**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 II**

**May 15, 2003**

**Prepared by**

**The National Toxicology Program (NTP) Interagency Center for the
Evaluation of Alternative Toxicological Methods (NICEATM)**

**Based on Standard Operating Procedure Recommendations from an**

**International Workshop Organized by the Interagency Coordinating
Committee**

**on the Validation of Alternative Methods (ICCVAM)**

**National Institute of Environmental Health Sciences (NIEHS)**

**National Institutes of Health (NIH)**

**U.S. Public Health Service**

**Department of Health and Human Services**

Test Method Protocol

The BALB/c 3T3 Neutral Red Uptake Cytotoxicity Test

A Test for Basal Cytotoxicity

**Phase II**

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

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

##  {#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 (9)*

**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**

**A. Facility Information**

1)  Name:

2)  Address:

3)  Study Director:

4)  Laboratory Technician(s):

5)  Scientific Advisor:

6)  Quality Assurance Director:

7)  Safety Manager:

8)  Facility Management:

**B. Test Schedule**

1)  Proposed Experimental Initiation Date:

2)  Proposed Experimental Completion Date:

3)  Proposed Report Date:

**V. test system**

> The NRU cytotoxicity assay procedure is a cell survival/viability
> chemosensitivity assay based on the ability of viable cells to
> incorporate and bind neutral red (NR), a supravital dye. NR is a weak
> cationic dye that readily penetrates cell membranes by non-ionic
> diffusion and accumulates intracellularly in lysosomes. Alterations of
> the cell surface or the sensitive lysosomal membrane lead to lysosomal
> fragility and other changes that gradually become irreversible. Such
> changes brought about by the action of xenobiotics result in a
> decreased uptake and binding of NR. It is thus possible to distinguish
> between viable, damaged, or dead cells, which is the basis of this
> assay.
>
> Healthy mammalian cells, when maintained in culture, continuously
> divide and multiply over time. A toxic chemical, regardless of site or
> mechanism of action, will interfere with this process and result in a
> reduction of the growth rate as reflected by cell number. Cytotoxicity
> is expressed as a concentration dependent reduction of the uptake of
> the NR after chemical exposure thus providing a sensitive, integrated
> signal of both cell integrity and growth inhibition.

## VI. DEFINITIONS {#vi.-definitions .unnumbered}

##  {#section-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}

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

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

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

c)  Water bath: 37ºC ± 1ºC

d)  Inverse phase contrast microscope

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

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

g)  Laboratory balance

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

i)  Shaker for microtiter plates

j)  Cell counter or hemocytometer

k)  Pipetting aid

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

m)  Cryotubes

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

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

p)  pH paper (wide and narrow range)

q)  Multichannel reagent reservoir

r)  Waterbath sonicator

s)  Magnetic stirrer

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

u)  Dry heat block (optional)

> \[Note: Tissue culture flasks and microtiter plates should be
> prescreened to ensure that they adequately support the growth of 3T3
> cells. Multi-channel repeater pipettes may be used for plating cells
> in the 96-well plates, dispensing plate rinse solutions, NR medium,
> and desorb solution. Do not use the repeater pipette for dispensing
> test chemicals to the cells.\]

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

a)  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)

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

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

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

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

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

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

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

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

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

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

l)  Glacial acetic acid, analytical grade

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

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

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

### 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.
>
> []{.mark}
>
> EXAMPLE: 0.25 g NR Dye powder in 100 mL H~2~O
>
> []{.mark}
>
> 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]{.underline}:
>
> 0.758 mL (3.3 mg NR dye/mL solution) NR Stock Solution

99 ml 99.242 mL NR Dilution Medium (pre-warmed to 37° C)

> The final concentration of the NR Medium is **[25 μg NR
> dye/mL]{.underline}** and aliquots will be prepared on the day of
> application.
>
> \[Note: The NR Medium shall be filtered (e.g., Millipore filtering,
> 0.2 -- 0.45 μm pore size) to reduce NR crystals. Aliquots of the NR
> Medium should be maintained at 37° C (e.g., in a waterbath) before
> adding to the cells and used within 30 min of preparation but also
> used within 15 min after removing from 37° C storage.\]

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

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

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

###  {#section-5 .unnumbered}

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

> 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, briefly 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. Repeat the rinsing procedure and
    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}

a)  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
    2.0 -- 3.0x10^4^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 2.0 -- 3.0x10^4^ cells/mL (=
    2.0 -- 3.0x10^3^ 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.

b)  Incubate cells for 24 ± 2 h (37ºC ± 1ºC, 90 % ± 5 % humidity, 5.0 %
    ± 1 % CO~2~/air) so that cells form a less than half (\< 50%)
    confluent monolayer. This incubation period assures cell recovery
    and adherence and progression to exponential growth phase.

c)  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 II if there is a change in the lot of cells used. Establish
    cells in culture and trypsinize cells as per **Section VII.C.4** for
    subculture. Resuspend cells in 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; Nigrosin) 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) and attempt to dissolve the
    chemical as specified in **Section** **VII.D.2.a**. If the test
    chemical does not dissolve in DMSO, weigh out approximately 100 mg
    test chemical in another glass tube and add enough ethanol to make
    the total volume approximately 0.5 mL (for 200 mg/mL) and attempt to
    dissolve the chemical as specified in **Section** **VII.D.2.a**. If
    the chemical is soluble in either solvent, no additional solubility
    procedures are needed.

> c\) If the chemical is NOT soluble in 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 {  | **1**    | **2**       | **3**      | **4**   | **5**  |
| #tier .un |          |             |            |         |        |
| numbered} |          |             |            |         |        |
+-----------+----------+-------------+------------+---------+--------+
| Total     | 0.5 mL   | 5 mL        | 50 mL      |         |        |
| Volume    |          |             |            |         |        |
|           |          |             |            |         |        |
| Chemical  |          |             |            |         |        |
| Dilution  |          |             |            |         |        |
| Medium    |          |             |            |         |        |
+-----------+----------+-------------+------------+---------+--------+
| Conc      | 20,000   | 2,000 μg/mL | 200 μg/mL  |         |        |
| entration | μg/mL    |             |            |         |        |
| of Test   |          | (2 mg/mL)   | (0.20      |         |        |
| Chemical  | (20      |             | mg/mL)     |         |        |
|           | mg/mL)   |             |            |         |        |
| (Add \~10 |          |             |            |         |        |
| mg to a   |          |             |            |         |        |
| tube. Add |          |             |            |         |        |
| enough    |          |             |            |         |        |
| medium to |          |             |            |         |        |
| equal the |          |             |            |         |        |
| first     |          |             |            |         |        |
| volume.   |          |             |            |         |        |
| Dilute to |          |             |            |         |        |
| s         |          |             |            |         |        |
| ubsequent |          |             |            |         |        |
| volumes   |          |             |            |         |        |
| if        |          |             |            |         |        |
| ne        |          |             |            |         |        |
| cessary.) |          |             |            |         |        |
+-----------+----------+-------------+------------+---------+--------+
| Total     |          | 0.5 mL      | 5 mL       | 50 mL   |        |
| Volume    |          |             |            |         |        |
| DMS       |          |             |            |         |        |
| O/Ethanol |          |             |            |         |        |
+-----------+----------+-------------+------------+---------+--------+
| Conc      |          | 200,000     | 20,000     | 2,000   |        |
| entration |          | μg/mL       | μg/mL      | μg/mL   |        |
| of Test   |          |             |            |         |        |
| Chemical  |          | (200 mg/mL) | (20 mg/mL) | (2      |        |
|           |          |             |            | mg/mL)  |        |
| (Add      |          |             |            |         |        |
| \~100 mg  |          |             |            |         |        |
| to a      |          |             |            |         |        |
| large     |          |             |            |         |        |
| tube. Add |          |             |            |         |        |
| enough    |          |             |            |         |        |
| DMSO or   |          |             |            |         |        |
| ethanol   |          |             |            |         |        |
| to equal  |          |             |            |         |        |
| the first |          |             |            |         |        |
| volume.   |          |             |            |         |        |
| Dilute to |          |             |            |         |        |
| s         |          |             |            |         |        |
| ubsequent |          |             |            |         |        |
| volumes   |          |             |            |         |        |
| if        |          |             |            |         |        |
| ne        |          |             |            |         |        |
| cessary.) |          |             |            |         |        |
+-----------+----------+-------------+------------+---------+--------+
| Total     |          |             |            |         | 50 mL  |
| Volume    |          |             |            |         |        |
| DMS       |          |             |            |         |        |
| O/Ethanol |          |             |            |         |        |
+-----------+----------+-------------+------------+---------+--------+
| Conc      |          |             |            |         | 200    |
| entration |          |             |            |         | μg/mL  |
| of Test   |          |             |            |         |        |
| Chemical  |          |             |            |         | (0.2   |
|           |          |             |            |         | mg/mL) |
| (Add \~10 |          |             |            |         |        |
| mg to a   |          |             |            |         |        |
| large     |          |             |            |         |        |
| tube. Add |          |             |            |         |        |
| enough    |          |             |            |         |        |
| DMSO or   |          |             |            |         |        |
| ethanol   |          |             |            |         |        |
| to equal  |          |             |            |         |        |
| 50 mL.)   |          |             |            |         |        |
+-----------+----------+-------------+------------+---------+--------+
| # Eq      | 10,000   | 1000 μg/mL  | 100 μg/mL  | 10      | 1      |
| uivalent  | μg/mL    |             |            | μg/mL   | μg/mL  |
| Concentra |          | (1 mg/mL)   | (0.1       |         |        |
| tion on C | (10      |             | mg/mL)     | (0.01   | (0.001 |
| ells  {#e | mg/mL)   |             |            | mg/mL)  | mg/mL) |
| quivalent |          |             |            |         |        |
| -concentr |          |             |            |         |        |
| ation-on- |          |             |            |         |        |
| cells .un |          |             |            |         |        |
| numbered} |          |             |            |         |        |
+-----------+----------+-------------+------------+---------+--------+

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

# *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 for 5 - 60
    min. This can be performed by warming tubes in a 37°C water bath or
    in a CO~2~ incubator at 37°C. The solution may be stirred during
    warming (stirring in a CO~2~ incubator will help maintain proper
    pH).

5.  Proceed to Tier 2 (and Tiers 3-5, if necessary of Table 2 and repeat
    procedures 2-4).

> b\) The preference of solvent for dissolving test chemicals is
> 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]{.underline}. If the
> laboratory has attempted all solubility testing without success, then
> the SMT will provide additional guidance for achieving test chemical
> solubility. The SMT anticipates that all validation study test
> chemicals will be tested in the NRU assays.

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

> \[Note: Preparation under red or yellow light is recommended to
> preserve chemicals that degrade upon exposure to light.\]
>
> **1. Test Chemicals in Solution**

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

b)  Prepare test chemical immediately prior to use. Test chemical
    solutions should not be prepared in bulk for use in subsequent
    tests. The solutions must not be cloudy nor have noticeable
    precipitate. Each stock dilution should have at least 1-2 mL total
    volume to ensure adequate solution for the test wells in a single
    96-well plate. The SMT may direct the Study Director to store an
    aliquot (e.g., 1 mL) of the highest 2X stock solution (e.g., low
    solubility chemicals) in a freezer (e.g., -70°C) for use in future
    chemical analyses.

c)  For chemicals dissolved in DMSO or ethanol, the final DMSO or
    ethanol concentration for application to the cells must be 0.5 %
    (v/v) in the vehicle controls and in all of the eight test
    concentrations.

d)  The stock solution for each test chemical should be prepared at the
    highest concentration found to be soluble in the solubility test.
    Thus, the highest test concentration applied to the cells in each
    range finding experiment is:

-   0.5 times the highest concentration found to be soluble in the
    solubility test, if the chemical was soluble in 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.

e)  The seven lower concentrations in the range finding experiment would
    then be prepared by successive dilutions that decrease by one log
    unit each. The following example illustrates the preparation of test
    chemical in solvent and the dilution of dissolved test chemical in
    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 (e.g., pH 0 - 14 to estimate and pH 5 --
> 10 to determine more precise value). The pH paper should be in contact
> with the solution for approximately one minute. Document the final pH
> (i.e., in the EXCEL template) 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 estimated IC~50~ value,
> avoiding too many [non-cytotoxic]{.underline} and/or [100
> %-cytotoxic]{.underline} concentrations. Experiments revealing less
> than one cytotoxic concentration on each side of the IC~50~ value
> shall be repeated, where possible, with a smaller dilution factor.
> Each experiment should have at least one cytotoxicity value ≥ 10.0 %
> and ≤ 50.0 % viability and at least one cytotoxicity value \> 50.0 %
> and ≤ 90.0 % viability. In addition, the dilution scheme shall be
> adjusted in subsequent replicate assays (i.e., definitive assays), if
> necessary, to increase the number of points on both sides of the
> IC~50~ in the 10-90% response range. (Taking into account pipetting
> errors, a progression factor of 1.21 is regarded the smallest factor
> achievable.)

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

> [Maximum Doses to be Tested in the Main Experiments]{.underline}
>
> If minimal or no cytotoxicity was measured in the dose range finding
> assay, a maximum dose for the main experiments will be established as
> follows:

-   For test chemicals prepared in 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.

```{=html}
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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 as shown in **Figure 2**.
>
> ***Figure 2. 96-Well Plate Configuration for Positive Control and Test
> Chemical Assays***

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

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

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

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

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

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

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

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

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

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

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

(C1 = highest, C8 = lowest)

> b = BLANKS (contain **no** cells)\
> VCb = VEHICLE CONTROL BLANK

#### 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.

```{=html}
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```
1)  The first method is to add each of the 2X dosing solutions into
    labeled, sterile reservoirs (e.g., Corning/Costar model 4870 sterile
    polystyrene 50 mL reagent reservoirs; or Corning/Transtar model 4878
    disposable reservoir liners, 8-channel; or other multichannel
    reservoirs).

2)  The second method utilizes a "dummy" plate (i.e., an empty sterile
    96-well plate) prepared to hold the dosing solutions immediately
    prior to treatment of the test plate (with cells). The test chemical
    and control dosing solutions should be dispensed into the dummy
    plate in the same pattern/order as will be applied to the plate
    containing cells. More volume than needed for the test plate (i.e.
    greater than 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. Do not use a multichannel repeater
> pipette for dispensing test chemical to the plates.

b)  After 24 h ± 2 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. Fifty microliters (50 µL) of
    dosing solution will be rapidly transferred from the 8-channel
    reservoir (or dummy plate) to the appropriate wells of the test
    plate using a single delivery multi-channel pipettor. For example,
    the VC may be transferred first (into columns 1, 2, 11, and 12),
    followed by the test article dosing solutions from lowest to highest
    dose, so that the same pipette tips on the multi-channel pipettor
    can be used for the whole plate. \[The Vehicle Control blank (VCb)
    wells (column 1, column 12, wells A2, A11, H2, H11) will receive the
    Vehicle Control dosing solutions (which should include any solvents
    used). Blanks for wells A3 -- A10 and H3 -- H10 shall receive the
    appropriate test chemical solutions for each concentration (e.g.,
    wells A3 and H3 receive C~1~ solution). \[The test chemical blanks
    in rows A and H will be used for their respective test chemical
    concentrations.\]

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 I of the Validation Study. If multiple sets of test chemical
    plates are set up, then clearly designate the positive control
    plates for each set; each set will be an individual entity. The mean
    IC~50~ ± two and a half standard deviations (SD) for the SLS
    acceptable tests from Phases Ia and Ib (after the removal of
    outliers) are the values that will be used as an acceptance
    criterion for test sensitivity for the 3T3 NRU assay. This plate
    will follow the same schedule and procedures as used for the test
    chemical plates (including appropriate chemical concentrations in
    the appropriate wells -- see sections VII.F.1 and F.2).

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

> After at least 46 h treatment, examine each plate under a phase
> contrast microscope to identify systematic cell seeding errors and
> growth characteristics of control and treated cells. Record any
> changes in morphology of the cells due to the cytotoxic effects of the
> test chemical, but [do not use these records for any quantitative
> measure of cytotoxicity]{.underline}. Undesirable growth
> characteristics of control cells may indicate experimental error and
> may be cause for rejection of the assay. Use the following Visual
> Observations Codes in the description of cell culture conditions.
>
> Visual Observations Codes

  -------------- --------------------------------------------------------
  **Note Code**  **Note Text**

                 

  1              Normal Cell Morphology

  2              Low Level of Cell Toxicity

  3              Moderate Level of Cell Toxicity

  4              High level of Cell Toxicity

  1P             Normal Cell Morphology with Precipitate

  2P             Low Level of Cell Toxicity with Precipitate

  3P             Moderate Level of Cell Toxicity with Precipitate

  4P             High level of Cell Toxicity with Precipitate

  5P             Unable to View Cells Due to Precipitate
  -------------- --------------------------------------------------------

#### 4. Measurement of NRU {#measurement-of-nru .unnumbered}

a)  Carefully remove (i.e., "dump") the medium with test chemical and
    rinse the cells very carefully with 250 µL pre-warmed D-PBS. Remove
    the rinsing solution by dumping and remove excess by gently blotting
    on sterile paper towels. Add 250 µL NR medium (to all wells
    including the blanks) and incubate (37ºC ± 1ºC, 90 % ± 5 % humidity,
    and 5.0 % ± 1 % CO~2~/air) for 3±0.1 h. Observe the cells briefly
    during the NR incubation (e.g., between 2 and 3 h -- Study
    Director's discretion) for NR crystal formation. Record observations
    in the Study Workbook. Study Director can decide to reject the
    experiment if excessive NR crystallization has occurred.

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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. Plates should be protected from light by using a cover
    during shaking.

f)  Plates should be still for at least five minutes after removal from
    the plate shaker (or orbital mixer). Observe the wells for bubbles.
    Measure the absorption (within 60 minutes of adding NR Desorb
    solution) of the resulting colored solution at 540 nm ± 10 nm in a
    microtiter plate reader (spectrophotometer), using the blanks as a
    reference. \[Note: Phases Ia and Ib data show the mean OD value for
    the plate blanks to be 0.057 ± 0.043 for 3T3 cells (± 2.5 standard
    deviations; data from 3 labs; N = 189). Use this range as a
    **[guide]{.underline}** for assessment of the blank values.\] 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 and a half (2.5) 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 left and the right mean of
> the VCs do not differ by more than 15.0 % from the mean of all VCs.

3.  A test meets acceptance criteria if:

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

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

4.  A test meets acceptance criteria if the r^2^ (coefficient of
    determination) value calculated for the Hill model fit (i.e., from
    PRISM software) is ≥ 0.90. A test does not meet acceptance criteria
    if the r^2^ value is \< 0.80. If the r^2^ value is ≥ 0.80 and \<
    0.90 ("gray zone"), then the SMT will evaluate the model fit and
    make the determination of whether or not the test meets the
    acceptance criteria and relate the information to the Study
    Director.

> \[Note: All acceptance criteria must be met for an assay to be
> considered acceptable.\]
>
> \[A corrected mean OD~540\ ±\ 10nm~ of 0.103 - 0.813 for the VCs is a
> target range but will not be a test acceptance criterion. Range
> determined from Phase Ib VC OD values from 3 laboratories (mean ± 2.5
> standard deviations, N = 98).\]
>
> 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 sections **VII.E.3.b** and **VII.F.5.a.3**). If
> this is not the case, and the concentration progression factor can be
> easily reduced, reject the experiment and repeat it with a smaller
> progression factor. In addition, the dilution scheme shall be adjusted
> in subsequent replicate assays, if necessary, to increase the number
> of points on both sides of the IC~50~ in the 10-90% response range.
> Numerical scoring of the cells (see **VII.F.3**) should be determined
> and documented in the Study Workbook.

###  {#section-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). The Study Director will use
good biological/scientific judgment for determining "unusable" wells
that will be excluded from the statistical analysis. This value is
compared with the mean NRU of all VC values (provided VC values have met
the VC acceptance criteria). Relative cell viability is then expressed
as percent of untreated VC. If achievable, the eight concentrations of
each chemical tested will span the range of no effect up to total
inhibition of cell viability. Data from the microtiter plate reader
shall be transferred to the Excel spreadsheet (template with macros
provided by the SMT) that will automatically determine cell viability
and perform statistical analyses (including determination of outliers).

> The concentration of a test chemical reflecting a 20 %, 50 %, and 80 %
> inhibition of cell viability (i.e., the IC~20~, IC~50~, and IC~80~) is
> determined from the concentration-response by applying a Hill function
> to the concentration-response data. Statistical software (e.g.,
> GraphPad PRISM 3.0) specified by the SMT shall be used to calculate
> IC~20~, IC~50~, and IC~80~ values (and the associated confidence
> limits) for each test chemical. In addition, the SMT shall provide
> guidelines for calculating ICx values and confidence limits. The
> Testing Facility shall report data using at least three (3)
> significant figures and shall forward the results from each assay to
> the SMT/biostatistician through the designated contacts in electronic
> format and hard copy upon completion of testing. The SMT will be
> directly responsible for the statistical analyses of the Validation
> Study data.

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

> 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.

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

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