**TEST METHOD PROTOCOL**

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

# 

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

**June 14, 2002**

**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

# The Normal Human Keratinocyte (NHK) Neutral Red Uptake Cytotoxicity Test

**A Test for Basal Cytotoxicity**

## I. 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.  **Determination of Positive Control Database**

> An historical database of IC~50~ values for the positive control
> chemical (Sodium Lauryl \[dodecyl\] Sulfate {SLS}) must be established
> and maintained by performing 10 concentration-response assays on the
> NHK cells before performing the NRU assay on test chemicals. Once the
> mean IC~50~ and the 95 % confidence interval (CI) of the IC~50~ of SLS
> are established, the values will be used as an acceptance criterion
> for test sensitivity for the NHK NRU assay.

B.  **NHK Neutral Red Uptake Cytotoxicity Test**

> After acceptable positive control mean IC~50~ and 95 % CI values have
> been established, the NHK NRU test will be performed to analyze the
> *in vitro* toxicity of test chemicals. This test will be used to
> determine IC~20~, IC~50~, and IC~80~ values for a 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 1*

**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**

-   Name:

-   Address:

-   Study Director:

-   Laboratory Technician(s):

-   Scientific Advisor:

-   Quality Assurance Director:

-   Safety Manager:

-   Facility Management:

**A. Test Schedule**

-   Proposed Experimental Initiation Date:

-   Proposed Experimental Completion Date:

-   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.

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

### 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.

> **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.

## 

## 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.
>
> *Clonetics/BioWhittaker* \[BioWhittaker, 8830 Biggs Ford Road,
> Walkersville, MD 21793-0127
>
> *BioWhittaker Europe* \[BioWhittaker Europe, S.P.R.L. Parc Industriel
> de Petit Rechain, B-4800 Verviers, BELGIUM\]

#### 2. Technical Equipment

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), 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)

### 

> \[Note: Tissue culture flasks and microtiter plates should be
> prescreened to ensure that they adequately support the growth of
> NHK.\]

#### 3. Chemicals, Media, and Sera

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, 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) with glucose)
    formulation containing calcium and magnesium cations, and
    supplemented with 1000 mg/L glucose)

g)  Fetal bovine serum (FBS)

h)  Neutral Red (NR) Dye -- tissue culture-grade; liquid form (e.g.,
    SIGMA N 2889); powder form (e.g., SIGMA N 4638)

i)  Dimethyl sulfoxide (DMSO), U.S.P analytical grade (Store under
    nitrogen @ -20ºC)

j)  Ethanol (ETOH), U.S.P. analytical grade (100 %, non-denatured for
    test chemical preparation; 95 % can be used for the desorb solution)

k)  Glacial acetic acid, analytical grade

l)  Hanks\' Balanced Salt Solution without Ca^2+^ or Mg^2+^ (CMF-HBSS)
    (e.g., Invitrogen \# 14170)

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

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

### B. Preparations of Media and Solutions

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

#### 

#### 1. Media

> 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 500ml of 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
>
> 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 4°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 concentration of
> calcium.
>
> 165 ul 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. If the liquid form is not available,
> the following formulation can be prepared.
>
> 0.4 g NR Dye powder in 100 ml of H~2~O
>
> Make up prior to use and store dark at room temperature. May store for
> up to two months.

#### 

#### 3. Neutral Red (NR) Medium

EXAMPLE:

> 1 ml (4mg NR dye/ml) NR Stock Solution\
> 79 ml KGM
>
> The final concentration of the NR Medium is 50 μg NR dye/ml.
>
> \[Note: The NR medium should be incubated overnight at 37ºC ± 1ºC and
> centrifuged at approximately 600 x g for 10 min (to remove NR
> crystals) before adding to the cells. Alternative procedures (e.g.,
> Millipore filtering) can be used as long as they guarantee that NR
> medium is free of crystals.\]

#### 

#### 4. Ethanol/Acetic Acid Solution (NR Desorb)

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

### C. Methods

### 

#### 1. Cell Maintenance and Culture Procedures

> 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 basis under a
> phase contrast microscope, and any changes in morphology or their
> adhesive properties must be noted in a Study Workbook (See **Section
> VII.F.3**)

#### 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 Cultures

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 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, 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)    |             |            |            |
|                            | (2500       | (5000      | (9000      |
| 1 flask each cell          | cm^2^)      | cm^2^)     | 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

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

(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 second rinse should
    be left on the cells for approximately 5 minutes. Discard the
    washing solution.

(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.

(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 of 0.8 - 1x10^4^cells/ml in Routine
    Culture Medium. Using a multi-channel pipette, dispense 250 µl PBS
    only into the peripheral wells (blanks) of a 96-well tissue culture
    microtiter plate. In the remaining wells, dispense 250 µl of the
    cell suspension (2x10^3^ -- 2.5x10^3^ cells/well). Prepare one plate
    per chemical to be tested.

(h) Incubate cells (37ºC ± 1ºC, 90 % ± 5.0 % humidity, and 5 % ± 1 %
    CO~2~/air) so that cells form a 30+ % 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)  Establish cells in culture and trypsinize cells as per **Section
    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). 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).

> **D. Establishing the Positive Control Database**
>
> An historical database of IC~50~ values for the positive control
> chemical (Sodium Lauryl \[dodecyl\] Sulfate {SLS}) must be established
> and maintained by performing 10 concentration-response assays on the
> NHK cells.

1.  **Positive Control Chemical Preparation**

> The positive control chemical (SLS) is prepared in the same manner as
> the test chemical (**Sections E.1 and E.2**) by following the
> instructions and substituting "test chemical" with "SLS."

2.  **Range Finder Experiment**

> Before initiating the 10 concentration-response assays, a range finder
> experiment will be performed using [eight]{.underline} concentrations
> of SLS by diluting the stock solution with a constant factor as per
> **Section E.3.a** and **E.3.b**. The eight chemical concentrations
> will be tested as per the test procedure outlined in **Section F** and
> analyzed as per procedures outlined in **Section G**.

3.  **Test Procedure**

> Once a range has been determined that satisfies the criteria in
> **Section E.3.b**, the definitive concentration-response assays shall
> use a ^6^√10 = 1.47 dilution scheme centered on the IC~50.~ The Test
> Facility will perform two tests per day on five different days. The 95
> % CI of the IC~50~ of SLS will be established and defined as an
> acceptance criterion for test sensitivity for the NHK NRU assay. The
> confidence intervals shall be calculated using the average of the
> individual IC~50~ values from each positive control assay performed.
> An example of an historical mean IC~50~ of SLS in NHK cultures is
> **4.4 μg/ml ± 0.97 μg/ml \[**two standard deviations\] (Triglia,
> 1989). All testing will follow the instructions in **Section F** using
> the 96-well plate configuration in Figure 1. The test meets acceptance
> criteria if the conditions in **Sections F.5.a.2** and **F.5.a.3** are
> met.
>
> ***Figure 1. 96-Well Plate Configuration for Positive Control and Test
> Chemical Assays***

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

  A     b     b     b      b      b      b      b      b      b      b      b     b

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

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

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

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

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

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

  H     b     b     b      b      b      b      b      b      b      b      b     b
  ----- ----- ----- ------ ------ ------ ------ ------ ------ ------ ------ ----- -----

> 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)

### 

### E. Preparation of Test Chemicals

> \[Note: Test chemical must be freshly prepared immediately prior to
> use. Each stock dilution should have at least 1-2 ml total volume to
> ensure adequate solution for the test wells in a single 96-well plate.
> The solutions must not be cloudy nor have noticeable precipitate. Test
> chemicals must be at room temperature before dissolving and diluting.
> Preparation under red or yellow light may be necessary, if rapid
> photodegradation is likely to occur.\]

#### 1. Dissolving Test Chemical

a)  Approximately 200,000 μg (200 mg) of the test chemical will be
    weighed into a glass tube and the weight will be documented.
    Assay-specific culture medium will be added to the vessel so that
    the concentration is 2,000,000 μg/ml (2000 mg/ml) and mixed using
    the mixing procedures outlined in **Section** **E.1.c**. If complete
    solubility is achieved, then additional solubility procedures are
    not needed. The test chemical can then be prepared and diluted for
    use in an assay. If only partial solubility is achieved, then add
    additional medium in the steps outlined in Table 1 until the
    concentration is a minimum of 200,000 μg /ml. If complete solubility
    at 200,000 μg/ml in culture medium can't be attained, then repeat
    the solubility steps in Table 1 and **Section E.1.c** using the
    other solvent(s) in the solubility hierarchy. Test chemicals that
    are only soluble in DMSO or ethanol will be prepared at 2,000,000
    μg/ml as the highest concentration of stock solution.

> Table 1 Determination of Solubility

+------------------------------------+----------+----------+----------+
| Solubility Data                    | Step 1   | Step 2   | Step 3   |
+------------------------------------+----------+----------+----------+
| Total volume of medium added (ml)  | 0.1      | 0.5      | 1.0      |
|                                    |          |          |          |
| Total volume of DMSO or ethanol    | 0.1      | 0.5      | 1.0      |
| added (ml)                         |          |          |          |
|                                    | ≥        | 400,000  | 200,000  |
| Approximate solubility (μg /ml)    | 2        |          |          |
|                                    | ,000,000 |          |          |
+------------------------------------+----------+----------+----------+

> [Example]{.underline}: If complete solubility is not achieved in 0.1
> ml medium (Step 1), then 0.4 ml is added to obtain a total volume of
> 0.5 ml (Step 2). No additional weighing of chemical is needed.
> Chemical and medium are again mixed in an attempt to dissolve.
>
> b\) Each test chemical will be prepared such that the highest test
> concentration applied to the cells in each range finding experiment is
> 100,000 μg/ml in culture medium (10,000 μg/ml if DMSO or ethanol is
> used). If 100,000 μg/ml in culture medium cannot be achieved, then the
> highest concentration attainable will be used. If the range finding
> experiment shows that 10,000 μg/ml is not high enough for the range of
> chemicals dissolved in DMSO or ethanol to meet the acceptance
> criteria, then higher concentrations will be used for the definitive
> experiment.
>
> .
>
> c\) The following mixing and solvent hierarchy will be followed in
> dissolving the test chemical:
>
> 1\) Dissolve test chemical in Treatment Medium.
>
> 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.
>
> If the test chemical doesn't dissolve (i.e., solution is cloudy or has
> precipitate) in the Treatment Medium, then follow the steps 1) through
> 4) using DMSO instead of Treatment Medium.
>
> If the test chemical doesn't dissolve in DMSO, then follow steps 1)
> through 4) using ethanol instead of DMSO.
>
> d\) For the range finding experiments, the highest 2x concentration of
> test chemical dissolved only in culture medium will be 200,000 μg/ml
> (200 mg/ml). The highest 2x concentration of test chemical first
> dissolved in DMSO or ethanol then transferred to culture medium will
> be 20,000 μg/ml (20 mg/ml). Dissolve test chemical in appropriate
> medium/solvent (at 200-fold the desired final test concentration in
> the case of DMSO or ethanol solvents, i.e., 20,000 μg/ml). The final
> solvent (DMSO or ethanol) concentration for application to the cells
> should be kept at a constant level of 0.5 % (v/v) in the vehicle
> controls and in all of the eight test concentrations. 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

1)  Label eight tubes 1 -- 8. Add 0.9 ml solvent (e.g., DMSO or ethanol)
    to tubes 2 \-- 8.

2)  Prepare stock solution of 2,000,000 μg test chemical/ml solvent in
    tube \# 1.

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

4)  Add 0.1 ml of 200,000 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., 20,000 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, dilute 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 8 2x concentrations for application
    to NHK cells. Each 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 10,000 μg/ml) in a
    total of 0.250 ml and the solvent concentration in the wells will be
    0.5% v/v.

> Check carefully to determine whether the chemical is still dissolved
> after the transfer from solvent stock solution to medium, and reduce
> the highest test concentration, if necessary. Document all test
> chemical preparations in the Study Workbook.

#### 2. pH of Test Chemical Solutions

> Measure the pH of the highest concentration of the test chemical in
> culture medium using pH paper. Document the pH and note the color of
> the medium for all dilutions. Do not adjust the pH.

#### 3. Concentrations of Test Chemical

#### 

> a\) Range Finder Experiment

#### Test eight concentrations of the test chemical/PC 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.). 

> b\) Main Experiment
>
> 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 (e.g.,
> ^6^√10 = 1.47; NOTE: this dilution factor will be used for the
> definitive positive control assays \[**Section VII.D.3\]**). Cover the
> relevant concentration range (≥10 % and ≤ 90 % effect) with at least
> three points of a graded effect, avoiding too many
> [non-cytotoxic]{.underline} and/or [100 %-cytotoxic]{.underline}
> concentrations. Experiments revealing less than three cytotoxic
> concentrations in the relevant range shall be repeated, where
> possible, with a smaller dilution factor. (Taking into account
> pipetting errors, a progression factor of 1.21 is regarded the
> smallest factor achievable.)

#### c) Test Chemical Dilutions

-   A factor of ^2^√10 = 3.16 could be used for covering a large range:

> (e.g., 1 ⇒3.16 ⇒10 ⇒31.6 ⇒100 ⇒316 ⇒1000 ⇒3160 µg/ml).

-   The simplest geometric concentration series (i.e., constant dilution
    / progression factor) are dual geometric series (e.g., a factor of
    2). These series have the disadvantage of numerical values that
    permanently change between logs of the series:

> (e.g., *log0*-2, 4, 8; *log1-* 16, 32, 64; *log2-* 128, 256, 512;
> *log3-* 1024, 2048,).

-   The decimal geometric series, first described by Hackenberg and
    Bartling (1959) for use in toxicological and pharmacological
    studies, has the advantage that independent experiments with wide or
    narrow dose factors can be easily compared because they share
    identical concentrations. Furthermore, under certain circumstances,
    experiments can even be merged together:

> 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 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.
>
> For an easier biometrical evaluation of several related concentration
> response experiments use decimal geometric concentration series rather
> than dual geometric series. 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.).*

-   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 definitive assay.

### F. Test Procedure

> 1\. The NHK NRU assay for test chemicals will use the 96-well plate
> configuration shown in Figure 1.

#### 2. Application of Test Chemical

a)  Two optional methods for rapidly applying the 2X dosing solutions
    onto the 96-well plates may be utilized. 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 and/or Corning/Transtar model 4878 disposable reservoir
    liners, 8-channel). 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. A third option, though not a recommended option,
    is to transfer test chemical solutions well by well using a single
    channel pipettor or repeat pipettor. This option will increase the
    amount of time needed for test chemical application. The use of a
    repeat pipettor increases the risk of dislodging cells from the
    culture plate.

b)  After 24 - 72 h incubation of the cells, remove Routine Culture
    Medium from the cells by careful inversion of the plate (i.e.,
    "dump") over an appropriate receptacle. Gently blot the plate on a
    sterile paper towel so that the monolayer is minimally disrupted. Do
    not use automatic plate washers for this procedure nor vacuum
    aspiration.

c)  Immediately add 125 µl of fresh Routine Culture Medium to each well.
    Add 125 µl of the appropriate concentration of test chemical, the
    PC, or the VC (see Figure 1 for the plate configuration).

d)  Incubate cells for 48 h ± 0.5 h (37ºC ± 1ºC, 90 % ± 5 % humidity,
    and 5.0 % ± 1 % CO~2~/air).

> e\) **[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 developing the positive control database. This plate
> will follow the same schedule and procedures as used for the test
> chemical plates.

#### 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 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

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 gentle tapping and
    blot the plate. 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 h.

b)  After incubation, remove the NR medium, and carefully rinse cells
    with 250 µl 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.

f)  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. Save raw data in the Excel format as provided by the
    Study Management Team.

#### 5. Quality Check of Assay

> a\) *Test Acceptance Criteria*
>
> 1\) A test meets acceptance criteria, if the IC~50~ for SLS is within
> the 95 % CI of the historical mean established by the Test Facility
> (as per **Section D**).
>
> 2\) A test meets acceptance criteria if the mean OD~540~ of VCs is ≥
> 0.3 and ≤ 1.1.
>
> 3\) A test meets acceptance criteria if the left and the right mean of
> the VCs do not differ by more than 15 % from the mean of all VCs.
>
> *b) Checks for Systematic Cell Seeding Errors*
>
> The [absolute value]{.underline} of optical density (OD~540~ of NRU)
> obtained in the untreated vehicle control may indicate whether the
> 2x10^3^ -- 2.5x10^3^ cells seeded per well have grown exponentially
> with normal doubling time during the assay. Historical optical
> densities observed during doubling time experiments can be used for
> comparison to determine exponential growth.
>
> 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.

### 

> c\) *Quality Check of Concentration-Response*
>
> The IC~50~ derived from the concentration-response of the test
> chemicals should be backed by at least three responses between 10 and
> 90 % inhibition of NRU. 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. Numerical scoring of the
> cells (see **Section F.3**) should be determined and documented in the
> Study Workbook.

### 

### G. Data 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 (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 provided by the Study Management Team for
> determining cell viability and performing statistical analyses.
>
> 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. It will not be necessary for the
> Testing Facilities to derive the equation since statistical software
> (e.g., GraphPad PRISM 3.0) specified by the Study Management Team
> 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
> Study Management Team 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 Study Management Team/biostatistician
> through the designated contacts in electronic format and hard copy
> upon completion of testing. The Study Management Team will be directly
> responsible for the statistical analyses of the Validation Study data.

## VIII. REFERENCES

> 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

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