p. 1

Ni -:w 3

Test R,1~,thod Prr3tocul 16r the 1Vf1h A'ez trcrl Red ljptrrl,e Cvtotoxicith-,3sscrs >

Phase III - L'ali<Jatiwl Stxclv:

November 4, 2003

TEST METHOD PROTOCOL

for the NHK Neutral Red Uptake Cytotoxicity Test

A Test for Basal Cytotoxicity for an In Vitro Validation Study

Phase III

November 4, 2003

Prepared by

The National Toxicology Program (NTP) Interagency Center for the Evaluation of

Alternative Toxicological Methods (NICEATM)

Based on Standard Operating Procedure Recommendations from an

International Workshop Organized by the Interagency Coordinating Committee

on the Validation of Alternative Methods (ICCVAM)

National Institute of Environmental Health Sciences (NIEHS)

National Institutes of Health (NIH)

U.S. Public Health Service

Department of Health and Human Services

i
p. 2

Test Met hod PrrJtocol, fnr the ~VHK Neutral Rcrl Uptake ('i7ntosic•itv ,4csar

Phase Ill - L'a1ie/crtiar Shrdv:

November 4. 200 ;

TEST METHOD PROTOCOL

The Normal Human Keratinocyte (NHK) Neutral Red Uptake

Cytotoxicity Test

A Test for Basal Cytotoxicity

Phase III

1. PURPOSE

The purpose of this study is to evaluate the cytotoxicity of test chemicals using the Normal Human

Keratinocyte (NHK) Neutral Red Uptake (NRU) cytotoxicity test . The data will be used to evaluate

the intra- and inter-laboratory reproducibility of the assay and effectiveness of the cytotoxicity assay

to predict the starting doses for rodent acute oral systemic toxicity assays. This test method protocol

outlines the procedures for performing the cytotoxicity test and is in support of the in vitro validation

study organized by NICEATM and the European Centre for the Validation of Alternative Methods

(ECVAM) and sponsored by NIEHS, U .S . Environmental Protection Agency, and ECVAM . This test

method protocol applies to all personnel involved with performing the cytotoxicity assay .

A. NHK Neutral Red Uptake Cytotoxicity Test

The NHK NRU test will be performed to analyze the in vitro toxicity of 60 blinded/coded test

chemicals. This test will be used to determine IC20, IC50, and ICgo values for the predetermined

set of test chemicals of varying toxicities.

11 . SPONSOR

A. Name: National Institute of Environmental Health Sciences (NIEHS); The

National Toxicology Program (NTP) Interagency Center for the

Evaluation of Alternative Toxicological Methods (NICEATM)

B. Address: P.O. Box 1223 3

Research Triangle Park, NC 27709

C. Representative: Named Representative

III. IDENTIFICATION OF TEST AND CONTROL SUBSTANCES

A. Test Chemicals : Blinded chemicals (60)

B. Controls: Positive: Sodium Lauryl Sulfate

Vehicle (Negative): Assay medium

Solvent (as directed) : Assay medium, DMSO, or ethanol as directed

by the Study Management Team, for preparation

of test chemicals

IV. TESTING FACILITY AND KEY PERSONNEL
p. 3

Test :'Weihocl Protocol for the NHK Neutral Red Uptake CV7oto.rrcitv Assa v

PhRare HI - Validation Studv:

November 4, 200 3

A. Facility Information

1) Name:

2) Address :

3) Study Director :

4) Laborato ry Technician(s):

5) Scientific Advisor:

6) Quality Assurance Director :

7) Safety Manager:

8) Facility Management:

B. Test Schedul e

1) Proposed Experimental Initiation Date :

2) Proposed Experimental Completion Date :

3) Proposed Report Date :

V. TEST SYSTEM

The NRU cytotoxicity assay procedure is a cell survival/viability chemosensitivity assay based on the

ability of viable cells to incorporate and bind neutral red (NR), a supravital dye . NR is a weak

cationic dye that readily penetrates cell membranes by non-ionic diffusion and accumulates

intracellularly in lysosomes. Alterations of the cell surface or the sensitive lysosomal membrane lead

to lysosomal fragility and other changes that gradually become irreversible . Such changes brought

about by the action of xenobiotics result in a decreased uptake and binding of NR . It is thus possible

to distinguish between viable, damaged, or dead cells, which is the basis of this assay .

Healthy mammalian cells, when maintained in culture, continuously divide and multiply over time. A

toxic chemical, regardless of site or mechanism of action, will interfere with this process and result in

a reduction of the growth rate as reflected by cell number . Cytotoxicity is expressed as a

concentration dependent reduction of the uptake of the NR after chemical exposure thus providing a

sensitive, integrated signal of both cell integrity and growth inhibition .

VI. DEFINITIONS

A. . Hill function: a four parameter logistic mathematical model relating the concentration of test

chemical to the response being measured in a sigmoidal shape .

Top - Bottom

Y = Bottom +

1 + 1 00og 1c50 - x)H i uslope

where Y= response, X is the logarithm of dose (or concentration), Bottom is the minimum

response, Top is the maximum response, logIC50 is logarithm of X at the response midway

between Top and Bottom, and HillSlope describes the steepness of the curve .

B. Documentation : all methods and procedures will be noted in a Study Workbook; logs will be

maintained for general laborato ry procedures and equipment (e .g., media preparation, test

chemical preparation, incubator function) ; all optical densi ty data obtained from the

spectrophotometer plate reader will be saved in electronic and paper formats; all calculations of

ICx values and other derived data will be in electronic and paper format ; all data will be archived .

I
p. 4

Test 1tlethoc/ Protocol for- the .M1WK Neutral Red Uptake C+-totoxicitr• Assa r

Phase III - L'alri/ation Stuc%- :

NuvernGer 4, 200 3

VII. PROCEDURES

A. Materials

[Note: Suggested brand names/vendors are listed in parentheses . Equivalents may be used unless

otherwise noted.]

1 . Cell Lines

Normal Human Epidermal Keratinocytes (NHK)

Non-transformed cells; from cryopreserved primary or secondary cells (Clonetics #CC-2507

or equivalent). Cells will be Clonetics NHK cells.

Cambrex [Cambrex Bio Science, 8830 Biggs Ford Road, Walkersville, MD 21793-0127

Cambrex Europe [Cambrex Bio Science Verviers, S .P.R.L. Parc Industriel de Petit Rechain,

B-4800 Verviers, BELGIUM]

2. Technical Equipment

[Note: Suggested brand names/vendors are listed in parentheses. Equivalents may be used .]

a) Incubator: 37°C f 1°C, 90 % ± 5 % humidity, 5 .0 % + 1% C02/air

b) Laminar flow clean bench (standard : "biological hazard")

c) Water bath: 37°C f 1°C

d) Inverse phase contrast microscope

e) Sterile glass tubes with caps (e.g., 5mL)

f) Centrifuge (optionally : equipped with microtiter plate rotor)

g) Laboratory balance

h) 96-well plate spectrophotometer (i.e ., plate reader) equipped with 540 nm ± 10 nm filter

i) Shaker for microtiter plates

j) Cell counter or hemocytometer

k) Pipetting ai d

1) Pipettes, pipettors (multi-channel and single channel; multichannel repeater pipette),

dilution bloc k

m) Cryotube s

n) Tissue culture flasks (75 - 80 em2, 25 cm2 )

o) 96-well flat bottom tissue culture microtiter plates (e .g., Nunc # 167 008 ;

Corning/COSTAR tissue culture-treated)

p) pH paper (wide and narrow range)

q) Multichannel reagent reservoi r

r) Waterbath sonicator

s) Magnetic stirrer

t) Antistatic bar ionizer/antistatic gun (optional for neutralizing static on 96-well plates)

u) Dry heat block (optional)

v) Adhesive film plate sealers (e .g ., Excel Scientific SealPlateTM ,Cat # STR-SEAL-PLT or

equivalent)

w) Vortex mixer

4
p. 5

Test .Vlethod Protoco( tor the ~VHK Neutral Red Uptake Cvtotaxieitv A sscr v

Phase 111- VcatiJation Stucfv :

;Vovetn6er 4, 200 ,

x) Filters/filtration devices

[Note: Tissue culture flasks and microtiter plates should be prescreened to ensure that they

adequately support the growth of NHK. Multi-channel repeater pipettes may be used for

plating cells in the 96-well plates, dispensing plate rinse solutions, NR medium, and desorb

solution. Do not use the repeater pipette for dispensing test chemicals to the cells . ]

3 . Chemicals, Media, and Ser a

a) Keratinocyte Basal Medium without Ca++ (KBM®, Clonetics CC-3104) that is completed

by adding the KBM® SingleQuots® (Clonetics CC-4131) to achieve the proper

concentrations of epidermal growth factor, insulin, hydrocortisone, antimicrobial agents,

bovine pituitary extract, and calcium (e .g., Clonetics Calcium SingleQuots®, 300 mM

CaCl2, Clonetics # CC-4202).

b) HEPES Buffered Saline Solution (HEPES-BSS) (e.g., Clonetics # CC-5022)

c) 0.025 % TrypsinlEDTA solution (e .g ., Clonetics # CC-5012)

d) Trypsin Neutralizing Solution (TNS) (e .g., Clonetics # CC-5002)

e) Phosphate Buffered Saline (PBS )

f) Dulbecco's Phosphate Buffered Saline (D-PBS) [formulation containing calcium and

magnesium cations; glucose optional] (for rinsing )

g) Neutral Red (NR) Dye - tissue culture-grade ; liquid form (e .g., SIGMA N 2889); powder

form (e.g., SIGMA N 4638)

h) Dimethyl sulfoxide (DMSO), U.S.P analytical grade (Store under nitrogen @-20°C)

i) Ethanol (ETOH), U.S.P. analytical grade (100 %, non-denatured for test chemical

preparation; 95 % can be used for the desorb solution)

j) Glacial acetic acid, analytical grade

k) Hanks' Balanced Salt Solution without CaZ+ or Mg 2+ (CMF-HBSS) (e.g., Invitrogen #

14170)

1) Distilled H20 or any purified water suitable for cell culture and NR desorb solution

(sterile)

m) Sterile/non-sterile paper towels (for blotting 96-well plates)

B. Preparations of Media and Solutions

[Note : All solutions (except NR stock solution, NR medium and NR desorb), glassware, pipettes,

etc., shall be sterile and all procedures should be carried out under aseptic conditions and in the

sterile environment of a laminar flow cabinet (biological hazard standard) . All methods and

procedures will be adequately documented.]

1 . Media

a) Routine Culture Medium/Treatment Medium

KBM® (Clonetics CC-3104) supplemented with KBM® SingleQuots® (Clonetics CC413 1) and Clonetics Calcium SingleQuots® (CC-4202) to make 500 mL medium. Final

concentration of supplements in medium are :

0.0001 ng/mL Human recombinant epidermal growth factor

5 μg/mL Insulin

0 .5 μg/mL Hydrocortisone

5
p. 6

Test d9~thotl Protnrol for the AHK Ventral Red Uptake Cvtotoxicitv :3ssa v

Phase III - Validation Sluov;;

November 4 . 2003 -

30 μg/mL Gentamicin

15 ng/mL Amphotericin B

0.10 mm Calcium

30 μg/mL Bovine pituitary extract

Complete media should be kept at 2-8°C and stored for no longer than two weeks.

NOTE:

KBM® SingleQuots® contain the following stock concentrations and volumes :

0.1 ng/mL hEGF 0.5 mL

5 .0 mg/mL Insulin 0.5 mL

0.5 mg/mL Hydrocortisone 0.5 mL

30 mg/mL Gentamicin, 15 ug/mL Amphotericin-B 0 .5 mL

7.5 mg/mL Bovine Pituitary Extract (BPE) 2 .0 mL

Clonetics Calcium SingleQuots® are 2 mL of 300mM calcium .

165 μl of solution per 500 mL calcium-free medium equals 0 .10 mM calcium in the

medium.

2 . Neutral Red (NR) Stock Solution

The liquid tissue culture-grade stock NR Solution will be the first choice for performing the

assay (e .g., SIGMA #N2889, 3 .3 mg/mL). Store liquid tissue culture-grade NR Stock

Solution at the storage conditions and shelf-life period recommended by the manufacturer .

If the liquid form is not available, the following formulation can be prepared.

EXAMPLE: 0.33 g NR Dye powder in 100 mL H2 0

The NR Stock Solution (powder in water) should be stored in the dark at room temperature

for up to two months.

3 . Neutral Red (NR) Medium

EXAMPLE:

1 .0 mL (3 .3 mg NR dye/mL) NR Stock Solution

99.0 mL Routine Culture Medium (pre-warmed to 37 C.)

The final concentration of the NR Medium is 33 u,g NR dye/mL and aliquots will be

prepared on the day of application .

[Note: The NR Medium shall be filtered (e .g., Millipore filtering, 0 .2 - 0.45 μm pore size)

used to reduce NR crystals. Aliquots of the NR Medium should be maintained at 37 C (e .g.,

in a waterbath) before adding to the cells and used within 30 min of preparation but also used

within 15 min after removing from 37 C storage .]

A
p. 7

Test Adc,thod Protorul for the A'HK Neutral Red t. f?tal-e Cvtutoxiriti- .4s.sa1-

Phase 11I - Validation Stuclv:

.'Y'ovemher 4. 200 3

4. Ethanol/Acetic Acid Solution (NR Desorb)

1% Glacial acetic acid solution

50 % Ethanol

49% H2O

C. Methods

1 . Cell Maintenance and Culture Procedures

NHK cells are routinely grown as a monolayer in tissue culture grade flasks (e .g., 25 cm2) at

37°C f 1°C, 90 % ± 5 % humidity, and 5.0 % f 1% C02/air. The cells should be examined

on a daily (i.e., on workdays) basis under a phase contrast microscope, and any changes in

morphology or their adhesive properties must be noted in a Study Workbook .

2 . Receipt of Cryopreserved Keratinocytes

Upon receipt of cryopreserved keratinocytes, the vial(s) of cells shall be stored in a liquid

nitrogen freezer until needed .

3 . Thawing Cells and Establishing Cell Culture s

a) Thaw cells by putting ampules into a water bath at 37°C for as brief a time as possible .

Do not thaw cells at room temperature or by hand . Seed the thawed cells into culture

flasks as quickly as possible and with minimal handling .

b) Slowly (taking approximately 1-2 min) add 9 mL of pre-warmed Routine Culture

Medium to the cells suspended in the cryoprotective solution and transfer cells into flasks

containing pre-warmed Routine Culture Medium (See Table 1).

c) Incubate the cultures at 37°C ± 1°C, 90 % ± 5 % humidity, 5 .0 % ± 1% C02/air until the

cells attach to the flask (within 4 to 24 h), at which time the Routine Culture Medium should

be removed and replaced with fresh Routine Culture Medium .

d) Unless otherwise specified, the cells should be incubated at 37°C ± 1°C, 90 % f 5 %

humidity, 5 .0 % + 1% C02/air and fed every 2-3 days until they exceed 50 % confluence

(but less than 80 % confluent) .

Table 1 . Guidelines for Establishing Cell Culture s

Cells/25 cm flask 6.25 x 10 1.25 x 105 2.25 x 105

(in approximately 5 niL) (2500/cm2) (5000/cm2) (9000/cm)

1 fl ask each cell concentration

Approximate Time to Subculture 96+ hours 72 - 96 hours 48 - 72 hours

Cells to 96-Well Plates 6 - 8 plates 6 - 8 plates 6 - 8 plates

Cell growth guidelines - actual growth of individual cell lots may vary .

4 . Subculture of NHK Cells to 96-Well Plates

7
p. 8

Test tt,fethod Protoco/ for the NHK atiaeatr, it Red Uptal-e Cs-tutoxiCitVAsscr v

Phase III - Validation Staedv

. 200 3 . November 4

[Note : It is important that cells have overcome the lag growth phase when they are used for

the test. Keratinocytes will be passaged only into the 96-well plates and will not be

subcultured into flasks for use in later assays ]

a) When the keratinocyte culture in a 25 cm2 flask exceeds 50 % confluence (but less than 80

% confluent), remove the medium and rinse the culture twice with 5 mL HEPES-BSS. The

first rinse may be left on the cells for up to 5 minutes and the second rinse should remain on

the cells for approximately 5 minutes. Discard the washing solutions.

b) Add 2 mL trypsin/EDTA solution to each flask and remove after 15 to 30 seconds. Incubate

the flask at room temperature for 3 to 7 min . When more than 50 % of the cells become

dislodged, rap the flask sharply against the palm of the hand.

c) When most of the cells have become detached from the surface, rinse the fl ask with 5 mL of

room temperature TNS . Ifmore than one flask is subcultured, the same 5 mL of TNS may

be used to rinse a total of up to two flasks.

d) Then rinse the flask with 5 mL CMF-HBSS and transfer the cell suspension to a centrifuge

tube .

e) Pellet the cells by centrifugation for 5 min at approximately 220 x g . Remove the

supernatant by aspiration .

f) Resuspend the keratinocyte pellet by gentle trituration (to have single cells) in Routine

Culture Medium . It is important to obtain a single cell suspension for exact counting .

Count a sample of the cell suspension using a hemocytometer or cell counter .

4

g) Prepare a cell suspension -1 .6 - 2 .0 x10 cells/mL in Routine Culture Medium. Using a

multi-channel pipette, dispense 125 μ1 Routine Culture Medium only into the peripheral

wells (blanks) of a 96-well tissue culture microtiter plate . In the remaining wells,

dispense 125 μl of the cell suspension (2x103 - 2.5x103 cells/well) . Prepare one plate per

chemical to be tested (see Figure 1, Section VILE .1).

h) Incubate cells (37°C f 1°C, 90 % ± 5 .0 % humidity, and 5 % f 1% C02/air) so that cells

form a 20+ % monolayer (-48-72 h). This incubation period assures cell recovery and

adherence and progression to exponential growth phase .

i) Examine each plate under a phase contrast microscope to assure that cell growth is

relatively even across the microtiter plate . This check is performed to identify

experimental and systemic cell seeding errors. Record observations in the Study

Workbook.

5 . Determination of Doubling Time

a) A cell doubling time procedure was performed on the initial lot of cells that was used in

the first cell culture assays of Phase la of the Validation Study. The doubling time only

needs to be determined in Phase III if there is a change in the lot of cells used . Establish

cells in culture and trypsinize cells as per Section VILC .4 for subculture . Resuspend

cells in appropriate culture medium . Use Table 1 to determine seeding densities .

R
P. 9

Test Method Protocol /iw the ArtfK :Veinal Red Uptake C's-totox?cih- A ssa v

Phose 111- Validation Stw]V~'

b) Seed five sets of cell culture vessels in triplicate for each cell type (e .g., 15 tissue culture

dishes [60mm x 15mm]). Use appropriate volume of culture medium for the culture

vessels. Note number of cells placed into each culture dish . Place dishes into the

incubators (37°C ± 1°C, 90 %± 5 % humidity, 5 .0 % ± 1% C02/air).

c) After 4-6 hours (use the same initial measurement time for each subsequent doubling

time experiment), remove three culture dishes and t rypsinize cells. Count cells using a

cell counter or hemocytometer. Cell viability may be determined by dye exclusion (e .g.,

Trypan Blue ; Nigrosin). Determine the total number of cells and document . Repeat

sampling at 24 hr, 48 hr, 72 hr, and 96 hr post inoculation. Change culture medium at 72

hr or sooner in remaining dishes if indicated by pH drop .

d) Plot cell concentration (per mL of medium) on a log scale against time on a linear scale .

Determine lag time and population doubling time . The doubling time will be in the log

(exponential) phase of the growth cu rve . Additional dishes and time are needed if the

entire growth curve is to be determined (lag phase, log phase, plateau phase).

D. Preparation of Test Chemicals

The Study Management Team will provide direction on the solvent to be used for each test

chemical. [Note: Preparation under red or yellow light is recommended to preserve chemicals

that degrade upon exposure to light.]

1 . Test Chemical in Solution

a) Allow test chemicals to equilibrate to room temperature before dissolving and diluting .

b) Prepare test chemical immediately prior to use . Test chemical solutions should not be

prepared in bulk for use in subsequent tests . Ideally, the solutions must not be cloudy nor

have noticeable precipitate . Each stock dilution should have at least 1-2 mL total volume

to ensure adequate solution for the test wells in a single 96-well plate . The SMT may

direct the Study Director to store an aliquot (e.g ., 1 mL) of the highest 2X stock solution

(e .g., low solubility chemicals) in a freezer (e .g., -70°C) for use in future chemical

analyses.

c) For chemicals dissolved in DMSO or ethanol, the final DMSO or ethanol concentration

for application to the cells must be 0 .5 %(v/v) in the vehicle controls and in all of the

eight test concentrations.

d) The stock solution for each test chemical should be prepared at the highest concentration

found to be soluble in the solubility test (Test Method Protocolfor Solubility

Determination). Thus, the highest test concentration applied to the cells in each range

finding experiment is :

0.5 times the highest concentration found to be soluble in the solubility test, if the

chemical was soluble in medium, o r

1/200 the highest concentration found to be soluble in the solubili ty test if the

chemical was soluble in ethanol or DMSO .

Q
p.10

Test Alethocl Protocol Jnr the NHK Neutral Rec! L%ptr.rl-e Cvtutnxieih- As .sav

Phase III - Validation Studt-

. 2003 : November -I

e) The seven lower concentrations in the range finding experiment would then be prepared

by successive dilutions that decrease by one log unit each . The following example

illustrates the preparation of test chemical in solvent and the dilution of dissolved test

chemical in medium before application to NHK cells.

Example : Preparation of Test Chemical in Solvent Using a Log Dilution Schem e

If DMSO was determined to be the preferred solvent at Tier 2 of the solubility test (i.e .,

200,000 μg/mL), dissolve the chemical in DMSO at 200,000 μg/mL for the chemical

stock solution .

1) Label eight tubes 1- 8 . Add 0.9 mL solvent (e .g., DMSO) to tubes 2 -- 8 .

2) Prepare stock solution of 200,000 Rg test chemical/mL solvent in tube # 1 .

3) Add 0 .1 mL of 200,000 ~t g/mL dilution from tube #1 to tube #2 to make a 1 :10

dilution in solvent (i .e ., 20,000 μg/mL).

4) Add 0.1 mL of 20,000 μg/mL dilution from tube #2 to tube #3 to make another 1 :10

dilution (i .e., 1 :100 dilution from stock solution) in solvent (i.e ., 2,000 μg/mL)

5) Continuing making serial 1 :10 dilutions in the prepared solvent tubes.

6) Since each concentration is 200 fold greater than the concentration to be tested, make

a 1 :100 dilution by diluting 1 part dissolved chemical in each tube with 99 parts of

culture medium ( e .g., 0.1 mL of test chemical in DMSO + 9 .9 mL culture medium) to

derive the eight 2X concentrations for application to NHK cells . Each 2X test

chemical concentration will then contain 1% v/v solvent. The NHK cells will have

0.125 mL of culture medium in the wells prior to application of the test chemical . By

adding 0.125 mL ofthe appropriate 2X test chemical concentration to the appropriate

wells, the test chemical will be diluted appropriately (e .g., highest concentration in

well will be 1,000 μg/mL) in a total of 0 .250 mL and the solvent concentration in the

wells will be 0.5% v/v.

7) A test article prepared in DMSO or ethanol may precipitate upon transfer into the

Routine Culture Medium . The 2X dosing solutions should be evaluated for

precipitates and the results recorded in the workbook . It will be permissible to test all

of the dosing solutions in the dose range finding assay and main experiments .

However, doses containing test article precipitates should be avoided and generally

will not be used in the ICx determinations for the definitive tests. Precipitates in 2X

dosing solutions are permissible for range finder tests but not for definitive tests .

Document all test chemical preparations in the Study Workbook.

2 . pH of Test Chemical Solution s

Prior to or immediately after application of the test chemical to the 96-well plate, measure the

pH of the highest 2X dosing concentration of the test chemical (i .e., Cl in the test plate, see

Figure 1) in culture medium. Use pH paper (e .g., pH 0 - 14 to estimate and pH 5 - 10 to

determine more precise value ; or Study Director's discretion). The pH paper should be i n

i n
p.11

Test Method 1'rotoiol jbr the ,VF(K Neutral Red t'ptul,e ('>>totoxicitv,4s .sa v

Phase III - Y'ali~fution Jtudv

3 : !Vo vemher 4, 200

contact with the solution for approximately one minute . Document the pH and note the color

of the 2X concentration medium (i .e ., in the EXCEL® template). Medium color for all

dosing dilutions should be noted in the workbooks . Do not adjust the pH.

3. Concentrations of Test Chemical

a) Range Finder Experiment

Test eight concentrations of the test chemical by diluting the stock solution with a

constant factor covering a large range. The initial dilution series shall be log dilutions

( e .g., 1 :10, 1 :100, 1 :1000, etc.).

If a range finder experiment does not generate enough cytotoxicity, then higher doses

should be attempted. If cytotoxicity is limited by solubility, then more stringent

solubility procedures to increase the stock concentration (to the maximum concentration

specified in Section VII.D.3 .b .) should be employed. Place the highest test chemical

concentration into an incubator (37°C ± 1°C, 90 % ± 5 % humidity, 5 .0 % ± 1% C02/air)

and stir or rock for up to 3 hours, if necessary, to facilitate dissolution . For stocks

prepared in medium, vessel caps should be loose to allow for CO2 exchange. Proceed

with dosing solution preparation and dosing .

If a range finding test produces a biphasic curve, then the doses selected for the

subsequent main experiments should cover the most toxic dose-response range (see

Example 1 - the most toxic range is 0 .001 - 0.1 μg/mL).

Example 1 - Biphasic Curve

Neutral Red Uptake

125%

100%

75%

Z

_m

> 50%

25 %

0%

0 .001 0 .010 0.100 1 .000 10 .000 100.000 1000.000 10000.000

Concentration (pg/mL)

b) Main Experimen t

[Note : After the range finding assay is completed, the definitive concentration-response

experiment shall be performed three times on three different days for each chemical (i.e .,

one plate per day per chemical). ]

Depending on the slope of the concentration-response curve estimated from the range

finder, the dilution/progression factor in the concentration series of the main experiment

11
p.12

Test Adz,thod Protocol jor the VHK Nerrtrnl Red C.~ptuke Cvtotox h- iti- Assav

Phase III - Validation Studr-

. 1(03 : November 4

should be smaller (e.g., dilution factor of 6410 = 1 .47). Cover the relevant concentration

range around the IC50 (> 0 % and < 100 % effect) preferably with several points of a

graded effect, but with a minimum of two points, one on each side of the estimated IC50

value, avoiding too many non-cytotoxic and/or 100 %-cytotoxic concentrations .

Experiments revealing less than one cytotoxic concentration on each side of the IC5 0

value shall be repeated, where possible, with a smaller dilution factor (see Section

VII.E.5.a.4). Each experiment should have at least one cytotoxicity value > 0 % and <

50.0 % viability and at least one cytotoxicity value > 50 .0 % and < 100 % viability . A

progression factor of 1 .21 [1 2 ~10] is regarded the smallest factor achievable and will be

the lowest dosing interval required.)

Determine which test chemical concentration is closest to the IC50 value (e .g., 50 %

cytotoxicity). Use that value as a central concentration and adjust dilutions higher and

lower in equal steps for the de finitive assay.

Maximum Doses to be Tested in the Main Experiments

If minimal or no cytotoxicity was measured in the dose range finding assay, a maximum

dose for the main experiments will be established as follows :

For test chemicals prepared in Routine Culture Medium, the highest test a rticle

concentration that may be applied to the cells in the main expe riments will be either

100 mg/mL, or the maximum soluble dose . Test chemical will be weighed into a

glass tube and the weight will be documented . A volume of Routine Culture Medium

will be added to the vessel so that the concentration is 200,000 μg/mL (200 mg/mL).

The solution is mixed using the mechanical procedures specified in Test Method

Protocol for Solubility Determination. If complete solubili ty is achieved in medium,

then 7 additional serial stock dosing solutions may be prepared from the 200 mg/mL

2X stock. If the test chemical is insoluble in medium at 200 mg/ml, proceed by

adding medium, in small incremental amounts, to attempt to dissolve the chemical by

using the sequence of mixing procedures specified in Test MethodProtocol for

Solubility Determination . More stringent solubili ty procedures may be employed if

needed based on results from the range finder experiment (Section VII.D.3.a.). The

highest soluble stock solution will be used to prepare the 7 additional serial stock

dosing solutions.

For test chemicals prepared in either DMSO or ethanol, the highest test a rticle

concentration that may be applied to the cells in the main experiments will be either

2.5 mg/mL, or less, depending upon the maximum solubility in solvent. Test

chemical will be weighed into a glass tube and the weight will be documented . A

volume of the appropriate solvent (determined from the original solubili ty test) will

be added to the vessel so that the concentration is 500,000 μg/mL (500 mg/mL). The

solution is mixed as specified in Test Method Protocol for Solubility Determination.

If complete solubility is achieved in the solvent, then 7 additional serial stock dosing

solutions may be prepared from the 500 mg/mL 200X stock. If the test chemical is

insoluble in solvent at 500 mg/ml, proceed by adding solvent, in small incremental

amounts, to attempt to dissolve the chemical by using the sequence of mixing

procedures. The highest soluble stock solution will be used to prepare the 7

additional serial stock dosing solutions.

If precipitates are obse rved in the 2X dilutions, continue with the experiment, make

the appropriate observations and documentation, and repo rt data to the SMT .

1?
p.13

Test .Liethod Protocol for the tVHK Arerrtral Red Uptake CVt0tOYiL'itVASSU V

Phase 111- Validation Studv

1 : November 4, 200-

c) Test Chemical Dilutions

The dosing factor of 3 .16 (= 2~10) divides a log into two equidistant steps, a factor of

2 .15 (= 3410) divides a decade into three steps. The factor of 1 .47 (= 6~10) divides a log

into six equidistant steps, the factor of 1 .78 (4~10) divides a log into four equidistant

steps, and the factor of 1 .21 (= 12410) divides the log into 12 steps.

EXAMPLE:

10 31.6 100

10 21.5 46.4 100

10 14.7 21.5 31.6 46.4 68.1 100

10 12.1 14.7 17.8 21 .5 26.1 31 .6 38.3 46.4 56.2 68 .1 82 .5 100

The technical production of decimal geometric concentration series is simple . An

example is given for factor 1 .47:

Dilute 1 volume of the highest concentration by adding 0 .47 volumes of diluent. After

equilibration, dilute 1 volume of this solution by adding 0 .47 volumes of diluent. . . (etc .).

E. Test Procedur e

1 . 96-Well Plate Configuratio n

The NHK NRU assay for test chemicals will use the 96-well plate configuration shown in

Figure 1.

VI
p. 14

Test ,L[ethocl Protocol for the VHK Neutral Red Uptake ('iootoxicitv,3ssa v

Phase 111- Validation stmjv:

November 4, 200 3

Figure 1. 96-Well Plate Configuration for Positive Control (PC) and Test Chemical Assays

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

A VCb VCb Czb . C2b, :Cab C,41~, C5b C6b C7b CSb- VCb VCb

B VCb VC1 Ct C2 C3 C4 C5 C6 C7 C8 VC2 VCb

C V~b VC1 C1 C2 C3 C4 C5 C6 C7 C8 VC2 VGb

D VCb VCI Ci C2 C3 C4 C5 C6 C7 C8 VC2 VCb

E VCb VCl CI C2 C3 C4 C5 C6 C7 C8 VC2 VCb

F VCb VC1 IC, C2 C3 C4 C5 C6 C7 C8 VC2 VCb

G VCb,~ VC1 C, C2 C3 C4 C5 C6 C7 C8 VC2 VCb

H VCb . VCb Qb Clb C3b C4b C4 b cbb C7ts- , Cab, VCb, VCb;

VC1 and VC2 = VEHICLE CONTROL

Ct -C8 = Test Chemicals or PC (SLS) at eight concentrations

(C1 = highest, C8 = lowest)

b = BLANKS (Test chemical or PC, but contain no cells)

VCb = VEHICLE CONTROL BLANK (contain no cells)

2 . Application of Test Chemica l

a) Two optional methods for rapidly applying the 2X dosing solutions onto the 96-well plates

may be utilized.

1) The first method is to add each of the 2X dosing solutions into labeled, sterile rese rvoirs

( e .g., Corning/Costar model 4870 sterile polys tyrene 50 mL reagent reservoirs or

Corning/Transtar model 4878 disposable reservoir liners, 8-channel ; or other

multichannel reservoirs).

2) The second method utilizes a "dummy" plate (i .e., an empty sterile 96-well plate)

prepared to hold the dosing solutions immediately prior to treatment of the test plate

(with cells) . The test chemical and control dosing solutions should be dispensed into the

dummy plate in the same pattern/order as will be applied to the plate containing cells .

More volume than needed for the test plate (i .e. greater than 125 μl/well) should be in the

wells of the dummy plate .

At the time of treatment initiation, a multi-channel micropipettor is used to transfer the 2X

dosing solutions, from the reservoirs or dummy plate, to the appropriate wells on the

treatment plate (as described in step c . below) . These methods will ensure that the dosing

solutions can be transferred rapidly to the appropriate wells of the test plate to initiate

treatment times and to minimize the range of treatment initiation times across a large number

14
p.15

Test Alethod Protocul tin the NHK laeartrcal fled Uptake Cvtvtoxtcitl; Assa y

Phase III - VulWaiiarr Stat(Jv

3 : N'overraher 4, 200

of treatment plates, and to prevent "out of order" dosing . Do not use a multichannel repeater

pipette for dispensing test chemical to the plates .

b) After 48 - 72 h (i.e., after cells attain 20+ % confluency [see Section Vll.C.4(h)]) incubation

of the cells, add 125 μl of the appropriate concentration of test chemical, the PC, or the VC

(see Figure 1 for the plate configuration) directly to the test wells . Do not remove Routine

Culture Medium for re-feeding the cells . The dosing solutions will be rapidly transferred

from the 8-channel reservoir (or dummy plate) to the test plate using a single delivery multichannel pipettor. For example, the VC may be transferred first (into columns 1, 2, 11, and

12), followed by the test article dosing solutions from lowest to highest dose, so that the same

pipette tips on the multi-channel pipettor can be used for the whole plate . [The Vehicle

Control blank (VCb) wells (column 1, column 12, wells A2, A11, H2, H11) will receive the

Vehicle Control dosing solutions (which should include any solvents used) . Blanks for wells

A3 - A10 and H3 - H10 shall receive the appropriate test chemical solution for each

concentration (e.g., wells A3 and H3 receive C, solution).] Incubate cells for 48 h ± 0.5 h

(37°C 7L 1°C, 90 % ± 5 % humidity, and 5.0 % + 1% C02/air) .

c) Positive Control: For each set of test chemical plates used in an assay, a separate plate of

positive control concentrations will be set up following the concentration range established in

the development of the positive control database in Phase I of the Validation Study . If

multiple sets of test chemical plates are set up, then clearly designate the positive control

plates for each set ; each set will be an individual entity . The Study Director will decide how

many test chemical plates will be run with a positive control plate . The mean IC50 ± two and

a half standard deviations (SD) for the SLS acceptable tests from Phases la, Ib, and II (after

the removal of outliers) are the values that will be used as an acceptance criterion for test

sensitivity for the NHK NRU assay . This plate will follow the same schedule and procedures

as used for the test chemical plates (including appropriate chemical concentrations in the

appropriate wells and meeting test acceptance criteria see Sections VII.E.1, E.2, and E.5).

3. Microscopic Evaluation

After at least 46 h treatment, examine each plate under a phase contrast microscope to

identify systematic cell seeding errors and growth characteristics of control and treated cells.

Record any changes in morphology of the cells due to the cytotoxic effects of the test

chemical, but do not use these records for any quantitative measure of c otoxicity .

Undesirable growth characteristics of control cells may indicate experimental error and may

be cause for rejection of the assay . Use the following Visual Observations Codes in the

description of cell culture conditions. Numerical scoring of the cells (see Section VILE.3)

should be determined and documented in the Study Workbook and in the appropriate section

of Addendum 11 of the EXCEL® study template .

15
p. 16

Test iVethod Protocol for the ,VIfK ;Ve rrtral Red Uptcrl c: C'rtutoxicit4- Assa n

Phase III - Vali,lation Sti«,fr:

November 4, 200;

Visual Observations Codes

Note Code Note Text

1 Normal Cell Morphology

2 Low Level of Cell Toxicity

3 Moderate Level of Cell Toxicity

4 High level of Cell Toxicity

1P Normal Cell Morphology with Precipitate

2P Low Level of Cell Toxicity with Precipitate

3P Moderate Level of Cell Toxicity with Precipitate

4P High level of Cell Toxicity with Precipitate

5P Unable to View Cells Due to Precipitate

4. Measurement of NRU

a) Carefully remove (i .e ., "dump") the Routine Culture Medium (with test chemical) and

rinse the cells very carefully with 250 μL pre-warmed D-PBS . Remove the rinsing

solution by dumping and remove excess by gently blotting on paper towels. Add 250 μL

NR medium (to all wells including the blanks) and incubate (37°C ± 1°C, 90 % ± 5 %

humidity, and 5.0 % f 1% C02/air) for 3±0 .1 h. Observe the cells briefly during the NR

incubation (e .g., between 2 and 3 h- Study Director's discretion) for NR crystal

formation. Record observations in the Study Workbook . Study Director can decide to

reject the experiment if excessive NR crystallization has occurred .

b) After incubation, remove the NR medium, and carefully rinse cells with 250 μL prewarmed D-PBS.

c) Decant and blot D-PBS from the plate . (Optionally: centrifuge the reversed plate.)

d) Add exactly 100 μL NR Desorb (ETOH/acetic acid) solution to all wells, including

blanks.

e) Shake microtiter plate rapidly on a microtiter plate shaker for 20 - 45 min to extract NR

from the cells and form a homogeneous solution . Plates should be protected from light

by using a cover during shaking.

f) Plates should be still for at least five minutes after removal from the plate shaker (or

orbital mixer). If any bubbles are observed, assure that they have been ruptured prior to

reading the plate . Measure the absorption (within 60 minutes of adding NR Desorb

solution) of the resulting colored solution at 540 nm ± 10 nm in a microtiter plate reader

(spectrophotometer), using the blanks as a reference . [Phases la and lb data show the

mean OD value for the plate blanks to be 0.055 ± 0.035 for NHK cells (± 2 .5 standard

deviations; data from 3 labs; N = 156). Use this range as a guide for assessment of the

blank values.] Save raw data in the Excel format as provided by the SMT .

5. Quality Check of Assay

a) Test Acceptance Criteria

tF
p.17

Phase 111- Validation Stwlv

Test Method Protoccrl the the NHK rlreutrirl Red Uptak

:

e

All acceptance criteria (i .e ., criteria 1, 2, and 3) must be met for a test to be acceptable .

1) The PC (SLS) ICso must be within two and a half (2 .5) standard deviations of the

historical mean established by the Test Facility (as per VILE.2 .c), and must meet

criteria 2 and 3, and must have an r2 (coefficient of determination) value calculated

for the Hill model fit (i .e ., from PRISM® software) > 0 .85.

2) The left and the right mean of the VCs do not differ by more than 15 % from the

mean of all VCs .

3) At least one calculated cytotoxicity value > 0 % and < 50 .0 % viability and at least

one calculated cytotoxicity value > 50 .0 % and < 100 % viability must be present .

Exception: If a test has only one point between 0 and 100 % and the smallest dilution

factor (i.e ., 1 .21) was used and all other test acceptance criteria were met, then the

test will be considered acceptable .

Stoaaing Rule for Insoluble Chemicals: If the most rigorous solubility procedures have

been performed and the assay cannot achieve adequate toxicity to meet the test acceptance

criteria after three definitive trials, then the Study Director may end all testing for that

particular chemical .

[Note: A corrected mean OD540 f tonm of 0 .205 - 1 .645 for the VCs is a target range but will

not be a test acceptance criterion . Range determined from Phase lb VC OD values from 3

laboratories (mean + 2 .5 standard deviations, N = 69).]

b) Checks for Systematic Cell Seeding Errors

To check for systematic cell seeding errors, untreated VCs are placed both at the left side

(row 2) and the right side (row 11 for the test plates) of the 96-well plate . Aberrations in

the cell monolayer for the VCs may reflect a volatile and toxic test article present in the

assay. If volatility is suspected, then proceed to Section VILE.6 .

Checks for cell seeding errors may also be performed by examining each plate under a

phase contrast microscope to assure that cell quantity is consistent.

6. Volatility of Test Chemicals

Highly volatile test chemicals may generate vapors from the treatment media during the test

chemical treatment incubation period . These vapors may become resorbed into the treatment

medium in adjacent wells, such that culture wells nearest the highest doses may become

contaminated by exposure to resorbed test article vapors . If the test chemical is particularly

toxic at the doses tested, the cross contamination may be evident as a significant reduction in

viability in the vehicle control cultures (i .e ., VC I) adjacent to the highest test chemical doses.

If potential test article volatility is suspected (e .g., for low density liquids) or if the initial

range finder test (non-sealed plate) results show evidence of toxic effects in the control

cultures (i.e ., > 15 % difference in viability between VC1 [column 21 and VC2 [column 11]),

then seal the subsequent test plates by the following procedure .

17
p.18

Test VIethod Pr•oto(ol jbr the Al-1K Neutral Red Uptak

_ ePha,se 111- L'alidatrorr S/rrdv

November 4, 1003

a) Plate Sealer Metho d

1) Plates and chemicals will be prepared as usual according to Sections VILD and

VII.E.

2) Immediately after the 96-well culture plate has been treated with the suspected

volatile chemical (Section VIl.E.2.b), apply the adhesive plate sealer (e .g., using a

hand, microplate roller, etc .) directly over the culture wells. Assure that the sealer

adheres to each culture well (well tops should be dry) . Place the 96-well plate cover

over the sealed plate and incubate the plate under specified conditions (Section

VIl.E.2.b). [Note: Do not jam the plate lid over the film to avoid deforming the

sealer and causing the sealer to detach from culture wells . Loose fit of the plate lid is

acceptable. ]

3) At the end of the treatment period, the plate sealer should be carefully removed to

avoid spillage. Continue with the NRU assay as per Section VILE.4.

F. Data Analysis

The Study Director will use good biological/scientific judgment for determining "unusable" wells

that will be excluded from the data analysis and provide explanations for the removal of any data

from the analysis.

A calculation of cell viability expressed as NRU is made for each concentration of the test

chemical by using the mean NRU of the six replicate values (minimum of four acceptable

replicates wells) per test concentration . This value is compared with the mean NRU of all VC

values. Relative cell viability is then expressed as percent of untreated VC . If achievable, the

eight concentrations of each chemical tested will span the range of no effect up to total inhibition

of cell viability. Data from the microtiter plate reader shall be transferred to the Excel®

spreadsheet template provided by the SMT. The template will automatically determine cell

viability, IC50 values by linear interpolation, and perform statistical analyses (including statistical

identification of outliers) . The template will also calculate the concentrations associated with 20

%, 50 %, and 80 % viability using the Hill slope and EC50 (i .e ., IC50) from the Hill function

analysis.

The Hill function analysis shall be performed using statistical software (e.g., GraphPad PRISM®

3 .0) and a template specified by the SMT to calculate IC20, IC5o, and IC80 values (and the

associated confidence limits) for each test chemical.

The Testing Facility shall report data using at least three (3) significant figures and shall forward

the results from each assay to the SMT through the designated contacts in electronic format and

hard copy upon completion of testing. The SMT will be directly responsible for the statistical

analyses of the Validation Study data .

VIlI. REFERENCES

Clonetics Normal Human Keratinocyte Systems Instructions for Use, AA-1000-4-Rev .03/00.

(http://www.clonetics.com).

1R
p.19

Test Rlclhod Protocul for the :VHK Neutral Red t~rake Cvtotoxicih~ Assa v

Phase I11- d"Crih lation Studv

: November 4 . 2(11) ;

Hackenberg, U . and H. Bartling. 1959 . Messen und Rechnen im pharmakologischen

Laboratorium mit einem speziellen Zahlensystem (WL24-System). Arch. Exp. Pathol. Pharmakol. 235: 437-463.

Triglia, D., P.T. Wegener, J. Harbell, K. Wallace, D. Matheson, and C. Shopsis. 1989.

Interlaboratory validation study of the keratinocyte neutral red bioassay from Clonetics

Corporation. In Alternative Methods in Toxicology, Volume 7. A .M. Goldberg, ed., pp. 357-365 . Mary Ann Liebert, Inc ., New York.

Test Method Protocol for Solubility Determination . In Vitro Cytotoxicity Validation Study . Phase III. August 29, 2003 . Prepared by The National Toxicology Program (NTP) Interagency Center

for the Evaluation of Alternative Toxicological Methods (NICEATM)

.

19
p. 20

Test Rkthoc/Protocol /br the iVHK 'Verttrcrl Red Uptcrl-e Cvtotoricitv .4ssa v

Phase 111- Y"alidatiorr Jhrdv :

November 4, ZOl);

IX. APPROVAL

SPONSOR REPRESENTATIVE DATE

(Print or type name)

Testing Facility STUDY DIRECTOR DATE

(Print or type name)

?n