 0.98
Replicate 3
1.13 1.08 0.96
IVT–CTR NRU Proficiency Study – November 2015 27/30
A-6.2. Lab F (Hep-G2)
Sample Replicate EC50 Mean STD CV% LCI UCI
A
1 17.43
14.36 2.679 18.65
16.97 17.91
2 12.48 12.09 12.89
3 13.18 12.75 13.62
B
1 21.15
18.92 2.205 11.657
20.62 21.68
2 16.74 16.33 17.16
3 18.87 18.44 19.31
C
1 17.27
16.68 2.04 12.23
16.81 17.75
2 14.41 14.08 14.75
3 18.36 17.84 18.91
Ratio A/B Ratio A/C Ratio B/C
Replicate 1
0.82 1.01 1.22
Replicate 2
0.75 0.87 1.16
Replicate 3
0.7 0.72 1.03
A-7.1. Lab G (CHO-K1)
Sample Replicate EC50 Mean STD CV% LCI UCI
A
1 72.1
69.86 5.69 0.08 - - 2 63.39
3 74.09
B
1 46.11
45.25 0.98 0.02 - - 2 44.19
3 45.46
C
1 46.94
44.13 2.46 0.06 - - 2 42.33
3 43.13
Ratio A/B Ratio A/C Ratio B/C
Replicate 1
1.56 1.54 0.98
Replicate 2
1.43 1.5 1.04
Replicate 3
1.72 1.72 1.05
IVT–CTR NRU Proficiency Study – November 2015 28/30
A-7.2. Lab G (Hep-G2)
Sample Replicate EC50 Mean STD CV% LCI UCI
A
1 14.94
27.14 15.80 0.58 - - 2 44.98
3 21.49
B
1 41.32
33.33 7.75 0.23 - - 2 32.83
3 25.85
C
1 21.2
28.46 9.73 0.34 - - 2 39.51
3 24.67
Ratio A/B Ratio A/C Ratio B/C
Replicate 1
0.36 0.7 1.95
Replicate 2
1.37 1.14 0.83
Replicate 3
0.83 0.87 1.05
A-8. Lab H (CHO-K1)
Sample Replicate EC50 Mean STD CV% LCI UCI
A
1 48.2/54.5/52.0/48.6 50.82 2.98 5.87 47.90 53.78
2 51.4/48.4/44.8/36.5 45.28 6.44 14.23 38.96 51.59
3 49.3/60.1/54.0/59.1 55.63 4.99 8.97 50.73 60.52
B
1 31.7/36.0/29.0/32.0 32.18 2.89 8.97 29.35 35.00
2 32.0/28.8/28.9/30.0 29.93 1.49 4.97 28.47 31.38
3 32.7/29.7/29.2/30.0 30.40 1.57 5.16 28.86 31.94
C
1 34.0/34.2/36.3/36.9 35.35 1.47 4.15 33.91 36.79
2 33.2/37.8/38.8/34.8 36.15 2.60 7.19 33.60 38.70
3 36.5/34.7/36.3/36.9 36.10 0.97 2.68 35.15 37.05
Ratio A/B Ratio A/C Ratio B/C
Replicate 1
1.58 1.44 0.91
Replicate 2
1.51 1.25 0.83
Replicate 3
1.83 1.54 0.84
IVT–CTR NRU Proficiency Study – November 2015 29/30
A-9.1. Lab I (CHO-K1)
Sample Replicate EC50 Mean STD CV% LCI UCI
A
1 63.30
59.90 11.05 18.45
62.02 64.61
2 68.85 67.77 69.94
3 47.55 46.74 48.38
B
1 50.97
46.79 6.37 13.61
50.14 51.81
2 49.94 49.27 50.62
3 39.46 38.87 40.06
C
1 53.83
51.78 6.58 12.70
52.87 54.82
2 57.08 56.29 57.88
3 44.42 43.73 45.11
Ratio A/B Ratio A/C Ratio B/C
Replicate 1
1.24 1.18 0.95
Replicate 2
1.38 1.21 0.87
Replicate 3
1.20 1.07 0.89
A-9.2. Lab I (A549)
Sample Replicate EC50 Mean STD CV% LCI UCI
A
1 158.16
163.84 15.80 9.64
153.52 162.95
2 181.69 177.27 186.22
3 151.65 148.07 155.32
B
1 160.02
160.42 13.44 8.38
156.33 163.79
2 174.06 170.75 177.42
3 147.18 143.93 150.50
C
1 146.58
151.94 11.66 7.67
142.67 150.59
2 165.32 161.81 168.91
3 143.94 140.43 147.53
Ratio A/B Ratio A/C Ratio B/C
Replicate 1
0.99 1.08 1.09
Replicate 2
1.04 1.10 1.05
Replicate 3
1.03 1.05 1.02
IVT–CTR NRU Proficiency Study – November 2015 30/30
A-10. Lab J (CHO-WBL)
Sample Replicate EC50 Mean STD CV% LCI UCI
A
1 46.80
66.93 17.57 26.25
46.14 47.47
2 74.81 72.47 77.24
3 79.18 76.34 82.12
B
1 46.82
56.85 10.19 17.92
46.14 47.51
2 67.19 65.28 69.17
3 56.53 55.45 57.63
C
1 42.66
56.90 12.47 21.92
40.78 44.63
2 65.90 64.71 67.11
3 62.14 60.95 63.34
Ratio A/B Ratio A/C Ratio B/C
Replicate 1
1.00 1.10 1.10
Replicate 2
1.11 1.14 1.02
Replicate 3
1.40 1.27 0.91
A-11. Lab K (A-549)
Sample Replicate EC50 Mean STD CV% LCI UCI
A
1 172.20
152.6 26.1 17.1 - - 2 123.00
3 162.50
B
1 140.50
134.5 9.3 7.0 - - 2 123.70
3 139.20
C
1 151.90
148.5 9.1 6.2 - - 2 138.10
3 155.40
Ratio A/B Ratio A/C Ratio B/C
Replicate 1
1.23 1.13 0.92
Replicate 2
0.99 0.89 0.90
Replicate 3
1.17 1.05 0.90