**TEST METHOD PROTOCOL**

**for the NHK Neutral Red Uptake Cytotoxicity Test**

#  {#section .unnumbered}

**A Test for Basal Cytotoxicity for an In Vitro Validation Study**

**Phase II**

**May 15, 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**

# TEST METHOD PROTOCOL {#test-method-protocol .unnumbered}

# The Normal Human Keratinocyte (NHK) Neutral Red Uptake {#the-normal-human-keratinocyte-nhk-neutral-red-uptake .unnumbered}

# Cytotoxicity Test {#cytotoxicity-test .unnumbered}

**A Test for Basal Cytotoxicity**

**Phase II**

## I. PURPOSE {#i.-purpose .unnumbered}

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 nine (9) blinded/coded test chemicals. This test will be used to
> determine IC~20~, IC~50~, and IC~80~ values for the predetermined set
> of test chemicals of varying toxicities.

**II. 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 12233

Research Triangle Park, NC 27709

**C.** **Representative**: *Named Representative*

**III. IDENTIFICATION OF TEST AND CONTROL SUBSTANCES**

**A.** **Test Chemicals**: *Blinded chemicals (9)*

**B.** **Controls**: Positive: Sodium Lauryl Sulfate

Vehicle (Negative): Assay medium

Solvent (as needed): Assay medium with appropriate solvent used to
prepare the test chemicals (**Section VII.E**)

**IV. testing facility and key personnel**

**A. Facility Information**

1)  Name:

2)  Address:

3)  Study Director:

4)  Laboratory Technician(s):

5)  Scientific Advisor:

6)  Quality Assurance Director:

7)  Safety Manager:

8)  Facility Management:

**B. Test Schedule**

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 {#vi.-definitions .unnumbered}

##  {#section-1 .unnumbered}

## A.*. Hill function*: a four parameter logistic mathematical model relating the concentration of test chemical to the response being measured in a sigmoidal shape. {#a..-hill-function-a-four-parameter-logistic-mathematical-model-relating-the-concentration-of-test-chemical-to-the-response-being-measured-in-a-sigmoidal-shape. .unnumbered}

> $$\begin{array}{r}
> Y = \text{Bottom} + \frac{\text{Top} - \text{Bottom}}{1 + \text{10}^{(\text{logIC50} - X)\text{HillSlope}}} \\
>
> \end{array}$$

### 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. {#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. .unnumbered}

> **B.** *Documentation*: all methods and procedures will be noted in a
> Study Workbook; logs will be maintained for general laboratory
> procedures and equipment (e.g., media preparation, test chemical
> preparation, incubator function); all optical density 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.

##  {#section-2 .unnumbered}

## VII. PROCEDURES {#vii.-procedures .unnumbered}

### A. Materials {#a.-materials .unnumbered}

> \[Note: Suggested brand names/vendors are listed in parentheses.
> Equivalents may be used unless otherwise noted.\]

###  {#section-3 .unnumbered}

#### 1. Cell Lines {#cell-lines .unnumbered}

> **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 {#technical-equipment .unnumbered}

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

a)  Incubator: 37ºC ± 1ºC, 90 % ± 5 % humidity, 5.0 % ± 1 % CO~2~/air

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

c)  Water bath: 37ºC ± 1ºC

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 aid

l)  Pipettes, pipettors (multi-channel and single channel; multichannel
    repeater pipette), dilution block

m)  Cryotubes

n)  Tissue culture flasks (75 - 80 cm^2^, 25 cm^2^)

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)

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q)  Multichannel reagent reservoir

r)  Waterbath sonicator

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s)  Magnetic stirrer

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

u)  Dry heat block (optional)

> \[Note: Tissue culture flasks and microtiter plates should be
> prescreened to ensure that they adequately support the growth of NHK.
> Multi-channel repeater pipettes may be used for plating cells in the
> 96-well plates, dispensing plate rinse solutions, NR medium, and
> desorb solution. Do not use the repeater pipette for dispensing test
> chemicals to the cells.\]

#### 3. Chemicals, Media, and Sera {#chemicals-media-and-sera .unnumbered}

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
    CaCl~2~, Clonetics \# CC-4202).

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

c)  0.025 % Trypsin/EDTA 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 @ -20ºC)

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 Ca^2+^ or Mg^2+^ (CMF-HBSS)
    (e.g., Invitrogen \# 14170)

l)  Distilled H~2~O or any purified water suitable for cell culture
    (sterile)

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

### B. Preparations of Media and Solutions {#b.-preparations-of-media-and-solutions .unnumbered}

> \[Note: All solutions (except NR stock solution, NR medium and NR
> desorb), glassware, pipettes, etc., shall be sterile and all
> procedures should be carried out under aseptic conditions and in the
> sterile environment of a laminar flow cabinet (biological hazard
> standard). All methods and procedures will be adequately documented.\]

####  {#section-4 .unnumbered}

#### 1. Media {#media .unnumbered}

> a\) Routine Culture Medium/Treatment Medium
>
> KBM (Clonetics CC-3104) supplemented with KBM SingleQuots (Clonetics
> CC-4131) 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 g/mL Insulin
>
> 0.5 g/mL Hydrocortisone
>
> 30 g/mL Gentamicin
>
> 15 ng/mL Amphotericin B
>
> 0.10 mM Calcium
>
> 30 g/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 l of solution per 500 mL calcium-free medium equals 0.10 mM
> calcium in the medium.

#### 2. Neutral Red (NR) Stock Solution {#neutral-red-nr-stock-solution .unnumbered}

> 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 H~2~O
>
> 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 {#neutral-red-nr-medium .unnumbered}

> EXAMPLE:
>
> 1.0 mL (3.3 mg NR dye/mL) NR Stock Solution

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

> The final concentration of the NR Medium is **[33 g NR
> dye/mL]{.underline}** and aliquots will be prepared on the day of
> application.
>
> \[Note: The NR Medium shall be filtered (e.g., Millipore filtering,
> 0.2 -- 0.45 m pore size) used to reduce NR crystals. Aliquots of the
> NR Medium should be maintained at 37 C (e.g., in a waterbath) before
> adding to the cells and used within 30 min of preparation but also
> used within 15 min after removing from 37 C storage.\]

#### 4. Ethanol/Acetic Acid Solution (NR Desorb) {#ethanolacetic-acid-solution-nr-desorb .unnumbered}

> 1 % Glacial acetic acid solution\
> 50 % Ethanol\
> 49 % H~2~O

### C. Methods {#c.-methods .unnumbered}

###  {#section-5 .unnumbered}

#### 1. Cell Maintenance and Culture Procedures {#cell-maintenance-and-culture-procedures .unnumbered}

> NHK cells are routinely grown as a monolayer in tissue culture grade
> flasks (e.g., 25 cm^2^) at 37ºC ± 1ºC, 90 % ± 5 % humidity, and 5.0 %
> ± 1 % CO~2~/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 {#receipt-of-cryopreserved-keratinocytes .unnumbered}

####  {#section-6 .unnumbered}

> Upon receipt of cryopreserved keratinocytes, the vial(s) of cells
> shall be stored in a liquid nitrogen freezer until needed.

####  {#section-7 .unnumbered}

#### 3. Thawing Cells and Establishing Cell Cultures {#thawing-cells-and-establishing-cell-cultures .unnumbered}

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 37ºC ± 1ºC, 90 % ± 5 % humidity, 5.0 % ± 1
    % CO~2~/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 37ºC ±
    1ºC, 90 % ± 5 % humidity, 5.0 % ± 1 % CO~2~/air and fed every 2-3
    days until they exceed 50 % confluence (but less than 80 %
    confluent).

> Table 1. Establishing Cell Cultures

+----------------------------+-------------+------------+------------+
| Cells/25 cm^2^ flask       | 6.25 x      | 1.25 x     | 2.25 x     |
|                            | 10^4^       | 10^5^      | 10^5^      |
| (in approximately 5 mL)    |             |            |            |
|                            | (           | (5         | (9         |
| 1 flask each cell          | 2500/cm^2^) | 000/cm^2^) | 000/cm^2^) |
| concentration              |             |            |            |
+----------------------------+-------------+------------+------------+
| Approximate Time to        | 96+ hours   | 72 - 96    | 48 - 72    |
| Subculture                 |             | hours      | hours      |
+----------------------------+-------------+------------+------------+
| Cells to 96-Well Plates    | 6 -- 8      | 6 -- 8     | 6 -- 8     |
|                            | plates      | plates     | plates     |
+----------------------------+-------------+------------+------------+

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

#### 4. Subculture of NHK Cells to 96-Well Plates {#subculture-of-nhk-cells-to-96-well-plates .unnumbered}

> \[Note: It is important that cells have overcome the lag growth phase
> when they are used for the test. Keratinocytes will be passaged only
> into the 96-well plates and will not be subcultured into flasks for
> use in later assays\]

a)  When the keratinocyte culture in a 25 cm^2^ 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 flask with 5 mL of room temperature TNS. If more 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.

g)  Prepare a cell suspension --1.6 -- 2.0 x10^4^cells/mL in Routine
    Culture Medium. Using a multi-channel pipette, dispense 125 µl
    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 (2x10^3^ -- 2.5x10^3^
    cells/well). Prepare one plate per chemical to be tested (see
    **Figure 2**, **Section VII.F.1**).

h)  Incubate cells (37ºC ± 1ºC, 90 % ± 5.0 % humidity, and 5 % ± 1 %
    CO~2~/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 {#determination-of-doubling-time .unnumbered}

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 Ia of
    the Validation Study. The doubling time only needs to be determined
    in Phase II if there is a change in the lot of cells used. Establish
    cells in culture and trypsinize cells as per **Section VII.C.4** for
    subculture. Resuspend cells in appropriate culture medium. Use
    **Table 1** to determine seeding densities.

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 (37ºC ± 1ºC, 90 % ± 5 % humidity, 5.0 % ± 1 % CO~2~/air).

c)  After 4-6 hours (use the same initial measurement time for each
    subsequent doubling time experiment), remove three culture dishes
    and trypsinize 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 curve. Additional dishes and time are needed if the
    entire growth curve is to be determined (lag phase, log phase,
    plateau phase).

### Solubility Test

> The preference of solvent for dissolving test chemicals is medium,
> DMSO, and then ethanol. Solubility shall be determined in a step-wise
> procedure that involves attempting to dissolve a test chemical at a
> relatively high concentration with the sequence of mechanical
> procedures specified in **Section** **VII.D.2.a.** If the chemical
> does not dissolve, the volume of solvent is increased so as to
> decrease the concentration by a factor of 10, and then the sequence of
> mechanical procedures in **Section** **VII.D.2.a** are repeated in an
> attempt to solubilize the chemical at the lower concentrations. For
> testing solubility in media, the starting concentration is 20,000 g/ml
> (i.e., 20 mg/mL) in Tier 1, but for DMSO and ethanol the starting
> concentration is 200,000 g/ml (i.e., 200 mg/mL) in Tier 2. Weighing
> out chemical for each solvent (i.e., media, DMSO, ethanol) can be done
> all at once, if convenient, but solubility testing (at each tier that
> calls for more than one solvent) is designed to be sequential - media,
> then DMSO, then ethanol -- in accordance with the solvent hierarchy
> (see **Figure 1**). This allows for testing to stop, rather than
> continue testing with less preferred solvents, if the test chemical
> dissolves in a more preferred solvent. For example, if a chemical is
> soluble in medium at a particular tier, testing may stop. Likewise, if
> a chemical is soluble in DMSO at any tier, testing need not continue
> with ethanol. However, since the issue of primary importance is
> testing the solvents and concentrations of test chemical required by
> any one tier, sequential testing of solvents may be abandoned if the
> lab can test more efficiently in another way.

1.  **Determination of Solubility**

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a)  Tier 1 begins with testing 20 mg/mL in Routine Culture Medium (see
    **Table 2**). Approximately 10 mg (10,000 g) of the test chemical
    will be weighed into a glass tube and the weight will be documented.
    A volume of Routine Culture Medium, approximately 0.5 mL, will be
    added to the vessel so that the concentration is 20,000 g/ml (20
    mg/mL). The solution is mixed as specified in **Section**
    **VII.D.2.a.** If complete solubility is achieved in media, then
    additional solubility procedures are not needed.

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b)  If the test chemical is insoluble in medium, proceed to Tier 2 by
    adding enough medium, approximately 4.5 mL, to attempt to dissolve
    the chemical at 2 mg/mL by using the sequence of mixing procedures
    specified in **Section VII.D.2.a**. If the test chemical dissolves
    in medium at 2 mg/mL, no further procedures are necessary. If the
    test chemical does NOT dissolve, weigh out approximately 100 mg test
    chemical in a second glass tube and add enough DMSO to make the
    total volume approximately 0.5 mL (for 200 mg/mL), and attempt to
    dissolve the chemical as specified in **Section VII.D.2.a**. If the
    chemical does not dissolve in DMSO, weigh out approximately 100 mg
    test chemical in another glass tube and add enough ethanol to make
    the total volume approximately 0.5 mL (for 200 mg/mL) and attempt to
    dissolve the chemical as specified in **Section VII.D.2.a**. If the
    chemical is soluble in either solvent, no additional solubility
    procedures are needed.

> c\) If the chemical is NOT soluble in media, DMSO, or ethanol at Tier
> 2, then continue to Tier 3 in **Table 2** by adding enough solvent to
> increase the volume of the three Tier 2 solutions by 10 and attempt to
> solubilize again using the sequence of mixing procedures in
> **Section** **VII.D.2.a.** If the test chemical dissolves, no
> additional solubility procedures are necessary. If the test chemical
> does NOT dissolve, continue with Tier 4 and, if necessary, Tier 5
> using DMSO and ethanol. Tier 4 begins by diluting the Tier 3 samples
> with DMSO or ethanol to bring the total volume to 50 mL. The mixing
> procedures in **Section** **VII.D.2.a** are again followed to attempt
> to solubilize the chemical. Tier 5 is performed, if necessary, by
> weighing out another two more samples of test chemical at \~10 mg each
> and adding \~50 mL DMSO or ethanol for a 200 g/mL solution, and
> following the mixing procedures in **Section** **VII.D.2.a**.
>
> [Example]{.underline}: If complete solubility is not achieved at
> 20,000 g/mL in Routine Culture Medium at Tier 1 using the mixing
> procedures specified in **Section** **VII.D.2.a,** then the procedure
> continues to Tier 2 by diluting the solution to 5 mL and mixing again
> as specified in **Section** **VII.D.2.a.** If the chemical is not
> soluble in medium, two samples of \~ 100 mg test chemical are weighed
> to attempt to solubilize in DMSO and ethanol at 200,000 g/mL (i.e.,
> 200 mg/mL). Solutions are mixed following the sequence of procedures
> prescribed in **Section** **VII.D.2.a** in an attempt to dissolve. If
> solubility is not achieved at Tier 2, then the solutions (media, DMSO,
> and ethanol) prepared in Tier 2 are diluted by 10 to test 200 g/mL in
> media, and 20,000 g/mL in DMSO and ethanol. This advances the
> procedure to Tier 3. Solutions are again mixed as prescribed in
> **Section** **VII.D.2.a** in an attempt to dissolve. If solubility is
> not achieved in Tier 3, the procedure continues to Tier 4, and to 5 if
> necessary (see **Figure 1** and **Table 2**).

Table 2 Determination of Solubility in Routine Culture Medium, DMSO, or
Ethanol

+---------------+----------+----------+---------+---------+---------+
| # TIER {#tier | **1**    | **2**    | **3**   | **4**   | **5**   |
|  .unnumbered} |          |          |         |         |         |
+---------------+----------+----------+---------+---------+---------+
| Total Volume  | 0.5 mL   | 5 mL     | 50 mL   |         |         |
|               |          |          |         |         |         |
| Medium        |          |          |         |         |         |
+---------------+----------+----------+---------+---------+---------+
| Concentration | 20,000   | 2,000    | 200     |         |         |
| of Test       | g/mL     | g/mL     | g/mL    |         |         |
| Chemical      |          |          |         |         |         |
|               | (20      | (2       | (0.20   |         |         |
| (Add \~10 mg  | mg/mL)   | mg/mL)   | mg/mL)  |         |         |
| to a tube.    |          |          |         |         |         |
| Add enough    |          |          |         |         |         |
| medium to     |          |          |         |         |         |
| equal the     |          |          |         |         |         |
| first volume. |          |          |         |         |         |
| Dilute to     |          |          |         |         |         |
| subsequent    |          |          |         |         |         |
| volumes if    |          |          |         |         |         |
| necessary.)   |          |          |         |         |         |
+---------------+----------+----------+---------+---------+---------+
| Total Volume  |          | 0.5 mL   | 5 mL    | 50 mL   |         |
| DMSO/Ethanol  |          |          |         |         |         |
+---------------+----------+----------+---------+---------+---------+
| Concentration |          | 200,000  | 20,000  | 2,000   |         |
| of Test       |          | g/mL     | g/mL    | g/mL    |         |
| Chemical      |          |          |         |         |         |
|               |          | (200     | (20     | (2      |         |
| (Add \~100 mg |          | mg/mL)   | mg/mL)  | mg/mL)  |         |
| to a large    |          |          |         |         |         |
| tube. Add     |          |          |         |         |         |
| enough DMSO   |          |          |         |         |         |
| or ethanol to |          |          |         |         |         |
| equal the     |          |          |         |         |         |
| first volume. |          |          |         |         |         |
| Dilute to     |          |          |         |         |         |
| subsequent    |          |          |         |         |         |
| volumes if    |          |          |         |         |         |
| necessary.)   |          |          |         |         |         |
+---------------+----------+----------+---------+---------+---------+
| Total Volume  |          |          |         |         | 50 mL   |
| DMSO/Ethanol  |          |          |         |         |         |
+---------------+----------+----------+---------+---------+---------+
| Concentration |          |          |         |         | 200     |
| of Test       |          |          |         |         | g/mL    |
| Chemical      |          |          |         |         |         |
|               |          |          |         |         | (0.2    |
| (Add \~10 mg  |          |          |         |         | mg/mL)  |
| to a large    |          |          |         |         |         |
| tube. Add     |          |          |         |         |         |
| enough DMSO   |          |          |         |         |         |
| or ethanol to |          |          |         |         |         |
| equal 50 mL.) |          |          |         |         |         |
+---------------+----------+----------+---------+---------+---------+
| # Equiv       | 10,000   | 1000     | 100     | 10 g/mL | 1 g/mL  |
| alent Concent | g/mL     | g/mL     | g/mL    |         |         |
| ration on Cel |          |          |         | (0.01   | (0.001  |
| ls  {#equival | (10      | (1       | (0.1    | mg/mL)  | mg/mL)  |
| ent-concentra | mg/mL)   | mg/mL)   | mg/mL)  |         |         |
| tion-on-cells |          |          |         |         |         |
|  .unnumbered} |          |          |         |         |         |
+---------------+----------+----------+---------+---------+---------+

> NOTE: The amounts of test chemical weighed and Routine Culture Medium
> added may be modified from the amounts given above, provided that the
> targeted concentrations specified for each tier are tested.

# *Figure 1. Solubility Flow Chart* {#figure-1.-solubility-flow-chart .unnumbered}

+--------+-------------------------------------------------------------+
| # TIER |                                                             |
|  1 {#t |                                                             |
| ier-1  |                                                             |
| .unnum |                                                             |
| bered} |                                                             |
+--------+-------------------------------------------------------------+
| STEP   | 20 mg/mL test chemical (TC) in 0.5 mL medium:               |
| 1:     |                                                             |
|        | -   if TC soluble, then **[STOP]{.underline}**.             |
|        |                                                             |
|        | -   if TC insoluble, then go to STEP 2.                     |
+--------+-------------------------------------------------------------+
| # TIER |                                                             |
|  2 {#t |                                                             |
| ier-2  |                                                             |
| .unnum |                                                             |
| bered} |                                                             |
+--------+-------------------------------------------------------------+
| STEP   | 2 mg/mL TC in medium -- increase volume from STEP 1 by 10   |
| 2:     | (i.e., to 5 mL)                                             |
|        |                                                             |
|        | -   if TC soluble, then **[STOP]{.underline}**.             |
|        |                                                             |
|        | -   if TC insoluble, then go to STEP 3.                     |
+--------+-------------------------------------------------------------+
| STEP   | 200 mg/mL TC in DMSO                                        |
| 3:     |                                                             |
|        | -   if TC soluble, then **[STOP]{.underline}**.             |
|        |                                                             |
|        | -   if TC insoluble, test at 200 mg/mL in ETOH.             |
|        |                                                             |
|        | -   if TC soluble, then **[STOP.]{.underline}**             |
|        |                                                             |
|        | -   If TC insoluble, go to STEP 4.                          |
+--------+-------------------------------------------------------------+
| **TIER |                                                             |
| 3**    |                                                             |
+--------+-------------------------------------------------------------+
| STEP   | 0.2 mg/mL TC in medium -- increase volume from STEP 2 by 10 |
| 4:     | (i.e., to 50 mL)                                            |
|        |                                                             |
|        | -   if TC soluble, then **[STOP]{.underline}**.             |
|        |                                                             |
|        | -   if TC insoluble, test at 20 mg/mL in DMSO -- increase   |
|        |     volume from STEP 3 by 10 (i.e., to 5 mL).               |
|        |                                                             |
|        | -   if TC soluble, then **[STOP.]{.underline}**             |
|        |                                                             |
|        | -   if TC insoluble, test at 20 mg/mL in ETOH -- increase   |
|        |     volume from STEP 3 by 10 (i.e., to 5 mL).               |
|        |                                                             |
|        | -   if TC soluble, then **[STOP.]{.underline}**             |
|        |                                                             |
|        | -   if TC insoluble, then go to STEP 5.                     |
+--------+-------------------------------------------------------------+
| **TIER |                                                             |
| 4**    |                                                             |
+--------+-------------------------------------------------------------+
| STEP   | 2 mg/mL TC in DMSO -- increase volume from STEP 4 by 10     |
| 5:     | (i.e., to 50 mL)                                            |
|        |                                                             |
|        | -   if TC soluble, then **[STOP.]{.underline}**             |
|        |                                                             |
|        | -   if TC insoluble, test at 2 mg/mL in ETOH -- increase    |
|        |     volume from STEP 4 by 10 (i.e., to 50 mL).              |
|        |                                                             |
|        | -   if TC soluble, then **[STOP.]{.underline}**             |
|        |                                                             |
|        | -   if TC insoluble, then go to STEP 6.                     |
+--------+-------------------------------------------------------------+
| **TIER |                                                             |
| 5**    |                                                             |
+--------+-------------------------------------------------------------+
| STEP   | 0.2 mg/mL TC in 50 mL DMSO                                  |
| 6:     |                                                             |
|        | -   if TC soluble, then **[STOP.]{.underline}**             |
|        |                                                             |
|        | -   if TC insoluble, test at 0.2 mg/mL in 50 mL ETOH        |
|        |                                                             |
|        | -   **[STOP]{.underline}**                                  |
+--------+-------------------------------------------------------------+

> **2. Mechanical Procedures**
>
> a\) The following hierarchy of mixing procedures will be followed to
> dissolve the test chemical:

1.  Add test chemical to solvent as in Tier 1 of **Table 2**.

> 2\) Gently mix. Vortex the tube (1 --2 minutes).
>
> 3\) If test chemical hasn't dissolved, use sonication for up to 5
> minutes.

4)  If sonication doesn't work, then warm solution to 37°C for 5 -- 60
    minutes. This can be performed by warming tubes in a 37°C water bath
    or in a CO~2~ incubator at 37°C. The solution may be stirred during
    warming (stirring in a CO~2~ incubator will help maintain proper
    pH).

> 5\) Proceed to Tier 2 (and Tiers 3-5, if necessary of Table 2 and
> repeat procedures 2-4).
>
> b\) The preference of solvent for dissolving test chemicals is medium,
> DMSO, and then ethanol. Thus, if a test chemical dissolves in more
> than one solvent at any one solubility-testing tier, then the choice
> of solvent follows this hierarchy. For example, if, at any tier, a
> chemical is soluble in medium and DMSO, but not ethanol, the choice of
> solvent would be medium. If the chemical were insoluble in medium, but
> soluble in DMSO and ethanol, the choice of solvent would be DMSO.
>
> After the lab has determined the preferred solvent for the test
> chemical and before proceeding to the cytotoxicity testing, the Study
> Director will discuss the solvent selection with the Study Management
> Team (SMT) of the validation study. [The SMT will relate what solvent
> should be used in the assay for each chemical]{.underline}. If the
> laboratory has attempted all solubility testing without success, then
> the SMT will provide additional guidance for achieving test chemical
> solubility. The SMT anticipates that all validation study test
> chemicals will be tested in the NRU assays.

## E. Preparation of Test Chemicals {#e.-preparation-of-test-chemicals .unnumbered}

> \[Note: Preparation under red or yellow light is recommended to
> preserve chemicals that degrade upon exposure to light.\]
>
> **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. 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.
    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, or

-   1/200 the highest concentration found to be soluble in the
    solubility test if the chemical was soluble in ethanol or DMSO.

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]{.underline}: Preparation of Test Chemical in Solvent Using a
> Log Dilution Scheme
>
> If DMSO was determined to be the preferred solvent at Tier 2 of the
> solubility test (i.e., 200,000 g/mL), dissolve the chemical in DMSO at
> 200,000 g/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 g test chemical/mL solvent in tube
    \# 1.

3)  Add 0.1 mL of 200,000 g/mL dilution from tube #1 to tube #2 to make
    a 1:10 dilution in solvent (i.e., 20,000 g/mL).

4)  Add 0.1 mL of 20,000 g/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 g/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 of the
    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 g/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 will be
    recorded in the workbook. It will be permissible to test all of the
    dosing solutions in the dose range finding assay only. Doses
    containing test article precipitates should be avoided, and will not
    be used in the ICx determinations for either the range finding
    experiments or the definitive tests.

> Document all test chemical preparations in the Study Workbook.

#### 2. pH of Test Chemical Solutions {#ph-of-test-chemical-solutions .unnumbered}

> Measure the pH of the highest concentration of the test chemical in
> culture medium using pH paper (e.g., pH 0 -- 14 to estimate and pH 5
> -- 10 to determine more precise value). The pH paper should be in
> contact with the solution for approximately one minute. Document the
> pH and note the color of the medium for all dilutions. Do not adjust
> the pH.

#### 3. Concentrations of Test Chemical {#concentrations-of-test-chemical .unnumbered}

####  {#section-8 .unnumbered}

> 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.).  {#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.-110-1100-11000-etc.. .unnumbered}

####  {#section-9 .unnumbered}

#### The data from any well that has precipitate will be excluded from any calculations. {#the-data-from-any-well-that-has-precipitate-will-be-excluded-from-any-calculations. .unnumbered}

> b\) Main Experiment
>
> \[Note: After the range finding assay is completed, the
> concentration-response experiment shall be performed [three times on
> three different days for each chemical (i.e., one plate per day per
> chemical)]{.underline}\]
>
> 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 should be smaller (^6^√10
> = 1.47). Cover the relevant concentration range (≥10 % and ≤ 90 %
> effect) preferably with three points of a graded effect, but with a
> minimum of two points, one on each side of the estimated IC~50~ value,
> avoiding too many [non-cytotoxic]{.underline} and/or [100
> %-cytotoxic]{.underline} concentrations. Experiments revealing less
> than one cytotoxic concentration on each side of the IC~50~ value
> shall be repeated, where possible, with a smaller dilution factor.
> Each experiment should have at least one cytotoxicity value ≥ 10.0 %
> and ≤ 50.0 % viability and at least one cytotoxicity value \> 50.0 %
> and ≤ 90.0 % viability. In addition, the dilution scheme shall be
> adjusted in subsequent replicate assays (i.e., definitive assays), if
> necessary, to increase the number of points on both sides of the
> IC~50~ in the 10-90% response range. (Taking into account pipetting
> errors, a progression factor of 1.21 is regarded the smallest factor
> achievable.)

Determine which test chemical concentration is closest to the IC~50~
value (e.g., 50 % cytotoxicity). Use that value as a central
concentration and adjust dilutions higher and lower in equal steps for
the definitive assay.

> [Maximum Doses to be Tested in the Main Experiments]{.underline}
>
> 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 article concentration that may be applied to the cells in the
    main experiments 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 g/mL (200 mg/mL).
    The solution is mixed as specified in **Section VII.D.2.a**. If
    complete solubility 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
    **Section VII.D.2.a**. The highest soluble stock solution will be
    used to prepare the 7 additional serial stock dosing solutions.

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```
-   For test chemicals prepared in either DMSO or ethanol, the highest
    test article 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 solubility test)
    will be added to the vessel so that the concentration is
    500,000 g/mL (500 mg/mL). The solution is mixed as specified in
    **Section VII.D.2.a**. 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 specified in **Section
    VII.D.2.a**. The highest soluble stock solution will be used to
    prepare the 7 additional serial stock dosing solutions.

#### c) Test Chemical Dilutions {#c-test-chemical-dilutions .unnumbered}

> The dosing factor of 3.16 (= ^2^√10) divides a log into two
> equidistant steps, a factor of 2.15 (= ^3^√10) divides a decade into
> three steps. The factor of 1.47 (= ^6^√10) divides a log into six
> equidistant steps, and the factor of 1.21 (= ^12^√10) 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.).*

### F. Test Procedure {#f.-test-procedure .unnumbered}

1.  **96-Well Plate Configuration**

> The NHK NRU assay for test chemicals will use the 96-well plate
> configuration shown in Figure 2.
>
> ***Figure 2. 96-Well Plate Configuration for Positive Control and Test
> Chemical Assays***

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

  A     VCb   VCb   C~1~b   C~2~b   C~3~b   C~4~b   C~5~b   C~6~b   C~7~b   C~8~b   VCb   VCb

  B     VCb   VC    C~1~    C~2~    C~3~    C~4~    C~5~    C~6~    C~7~    C~8~    VC    VCb

  C     VCb   VC    C~1~    C~2~    C~3~    C~4~    C~5~    C~6~    C~7~    C~8~    VC    VCb

  D     VCb   VC    C~1~    C~2~    C~3~    C~4~    C~5~    C~6~    C~7~    C~8~    VC    VCb

  E     VCb   VC    C~1~    C~2~    C~3~    C~4~    C~5~    C~6~    C~7~    C~8~    VC    VCb

  F     VCb   VC    C~1~    C~2~    C~3~    C~4~    C~5~    C~6~    C~7~    C~8~    VC    VCb

  G     VCb   VC    C~1~    C~2~    C~3~    C~4~    C~5~    C~6~    C~7~    C~8~    VC    VCb

  H     VCb   VCb   C~1~b   C~2~b   C~3~b   C~4~b   C~5~b   C~6~b   C~7~b   C~8~b   VCb   VCb
  ----- ----- ----- ------- ------- ------- ------- ------- ------- ------- ------- ----- -----

> VC = untreated VEHICLE CONTROL (mean viability set to 100 %)

C~1~ -- C~8~ = Test Chemicals or Positive Control (SLS) at eight
concentrations

> (C1 = highest, C8 = lowest)
>
> b = BLANKS (contain **no** cells)\
> VCb = VEHICLE CONTROL BLANK

###  {#section-10 .unnumbered}

#### 2. Application of Test Chemical {#application-of-test-chemical .unnumbered}

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

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```
1)  The first method is to add each of the 2X dosing solutions into
    labeled, sterile reservoirs (e.g., Corning/Costar model 4870 sterile
    polystyrene 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 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 \[see
    Section VII.C.4(h)\]) incubation of the cells, add 125 µl of the
    appropriate concentration of test chemical, the PC, or the VC (see
    Figure 2 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
    multi-channel 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. \[The
    Vehicle Control blank (VCb) wells (column 1, column 12, wells A2,
    A11, H2, H11) will receive the Vehicle Control dosing solutions
    (which should include any solvents used). Blanks for wells A3 -- A10
    and H3 -- H10 shall receive the appropriate test chemical solution
    for each concentration (e.g., wells A3 and H3 receive C~1~
    solution). The test chemical blanks in rows A and H will be used for
    their respective test chemical concentrations.\] Incubate cells for
    48 h ± 0.5 h (37ºC ± 1ºC, 90 % ± 5 % humidity, and 5.0 % ± 1 %
    CO~2~/air).

c)  **[Positive Control]{.underline}**: 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 mean
    IC~50~ ± two and a half standard deviations (SD) for the SLS
    acceptable tests from Phases Ia and Ib (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 -- see sections VII.F.1 and F.2)..

#### 3. Microscopic Evaluation {#microscopic-evaluation .unnumbered}

> 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 cytotoxicity]{.underline}. 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.
>
> 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 {#measurement-of-nru .unnumbered}

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 sterile paper towels. Add 250 µL NR
    medium (to all wells including the blanks) and incubate (37ºC ± 1ºC,
    90 % ± 5 % humidity, and 5.0 % ± 1 % CO~2~/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 pre-warmed 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). Observe the wells for bubbles.
    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. \[Phases Ia and Ib data show the mean OD value for the
    plate blanks to be 0.055 0.035 for NHK cells ( 2.5 standard
    deviations; data from 3 labs; N = 156). Use this range as a
    **guide** for assessment of the blank values.\] Save raw data in the
    Excel format as provided by the SMT.

#### 5. Quality Check of Assay {#quality-check-of-assay .unnumbered}

> a\) Test Acceptance Criteria

1.  A test meets acceptance criteria, if the IC~50~ for SLS is within
    two and a half (2.5) standard deviations of the historical mean
    established by the Test Facility (as per **VII.F.2.c**).

2.  A test meets acceptance criteria if the left and the right mean of
    the VCs do not differ by more than 15.0 % from the mean of all VCs.

> 3\) A test meets acceptance criteria if:

-   at least one calculated cytotoxicity value ≥ 10.0 % and ≤ 50.0 %
    viability and

-   at least one calculated cytotoxicity value \> 50.0 % and ≤ 90.0 %
    viability.

> 4\) A test meets acceptance criteria if the r^2^ (coefficient of
> determination) value calculated for the Hill model fit (i.e., from
> PRISM software) is ≥ 0.90. A test does not meet acceptance criteria if
> the r^2^ value is \< 0.80. If the r^2^ value is ≥ 0.80 and \< 0.90
> ("gray zone"), then the SMT will evaluate the model fit and make the
> determination of whether or not the test meets the acceptance criteria
> and relate the information to the Study Director.
>
> \[Note: All acceptance criteria must be met for an assay to be
> considered acceptable.\]
>
> \[A corrected mean OD~540\ ±\ 10nm~ of 0.205 - 1.645 for the VCs is a
> target range but will not be a test acceptance criterion. Range
> determined from Phase Ib VC OD values from 3 laboratories (mean ± 2.5
> standard deviations, N = 69).\]
>
> 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.
>
> 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.

###  {#section-11 .unnumbered}

> c\) Quality Check of Concentration-Response
>
> The IC~50~ derived from the concentration-response of the test
> chemicals should be backed by preferably three responses ≥ 10 and ≤ 90
> % inhibition of NRU and at least two responses, one on either side of
> the IC~50~ value (see sections **VII.E.3.b and VII.F.5.a.3**). If this
> is not the case, and the concentration progression factor can be
> easily reduced, reject the experiment and repeat it with a smaller
> progression factor. In addition, the dilution scheme shall be adjusted
> in subsequent replicate assays, if necessary, to increase the number
> of points on both sides of the IC~50~ in the 10-90% response range.
> Numerical scoring of the cells (see **VII.F.3**) should be determined
> and documented in the Study Workbook.

###  {#section-12 .unnumbered}

### G. Data Analysis {#g.-data-analysis .unnumbered}

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. The Study Director will use good biological/scientific
judgment for determining "unusable" wells that will be excluded from the
statistical analysis. This value is compared with the mean NRU of all VC
values (provided VC values have met the VC acceptance criteria).
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 with macros provided by the SMT) that will
automatically determine cell viability and perform statistical analyses
(including determination of outliers).

> The concentration of a test chemical reflecting a 20 %, 50 %, and 80 %
> inhibition of cell viability (i.e., the IC~20~, IC~50~, and IC~80~) is
> determined from the concentration-response by applying a Hill function
> to the concentration-response data. Statistical software (e.g.,
> GraphPad PRISM 3.0) specified by the SMT shall be used to calculate
> IC~20~, IC~50~, and IC~80~ values (and the associated confidence
> limits) for each test chemical. In addition, the SMT shall provide
> guidelines for calculating ICx values and confidence limits. The
> Testing Facility shall report data using at least three (3)
> significant figures and shall forward the results from each assay to
> the SMT/biostatistician 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.

## VIII. REFERENCES {#viii.-references .unnumbered}

> Clonetics Normal Human Keratinocyte Systems Instructions for Use,
> AA-1000-4-Rev.03/00. ([http://www.clonetics.com)]().

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.

## IX. APPROVAL {#ix.-approval .unnumbered}

> **\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_
> \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_**
>
> **SPONSOR REPRESENTATIVE DATE**
>
> **(Print or type name)**
>
> **\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_
> \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_**
>
> **Testing Facility STUDY DIRECTOR DATE**
>
> **(Print or type name)**
>
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