Sumitomo Chemicals Co. Ltd. Ver.09E

1
2
3
4
5
6
7
8

Hand1-Luc Embryonic Stem Cell Test
(Hand1-Luc EST)

9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42

Draft protocol ver. 09 E
March, 9th, 2016

Environmental Health Science Laboratory,
Sumitomo Chemical Co., Ltd.
Noriyuki Suzuki, Ph.D.
Florian Le Coz, M.Sc.
Hirohisa Nagahori, Ph.D.
Koichi Saito, Ph.D.

1

Sumitomo Chemicals Co. Ltd. Ver.09E

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34

TABLE OF CONTENTSINTRODUCTION ............................................................................................ 3
MATERIALS AND METHODS ....................................................................... 3
EXPERIMENT 1 .................................................................................... 3
1-1 Measurement of actual value of pKa or in silico method .......... 3
EXPERIMENT 2........................................................................................ 4
2-1 Measurement of ID50 and IC50 values in the Hand1-Luc EST ..... 4
A. Materials ........................................................................................ 4
Cells (Hand1-ES cells) ....................................................................... 4
Reagents and preparation methods ................................................... 4
Equipments ........................................................................................ 6
Expendable supplies .......................................................................... 6
B. Experimental Methods .................................................................. 7
Thawing of Hand1-ES cells ................................................................ 7
Maintenance of cells .......................................................................... 7
Preparation of test chemicals ........................................................... 8
Serial dilution of test chemicals and arrangement of plate format ...... 9Dose finding study ........................................................................... 10
Definitive study ................................................................................ 13
Data analysis ................................................................................... 13
Acceptance criteria .......................................................................... 14
EXPERIMENT 3 (optional prediction with PM2) ................................ 15
3-1 Measurement of metabolic stability of test chemicals ............. 15
JUDGMENT (Prediction of test chemicals) .............................................. 16
OPTION........................................................................................................ 17
UPDATE RECORD ...................................................................................... 192

Sumitomo Chemicals Co. Ltd. Ver.09E

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24

INTRODUCTION
Hand1-Luc Embryonic Stem Cell Test (Hand1-Luc EST) is a novel short term
test for predicting embryonic toxic chemicals using transgenic engineering
mouse embryonic stem (ES) cells with consideration of pharmacokinetic
property of test chemicals. This protocol describes how to prepare, determine
the pharmacokinetic property and analyze cytotoxicity and differentiation toxicity
of chemicals．

MATERIALS AND METHODSEXPERIMENT1
1-1 Measurement of actual value of pKa or in silico methods
Actual value can be measured by titration, spectrophotometric andconductometric methods. Detailed protocol is shown in OECD Test Guideline(112,
http://www.oecd.org/env/ehs/testing/oecdguidelinesforthetestingofchemicals.htm)
The value can be calculated by commercial software or available tools on theinternet such as ADMET Predictor, ACD/labs or Mervin(http://www.chemaxon.com/marvin/sketch/index.jsp).

3

Sumitomo Chemicals Co. Ltd. Ver.09E

1
2
3
4
5
6

EXPERIMENT 2
2-1 Measurement of ID50 and IC50 values in the Hand1-Luc EST
Schema of the Hand1-Luc EST procedure is described in Figure 1.
Dose finding
study

Definitive
study

Chemical preparation(diluted with ratio 1:10)

Chemical preparation
carry out with a smaller dilution
factor covering the relevant dose
response

Cell preparation

Cell preparation

Addition of thechemicals to the plate

Addition of thechemicals to the plate

Measurement of
2-endpoints

Measurement of
2-endpoints

Pre-culture of the cells
Passage of the cells
Vehicle selection

Day 0

Day 5

7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32

Data analysis

Data analysis

Figure.1 Outline of the Hand1-Luc EST

A. Materials
Cells (Hand1-ES)
Transgenic mouse embryonic stem (ES) cell, named as Hand1-ES,
containing firefly luciferase genes under the control of Hand1 (heart andneural crest derivatives expressed transcript 1) promoters were establishedby Sumitomo Chemical Co., Ltd. as a previous method with minor
modification (Suzuki et al., Toxicol. Sci., 124, 460–471, 2011). An embryonicstem cell line (named as KOB1-ES) from C57/B6 mouse established by
Sumitomo Chemical Co. Ltd. was used for development of the Hand1-ES
cells.
Reagents and preparation methods
For maintenance of Hand1-ES cells
 StemMedium (Serum Free Media for Mouse ES Cell) (DS Pharma
Biomedical, Cat. DSRK100)▲Note Dissolved Dispensed medium is stored at -20 °C (in an appropriate
volume in tubes).
 Gelatin from porcine, type A (SIGMA, Cat. G2500)
 ESGRO mLIF (107 units/mL) (Millipore, Cat. ESG1107)
4

Sumitomo Chemicals Co. Ltd. Ver.09E

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33

34
35

 Distilled water (SIGMA, Cat. W3500-500mL)
 0.25 % Trypsin /1mM EDTA solution (Nacalai tesque, Cat.35554-64)
 50 mg/mL Geneticin Disulfate (G418) solution (Nacalai tesque, Cat.
16513-84)
 Trypan blue solution (SIGMA, Cat.T8154-20ML or equivalents, regardlessof makers, are acceptable)
For chemical exposure, positive control, solvents
 5-Fluorouracil (5-FU) (SIGMA, Cat. F6627-1G, purity 99 %) Dimethyl sulfoxide (DMSO)（MP Biomedicals, Inc. (WAKO Pure Chemicals),Cat. 594-03771）
 PBS(-) (Invitrogen, Cat No. 10010-023 (500mL))
For differentiation or cytotoxicity assay
 DMEM (Invitrogen, Cat. 11965-092)
 FBS (Hyclone, Cat 533-90935, Lot. KTC30729)▲Note The quality of the serum is very important for mESC growthand differentiation. Several lots of serum have to be tested before
performing the assay.
 GlutaMaxI solution (100×conc.) (Invitrogen, Cat. 35050-061)
 100 mM Non-Essential Amino Acid solution (NEAA) (Invitrogen, Cat.
11140-050)
 Penicillin-Streptomycin, liquid (PS solution) (Nacalai tesque, Cat.
26252-94)
 2-Mercaptoethanol (2-ME) (Nacalai tesque, Cat. 21438-82)
 Steady-Glo® Luciferase Assay System（Promega, Cat No. E2510, E2520
and E2550）▲Note Dissolved reagents stored at -80 °C (in an appropriate volume intubes).
 CellTiter-FluorTM Cell Viability Assay（Promega, Cat No. G6080, G6081 andG6082）
Maintenance medium (Culture medium for maintenance of Hand1-ES cells)
Content
VolumeStemMedium
10 mL
100mM 2-ME solution
10 L
ESGRO (107unit/mL)
1 L
50 mg/mL G418 solution
20 L
Note
- Supplemented medium is stored for no longer than 1 week at 4 °C.
- Before use, supplemented medium should be prewarmed to 37 °C ina water bath or incubator.
Assay medium
Content

Volume5

Sumitomo Chemicals Co. Ltd. Ver.09E

1
2

3
4

DMEM
82 mL
FBS (Heat inactivated)
15 mL
GlutaMaxI solution (100× conc.)
1 mL
100mM NEAA solution
1 mL
PS solution
1 mL
100mM 2-ME solution
100 L
Note- Supplemented medium is stored for no longer than 2 weeks at 4 °C.
- Before use, supplemented medium should be prewarmed to 37 °C ina water bath or incubator.
0.1% Gelatin solution
Content
GelatinDistilled water

Volume0.5 g
500 mL

Note- Autoclaved solution is stored for no longer than 2 months at 4 °C.
- 60mm dishes should be coated with gelatin solution (37°C, over
30min).
2-Mercaptoethanol (2-ME) solution
Content
2-ME (14.3 M solution)
Distilled water

Volume
70 L
10 mL

Note
Stock solutions are stored at -80 °C or prepared at time of use.
5
6

7
8
9
10
11
12
13
14
15
16
17
18
19
20

Fetal Bovine Serum, Heat-InactivatedContent
FBS
Note
Incubate at 56 °C for 30 min and store at -20 °C (appropriate volume intubes).
Dissolved FBS is kept at 4 °C and use within 1 month.

Equipments
 Measuring device of micro-plate type luminometer Measuring device of micro-plate type fluorometer Plate shaker
 Water bath
 CO2 incubator (5%, 37°C)
 Single and 8 channel micropipette Appropriate cell counter (hemocytometer)
Expendable supplies 60 mm Cell culture dish (BD Falcon, Cat.353004)
 Storage Plate 96 well Round Bottom (Corning, Cat.3359)
6

Sumitomo Chemicals Co. Ltd. Ver.09E

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50

 96 well Assay Blocks 2mL/well (Corning, Cat.3960)
 PrimeSurface® 96well white plate (Sumitomo Bake Lite, Co., Ltd.
MS-9096W）
 Plate seal (for PrimeSurface® 96well white plate) (Watson, Cat.
547-KTS-HC)
 Plate seal “TopSeal-A+” (PerkinElmer No. 6050185)
 Cell strainer (40m, BD Falcon, Cat.352340)
 Reservoir
 Pipette
B. Experimental Methods
Thawing of Hand1-ES cells
1.
2.
3.

4.
5.

6.

Coat the 60 mm dishes with the 0.1 % gelatin solution. Add 5 ml of thegelatin solution in the dishes and incubate at 37 °C during 30 min.
Thaw frozen cells in 37 °C water bath, and add to the 9 mL of
maintenance medium.
Centrifuge the tube at 900 to 1,400 rpm for 5 min at room temperature,
discard supernatant and resuspend the cells in 5 mL of maintenancemedium.
Count cell number using aliquot of solution.
Remove the gelatin solution from the dishes (step 1) and seed thecells at a concentration of approx. 0.5 to 1.0 ×106 cells/ gelatin-coated
60 mm dish in 5mL of maintenance medium.
Incubate the cells at 37°C in a humidified atmosphere with 5% CO2.

Maintenance of cells
Grown cells should be passaged 2 or 3 days after thawing.
1.
Prepare the 60 mm dishes coated with the 0.1 % gelatin solution
before maintenance of cells.
2.
Remove the supernatant from culture dish, wash with 5 mL of PBS(-).
3.
Add 2 mL of 0.25% trypsin/1 mM EDTA and remove immediately.
Then, cells are incubated at 37 °C for 1 to 2 min.
4.
Add to 2 mL of maintenance medium to the dish, suspend the cells
using micropipette (1000 L).
5.
Count cell number
6.
Remove the gelatin solution from the dishes (step 1)
7.
For assay, seed the cells at a concentration of 2.0×106cells/
gelatin-coated 60 mm dish in 5mL of maintenance medium. Incubate
the cells at 37°C with 5% CO2 for 1 day.
8.
For passage, seed the cells at a concentration of 0.2, 0.5 and1.0×106cells/ gelatin-coated 60 mm dish in 5mL of maintenancemedium. Incubate the cells at 37°C with 5% CO2 for 2 or 3 days. Cells
should be used until the cells reach 80–90% confluence.
▲Note;- Cells should be passed until the cells reach 80–90% confluence.
- Cells should be used within 2 passages.
7Sumitomo Chemicals Co. Ltd. Ver.09E

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50

Preparation of test chemicals, choice of solvent and precipitation evaluation
Test chemicals are dissolved in an appropriate solvent such as PBS(-) andDMSO. The recommended maximum final concentrations are 1 % (v/v) for
PBS(-) or 0.1 % (v/v) for DMSO. The chemicals must be weighed anddissolved in solvent before each experiment, including 5-FU for the
positive-reference control. The highest test concentration of any chemicals is1000 g/mL.
Dilution series of 7 concentrations of 5-FU (0.0003, 0.0006, 0.0013, 0.0025,
0.005, 0.01 and 0.02 mg/mL; with a common ratio of 1:2) as positive-reference
control is dissolved from a 0.02 mg/mL prepared stock solution in PBS(-).
A flowchart for selection of appropriate vehicle, preparation of chemicals andprecipitation evaluation is shown in Figure 2.
Dissolve the test chemical first in PBS(-) to obtain a concentration of 100
mg/mL. For example, weigh 10 mg of the test chemical in an appropriate tube
and add PBS(-) up to 100 L. If the chemical is not soluble at 100 mg/mL,
dilute with a common ratio of 1:2 to obtain a concentration of 50 mg/mL. If thechemical is not soluble at 50 mg/mL, dilute with a common ratio of 1:2 toobtain a concentration of 25 mg/mL. Other final volumes are also possible but
the minimum required one should be 100 L depending on the needs of test
chemical.
If the chemical is not soluble at 25 mg/mL in PBS(-), the chemical should be
dissolved in DMSO at 1000 mg/mL. For example, weigh 100 mg of the test
chemical in an appropriate tube and add DMSO up to 100L. At this step, aprecipitation test should be undertaken as followed: add 1μl of the
DMSO-dissolved chemical in the tube containing 999μl of assay medium. Mix
the medium with the chemical thoroughly by vortex. If the precipitate is notobserved at 1000μg/ml, check water solubility over 1000 μg/ml. then 1000
μg/ml should be the highest concentration tested.
If the chemical is not soluble at 1000 mg/mL, the highest soluble
concentration should be determined by diluting the solution from 500 mg/ml at
a common ratio of two (250 mg/ml -> 125 mg/ml if needed) with DMSO. If thechemical is not soluble at 250 mg/mL in DMSO, prepare solution of test
chemical using PBS(-) or DMSO with 2-fold dilution factor (ex; 125 mg/mL in
DMSO or 12.5 mg/mL in PBS(-) if needed). Other final volumes are also
possible but the minimum required one should be 100 L depending on theneeds of test chemical.
Sonication and vortexing may be used if needed，and attempt to dissolve thechemical for at least 5 minutes.
For each highest concentration where the chemical is soluble, theprecipitation test should be provided. For example, if the chemical is soluble in
DMSO at 500 mg/mL, then from this concentration and with a 2-fold dilution
factor, the possibility of precipitation in the assay medium should be verified.
For this purpose, add 1 μL of the DMSO dissolved chemical in the tube
containing 999 μl of assay medium. Mix the medium with the chemicalthoroughly by vortex.
If a precipitate appears on the tips of the 8 channel micropipette or remains in
8Sumitomo Chemicals Co. Ltd. Ver.09E

1
2
3
4
5
6
7
8

the medium after mixing, then the corresponding concentrations should not betaken into account for the experiment. For example, if the last precipitate isobserved at 250μg/mL then the highest concentration used for the experimentwill be 125μg/mL.
For chemicals that do not dissolve in PBS, the solubility in DMSO should be
carefully evaluated especially for liquid chemicals where small and difficult tosee bubbles may appear.
Stock solutionof chemicals
Prepare 100 mg/mL solution oftest chemical using PBS(-).

Highest concentrationof test chemicals
in the assays

soluble100 mg/mL
in PBS(-)

Not necessary

solubleFinal 1000 g/mL

soluble50 mg/mL
in PBS(-)

Not necessary

solubleFinal 500 g/mL

soluble25 mg/mL
in PBS(-)

Not necessary

solubleFinal 250 g/mL

solubleYes
1000 mg/mL
in DMSO
insoluble

solubleFinal 1000 g/mL

solubleYes
500 mg/mL
in DMSO
insoluble

solubleFinal 500 g/mLYes

solubleFinal 250 g/mL

insoluble
Prepare 50 mg/mL solution oftest chemical using PBS(-).

Precipitation
Evaluation in
assay medium

insoluble
Prepare 25 mg/mL solution oftest chemical using PBS(-).

insoluble
Prepare 1000 mg/mL solutionof test chemical using DMSO.

insoluble
Prepare 500 mg/mL solution oftest chemical using DMSO.

insoluble
Prepare 250 mg/mL solution oftest chemical using DMSO.

insoluble

9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30

soluble250 mg/mL
in DMSO

Prepare solution of test chemical using PBS(-) or DMSO
with 2-fold dilution factor(ex. 12.5 mg/mL in PBS(-) or 125 mg/mL in DMSO)

Figure 2

Vehicle selection and preparation of test chemicals

Serial dilution of test chemicals and arrangement of plate format
Generally, the test chemicals should be prepared as follows;- Maximum concentration (final concentration): 1000 g/mL (depend on thesolubility of test chemicals)- Serially dilute with the same solvent at a common ratio of 1:10, basically inthe dose finding assay. Carry out the definitive study with smaller dilution
factor (ex; 1:1.5, 1:2, 1:3 or 1:5) covering the relevant range of dose
response and as much as possible and if the gap between the IC 50 and
ID50 allows it, the lowest common ratio should be chosen. If IC 50 and ID50
values are close to each other, the IC50 value should be situated in the
middle of the new concentration range.
1.

Prepare dissolved 5-FU as positive-reference control and test
chemicals according to the procedure (described in section“Preparation of test chemicals”) using 96 well microplates (Corning,
Cat. 3359) (cf. Figure 3, row 1A - 1H, 2A - 2H) before assay.
9Sumitomo Chemicals Co. Ltd. Ver.09E

1
Positive
Test
controlchemical(5-FU)
1

2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18

3

A

PBS(-)

B0 . 0 0 0 3 m g /CmoLn c . 1

C

0 . 0 0 0 6 m g /CmoLn c . 2

D

0 . 0 0 1 3 m g /CmoLn c . 3

E

0 . 0 0 2 5 m g /CmoLn c . 4

F0.005 mg/m
CLo n c . 5

G0.01 mg/mL
Conc.6

H

0.02 mg/mL
Conc.7

4

5

6

7

8

9

10 11 12VC
ratio 1:10
ratio 1:10
ratio 1:10
ratio 1:10
ratio 1:10
ratio 1:10

VC: Vehicle control (DMSO or PBS(-))
Figure 32.

3.

Serial dilution and arrangement of test chemicals (1)Add 20 L of diluted 5-FU to the well of 96 well Assay Block containing
980 L of assay medium using an 8 channel micropipette (cf. Figure 4,
row 1A – 1H).
Add 20L of chemicals to the well of 96 well Assay Block containing
980 L of assay medium using an 8 channel micropipette if chemicals
are dissolved in PBS(-) (cf. Figure 4, row 2A – 2H).
Add 2 L of chemicals to the well of 96 well Assay Block containing
998 L of assay medium using an 8 channel micropipette if chemicals
are dissolved in DMSO. Store solution at room temperature (20 30°C) until next steps.
1

19
20
21
22
23
24
25
26
27
28
29
30
31
32

2

A
B
C
D
E
F
G
H

Figure 4

2

3

4

5

6

7

8

9

10 11 12← 9 8 L
0 o r 9 9L8 o f a s s a y m e d i u m

Serial dilution and arrangement of test chemicals (2)Dose finding study
Preparation procedure of cells for the assays.
1.
2.

Dispense 100 L of PBS(-) into the peripheral wells and 100 L of
assay medium into row 11B – 11G (cf. Figure 5).
Prewarm assay medium and PBS(-) at 37 °C. 0.25% Trypsin /1mM
EDTA solution is kept at room temperature (20 - 30°C) by bathing inwater or ambient air.
10

Sumitomo Chemicals Co. Ltd. Ver.09E

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20

3.
4.

Remove the supernatant from culture dish, wash with 5 mL of PBS(-).
Add 1 - 2 mL of 0.25% trypsin/1 mM EDTA and remove immediately.
Then, cells are incubated at 37 °C for 1 - 2 min.
5. Add to 2 mL of assay medium to the dish, and suspend the cells using
a micropipette (1000L).
6. Use cell strainer with 50mL centrifuge tubes to obtain more uniform
single-cell suspensions.
7. Check viability by staining an aliquot of the cell suspension with
Trypan blue.
▲Note A viability of 90 % or higher is acceptable.
8. Count cell number.
9. Dilute the cells in assay medium at cell density of 15,000 cells/mL (750cells /50 l/well).
10. Transfer the cells to a reservoir, and dispense 50 L of cell suspension
to PrimeSurface® 96well white plate (Sumitomo Bake Lite, Co. Ltd.,
MS-9096W） (cf. Figure 5; row 2B – 10G in the plates for 5-FU or test
chemicals).
11. Incubate the cells at 37°C with 5% CO2.

1

2

3

4

5

6

7

8

9

10

11

12

A
B
C
D
E
F
G
H
: Cells

21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37

: 100uL of PBS(-): 100uL of assay medium

Figure 5

Preparation of the cells for the assay

Treatment of the test chemicals for the assays
1. After more than 2 hrs of incubation, mix prepared solution of 5-FU
and test chemicals by pipetting thoroughly.
2. Add 50 Lof assay medium to a plate (cf. Figure 6; row 2B –2G).
3. Dispense 50 L of solution of chemicals to 96 well-plate containing
the cells using an 8 channel micropipette (cf. Figure 6; row 3B–10G).
4. Shake the plate with a plate shaker for a few seconds.
5. Incubate the cells at 37°C with 5% CO2.

11

Sumitomo Chemicals Co. Ltd. Ver.09E

1
A
B
C
D
E
F
G
H

2

3

4

MC

VC

conc.1 conc.2 conc.3 conc.4 conc.5 conc.6 conc.7

MC

VC

conc.1 conc.2 conc.3 conc.4 conc.5 conc.6 conc.7

MC

VC

conc.1 conc.2 conc.3 conc.4 conc.5 conc.6 conc.7

MC

VC

conc.1 conc.2 conc.3 conc.4 conc.5 conc.6 conc.7

MC

VC

conc.1 conc.2 conc.3 conc.4 conc.5 conc.6 conc.7

MC

VC

conc.1 conc.2 conc.3 conc.4 conc.5 conc.6 conc.7: Cells

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40

5

6

7

8

9

10

11

12

: 100uL of PBS(-): 100uL of assay medium

Figure 6 Arrangement of chemicals and vehicles for assay

Measurement of fluorescence and luminescence▲Note Switch on measuring equipments 30 min before starting themeasurement.
120 hours after seeding the Hand1-ES cells, measure the two following
endpoints as described below:
For cytotoxicity assay
1. Completely thaw the CellTiter-Fluor™ Cell Viability Assay components
in a 37°C water bath. Vortex the GF-AFC substrate (▲Note avoid
exposure to light) to ensure homogeneity, then briefly centrifuge forcomplete substrate volume recovery.
2. Transfer the GF-AFC Substrate into the Assay Buffer container toform a complete reagent (cf; Assay buffer 1mL : GF-AFC substrate 10
L) . Mix by vortexing the contents until the substrate is thoroughly
dissolved.
3. Add 10 L of CellTiter-Fluor™ complete reagent to each well (cf.
Figure 6; row 2B - 11G).
4. Mix briefly by orbital shaking, then incubate for at least 30 minutes at
37°C. Seal the surface of the plates.
▲Note Don’t incubate longer than 1hour, and be sure to shield plates
from ambient light.
5. Measure resulting fluorescence using a fluorometer (380–400nmEx/505nmEm).
▲Note You may need to adjust instrument gains.
For differentiation assay
1. Thaw the Steady-Glo® Luciferase Assay System and equilibrate to
25 °C prior to use (▲Note avoid exposure to light).
2. Dispense 100 L of Steady-Glo® Luciferase Assay System to eachwell of 96 well plates by an 8 channel micropipette (cf: Figure 6; row
2B – 11G).
3. Shake the plates gently for over 30 min at room temperature (20 –30 °C) with a plate shaker. Seal the surface of the plates.
12

Sumitomo Chemicals Co. Ltd. Ver.09E

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51

4.

▲Note Don’t incubate longer than 1hour, and be sure to shield plates
from ambient light.
Place the plate in luminometer. Luminescence is measured with theappropriate time (cf. 1sec for validation study) according to the
manufacturer’s instructions.

Definitive study
Calculate endpoints according to Steps “Data analysis”. For the definitive
study, choose seven dilutions. Carry out the definitive study with a smaller
dilution factor covering the relevant range of dose response according todose finding study (see Section of Experimental validity of definitive
studies)

Data analysis
Use an Excel (Microsoft) spreadsheet for data recording (Result format ver.
08E). Calculation method and the acceptance criteria for these assays are
as follows;
Determination of ID50 and IC50 values
Relative viabilities (surviving rate) or relative activities for eachconcentration of test chemicals against vehicle control can be calculatedusing Excel (Microsoft) spreadsheet. Calculation formulas are described asfollows;
For differentiation assay
a : Mean values of vehicle control
b : Mean values of test chemicals
c : Mean values of backgroundd : NET values of vehicle control : a-ce : NET values of test chemicals : b-c
A : Relative activity of vehicle control : 100 (%)
B : Relative activity of test chemicals : e/d X 100 (%)
For cytotocxicity assay
a : Mean values of vehicle control
b : Mean values of test chemicals
c : Mean values of backgroundd : NET values of vehicle control : a-ce : NET values of test chemicals : b-c
A : Relative viability of cells for vehicle control : 100 (%)
B : Relative viability of cells for test chemicals : e/d X 100 (%)
Inhibition of differentiation is expressed as the concentration of the test
chemical that reduced the luminescence by 50% (ES-ID50, calculated fromthe concentration-response curve). Cytotoxicity is expressed as theconcentration of the chemical reducing the viability of cells to 50% of thecontrol level (ES-IC50 determined from concentration-response curves).
The IC50 and ID50 values relative to solvent controls were derived from13

Sumitomo Chemicals Co. Ltd. Ver.09E

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41

computational regression analysis of concentration response curves with athree parameter logistic function using an Excel (Microsoft) spreadsheet fordata recording (Result format ver.08E).
The full concentration-response curve is required for the calculation of the
ID50 and IC50 values, but this may not always be achievable or practical due
to limitations of the test concentration range (for example due to cytotoxicity
or solubility problems).

Acceptance criteria
Quality controli) Quality control of cell condition
Check viability of the cells by staining an aliquot of the cell suspension with
Trypan blue. A viability of 90% or greater is acceptable.
ii) Quality control of differentiation and cytotoxicity assays
To verify the cell growth and cell differentiation, the comparison of themedium control (MC) and background (BG) is used.
- The Lower limit of the 95% confidence intervals of the ratio of MC / BG
should be above 1 for the cytotoxicity assay- The Lower limit of the 95% confidence intervals of the ratio of MC / BG
should be above 10 for differentiation assayiii) Performance standard of the assay
The quality of the assay must be controlled using 5-FU as apositive-reference chemical.
-The range of ID50 for 5-FU should be within 0.003 and 0.067 g/mL(according to phase 0 study).
-The range of IC50 for 5-FU should be within 0.003 and 0.065 g/mL(according to phase 0 study).
iv) Quality control for effect of vehicle
To verify the effect of the vehicle, the comparison of the medium control(MC) and vehicle control (VC) is used.
- The Lower limit of the 95% confidence intervals of the ratio of VC / MCshould be above 0.2 for the cytotoxicity and differentiation assays.
- In addition to the above criterion
CV (SD/mean X 100%) of VC should be below 100%.

14

Sumitomo Chemicals Co. Ltd. Ver.09E

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24

EXPERIMENT 3 (for optional prediction with PM2)
3-1 Measurement of metabolic stability of test chemicals
Liver S9 fractions
Rat liver S9 fractions are commercially available (Male, pool of 400 livers,
XenoTech). The brief procedure for preparation is as follows. A portion of liverfrom male SD rats is homogenized in 4 volumes of Tris/HCl buffer (pH7.4) with ahomogenizer. Liver homogenate is centrifuged at 9,000 × g for about 20 min at
4 °C to separate S9 fractions. The protein level in the S9 fraction can bemeasured by commercially available protein assay kit.
Metabolic stability
Substrate solution is prepared by mixing 100 µM substrate in acetonitrile with
100 fold volume of 6 mM β-NADPH (Oriental Yeast) in 125 mM potassium
phosphate buffer (pH7.4). The reaction is initiated by mixing 50 µL of substratesolution with 50 µL of diluted liver S9 fractions (0.5 mg protein/mL) in 125 mM
potassium phosphate buffer (pH7.4). After incubation for 35 min at 37 ºC, thereaction was stopped by addition of 400 µL methanol. After centrifugation at
3,000 g for 10 min, the supernatant is analyzed by the triple quadrupole LC/MS
and the peak area of the target ion is calculated. The control sample is preparedwithout liver S9 fractions. The metabolic stability is expressed as the ratio of
peak area with liver S9 to that of control.

15

Sumitomo Chemicals Co. Ltd. Ver.09E

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21

22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40

JUDGMENT
In the Hand1-Luc EST, the embryotoxic potency of test chemicals waspredicted using following equation. a biostatistically based prediction model
shown in Figure 7.
Discriminant equation
Prediction Model 1(PM1)
Score = 1.474 × log (1) + 0.714 × log (2) − 1.109
1, log(IC50/ID50) ; 2, log(Maximum dose/IC50)
Probability: inverse logit transform of the score: probability ≥ 0.52
: probability < 0.52

Positive
Negative

Majority judgment of 3 replicates (Phase 2a)
Dose

Definitive

Definitive

Definitive

Finalfinding

study1study2study3

judgment

Case 1

-

P

P

-

P

Case 2

-

N

P

P

P

Case 3

-

N

P

N

N

Case 4

-

N

N

-

N

Case 5

-

P

N

P

P

Case 6

-

P

N

N

N

The dose finding is not considered for the evaluation. If both first definitive
studies conclude negativity or positivity, then only two definitive studies are
required.
However, if definitive study 1 and definitive study 2 have 2 different conclusions,then a 3rd definitive study will be required.
Experimental validity of definitive studies:
If the IC50 and ID50 values of DS1 compared to the IC50 and ID50 values of DS2
are different from a factor 7, then one more test should be done and the 2closest results should be taken into account.
(Example1: If DS1, IC50=10 μg/ml, DS2, IC50=125 μg/ml and DS3, IC50=28 μg/ml
then only the results of DS1 and DS3 should be analyzed.
Example 2: If DS1, IC50=10 μg/ml, DS2, IC50=12 μg/ml and DS3, IC50=128μg/ml then only the results of DS1 and DS2 should be analyzed)
If, again, IC50 and ID50 values of DS3 are higher or lower than a factor 7
compared to DS1 and DS2, then one more last test should be performed. Thisevaluation should be done disregarding the positivity or negativity judgment.
16

Sumitomo Chemicals Co. Ltd. Ver.09E

1
2
3
4
5

OPTION (Experiment 2)
In the Hand1-Luc EST, prediction of the embryotoxic potency of test chemicals
can be improved optionally by measuring metabolic stability (to be submitted).

17

Sumitomo Chemicals Co. Ltd. Ver.09E

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51

UPDATE RECORD
Ver.01E, 2013, Feb, 20th distributions
Ver.02E, 2013, April, 30th distributions
Major modification- Plate format for the assays- Positive reference chemicals- Preparation of test chemicals- Maximum final concentrations of vehicle (PBS(-) and DMSO)- Quality control and acceptability criteria
Ver.03E, 2013, July, 18th distributions- Plate format for the assays- Detection method for cytotoxicity
Ver.04E, 2013, July 23th distributions- Minor modification- Tentative acceptance criteria
Ver.05-1 E, 2013, November 6th distributions
Major modification- Measurement at 120 hr- Tentative acceptance criteria or phase 1 study.
Ver.06-1 E, 2014, March 10th distributions
Major modification:- Measurement of the water solubility, suppression of the measurementof the logP
- Inclusion of a precipitation evaluation step
- Modification of the prediction model-Tentative acceptance criteria modified for phase 2.
Ver.07 E, 2014, May 14th distributions
Minor modification:- Measurement of the water solubility- Modification of the prediction model
Ver.08 E, 2015, June 26th distributions
Minor modification:- According the suggestion of VMT member, calculation method of IC50
and ID50 values using a three parameter logistic function instead of
2-parameter- Modification of the prediction model
Ver.09 E, 2016, March 9th distribution
Minor modifications:- Correction English figure 1- Correction on the top seal product reference number (expendable
supplies section), that has been updated by the supplier.
18

Sumitomo Chemicals Co. Ltd. Ver.09E

1
2
3
4
5
6
7
8
9

- Precisions about dissolution of liquid chemicals in solvent- Figure 2 corrected with insertion of evaluation of precipitation in theassay medium- Precision on dilution ratio that should be chosen for the definitive
studies- Definitive studies’ quality control: IC50 and ID50 of DS1 and theones in DS2 are significantly different.
- Correction of the prediction model.
- Correction of optional experiment for PM2

19