Validation Article IV. CF assay

Altern. Animal Test. EXperiment. 5, 74-86 (1998)

Validation Study on Five Cytotoxicity Assays by JSAAE
IV.
Details of the Colony Formation Assay
Noriho Tanaka1, Masumi Asakura2, Chiharu Hattori3, Akira Hayasaka4, Makoto
Hayashi5, Takashi Hayashi6, Hiroshi Hori7, Hiroyoshi Hoshi8, Ikuo Imazeki4,
Takuya Ishibashi7, Hiroshi Itagaki9, Miyako Kakuma10, Shinya Kaneda11, Mayako
Kato1, Akio Kawakami12, Akihiko Kido13, Michiyo Kitazawa14, Hajime Kojima15,
Daijyo Maki16, Chihomi Mitsuoka16, Satoru Miyazaki17, Fumio Mizuno13, Matsuko
Moriyasu16, Madoka Nakajima14, Nahoko Nakano16, Shinobu Nakamura16,
Tamotsu Nishitomi13, Tadao Ohno*18, Takashi Omori19, Hiroshi Ono1, Makoto
Ono10, Yuko Osanai10, Kaoru Saijo18, Tetsuji Sasaki18, Hidetaka Sato20, Shinichiro
Sato21, Hiroyasu Shimada3, Kazuyuki Shimono11, Hideaki Sugawara17, Yasuko
Sugiki 22, Shigeru Sugimoto 20, Junko Suzuki 20, Ken Takahashi 23, Minoru
Takizawa24, Jirou Taniya25, Noriko Teramoto18, Toshiyuki Tsuchiya26, Motoo
Uejima11, Hayao Ueno27, Yuji Ugai14, Shinobu Wakuri1, Xinhai Wang18, Masanori
Yoshida27, Isao Yoshimura19, Kouichi Yuhki25
1

Hatano Research Institute, Food and Drug Safety Center, 729-5 Ochiai, Hadanoshi, Kanagawa 257, 2Japan Bioassay Laboratory, 2445 Hirasawa, Hadano-shi,
Kanagawa 257, 3Developmental Research Laboratory, Daiich PharmaceuticalCo., Ltd., 1-16-13 Kita-Kasai, Edogawa-ku, Tokyo 134, 4Chugai PharmaceuticalCo., Ltd., 3-41-8 Takada, Toshima-ku, Tokyo 171, 5Division of Genetics and Mutagenesis, National Institute of Health Sciences (NIHS), 1-18-1 Kamiyoga,
Setagaya-ku, Tokyo 158, 6Ina Research Inc., 8047 Nishiminowa, Ina-shi, Nagano399-45, 7Tsuruga Institute of Biotechnology, Toyobo Co., Ltd., 10-24 Toyo-cho,
Tsuruga-shi, Fukui 914, 8Research Institute for the Functional Peptides, 11-26
Minamisanban-cho, Yamagata-shi 990, 9Shiseido Safety & Analytical ResearchCenter,1050 Nippa-cho, Kohoku-Ku, Yokohama-shi 223, 10Beauty Care Products
Laboratory, Nagase, Co., Ltd., 5-1 Kobuna-cho, Nihonbashi, Chuo-ku, Tokyo
103, 11Naruto Research Institute, Otsuka Pharmaceutical Factory, Inc., 115
kuguhara, Tateiwa, Muya-cho, Naruto-shi, Tokushima 772, 12Safety Research* Correspondence : The Working Group of JSAAE for the
1st Validation Study on Cytotoxicity Assays, c/o T.
Ohno, RIKEN Cell Bank, 3-1-1 Koyadai, Tsukuba Science City, Ibaraki 305, Japan
Key words: alternatives, colony formation assay, cytotoxicity assay, inter-laboratory validation

Abbreviations: CF, colony formation; ED50, 50% effective dose; JSAAE, Japanese Society of Alternatives to++++Animal Experiments; PBS(-), Ca , Mg -freeDulbecco’s phosphate-buffered saline; PFD, power for
distinction.
Received: July 4, 1997. Accepted: September 10, 1997.

– 74 –

Laboratories, Yamanouchi Pharmaceutical Co., Ltd., 1-1-8 Azusawa, Itabashiku, Tokyo 174, 13Kashima Laboratory, Mitsubishi-kagaku Institute of Enviromental
and Toxicological Sciences, 14 Sunayama, Hasaki-machi, Kashima-gun, Ibaraki
314-02, 14Biosafety Research Center,Foods, Drugs and Pesticides, 582-2 Arahama,
Shioshinden, Fukude-cho, Iwata-gun, Shizuoka 437-12, 15Biochemical ResearchInstitute, Nippon Menard Cosmetic Co., Ltd., 2-7 Torimi-cho, Nishi-ku, Nagoyashi 451, 16Panapharm Laboratories Co., Ltd., 1285 Kurisaki-cho, Uto-shi,
Kumamoto 869-04, 17Life Science Research Information Laboratory, The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako-shi,
Saitama 351-01, 18RIKEN Cell Bank, The Institute of Physical and Chemical
Research (RIKEN), 3-1-1 Koyadai, Tsukuba Science City, Ibaraki 305, 19Department of Management Science, Faculty of Engineering, Science University of Tokyo, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162, 20Japan Food Research Laboratories, 6-11-10 Nagayama, Tama-shi, Tokyo 206, 21Division of Radiation Medicine,National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba-shi
263, 22Menicon Co., Ltd., 5-1-10 Takamoridai, Kasugai-shi, Aichi 487, 23The Institute of Environmental Toxicology, 4321 Uchimoriya-cho, Mitsukaido-shi,
Ibaraki 303, 24Hakko Electric Machine Works Co. Ltd., 3055 Togura, Toguramachi, Hanishina-gun, Nagano 389-08, 25Koken Bioscience Institute, Koken Co.
Ltd., 2-11-12 Nakane, Meguro-ku, Tokyo 152, 26Safety Evaluation Center, Central Research Laboratory, Showa Denko K. K., 1-1-1 Ohnodai, Midori-ku, Chibashi 267, 27Takeda Analytical Research Laboratories, Ltd., 2-17-85 Juso-honmachi,
Yodogawa-ku, Osaka 532, Japan
Abstract
The inter-laboratory validation study on 5 cytotoxicity assays conducted by
JSAAE has been described in the preceding articles in this issue. Presented here
are precise data and the protocols on the colony formation (CF) assay with twocell lines, HeLa S3 (SC) that is common to the other 4 assays and BALB/3T3 thatis frequently used in this assay. The CF assay showed high performance rates
with both HeLa S3 (SC) and BALB/3T3 A31-1-1 cell lines. Almost all submitted
data files were acceptable before ED50 calculation in this assay. Variations on
negative controls in the assay revealed technical differences among laboratories
and the characteristics of cells applied. The CF assay with BALB/3T3 A31-1-1
cells resulted in the lowest median values of log(ED50) for Tween 20, Tween 80
and cetylpyridinium chloride monohydrate. For propylene glycol assayed with
HeLa S3 (SC) cells, the lowest median value of log(ED50) was observed. Thus
the CF assay was able to detect with the highest sensitivity the least toxic chemical (#4) and the most severely toxic chemical. CF assay with HeLa S3 (SC) cells
gave the largest power for distinction when observing the most and the least toxic
chemicals. However, the toxicities of moderately toxic chemicals were not sharply
distinguishable from each other. Comparing the mean hinge-spreads, CF assay– 75 –

Validation Article IV. CF assay

with BALB/3T3 A31-1-1 cells showed the largest variation of the log(ED50)
values among all assay systems examined in this study. These results suggest that
the CF assay is a highly sensitive assay but is influenced by many factors such as
culture conditions and techniques of cell handling.
Introduction
As described in the preceding papers (seeValidation Article I in this issue), the JapaneseSociety of Alternatives to Animal Experiments(JSAAE) organized a first step inter-laboratory
validation study on 5 cytotoxicity assays in October 1992, since a battery of appropriate cytotoxicity assays is expected to be the core alternative to the in vivo Draize test (Watanabe et al.,
1989; Ohno et al., 1995).
Colony formation (CF) assay measures reproductive death through clonal cell growth. The
CF assay is known to be a highly sensitive
method because it involves long-term exposure
to chemicals and requires only low cell density(Clemedson et al., 1996a and b; Sasaki andTanaka, 1991; Sasaki et al., 1991). For these reasons, the CF assay has been adopted in the Japanese guidelines for medical devices (Tsuchiya
et al., 1993). On the other hand, it has some disadvantages, e.g., the assay takes about 7-14 days
to complete and needs much incubator space.
This report supplements Validation Article I (thefirst article in this issue). We describe here precise results of the CF assay carried out in thevalidation study, including the protocols, representative hand-plotted dose-response curves,
extra ordinal data files, ED50 values on eachaccepted data files, and discussions.

masked reference chemical). The symbol # andnumbers in parentheses are double-mask codes
given to these chemicals by the Chemical Bankand the Working Group before their transfer toeach laboratory. The two samples (#1 and #7)
were the same chemical and were supplied todetermine intra-laboratory variation of the finaldata.
In addition to the common HeLa S3 (SC) cells
from human cervix carcinoma, BALB/3T3 A311-1 cells from mouse embryo were used in thisCF assay. Both cell lines were supplied fromRIKEN Cell Bank, Tsukuba Science City.
Data analysis
The submitted data files were first inspectedmanually to detect simple recording errors. Thenhand-plotted dose-response curves drawn fromthe mean values at each chemical concentration
were checked visually. The ED50 values of possibly acceptable candidate data files (see Validation Article I, Tables 3 and 5) were analyzed
by computer-assisted LAP-JSAAE program (seeValidation Article II) which is based on calculation of dose-response by the non-linear leastsquares method. Finally, general statistical analyses were applied to the calculated data.
Protocol of the CF assay

Materials and Methods
Chemicals and Cell Lines
The six chemicals tested and their allocation
to 7 coded samples were described in the preceding paper (see Validation Article I). They were
Tween 20 (#1, non-irritant), Tween 80 (#2, nonirritant), sucrose fatty acid ester (#3, irritant),
propylene glycol (#4, non-irritant), cetylpyridinium chloride monohydrate (#5, severe irritant), sodium lauryl sulphate (#6, moderate irritant) and Tween 20 (#7, same as #1, an intrinsic

1. Materials
1-1. Cultures(1) Culture medium
1) HeLa S3 (SC) cells: Eagle’s minimum essential medium (MEM, autoclavable, containing 1.8 mg/ml of NaHCO3 and pH 7.3- 7.6) + 10% (v/v) calf serum (CS).
2) BALB/3T3 A31-1-1 cells: MEM (autoclavable, containing 1.8 mg/ml of NaHCO3
and pH 7.3 - 7.6) + 10% (v/v) fetal bovine
serum (FBS).

– 76 –

(2) Subculture for maintenance
HeLa S3 (SC) cells used in the assay werein log phase. Subculture was performed withthe dissociation medium consisting of 0.05%
trypsin and 0.02% EDTA dissolved in Ca++,
Mg++-free Dulbecco's phosphate-buffered saline (PBS(-)). After dissociation of the cells,
2x105 cells were seeded into a 60-mm dishcontaining 5 ml of culture medium. HeLa S3(SC) cells were subcultured every 3 - 4 days.
BALB/3T3 A31-1-1 cells were subcultured at 7 day intervals. The cells (100-150 x
104 cells/ 60-mm dish) in log phase weretreated with 0.25% trypsin in PBS(-), then 1015 x 104 cells were seeded in a 60-mm dishcontaining 5 ml of culture medium.
1-2. Reagents(1) PBS(-)(2) Dissociation medium: 0.05% trypsin and
0.02% EDTA dissolved in PBS(-), 0.25%
trypsin in PBS(-).
(3) Methanol(4) Giemsa solution diluted to approximately
5-10%
2. Test chemical preparation
Test chemicals #1, 2, 4, 5, 6 and 7 were dissolved in PBS(-), sterilized by membrane filtration and then serially diluted with PBS(-).
Chemical #3 was dissolved in DMSO. The final
concentrations of organic solvents in the culture
medium were kept at 0.5% or less.
Since the concentration range employed was
different for each chemical, at first the practically attainable maximum concentration wasdetermined and diluted serially, then the assay
was carried out with a wide dose range covering
approximately 5-6 orders of magnitude. From
the results of this preliminary test, the maximum
concentration was set at the lowest concentration able to kill 100% of the cells, and this concentration was then diluted serially. The 2 to 3
orders of magnitude of the test concentration
range were covered by this serial dilution. Finally the assay was repeated to be able to obtain
more than 3 points with a cytotoxic effect occurring between 20-80% of the maximum cytotoxicity.

3. Procedure for the CF assay
3-1. Cell seeding(1) Cells at approximately 80% confluence wereselected, i. e. logarithmically growing cells.
After discarding the culture medium andwashing the cells with 2 ml of PBS(-), thecells were dissociated by adding 2 ml of the
dissociation medium that was immediately
aspirated. After remaining for approximately
5 min at room temperature, cells were suspended in 5 ml of PBS(-) and transferred to a
15-ml centrifuge tube containing 5 ml of theculture medium. The cells were collected bycentrifugation at 1000 rpm (140 x g) for 3
min.
(2) For HeLa S3 (SC) cells: A cell suspension
containing 25 cells/ml in culture medium was
prepared. An aliquot (4 ml) of this suspension, was added to each 60-mm dish, i.e., 100
cells/dish. To avoid uneven attachment of thecells, the dish was kept undisturbed for 20
min at room temperature. Then the cells were
cultured for 24 hr in a CO2-incubator at 37°C.
For BALB/3T3 A31-1-1 cells: The cells
were plated in 60-mm dishes in the samemanner as HeLa S3 (SC) cells.
3-2. Treatment
After the 24-hr incubation, 20 µl of the test
chemical solution was added to each dish withvery gentle agitation. The dishes were cultured
without medium change for a further 13 days
with HeLa.S3 (sc) cells or 7 days with BALB/
3T3 A31-1-1 cells.
3-3. Fixing and Staining(1) The culture medium was discarded.
(2) Cells were fixed by adding 3-4 ml of methanol for at least 5 min.
(3) After fixing, cells were stained with the Giemsa solution for at least 15 min.
(4) The solution was discarded and the cells wererinsed with tap water.
(5) Colonies which consisted of more than 50
cells in each dish were counted.
3-4. Worksheet for data collection
A sample of a worksheet (Fig. 1) for data collection was shown to each laboratory. However,– 77 –

Validation Article IV. CF assay

Fig. 1. An example of the worksheet for data collection for the CF assay
Note that each laboratory was allowed to make their own style of the worksheet with fixed
positions of raw data recording. These worksheets were formed on personal computer softwares.
All the worksheets were first transferred to MS-DOS text files and incorporated to the software
Excel version 4.0, and then recalculated.

each laboratory was allowed to make their own
style of the worksheet with fixed positions for
raw data recording. These worksheets wereformed on personal computer software. All theworksheets were first transferred to MS-DOS
text files and incorporated to the software Excel– 78 –

version 4.0, and then recalculated.
4. Calculation of cytotoxicity
Plating efficiency (PE) was defined as: (number of colonies/number of cells plated) x 100%.
Cytotoxicity was expressed in percentage of relative viability (or survival) which was defined as(mean PE in the treated dishes at a chemical concentration / mean PE in the control dishes) x
100%. This value was submitted for the ED50
calculation in the LAP-JSAAE program.
Results and Discussion
Quality of raw data
To understand the characteristics of raw data
and resulting dose-response curves in all submitted data files, hand-plotted dose-response
curves* on mean values at each chemical concentration were drawn. At this preliminary step,
we found that Lab-12 and Lab-35 had carriedout the assay using FBS instead of CS for HeLa
S3 (SC) cells; that is a violation of the protocol
for this assay. Their data files (7 files for eachlaboratory) were not accepted for further analyses. No other violation was found in the CF assay (see Validation Article I, Table 3).
Fig. 2 illustrates two examples of the handplotted dose-response curves of CF assays. Titles
indicate the cell line used and the code numberof tested chemical. The plots obtained provided
information difficult to assume from calculated
ED50 values and accompanying outputs fromthe logistic analysis program, LAP-JSAAE. CF
assays with HeLa S3 (SC) and BALB/3T3 A311-1 cells showed clear S-shape curves as compared with other assays. As shown in Fig. 2a,the dose-response curves drawn from the data
file of Lab-36 and Lab-24 were apparently outliers. The ED50 derived from the data file of Lab36 was an extremely high value. After the meeting on raw data confirmation, Lab-36 was allowed to amend their simple error in recording
the data of HeLa S3 (SC) to 1/200 of that which
they initially submitted.
* Copies of all the hand-plotted dose-response curves areavailable on request.

The data on chemical #1 submitted from Lab24 showed an extremely low ED50 value in theassay with HeLa S3 (SC) cells compared withthe value for chemical #7 which is the samechemical, Tween 20. As shown in Fig 2b, thehand-plotted dose-response curves of the CF
assay with BALB/3T3 A31-1-1 cells varied considerably depending on the laboratory. BALB/
3T3 A31-1-1 cells showed a wide range of toxicity responses with chemical #5, cetylpyridinium chloride monohydrate, the most toxic
among the 6 chemicals used in the present validation. Extra ordinal dose-response curves were
evident from the data files of Lab-24 and Lab31 (Fig 2b).
We excluded data files containing any abnormality according to the flow chart as described(see Fig. 1 in Validation Article I), then acceptable data files were analyzed by the LAP-JSAAE
program. The data files on BALB/3T3 A31-1-1
cells submitted from Lab-36, which were once
accepted after amendment as described above
for HeLa S3 (SC) cells, were finally rejected by
the LAP-JSAAE program.
Characteristics of submitted data files andlog(ED50) values
Fig. 3 illustrates the characteristics of accepted data files of CF assays with log(ED50)
values. Among the 24 laboratories that participated initially in CF assay, two (Lab-43 and Lab47) did not submit any data for the assay witheither cell line, and three (Lab-4, Lab-17 andLab-31) did not submit any data with HeLa S3(SC) cells. The ratio of ED50s of chemical #1
and #7 exceeded 5-fold in Lab-24 and, therefore, all the data files for chemical #1 - #7 as aset was rejected (see Validation Article I, Table
6).
The data of Lab-4, Lab-12 and Lab-36 wereincomplete. Many data files on the chemical #4,
propylene glycol, in the assay with both cell lines
were lacking. This may be due to extremely low
toxicity of chemical #4. The protocols for the
CF assay required that the maximum concentration of organic solvents should be 0.5% or less.
The solvent concentrations used for chemical #4– 79 –

Fig. 2. Hand-plotted dose response curves of the CF assays at the submission of data files
Hand-plotted dose response curves were drawn to understand the gross characteristics of data files at their submission to The Working Group. a (this page), CF assays with chemical#1 (Tween 20) on HeLa S3 (SC) cells. b (next page), CF assays with chemical #5 (cetylpyridinium chloride monohydrate) on BALB/3T3 A31-1-1 cells. Points represent the mean of
viability for an observed concentration in an assay. The figure in an open symbol indicates the laboratory number.

Validation Article IV. CF assay– 80 –

– 81 –

Validation Article IV. CF assay

Fig. 3. Log(ED50) values and visualized characteristics of submitted data files
Open boxes represent the files finally accepted for comparison of inter-laboratory variation of log(ED50) values without any difficulty in obtaining ED50 values. Asterisks indicate that the log(ED50) value became an
outlier in the box-wisker plot analysis shown in Fig. 5
No data file was submitted.
A data file(s) was submitted but not useful because of severe violation of the protocol.
A check code was found in data files of chemical #1 or #7; then all corresponding data file sets were discarded.
The ratio of ED50s of chemical #1 and chemical #7, or vise versa, was over 5.
A check code was found in data files of chemical #2, #3, #4, #5, or #6.
Data files accepted after amending simple recording errors on reported concentration of chemicals after the
discussion meeting held on November 30, 1994.
Definition of error codes are;Code-A : Calculation of an ED50 value failed because of wide variation of data.
Code-C : No observed point was found in the data file bewteen 20-80% of the maximum effect.
Code-E : Of the 95% confidence limits of ED50, the upper limit was over 100 times that of the lower limit.
Code-F : RMS is 10 or more. RMS indicates the degree of deviation of the observed dose-response relationshipfrom the logistic model. See details in Validation Article II in this issue.

– 82 –

Hela S3 (SC)BALB/3T3 A31-1-1

60

80
70

50

60
No. of dishes

No. of dishes

40
30
20

50
40
30
20

10

90

100

80

70

60

50

40

30

20

10

120

110

100

90

80

70

60

50

40

30

20

0

0

10

0

0

10

Plating efficiency (%)

Plating efficiency (%)

Fig. 4. Distribution of plating efficiencies in negative controls obtained from raw data of the CF assay
a, left, HeLa S3 (SC) cells. b,right, BALB/3T3 A31-1-1 cells.

were not enough to observe cytotoxic responses.
The data files of chemical #4 were accepted fromonly 8 laboratories per cell line.
Due to protocol violation, data files from Lab12 and Lab-35 on HeLa S3 (SC) cells were excluded from analysis. Overall, for assays on
HeLa S3 (SC) cells and BALB/3T3 A31-1-1
cells, performance rates were calculated to be
77% and 89%, respectively (see Validation Article I, Table 3).
The capital letters in shaded boxes in Fig. 3
are check codes attached by the LAP-JSAAE
program for calculation of ED50 values. Mostof the rejected files on chemical #4 were marked
with the code C and code A. There was no data
file marked with the check code B and D. Thus,
a total of 100 files for HeLa S3 (SC) cells and
126 files for BALB/3T3 A31-1-1 cells were finally accepted for further box-whisker-plot
analysis (Fig. 4).
Technical stability of the CF assay in participating laboratories
Among the 24 laboratories that participatedinitially, 15 received the technology transfer (seeTable 2 of Validation Article I in this issue). In
one of these laboratories, the recipient did notconduct the CF assay but instead a technician
did the work. Six laboratories (Lab-5, -12, -17, 24, -28, and -33) did not receive the technology
transfer but performed the assay. Lab-43 and 47 did receive the technology transfer but did

not submit any data.
To determine technical stability of the CF
assay within a laboratory, the variation in thenegative controls was calculated on each assay(Table 1). With HeLa S3 (SC) cells, Lab-45 had
carried out the assays with the largest variation
for negative controls. Their coefficient of variation was 49.6% (see CV-1 in Table 1) in the 4assays for the 7 chemicals with the average plating efficiency 71.6%; the plating efficiency of
their negative controls varied from 18.5 to 91.6%(Table 2, see Mean). Within assay dish lots, thecoefficients of variation were between 8.6% and14.5% with a mean of 11.1% for the 4 assays(see CV-2 in Table 2). This tendency, i.e., thelarger variation among assays for different
chemicals and the smaller variation among thedishes in an assay, was not necessarily the casefor the negative controls observed in other laboratories.
With HeLa S3 (SC) cells, the mean value ofplating efficiencies of all participants was 70.1%(Table 1). With BALB/3T3 A31-1-1 cells, themean value of plating efficiencies of all participants was slightly less at 53.8% (Table 1).
When numbers of dishes with a range of negative control plating efficiency values were plotted from the CF assays with HeLa S3 (SC) and
BALB/3T3 A31-1-1 cells, characteristic distributions were observed for the two cell lines (Fig.
4). With HeLa S3 (SC) cells, a bimodal distribution was observed with two peaks at 10% and
80% plating efficiencies, while normal distribu-– 83 –

Validation Article IV. CF assay

Table 1. Variation of the negative controls in CF assays

Lab.
No.

5
10
11
12
13
14
17
20
24
27
28
30
31
33
35
36
37
39
40
45
46

HeLa S3 (SC) cells
Assays

PEa
(%)

1
7
3

BALB/3T3 A31-1-1 cells

SDb

CV-1c CV-2d Assays PE
(%)
(%)
(%)

47.4
90.6
87.2

3.9
5.7

4.3
6.5

5.8
10.8

6
5

99.0
19.3

4.5
0.7

4.5
3.6

11.1
5.5

4

86.2

2.4

2.8

9.2

4
1
3

73.1
93.5
61.6

6.4

8.8

9.7

6.6

10.7

15.2

4

81.1 13.4

16.5

8.5

6
3
6
1
4
3

20.6 2.8
74.2 10.5
40.7 1.0
93.8
71.6 35.5
82.4 4.5

13.6
14.2
2.5

18.3
7.1
6.3

49.6
5.5

Mean

70.1 25.4

11.0

SD

CV-1
(%)

CV-2
(%)

2
6
3
5
6
5
2
5
4
4
1
3
5
3
3

44.6
68.3
46.2
41.4
44.5
39.6
35.6
72.4
81.5
52.5
48.5
64.7
85.5
51.7
55.8

1.4
7.9
9.6
9.8
9.4
1.9
0.9
7.9
17.2
0.8

3.1
11.6
20.8
23.7
21.1
4.8
2.5
10.9
21.1
1.5

6.3
7.7
14.0
6.1
14.1
4.3
6.6
10.2
6.2
20.6

4.9
6.6
15.8
2.1

7.6
7.7
30.6
3.8

12.2
8.0
12.6
10.8

11.1
8.7

3
6
2
4
4

51.1
65.7
27.4
39.9
59.7

5.7
9.4
2.1
14.7
7.0

11.2
14.3
7.7
36.8
11.7

5.4
5.1
8.6
15.0
8.4

9.8

Mean

53.8

15.3

13.3

9.6

a Mean plating efficiency of the negative controls in the assays
b Standard deviationc Coefficient of variation among independent assays calculated from the mean negative control
value of each assay
d Coefficient of variation among the negative control dishes within an assay: Mean of the assays

tion with a peak at 50% was observed with
BALB/3T3 A31-1-1 cells. In the CF assay, theplating efficiency of BALB/3T3 A31-1-1 cells
was greatly affected by serum lot. Therefore, weused the same lot of FBS with BALB/3T3 A311-1 cells but did not use the same lot of CS for
HeLa S3 (SC) cells. This is one of the possible
reasons for the normal distribution profile foundwith the plating efficiencies of BALB/3T3 A311-1 cells, since almost the same participants performed the CF assay using both HeLa S3 (SC)and BALB/3T3 A31-1-1 cells.

Effect of cell lines
As shown in the box-whisker-plots oflog(ED50) (Fig. 5), BALB/3T3 A31-1-1 cells exhibited higher sensitivity than HeLa S3 (SC) cells
to cytotoxic chemicals except for chemical #4
in the CF assay. Nine and five outliers were observed in the CF assays with HeLa S3 (SC) cells
and BALB/3T3 A31-1-1 cells, respectively (Fig.
5). As mentioned in Validation Article I in thisissue, the CF assay with HeLa S3 (SC) cells gavethe largest "power for distinction" (PFD) of 22,
while this assay with BALB/3T3 A31-1-1 cells– 84 –

Table 2. Intra-laboratory variation of the negative controls in CF assays with
HeLa S3 (SC) cells carried out in Lab-45.

Plating efficiency in each dish (%)
Assay forChemical D-1

D-2

D-3

D-4

D-5

D-6

#1, 6, 7
#2
#4
#5

95
89
79
19

89
88
82
18

78
89
80
15

101
94
21

88
88
20

93
71
118
18

Meand
SDd
CV-1e

D-7

89
20

D-8

Meana

SDb

CV-2c

103
17

88.8
87.7
91.6
18.5

7.6
9.6
13.3
1.9

8.6
10.9
14.5
10.4

71.6

11.1

35.5
49.6

a Mean plating efficiency of the negative controls in each assay
b Standard deviationc Coefficient of variation (%) among the negative control dishes within an assay
d Calculated from the four assays
e Coefficient of variation among independent assays calculated from the mean negative
control value of each assay

gave the value of 13.4. The value of PFD will be
helpful as an indicator to distinguish cytotoxicities of individual chemicals tested in the assay.
Conclusion
As has been described in the first article ofthis issue, the CF, CV, MTT, and NR assays arerecommendable from the view point of performance of the assays. From the view point of inter-laboratory variation, the CF assay with
BALB/3T3 A31-1-1 cells was not necessarily thebest one. However, the CF assay with HeLa S3(SC) cells was the best from the view point of
the "power for distinction" of toxicities amongchemicals.
Acknowledgment
This work was supported in part by a fundfrom JSAAE and in part by Special Coordination Funds for Promoting Science and Technology from The Science and Technology Agencyof Japan.

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Validation Article IV. CF assay

Log(ED50)
-8

-7

-6

-5

-4

-3

-2

-1

0

1

2

3

♯1
HeLa S3 (SC)BALB/3T3 A31-1-1

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♯7
33

HeLa S3 (SC)BALB/3T3 A31-1-1

3♯2
HeLa S3 (SC)BALB/3T3 A31-1-1

3♯3
HeLa S3 (SC)BALB/3T3 A31-1-1

3

♯4
3
3

3

HeLa S3 (SC)BALB/3T3 A31-1-1♯5
3

HeLa S3 (SC)BALB/3T3 A31-1-1

3

3
3

♯6
3

HeLa S3 (SC)BALB/3T3 A31-1-1

Fig. 5. Box-whisker-plots of results of the CF assay
Asterisks indicate outliers of the corresponding box-whisker-plots.

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