**TEST METHOD PROTOCOL**

**for the BALB/c 3T3 Neutral Red Uptake Cytotoxicity Test**

#  {#section .unnumbered}

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

**Phase Ib**

**November 15, 2002**

**Revised November 22, 2002**

Revised by IIVS Nov. 26, 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 BALB/c 3T3 Neutral Red Uptake Cytotoxicity Test

A Test for Basal Cytotoxicity

**Phase Ib**

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

The purpose of this study is to evaluate the cytotoxicity of test
chemicals using the BALB/c 3T3 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 supports 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.  **BALB/c 3T3 Neutral Red Uptake Cytotoxicity Test**

> The 3T3 NRU test will be performed to analyze the *in vitro* toxicity
> of three (3) 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.

##  {#section-1 .unnumbered}

**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 (3)*

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

Vehicle (Negative): Assay medium (DMEM containing 5% NBCS,

4 mM L-Glutamine, 100 IU/mL Penicillin,

100 µg/mL Streptomycin)

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

**IV. testing facility and key personnel**

1)  Name:

2)  Address:

3)  Study Director:

4)  Laboratory Technician(s):

5)  Scientific Advisor:

6)  Quality Assurance Director:

7)  Safety Manager:

8)  Facility Management:

**A. 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-2 .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.

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

### 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.

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

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

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

> BALB/c 3T3 cells, clone 31
>
> [CCL-163]{.underline}, LGC Reference Materials, Customer Service,
> Queens Road, Teddington, Middlesex, TW110LY, UK
>
> [CCL-163]{.underline}, American Type Culture Collection \[ATCC\],
> Manassas, VA, USA)

#### 2. Technical Equipment {#technical-equipment .unnumbered}

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

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

2)  Laminar flow clean bench/cabinet (standard: \"biological hazard\")

3)  Water bath: 37ºC ± 1ºC

4)  Inverse phase contrast microscope

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

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

7)  Laboratory balance

8)  96-well plate spectrophotometer (i.e., plate reader) equipped with
    540 nm ± 10 nm filter

9)  Shaker for microtiter plates

10) Cell counter or hemocytometer

11) Pipetting aid

12) Pipettes, pipettors (multi-channel and single channel), dilution
    block

13) Cryotubes

14) Tissue culture flasks (e.g., 75 - 80 cm^2^, 25 cm^2^)

15) 96-well flat bottom tissue culture microtiter plates (e.g., Nunc \#
    167 008; Falcon tissue culture-treated)

16) pH paper (wide and narrow range)

> \[Note: Tissue culture flasks and microtiter plates should be
> prescreened to ensure that they adequately support the growth of 3T3
> cells.\]

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

-   Dulbecco's Modification of Eagle's Medium (DMEM) without
    L-Glutamine; should have high glucose \[4.5gm/l\] (e.g., ICN-Flow
    Cat. No. 12-332-54)

-   L-Glutamine 200 mM (e.g., ICN-Flow \# 16-801-49)

-   New Born Calf Serum (NBCS or NCS) (e.g., Biochrom \# SO 125)

-   0.05 % Trypsin/0.02 % EDTA solution (e.g., SIGMA T 3924, ICN-Flow,
    \# 16891-49)

-   Phosphate buffered saline (PBS) without Ca^2+^ and Mg^2+^(for
    trypsinization)

-   Hanks' Balanced Salt Solution (HBSS) without Ca^2+^ and
    Mg^2+^(CMF-HBSS)

-   Dulbecco's Phosphate Buffered Saline (D-PBS) \[formulation
    containing calcium and magnesium cations; glucose optional\] (for
    rinsing)

-   Penicillin/streptomycin solution (e.g. ICN-Flow \# 16-700-49)

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

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

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

-   Glacial acetic acid, analytical grade

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

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

> \[Note: Due to lot variability of NBCS/NCS, first check a lot for
> growth stimulating properties with 3T3 cells (approximately 20-24 h
> doubling time) and then reserve a sufficient amount of NBCS/NCS. May
> use pre-tested serum lot from Phase Ia of the validation study if the
> serum has been stored under appropriate conditions and shelf-life has
> not expired.\]

### 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-3 .unnumbered}

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

> DMEM (buffered with sodium bicarbonate) supplemented with (final
> concentrations in DMEM are quoted):
>
> a\) for freezing (Freeze Medium); contains 2X concentration of
> NBCS/NCS and DMSO of final freezing solution\
> 40 % NBCS/NCS\
> 20 % DMSO
>
> b\) for routine culture (Routine Culture Medium)\
> 10 % NBCS/NCS\
> 4 mM Glutamine
>
> c\) for solubility testing and test chemical dilution (Chemical
> Dilution Medium)\
> 4 mM Glutamine\
> 200 IU/mL Penicillin\
> 200 µg/mL Streptomycin

d)  for dilution of NR stock solution (NR Dilution Medium)

> 5 % NBCS/NCS\
> 4 mM Glutamine\
> 100 IU/mL Penicillin\
> 100 µg/mL Streptomycin
>
> \[Note: The Chemical Dilution Medium with test chemical will dilute
> the serum concentration of the Routine Culture Medium in the test
> plate to 5 %. Serum proteins may mask the toxicity of the test
> substance, but serum cannot be totally excluded because cell growth is
> markedly reduced in its absence.\]
>
> Completed media formulations should be kept at approximately 2-8° C
> and stored for no longer than two weeks.

#### 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.

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

#### 3. Neutral Red (NR) Medium {#neutral-red-nr-medium .unnumbered}

> EXAMPLE:
>
> 1 mL (3.3 mg NR dye/mL) NR Stock Solution

99 ml 99 mL NR Dilution Medium (pre-warmed to 37° C)

> The final concentration of the NR Medium is 33 μg NR dye/mL.
>
> \[Note: The NR medium may be centrifuged at approximately 600 x g for
> 10 min (to remove NR crystals). The NR Medium shall be filtered (e.g.,
> Millipore filtering, 0.2 -- 0.45 μm pore size) to reduce NR crystals.
> The temperature of the NR Medium should be maintained at 37° C (e.g.,
> in a waterbath) before adding tothe cells and will be used within 15
> minutes after removing from 37° C storage. Aliquots of NR Medium can
> be made on the day of testing and maintained at 37° C for later use.\]

#### 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}

> BALB/c 3T3 cells are routinely grown as a monolayer in tissue culture
> grade flasks (e.g., 75 - 80 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 noted in a Study
> Workbook.

#### 2. Receipt of Cryopreserved BALB/c 3T3 Cells {#receipt-of-cryopreserved-balbc-3t3-cells .unnumbered}

> Upon receipt of cryopreserved BALB/c 3T3 cells, the vial(s) of cells
> shall be stored in a liquid nitrogen freezer until needed.

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

#### 3. Thawing Cells {#thawing-cells .unnumbered}

> Thaw cells by putting ampules into a water bath at 37°C ± 1ºC. Leave
> for as brief a time as possible.

a)  Resuspend the cells in pre-warmed Routine Culture Medium and
    transfer into pre-warmed Routine Culture Medium in a tissue-culture
    flask.

b)  Incubate at 37ºC ± 1ºC, 90 % ± 5 % humidity, and 5.0 % ± 1 %
    CO~2~/air.

c)  When the cells have attached to the bottom of the flask (within 4 to
    24 h), decant the supernatant and replace with fresh pre-warmed
    (37ºC) medium. Culture as described above.

d)  Passage at least two times before using the cells in a cytotoxicity
    test.

> A fresh batch of frozen cells from the stock lot of cells should be
> thawed out and cultured approximately every two months. This period
> resembles a sequence of about 18 passages.

#### 4. Routine Culture of BALB/C 3T3 Cells {#routine-culture-of-balbc-3t3-cells .unnumbered}

> When cells exceed 50 % confluence (but less than 80 % confluent) they
> should be removed from the flask by trypsinization:

a)  Decant medium, rinse cultures with 5 mL PBS or Hanks' BSS ([without
    Ca^2+^, Mg^2+^)]{.underline} per 25 cm^2^ flask (15 mL per 75 cm^2^
    flask). Wash cells by gentle agitation to remove any remaining serum
    that might inhibit the action of the trypsin.

b)  Discard the washing solution.

c)  Add 1-2 mL trypsin-EDTA solution per 25 cm^2^ to the monolayer for a
    few seconds (e.g., 15-30 seconds).

d)  Remove excess trypsin-EDTA solution and incubate the cells at room
    temperature.

e)  After 2-3 minutes (min), lightly tap the flask to detach the cells
    into a single cell suspension.

#### 5. Cell Counting {#cell-counting .unnumbered}

> After detaching the cells, add 0.1-0.2 mL of pre-warmed (37ºC) Routine
> Culture Medium/cm^2^ to the flask (e.g., 2.5 mL for a 25 cm^2^ flask).
> Disperse the monolayer by gentle trituration. It is important to
> obtain a single cell suspension for exact counting. Count a sample of
> the cell suspension obtained using a hemocytometer or cell counter
> (e.g., Coulter counter).

#### 6. Subculture of Cells {#subculture-of-cells .unnumbered}

###  {#section-7 .unnumbered}

> After determination of cell number, the culture can be sub-cultured
> into other flasks or seeded into 96-well microtiter plates. BALB/c 3T3
> cells are routinely passaged at suggested cell densities as listed in
> the table (approximate doubling time is 20-24 h). The individual
> laboratories will need to determine and adjust the final density to
> achieve appropriate growth.
>
> Table 1. Cell Densities for Subculturing

  --------------- --------------- -------------------- --------------------
  Days in Culture Seeding Density Total Cells per 25   Total Cells per 75
                  (cells/cm^2^)   cm^2^ flask          cm^2^ flask

  2               16800           4.2 x 10^5^          1.26 x 10^6^

  3               8400            2.1 x 10^5^          6.3 x 10^5^

  4               4200            1.05 x 10^5^         3.15 x 10^5^
  --------------- --------------- -------------------- --------------------

> \[Note: It is important that cells have overcome the lag growth phase
> when they are used for the test.\]

#### 7. Freezing Cells (procedure required only if current stock of cells is depleted) {#freezing-cells-procedure-required-only-if-current-stock-of-cells-is-depleted .unnumbered}

###   {#section-8 .unnumbered}

> Stocks of BALB/c 3T3 cells can be stored in sterile, freezing tubes in
> a liquid nitrogen freezer. DMSO is used as a cryoprotective agent.

a)  Centrifuge trypsinized cells at approximately 200 x g.

b)  Suspend the cells in cold Routine Culture Medium (half the final
    freezing volume) so a final concentration of 1-5x10^6^ cells/mL can
    be attained.

c)  Slowly add cold Freeze Medium to the cells so that the solvent will
    equilibrate across the cell membranes. Bring the cell suspension to
    the final freezing volume. The final cell suspension will be 10 %
    DMSO. Aliquot the cell suspension into freezing tubes and fill to
    1.8 mL.

d)  Place the tubes into an insulated container (e.g., styrofoam trays)
    and place in a freezer (-70 to -80°C) for 24 h. This gives a
    freezing rate of approximately 1°C/min. The laboratory needs to
    ensure that the freezing protocol is applicable to the 3T3 cells and
    that the cells are viable when removed from cryopreservation.

e)  Place the frozen tubes into liquid nitrogen for storage.

#### 8. Preparation of Cells for Assays {#preparation-of-cells-for-assays .unnumbered}

1.  Cultured cells that are going to be used in seeding the 96-well
    plates should be fed fresh medium the day before subculturing to the
    plates. On the day of plate seeding, prepare a cell suspension of
    2.5x10^4^cells/mL in Routine Culture Medium. Using a multi-channel
    pipette, dispense 100 µl Routine Culture Medium only into the
    peripheral wells (blanks) of a 96-well tissue culture microtiter
    plate (See **Section VII.F.1**). In the remaining wells, dispense
    100 µl of a cell suspension of 2.5x10^4^ cells/mL (= 2.5x10^3^
    cells/well). The seeding density should be noted to ensure that the
    cells in the control wells are not overgrown after three days (i.e.,
    24 h incubation in step **b** and 48 h exposure to test chemicals).
    Prepare one plate per chemical to be tested.

2.  Incubate cells for 24 [+]{.underline} 1 h (37ºC ± 1ºC, 90 % ± 5 %
    humidity, 5.0 % ± 1 % CO~2~/air) so that cells form a less than half
    confluent monolayer. This incubation period assures cell recovery
    and adherence and progression to exponential growth phase.

3.  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.

#### 9. 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 Ib 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 NR Dilution Medium (5 % NBCS/NCS).
    Seed cells at 4200 cells/cm^2^.

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) if Study Director sees a need. Use appropriate
    size exclusion limits if using a Coulter counter. Determine the
    total number of cells and document. Repeat sampling at 24 h, 48 h,
    72 h, and 96 h post inoculation. Change culture medium at 72 h 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. 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 medium, the starting concentration is 20,000
> μg/ml (i.e., 20 mg/mL) in Tier 1, but for DMSO and ethanol the
> starting concentration is 200,000 μg/ml (i.e., 200 mg/mL) in Tier 2.
> Weighing out chemical for each solvent (i.e., medium, 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 - medium, 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**

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

```{=html}
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```
b)  If the test chemical is insoluble in Chemical Dilution 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 Chemical Dilution 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). In another glass tube, also
    add approximately 100 mg test chemical to enough ethanol to make the
    total volume approximately 0.5 mL (for 200 mg/mL). Mix both
    solutions as specified in **Section** **VII.D.2.a** in an attempt to
    solubilize the test chemical. If the chemical is soluble in either
    solvent, no additional solubility procedures are needed.

> c\) If the chemical is NOT soluble in Chemical Dilution Medium, 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
> μg/mL solution, and following the mixing procedures in **Section**
> **VII.D.2.a**.
>
> [Example]{.underline}: If complete solubility is not achieved at
> 20,000 μg/mL in Chemical Dilution 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 Chemical Dilution Medium, two samples of \~ 100 mg test
> chemical are weighed to attempt to solubilize in DMSO and ethanol at
> 200,000 μg/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 (Chemical Dilution Medium, DMSO, and ethanol) prepared in
> Tier 2 are diluted by 10 so as to test 200 μg/mL in media, and 20,000
> μg/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 Chemical Dilution Medium, DMSO,
or Ethanol

+---------------+----------+----------+---------+---------+---------+
| # TIER {#tier | **1**    | **2**    | **3**   | **4**   | **5**   |
|  .unnumbered} |          |          |         |         |         |
+---------------+----------+----------+---------+---------+---------+
| Total Volume  | 0.5 mL   | 5 mL     | 50 mL   |         |         |
|               |          |          |         |         |         |
| Chemical      |          |          |         |         |         |
| Dilution      |          |          |         |         |         |
| Medium        |          |          |         |         |         |
+---------------+----------+----------+---------+---------+---------+
| Concentration | 20,000   | 2,000    | 200     |         |         |
| of Test       | μg/mL    | μg/mL    | μg/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       |          | μg/mL    | μg/mL   | μg/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       |          |          |         |         | μg/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      | 1 μg/mL |
| alent Concent | μg/mL    | μg/mL    | μg/mL   | μg/mL   |         |
| ration on Cel |          |          |         |         | (0.001  |
| ls  {#equival | (10      | (1       | (0.1    | (0.01   | mg/mL)  |
| ent-concentra | mg/mL)   | mg/mL)   | mg/mL)  | mg/mL)  |         |
| tion-on-cells |          |          |         |         |         |
|  .unnumbered} |          |          |         |         |         |
+---------------+----------+----------+---------+---------+---------+

> NOTE: The amounts of test chemical weighed and Chemical Dilution
> 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 Chemical Dilution     |
| 1:     | Medium:                                                     |
|        |                                                             |
|        | -   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. This can
> be performed by warming 5 mL tubes in a 37°C water bath for at least
> 5-10 minutes before evaluating solubility. Warm larger vessels for at
> least 15-20 minutes in a 37°C water bath before evaluating solubility.
>
> 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
> Chemical Dilution Medium, DMSO, and then ethanol. Thus, if (all
> solvents for a particular tier are tested simultaneously and) a test
> chemical dissolves in more than one solvent, then the choice of
> solvent follows this hierarchy. For example, if, at any tier, a
> chemical is soluble in Chemical Dilution 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.

## 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 Chemicals in Solution**

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

b)  Prepare test chemical immediately prior to use. 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.

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 Chemical Dilution
    Medium, or

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

4.  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
    Chemical Dilution Medium before application to 3T3 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 μg/mL), dissolve the chemical in DMSO
> at 200,000 μg/mL for the chemical stock solution.

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

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

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

4)  Add 0.1 mL of 20,000 μg/mL dilution from tube #2 to tube #3 to make
    another 1:10 dilution (i.e., 1:100 dilution from stock solution) in
    solvent (i.e., 2,000 μg/mL)

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

6)  Since each concentration is 200 fold greater than the concentration
    to be tested, make a 1:100 dilution by diluting 1 part dissolved
    chemical in each tube with 99 parts of Chemical Dilution Medium
    (e.g., 0.1 mL test chemical in DMSO + 9.9 mL Chemical Dilution
    Medium) to derive the eight 2X concentrations for application to 3T3
    cells. Each 2X test chemical concentration will then contain 1 % v/v
    solvent. The 3T3 cells will have 0.05 mL Routine Culture Medium in
    the wells prior to application of the test chemical. By adding 0.05
    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 μg/mL) in a total
    of 0.1 mL and the solvent concentration in the wells will be 0.5%
    v/v.

7)  A test article prepared in Chemical Dilution Medium, 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. 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-9 .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-10 .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 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. 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
>
> 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 Chemical Dilution 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 Chemical Dilution Medium will be
    added to the vessel so that the concentration is 200,000 μg/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 μg/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 3T3 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     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)

#### 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. 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 50 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.

b)  After 24 h ± 1 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 50 µL of fresh pre-warmed Routine Culture Medium to
    all of the wells, including the blanks. Add 50 µL of Chemical
    Dilution Medium to the blank wells. Then add 50 µL Chemical Dilution
    Medium containing either the appropriate concentration of test
    chemical, the PC, or the VC (see **Figure 2** for the plate
    configuration). The solutions will be transferred from the dummy
    plate to the test plate by adding the vehicle control first then
    lowest to highest dose so that the same pipette tips on the eight
    channel pipettor can be used for the whole plate.

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 the development of the positive control database in
    Phase Ia of the Validation Study. The mean IC~50~ and ± two standard
    deviations (SD) of the IC~50~ of SLS (mutually agreed upon by the
    Testing Facility and the SMT) are the values that will be used as an
    acceptance criterion for test sensitivity for the 3T3 NRU assay.
    This plate will follow the same schedule and procedures as used for
    the test chemical plates.

#### 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 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., at 1, 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.

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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.

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. \[Phase Ia data show the mean OD value for the plate
    blanks to be 0.051 ± 0.022 for 3T3 cells (± two standard deviations;
    data from 3 labs; N = 59). Use this value as a
    **[guide]{.underline}** for assessment of the blank values.\] Save
    raw data in the Excel format as provided by the SMT.

###  {#section-11 .unnumbered}

#### 5. Quality Check of 3T3 NRU Assay {#quality-check-of-3t3-nru-assay .unnumbered}

> a\) Test Acceptance Criteria
>
> 1\) A test meets acceptance criteria, if the IC~50~ for SLS (PC) is
> within ± two (2) standard deviations of the historical mean
> established by the Test Facility (as per **VII.F.2.e**).
>
> 2\) A test meets acceptance criteria if the corrected mean OD~540~ of
> VCs is ≥ 0.30 and ≤ 0.80.

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.

4.  A test meets acceptance criteria if a minimum of two points, one on
    each side of the IC~50~ value, are determined and fall within the
    range ≥10 % and ≤ 90 % effect.

> \[Note: All acceptance criteria must be met for an assay to be
> considered acceptable.\]
>
> 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-12 .unnumbered}

> c\) Quality Check of Concentration-Response
>
> The IC~50~ derived from the concentration-response of the test
> chemicals will 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 **VII.E.3.b**). 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-13 .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 replicate well) per test
concentration (blanks will be subtracted). 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 SMT 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. 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}

> 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.

Spielmann, H., S. Gerner, S. Kalweit, R. Moog, T. Wirnserberger, K.
Krauser, R. Kreiling, H. Kreuzer, N.P. Luepke, H.G. Miltenburger, N.
Müller, P. Murmann, W. Pape, B. Siegmund, J. Spengler, W. Steiling, and
F.J. Wiebel. 1991. Interlaboratory assessment of alternatives to the
Draize eye irritation test in Germany. Toxicol. *In Vitro* 5: 539-542.

##  {#section-14 .unnumbered}

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

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