Technical Report Neutral Red Uptake Assay Proficiency Study · 2016-04-26 · IVT–CTR NRU Proficiency Study – November 2015 3/30 1. Summary A Neutral Red Uptake (NRU) assay proficiency

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


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.


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



B1: Cigarette B Replicate 1



C1: Cigarette C Replicate 1



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.


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>


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.


Figure 1 - Reported vs. determined EC50. Sample A. CHO cells

Figure 2 - Reported vs. determined EC50. Sample B. CHO cells


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


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


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


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


LAB A LAB G

LAB B LAB H

LAB D LAB I

LAB E LAB J

LAB F

CHO Cells


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


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


LAB F

LAB G


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


LAB I

LAB K


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)


20

30

40

50

60

70

80

me

an

EC

50 (

µg

/mL

)

Sample A


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


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)


0,0

2,5

5,0

7,5

10,0

12,5

LA

B S

TD

EV

Sample A


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


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.


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


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


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.


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


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


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


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


• 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

http://iccvam.niehs.nih.gov/methods/acutetox/acutetox.htm


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)


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 - -


Replicate 1

1.40 1.26 0.90

Replicate 2

1.64 1.14 0.70

Replicate 3

1.63 1.31 0.80


A-2.2. Lab B (Balb/c)


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 - -


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)


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


Replicate 1

1.29 1.21 0.94

Replicate 2

1.35 1.17 0.86

Replicate 3

1.42 1.19 0.84


A-4.1. Lab D (CHO)


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


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)


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


Replicate 1

1.02 1.44 1.42

Replicate 2

1.22 1.13 0.92

Replicate 3

1.17 1.38 1.17


A-5. Lab E (CHO-K1)


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


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)


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


Replicate 1

1.14 1.17 1.03

Replicate 2

1.06 1.04 0.98

Replicate 3

1.13 1.08 0.96


A-6.2. Lab F (Hep-G2)


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


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)


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


Replicate 1

1.56 1.54 0.98

Replicate 2

1.43 1.5 1.04

Replicate 3

1.72 1.72 1.05


A-7.2. Lab G (Hep-G2)


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


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)


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


Replicate 1

1.58 1.44 0.91

Replicate 2

1.51 1.25 0.83

Replicate 3

1.83 1.54 0.84


A-9.1. Lab I (CHO-K1)


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


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)


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


Replicate 1

0.99 1.08 1.09

Replicate 2

1.04 1.10 1.05

Replicate 3

1.03 1.05 1.02


A-10. Lab J (CHO-WBL)


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


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)


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


Replicate 1

1.23 1.13 0.92

Replicate 2

0.99 0.89 0.90

Replicate 3

1.17 1.05 0.90

Documents

Technical Report Neutral Red Uptake Assay Proficiency Study · 2016-04-26 · IVT–CTR NRU Proficiency Study – November 2015 3/30 1. Summary A Neutral Red Uptake (NRU) assay proficiency