2.0 TEST METHOD PROTOCOL COMPONENTS OF THE 3T3 AND NHK *IN VITRO* NRU
TEST METHODS 2-3

2.1 Overview of the 3T3 and NHK NRU Test Methods 2-4

> 2.1.1 The 3T3 NRU Test Method 2-7
>
> 2.1.2 The NHK NRU Test Method 2-8
>
> 2.1.3 Measurement of NRU for both 3T3 and NHK Test Methods 2-9

2.2 Descriptions and Rationales of the 3T3 and NHK NRU Test Methods 2-9

> 2.2.1 Materials, Equipment, and Supplies 2-9
>
> 2.2.2 Reference Substance Concentrations/Dose Selection 2-12
>
> 2.2.3 NRU Endpoints Measured 2-14
>
> 2.2.4 Duration of Reference Substance Exposure 2-15
>
> 2.2.5 Known Limits of Use 2-16
>
> 2.2.6 Nature of Response Assessed 2-17
>
> 2.2.7 Appropriate Vehicle, Positive, and Negative Controls 2-18
>
> 2.2.8 Acceptable Ranges of Control Responses 2-19
>
> 2.2.9 Nature of Experimental Data Collected 2-20
>
> 2.2.10 Type of Media for Data Storage 2-21
>
> 2.2.11 Measures of Variability 2-21
>
> 2.2.12 Methods for Analyzing NRU Data 2-22
>
> 2.2.13 Decision Criteria for Classification of Reference Substances
> 2-23
>
> 2.2.14 Information and Data Included in the Test Report 2-23

2.3 Basis for Selection of the *In Vitro* NRU Cytotoxicity Test Methods
2-25

> 2.3.1 *Guidance Document* Rationale for Selection of *In Vitro* NRU
> Cytotoxicity Test Methods 2-25
>
> 2.3.2 *Guidance Document* Rationale for Selection of Cell Types 2-26

2.4 Proprietary Components of the 3T3 and NHK NRU Test Methods 2-28

2.5 Basis for Number of Replicate and Repeat Experiments for the 3T3 and
NHK NRU Test Methods 2-28

2.6 Basis for Modifications to the 3T3 and NHK NRU Test Method\
Protocols 2-29

> 2.6.1 Phase Ia: Laboratory Evaluation Phase 2-29
>
> 2.6.2 Phase Ib: Laboratory Evaluation Phase 2-33
>
> 2.6.3 Phase II: Laboratory Qualification Phase 2-37
>
> 2.6.4 Phase III: Laboratory Testing Phase 2-44

2.7 Differences in 3T3 and NHK NRU Test Method Protocols and the
*Guidance Document* Standard Protocols 2-45

> 2.7.1 Optimization of the *Guidance Document* Protocols Prior to
> Initiation\
> of the Study 2-45
>
> 2.7.2 Optimization of the *Guidance Document* Protocols During the
> Study ..2-47

2.8 Overview of the Solubility Protocol 2-48

2.9 Components of the Solubility Protocol 2-50

> 2.9.1 Medium, Supplies, and Equipment Required 2-50
>
> 2.9.2 Data Collection 2-51
>
> 2.9.3 Variability in Solubility Measurement 2-52
>
> 2.9.4 Solubility and the 3T3 and NHK NRU Test Methods 2-52
>
> 2.9.5 Methods for Analyzing Solubility Data 2-53

2.10 Basis of the Solubility Protocol 2-53

> 2.10.1 Initial Solubility Protocol Development 2-53
>
> 2.10.2 Basis for Modification of the Phase II Protocol 2-54

2.11 Summary 2-55

**2.0 TEST METHOD PROTOCOL COMPONENTS OF THE 3T3 AND NHK *IN VITRO* NRU
TEST METHODS**

The *Guidance Document* (ICCVAM 2001b) recommended that the following
conditions be incorporated into any *in vitro* cytotoxicity protocol
used to predict *in vivo* acute lethality:

-   use a cell line (or primary cells) that divides rapidly

-   use an initial seeding density that allows rapid growth throughout
    the exposure period

-   apply reference substances only on cells in the exponential phase of
    growth

-   use a reference substance exposure period at least the duration of
    one cell cycle

-   use appropriate positive and vehicle control substances for which
    cytotoxicity, or lack of cytotoxicity, has been well characterized
    by the performing laboratory

-   use solvents only at levels previously shown not to cause
    cytotoxicity to the cell system over the entire period of the assay

-   use a well established measurement endpoint that has good
    interlaboratory reproducibility

-   use tests compatible with 96-well plates and apparatus (i.e.,
    spectrophotometers) that allow a quick and precise measurement of
    the endpoint

-   use a progression factor in the concentration-response experiment
    that yields graded effects between no effect and total cytotoxicity

**Section 2.1** provides descriptions of the protocol applications to
the NICEATM/ECVAM *In Vitro* Cytotoxicity Validation Study. **Section
2.2** provides details for performing the 3T3 and NHK NRU test methods
and explains the rationale for various test method components. The basis
for the selection of these *in vitro* cytotoxicity test methods is given
in **Section 2.3** and proprietary aspects associated with this study
are described in **Section 2.4**. **Section 2.5** discusses the basis
for replicate and repeat tests. **Section 2.6** details the
modifications and revisions made throughout all phases leading to the
development of the final protocol used in Phase III of this validation
study. **Section 2.7** shows the differences between the test methods
used in this study and the test methods outlined in the *Guidance
Document*. **Sections 2.8** and **2.9** provide details on the
solubility protocol for the reference substances used in to validate the
two *in vitro* NRU cytotoxicity test methods.

These test method protocols were provided to the three cytotoxicity
testing laboratories that participated in the NICEATM/ECVAM study (see
**Section 5.6.3** for additional laboratory information):

-   ECBC: The U.S. Army Edgewood Chemical Biological Center

-   FAL: Fund for the Replacement of Animals in Medical Experiments
    (FRAME) Alternatives Laboratory

-   IIVS: Institute for *In Vitro* Sciences

A fourth laboratory was used (BioReliance Corporation, Rockville, MD) to
procure and distribute the coded reference substances and to perform
solubility tests on all validation study reference substances prior to
distribution to the cytotoxicity testing laboratories.

**2.1 Overview of the 3T3 and NHK NRU Test Methods**

The authors of the *Guidance Document* (ICCVAM 2001b) developed and
presented a proposed 3T3 NRU protocol for use in a validation study
based on the BALB/c 3T3 Cytotoxicity Test, INVITTOX Protocol No. 46
(available at the FRAME-sponsored INVITTOX database
\[<http://embryo.ib.amwaw.edu.pl/invittox/>\]) which in turn was based
on the Borenfreund and Puerner (1985) protocol, as elaborated on in
Spielmann et al. (1991) and Spielmann et al. (1996).

The *Guidance Document* protocol also included revisions based on
experience with a modification of another test, the 3T3 NRU
Phototoxicity Test, INVITTOX Protocol No. 78, also available at the
FRAME database. The Registry of Cytotoxicity (RC) regression for
prediction of acute oral systemic rodent (rat and mouse) toxicity (Halle
1998; Spielmann et al. 1999) was included as the prediction model (see
**Section 1.1.2**). The RC is a database of acute oral LD~50~ values for
rats and mice obtained from RTECS^®^ and IC~50~ values from *in vitro*
cytotoxicity assays using multiple cell lines and cytotoxicity endpoints
for chemicals with known molecular weights.

The NHK NRU test method protocol in the *Guidance Document* was based on
a NRU test method by Borenfreund and Puerner (1984) using human
epidermal keratinocytes (Heimann and Rice 1983) and was obtained from
IIVS. Formulations for the media and solutions and general NHK cell
culture techniques correspond to Clonetics^^ products from the CAMBREX
Corporation. The authors of the *Guidance Document* expanded the IIVS
protocol by adding details on equipment, media and reagent components,
and experimental procedure.

The test method protocol components for the *in vitro* NRU cytotoxicity
test methods used in the NICEATM/ECVAM study are very similar for both
the 3T3 and the NHK cells (see **Figure 2-1**). The following procedures
are common to both cell types:

-   preparation of reference substances and positive control

-   cell culture environmental conditions

-   determination of test substance solubility

-   96-well plate configuration for testing samples

-   range finder and definitive tests (48-hour exposure to the reference
    substance)

-   microscopic evaluation of cell cultures for toxicity

-   measurement of NRU

-   data analysis

The main differences in the test methods are:

-   the conditions of propagation of the cells in culture

-   the cell growth medium components

-   the application of reference substances to the 96-well plate (i.e.,
    different volumes of reference substance solution)

The nature of the NRU response is described in **Section 1.3.1**.
**Figure 2-1** provides an overview to the major steps for performance
of the *in vitro* NRU cytotoxicity test methods.

**Figure 2-1 Major Steps for Performance of the NRU Test Methods in
the**

***In Vitro* Cytotoxicity Validation Study**

\(1\) 3T3 cells or NHK cells are seeded into 96-well plates to form a
sub-confluent monolayer (24 hours for 3T3 cells, 48-72 hours for NHK
cells)

⇓

\(2\) Culture medium is removed (for 3T3 cells only)

⇓

> \(3\) Reference substances in treatment medium are added to the cells;
> cells are exposed for 48 hours to the reference substance over a range
> of eight (8) concentrations

⇓

\(4\) Cells are evaluated microscopically for toxicity based on
morphological alterations

⇓

\(5\) Treatment medium is removed; cells are washed once with Dulbecco's
Phosphate Buffered Saline (D-PBS); Neutral Red (NR) dye medium is added
(3T3 cells: 25 μg/mL NR dye; NHK cells: 33 μg/mL NR dye); plates are
incubated for 3 hours

⇓

\(6\) NR medium is discarded; cells are washed once with D-PBS; NR
desorbing fixative is added to the plates

⇓

\(7\) Plates are shaken for 20 minutes

⇓

\(8\) NR absorption is measured at optical density (OD) 540 ± 10 nm

⇓

\(9\) NRU is calculated as the % of control values to define IC~20~,
IC~50~, and IC~80~ reference substance concentrations (µg/mL)[^1]

2.1.1 [The 3T3 NRU Test Method]{.underline}

*Initiating and Subculturing of 3T3 Cells*

(CCL-163, 3T3 BALB/c mouse fibroblast, clone 31, American Type Culture
Collection \[ATCC\], Manassas, VA, USA)

Cryopreserved 3T3 cells are thawed, resuspended in a routine culture
medium containing Dulbecco's Modification of Eagle's Medium (DMEM)
supplemented with non heat-inactivated 10% newborn calf serum (NCS),
transferred into tissue culture flasks (25 or 75 - 80 cm^2^), and
incubated at 37°C ± 1°C, 90% ± 5% humidity, and 5.0% ± 1% CO~2~/air.
When cells reach 50 -- 80% confluency (as estimated from a visual
inspection of cell density), they are removed from the flask by
trypsinization. A single-cell suspension is added to new flasks for
propagation and the cells are passaged/subcultured at least two times
before seeding into 96-well plates for test assays. Subsequent passages
may be maintained in culture for approximately two months (\~18
passages) and used in NRU test methods. A new frozen ampule is thawed
when needed and the above procedures are repeated. The protocols provide
cell culture density guidelines for subculturing the cells and each
laboratory determines the final seeding densities to achieve appropriate
growth.

*Preparation of Cells for 96-well Plate Assays*

After achieving appropriate subculturing of cells, 100 µL of the cell
suspension (2.0 -- 3.0x10^3^ cells/well) are placed in the appropriate
wells and 100 µL of cell-free culture medium are dispensed into the
peripheral wells (blanks). One plate per reference substance is
prepared. The cells are incubated for 24 ± 2 hours and checked to be
sure that approximately a half-confluent monolayer is attained at the
time of reference substance application.

*Reference Substance Application*

After the appropriate incubation period, medium is removed and 50 µL of
the routine culture medium with 10% NCS are added to each well. Then, 50
µL treatment medium containing the appropriate reference substance
concentrations are added for a final concentration of 5% NCS. The cells
are incubated for 48 ± 0.5 hours. At the end of the incubation period,
the cells are microscopically evaluated for changes in morphology and
their appearance is documented (as per Visual Observation Codes in the
protocol) prior to measurement of the NRU of the cells.

2.1.2 [The NHK NRU Test Method]{.underline}

*Initiating and Subculturing of NHK Cells*

(pooled primary neonatal foreskin cells, Clonetics^®^ \# CC-2507, lot \#
1F0490N, CAMBREX Bio Science Walkersville, Inc., Walkersville, MD, USA)

Cryopreserved cells are thawed, resuspended in keratinocyte complete
growth medium, transferred into tissue culture flasks (25 cm^2^ without
fibronectin-collagen coating), and incubated at 37°C ± 1°C, 90% ± 5%
humidity, and 5.0% ± 1% CO~2~/air. When cells reach 50 -- 80% confluency
(as estimated from a visual inspection of cell density), they are
removed from the flask by trypsinization and prepared for subculturing
into the 96-well plates. Keratinocytes are not subcultured beyond the
second passage. Additional frozen ampule(s) are thawed as needed. The
protocols provide cell culture density guidelines for establishing the
cells out of cryopreservation and each laboratory determines the final
seeding densities to achieve appropriate growth.

*Preparation of Cells for 96-well Plate Assays*

After appropriate subculturing of cells is achieved, 125 µL of the cell
suspension (2.0 -- 2.5x10^3^ cells/well) are placed in the appropriate
wells and 125 µL of cell-free culture medium are dispensed into the
peripheral wells (blanks). One plate per reference substance is
prepared. The cells are incubated for \~ 48 - 72 hours and checked to be
sure that a monolayer of 20+% confluency (e.g., 20 -- 50% confluency) is
attained at the time of reference substance application.

[]{.mark}

*Reference Substance Application*

After the appropriate incubation period, 125 µL of the culture medium
containing the appropriate reference substance concentrations are added
to the test wells (the existing 125 µL of culture medium is not
removed). The cells are incubated for 48 ± 0.5 hours. At the end of the
incubation period, the cells are microscopically evaluated for changes
in morphology and their appearance is documented (as per Visual
Observation Codes in the protocol) prior to measurement of the NRU of
the cells.

[]{.mark}

2.1.3 [Measurement of NRU for both 3T3 and NHK Test Methods]{.underline}

The treatment medium is removed from the 96-well plates, the cells are
rinsed with phosphate buffered saline (PBS), 250 μL NR dye medium is
added to the wells (25 µg NR/mL concentration for 3T3 cells, 33 µg NR/mL
concentration for NHK cells), and the plates are incubated (37°C ± 1°C,
90% ± 5% humidity, and 5.0% ± 1% CO~2~/air) for three hours. After
incubation, the NR medium is removed, the cells are rinsed with PBS, and
the desorb solution is applied. The plates are shaken on a microtiter
plate shaker for 20 to 45 minutes to extract NR from the cells and form
a homogeneous solution. The absorption (i.e., OD measurement) of the
resulting colored solution is measured (within 60 minutes of adding the
desorb solution) at 540 nm ± 10 nm in a spectrophotometric microtiter
plate reader, using the blanks as reference. Data from the plate reader
is transferred to a Microsoft^®^ EXCEL^®^ (Microsoft Corporation,
Redmond, WA, USA) spreadsheet template (hereafter know as EXCEL^®^
template) designed by the SMT and laboratories for statistical analyses
for this study.

**2.2 Descriptions and Rationales of the 3T3 and NHK NRU Test Methods**

The protocols used in Phases I, II, and III of the validation study
(**Appendices B and C**) are modifications of the protocols reported in
the *Guidance* *Document* (ICCVAM 2001b, **Appendix D**). The SMT and
the cytotoxicity laboratories provided comments and recommendations in
the development of these protocols. The following information is
specific to the NICEATM/ECVAM validation study.

2.2.1 [Materials, Equipment, and Supplies]{.underline}

*3T3 Cells*

3T3 cells (see **Section 2.1.1**), an immortalized mouse fibroblast cell
line, were procured from the ATCC by IIVS at passage number 64. IIVS
placed the cells in culture to expand the number of cells and
cryogenically-preserved them as a pool at passage number 69. ECBC and
FAL received frozen ampules of cells at passage number 69 from IIVS,
propagated the cells, and cryopreserved multiple ampules of cells at a
slightly higher passage number to establish a working cell bank (for
each laboratory) for use throughout the study.

*NHK Cells*

These normal human epidermal keratinocytes are primary neonatal foreskin
cells pooled from several donors and were obtained from CAMBREX Bio
Science Walkersville, Inc. (**see Section 2.1.2**). IIVS reserved the
specific lot of pooled cells (stored at CAMBREX) for use throughout the
study by all laboratories. At each laboratory, cryopreserved NHK cells
are thawed from a cryogenic ampule, seeded into culture flasks,
propagated according to protocol, then trypsinized and seeded into
96-well plates. NHK cells are passaged only once (to the 96-well plates)
and each new assay begins with fresh cells from the cryogenically
preserved working bank if NHK cells in the culture flasks are too
confluent according to protocol guidelines.

*Tissue Culture Materials and Supplies*

The 3T3 and NHK NRU test methods require general tissue culture
materials and supplies (see **Appendices B-1 and B-2** \[protocols\] for
formulations and concentrations of solutions and media). Both test
methods use the same materials for solubility testing (**Section
2.8.1**). Freshney (2000) provides information on all aspects of cell
culture including materials, supplies, and equipment needed. The
following materials are needed for both test methods:

-   trypsin (i.e., 0.05% trypsin)

-   PBS

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

-   NR dye

-   glacial acetic acid

-   dimethyl sulfoxide (DMSO)

-   ethanol (ETOH)

-   distilled water

*Culture Medium*

Medium for 3T3 cells consists of DMEM containing high glucose (4.5 gm/L)
and supplemented with non heat-inactivated NCS, L-glutamine, penicillin,
and streptomycin. The culture medium for NHK cells consists of
Clonetics^®^ keratinocyte basal medium (KBM^®^) supplemented with KBM^®^
SingleQuots^®^ (epidermal growth factor, insulin, hydrocortisone,
antimicrobial agents, bovine pituitary extract) and Calcium
SingleQuots^®^ (calcium)\[all from CAMBREX Corporation\].

*Cell Culture Materials*

Laboratory items needed include the following:

-   sterile, disposable tissue culture plasticware (e.g., 25 cm^2^ - 75
    cm^2^ flasks, multiwell/microtiter plates \[96-well\], petri dishes)
    \`

-   cryogenic ampules

-   pipettes, pipette tips

-   multichannel solution reservoirs

-   centrifuge tubes

-   microporous sterilization filters

-   general plastic containers

-   glass tubes (for preparation of reference substance dilutions)

*Equipment*

Performance of the NRU test methods requires a laboratory equipped with
a designated cell culture area. Essential equipment for cell culture
work and the NRU test method includes:

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

-   laminar flow clean bench/cabinet (standard: \"biological hazard\")

-   water bath (37ºC ± 1ºC)

-   inverted phase contrast microscope

-   centrifuge (capable of 220 x g)

-   laboratory balance (capable of measuring to 10 mg)

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

-   shaker for microtiter plates

-   cell counter or hemocytometer

-   pipetting aid

-   pipettes, pipettors (multi-channel and single channel, multichannel
    repeater pipette)

-   waterbath sonicator

-   refrigerator

-   freezer

-   cryostorage container (liquid nitrogen).

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-   magnetic stirrer

-   antistatic bar ionizer

-   personal computer

-   osmometer

-   pH meter

#### 

2.2.2 [Reference Substance Concentrations/Dose Selection]{.underline}

Each laboratory prepares the reference substance immediately prior to
testing (i.e., same day as test). Bulk solutions are not prepared for
subsequent testing. The highest concentration of dissolved reference
substance is identified using the solubility protocol and designated as
the 2X stock solution. All reference substance dilutions for the assay
are serially derived from the stock solution (see **Appendix D**
\[*Guidance Document*\] for serial dilution methods).

*Range Finder Test*

A range finder test is the initial 3T3 and/or NHK NRU test method
performed to determine starting doses for the main (definitive) test.
The range finder test uses eight concentrations of the reference
substance prepared by diluting the stock solution in log dilutions to
cover a large concentration range. The highest concentrations applied to
the cells are 10 mg/mL for reference substances dissolved in culture
medium and 1 mg/mL in medium for reference substances dissolved in DMSO,
unless precluded by the solubility of the reference substance. ETOH was
not used as a solvent in NRU test methods for any of the 72 reference
substances in the NICEATM/ECVAM study.

If a range finder test does not generate enough cytotoxicity, then a
second range finder test is conducted at higher doses, unless precluded
by solubility. If solubility is an issue, then more stringent solubility
procedures are employed to increase the stock concentration (to the
maximum concentration specified in **Appendices B-1 and B-2**). If the
test produces a biphasic response curve for NR uptake, then the doses
selected for the subsequent definitive tests (concentration-response
assays) cover the most toxic dose-response range that includes the range
where 50% toxicity is first exceeded (see **Section 2.6.3** -- *Unusual
Dose-Response Curves*).

*Definitive Test*

In the following, because of its capacity to determine the IC~50~ value
of a test compound, the main test of the *3T3 and/or NHK* NRU test
method will be referred to as the definitive test. The concentration
closest to the calculated IC~50~ value in the range finder test can
serve as the midpoint of the eight concentrations tested in a definitive
test. In the absence of other information (e.g., knowledge of slope for
the toxicity curve), the recommended dilution factor is 1.47 (^6^√10),
which divides a log into six equidistant steps (e.g., 10, 14.7, 21.5,
31.6, 46.4, 68.1, 100), as a starting dilution series. A progression
factor of 1.21 (^12^√10) is regarded the smallest factor achievable and
was the lowest dosing interval allowed in the validation study. The
positive control chemical is tested similarly to the reference
substances in the definitive test.

A successful definitive test is one that meets all of the test
acceptance criteria as outlined in the protocol. Definitive tests were
repeated as per the protocols if the test failed to meet all test
criteria. **Section 2.5** addresses the basis for replicate testing.

If minimal or no cytotoxicity is measured in the dose range finding
test, the maximum dose for a definitive test is as follows:

-   Reference Substances Prepared in NHK or 3T3 Medium: the highest
    reference substance concentration applied to the cells in the
    definitive test is either 100 mg/mL (using 200 mg/mL 2X stock) or
    the maximum soluble dose. A review of the RC chemicals used in this
    study showed that, among water-soluble chemicals, glycerol had the
    highest reported IC~50~ value (57 mg/mL). To capture this value
    during testing and that of other relatively non-toxic chemicals, the
    100 mg/mL upper concentration limit was established.

-   Reference Substances Prepared in DMSO: the highest test article
    concentration applied to the cells in the definitive test is either
    2.5 mg/mL, or the maximum soluble dose.

2.2.3 [NRU Endpoints Measured]{.underline}

*Neutral Red Uptake and Measurement*

After cells are exposed to the reference substance or the positive
control chemical for the specified period, 3T3 or NHK cells are
incubated with the NR dye for three hours, the dye is eluted from the
lysosomes using a desorb solution, and the OD of the resulting
colorimetric endpoint is measured using a spectrophotometric microtiter
plate reader. The OD values are a reflection of the NRU by the cells.
The greater the OD value is, the greater the NRU and the higher the
percent viability[^2] of the cells is in reference to the vehicle
control (VC) wells. These OD data are transferred to the EXCEL^®^
template. The mean OD values of the six replicate values (six wells
\[minimum of four\] in the 96-well plate) per test concentration are
used to determine relative cell viability by calculating its percentage
of the mean NRU of all VC values on the same plate.

*Determination of IC~50,~ IC~20,~ and IC~80~ Values*

The IC~50~ values are determined from the concentration response using a
Hill function which is a four parameter logistic mathematical model
relating the concentration of the reference substance to the response
(typically following a sigmoidal shape). Information on modifications to
the Hill function used in later phases of the validation study may be
found in **Section 2.6.3**.

Data from the EXCEL^®^ template were transferred to a template designed
by the SMT for a commercially available statistical software program
(GraphPad PRISM^®^ 3.0, GraphPad Software, Inc., San Diego, CA, USA --
hereafter known as PRISM^®^ template) to generate the inhibitory
concentrations IC~50~, IC~20~, and IC~80~ reported as μg/mL of reference
substance in solution. IC~20~ and IC~80~ data were collected for
potential use in designing a prediction model for estimating human
lethal blood concentrations.

2.2.4 [Duration of Reference Substance Exposure]{.underline}

The SMT and laboratory representatives reevaluated the reference
substance exposure duration recommended in the *Guidance Document*
(ICCVAM 2001b) before initiating the NICEATM/ECVAM study. The *Guidance
Document* recommends an exposure of 24 hours for the 3T3 cells and 48
hours for the NHK cells. The results from a cytotoxicity study by
Riddell et al. (1986) show large differences in cytotoxicity in 3T3
cells induced by some chemicals depending on whether an exposure
duration of 24 or 72 hours was used. IIVS conducted studies to evaluate
the effect of exposure duration (24, 48, and 72 hours) on the
sensitivity of 3T3 cells to six chemicals selected from the list in
Riddell 1986. Since the closest fit to the RC regression line (Halle
2003) occurred when 48-hour exposure duration was used, this exposure
duration is used in the standardized protocol for 3T3 cells (see
**Appendix E**). In addition, IIVS evaluated the sensitivity of NHK
cells to the same six chemical using exposure durations of 48 and 72
hours. To make a comparison with the RC regression, the 11 chemicals
recommended by the *Guidance Document* were tested in both cell types
using the same exposure durations. IIVS scientists concluded that the
optimum exposure duration for both cell types was 48 hours (Curren et
al. 2003). The SMT concurred and revised the exposure duration in the
3T3 protocol to 48 hours.

2.2.5 [Known Limits of Use]{.underline}

*Solubility/Volatility*

*In vitro* cytotoxicity test methods are inadequate for substances that
cannot be dissolved in media, DMSO, or ETOH at a sufficiently high
concentration to induce cytotoxicity in excess of 50%. Some reference
substance dilutions in this study had precipitates in various 2X
concentrations prior to dilution for application to the test plates.
Precipitates were observed in a number of test plates after addition of
solutions to the cultures and at the end of testing (1X solutions \[see
**Section 3.5** and **Table 5-11**\]). Volatility was detected for a
number of reference substances during the range finder tests by
observance of cross contamination of wells (i.e., high cytotoxicity in
some VC wells). Some volatility was controlled by using plate sealers
during the definitive tests (see **Section 2.6.3** -- *Testing Volatile
Reference Substances*). Plate sealers could be used during the range
finder tests if the laboratory suspected that the reference substance
might be volatile. However, use of plate sealers requires additional
laboratory skills and highly volatile reference substances are difficult
to test even with the use of plate sealers. Additionally, some test
substances (e.g., organic solvents) may react chemically with the
plastic plate sealers. Also, chemicals that are unstable or exothermic
in water cannot be adequately tested with these test methods.

*Biokinetic Determinations*

The Workshop report (ICCVAM 2001a) provides discussions on the role of
the kinetics of a chemical *in vivo* vis a vis its acute systemic
toxicity.

> "Results obtained from *in vitro* studies in general are often not
> directly applicable to the *in vivo* situation. One of the most
> obvious differences between the situation *in vitro* and *in vivo* is
> the absence of processes regarding absorption, distribution,
> metabolism and excretion (i.e., biokinetics) that govern the exposure
> of the target tissue in the intact organism. The concentrations to
> which *in vitro* systems are exposed may not correspond to the actual
> situation at the target tissue after *in vivo* exposure. In addition,
> the occurrence of metabolic activation and/or saturation of specific
> metabolic pathways or absorption and elimination mechanisms may also
> become relevant for the toxicity of a compound *in vivo*. This may
> lead to misinterpretation of *in vitro* data if such information is
> not taken into account. Therefore, predictive studies on biological
> activity of compounds require the integration of data on the
> mechanisms of action with data on biokinetic behavior."

Biokinetic determinations were not specifically addressed in this study.

*Organ-Specific Toxicity*

The Workshop report also addresses concerns about which *in vitro* test
methods can adequately predict organ-specific toxicity and identifies
the organ systems in which failure after acute exposure could lead to
lethality (liver, central nervous system, kidney, heart, lung, and
hematopoietic system). Each system is reviewed individually and a
five-step *in vitro* testing scheme (as opposed to a single *in vitro*
test method) that could act as a test battery that may eventually be
used as a replacement for *in vivo* acute toxicity testing is proposed.

-   Step 1 of the proposed *in vitro* scheme recommends performing a
    physico-chemical characterization and biokinetic modeling.

-   Step 2 promotes the use of a basal cytotoxicity test method (e.g.,
    3T3 and NHK NRU test methods).

-   Step 3 calls for a test to determine the potential that metabolism
    will mediate the basal cytotoxicity effect.

-   Step 4 is to assess the test substance's effect on energy
    metabolism.

-   Step 5 is to assess the ability of the substance to disrupt
    epithelial cell barrier function (ICCVAM 2001a).

[]{.mark}

Organ-specific toxicity and metabolic effects were not tested in this
study.

2.2.6 [Nature of Response Assessed]{.underline}

Neutral red is a weakly cationic, water-soluble dye that stains living
cells by readily diffusing through the plasma membrane and concentrating
in lysosomes. The intensity of the dye in culture is directly
proportional to the number of living cells. In addition, since altering
the cell surface or the lysosomal membrane by a toxicological agent
causes lysosomal fragility and other adverse changes that gradually
become irreversible, cell death and/or inhibition of cell growth
decreases the amount of neutral red taken up by the culture (see
**Section 1.3.1**).

[]{.mark}

2.2.7 [Appropriate Vehicle, Positive, and Negative Controls]{.underline}

*Positive Control (PC)*

The *Guidance Document* recommended sodium lauryl sulfate (SLS, Chemical
Abstracts Service Reference Number \[CASRN\] 151-21-3) as an appropriate
PC chemical for *in vitro* cytotoxicity test methods (ICCVAM 2001b). SLS
is frequently used for this purpose and historical data are available
(e.g., Spielmann et al. 1991). A PC test plate was included with each
run of any 3T3 and/or NHK NRU test method assay and was treated the same
as any reference substance assay plate.

### 

The acceptable range for the PC IC~50~ was based on the statistical
approach recommended in the *Guidance Document*. Initially, in Phase Ia
of the validation study, the 3T3 and NHK tests were considered
acceptable if the IC~50~ was within the 95% confidence interval of an
historical mean IC~50~ value. The SMT decided that the test acceptance
criterion for the IC~50~ for Phase III of the validation study (for both
cell types) was 2.5 standard deviations of the mean SLS IC~50~ data
obtained during Phases I and II. The exception to this was the FAL NHK
data, where only the Phase II data were used as the basis for
establishing the acceptable range for the PC. SLS data produced at FAL
during Phase I was not used due to a protocol change in culturing the
cells (see **Section 2.6.2** -- *Resultant protocol changes for Phase
II*). The historical mean, standard deviation, and acceptance limits
were determined separately for each laboratory (see **Table 5-2**).

#### 

*Vehicle Control (VC)*

For the NICEATM/ECVAM validation study, the VC consisted of complete
DMEM (see **Appendix B-1**) for 3T3 cells and complete Clonetics^®^
KBM^®^ (see **Appendix B-2**) for NHK cells for reference substances
dissolved in medium. For reference substances dissolved in DMSO, the VC
consisted of medium with the same amount of solvent as that used in the
reference substance concentrations that are applied to the 96-well test
plate (i.e., 0.5 % \[v/v\]).

*Negative Control*

A negative control was not incorporated into the NRU test methods. The
SMT and study directors decided that the vehicle control would be used
in place of a negative control.

2.2.8 [Acceptable Ranges of Control Responses]{.underline}

The *Guidance Document* established the use of the [absolute
value]{.underline} of the OD~540~ value of NRU obtained in the untreated
VC to indicate whether the cells seeded in the 96-well plate have grown
exponentially with a normal doubling time during the assay. A mean
OD~540~ ≥ 0.3 was recommended as the acceptable range of VC responses
and was made a test acceptance criterion for both cell types. Protocols
for Phases II and III provide a range of OD values for use as guidance
in each phase of the study.

**Table 2-1 Vehicle Control OD~540~** Ranges

  ----------- ------------------- --------------------- ----------------------
  **Phase**   **OD~540~ Range -   **OD~540~ Range -     **Notes**
              3T3**               NHK**                 

  Ia          ≥ 0.3 and ≤ 1.1     ≥ 0.3 and ≤ 1.1       Test Acceptance
                                                        Criterion

  Ib          ≥ 0.30 and ≤ 0.80   ≥ 0.60 and ≤ 1.70     Test Acceptance
                                                        Criterion

  II          ≥ 0.103 and ≤ 0.813 ≥ 0.35 and ≤ 1.50     Target Range (not
                                                        criterion)

  III         ≥ 0.103 and ≤ 0.813 ≥ 0.205 and ≤ 1.645   Target Range (not
                                                        criterion)
  ----------- ------------------- --------------------- ----------------------

In Phase III, 99.5% (914/919) of all 3T3 mean VC OD values and 97%
(913/944) of all NHK mean VC OD values were within the target range.
Most OD values out of the ranges were from range finding tests and were
usually the result of volatile reference substances affecting the VC
cells nearest the highest reference substance concentration.

*VCs as Quality Control*

To check for systematic cell seeding errors and potential volatility
issues, untreated VCs were placed both at the left side (row 2) and the
right side (row 11) of the 96-well plate (see **Appendices B-1 and
B-2**). Volatile reference substances generally affect the left side VC
(closest to the highest reference substance concentration). The test
acceptance criterion was that the left and the right mean of the VCs did
not differ by more than 15% from the mean of all VCs. This criterion was
used in all phases of the study for reference substances and PC test
plates.

2.2.9 [Nature of Experimental Data Collected]{.underline}

Each laboratory maintained a Study Workbook to document all aspects of
this study. All raw data from cell culture procedures (e.g., cell
growth, application of reference substances, NRU test method, etc.) and
all solubility studies were recorded in the workbook.

*NRU OD Measurements*

At the conclusion of the NRU desorb step, the OD of the resulting
colored solution in each well of the 96-well plates was measured at 540
± 10 nm in a spectrophotometric microtiter plate reader. Raw OD data
from the plate reader was transferred to the EXCEL^®^ template. The
template converts the raw data (six wells/reference substance
concentration) to derived data by subtracting the mean blank value (two
wells/reference substance concentration) associated with each reference
substance concentration. The VCs had a total of 12 test wells and 20
blanks. The corrected OD values were referenced to the mean VC OD value
and a relative viability (% of VC) was determined for each test well.
The percent viability values was then transferred to the PRISM^®^
template for calculation of the IC~20~, IC~50~, and IC~80~ values.

*Type of Data Collected*

Originals of the raw data (the Study Workbook and computer printouts of
absorbance readings from the plate reader) and copies of other raw data
such as instrument logs were collected and archived under the direction
of the Study Director according to Good Laboratory Practice
(GLP)-compliant procedures.

The Study Director/technicians entered the following information to the
EXCEL^®^ template:

-   raw data: OD values from microtiter plate reader

```{=html}
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```
-   testing identification for: test facility, chemical code, study
    number, 96-well plate number, experiment number

-   reference substance preparation: solvent used, solvent concentration
    in dosing solutions, highest stock concentration, dilution factor,
    pH of 2X dosing solutions, medium clarity/color, presence/absence of
    precipitate in 2X solutions, PC concentration range

-   cell line/type: cell supplier, lot number, cryopreserved passage
    number, passage number in assay

-   cell culture conditions: medium/supplements and supplier and lot
    numbers, serum concentrations

-   test acceptance criteria: acceptable number of values on each side
    of the IC~50~ (i.e., number of points \> 0 and ≤ 50% viability and
    \> 50 and \< 100% viability), acceptable % difference for the VCs,
    acceptable Hill function R^2^ value (coefficient of determination)
    for the PC, and calculated IC~50~ concentration for the PC

-   timeline: dates for cell seeding, dose application, OD~540~
    determination

-   test results: mean corrected OD~540~ value, Hill function R^2^
    value, logs of IC~20~, IC~50~, and IC~80~ (PRISM^®^ template
    presents data as logs of the IC~x~; EXCEL^®^ converts values to
    IC~x~ in μg/mL)

-   visual observations: protocol codes for cell culture conditions for
    all reference substance concentrations (i.e., relative level of cell
    cytotoxicity, cell morphology, presence of precipitate)

2.2.10 [Type of Media for Data Storage]{.underline}

Raw data from the NRU cytotoxicity test methods was saved in the
EXCEL^®^ template file format provided by the SMT for further analysis
of the concentration-response (percent viability calculations). The
derived test method data were stored electronically. All EXCEL^®^ and
PRISM^®^ files were copied and transferred to compact disks. NICEATM and
the laboratories printed copies of all data sheets (stored at NICEATM
and at the testing facilities). Copies were also included in the final
reports.

2.2.11 [Measures of Variability]{.underline}

Each 96-well plate used in the NRU test methods has three main measures
of variability.

1)  Each plate contains VCs on each end of the plate (columns 2 and 11).
    The percent difference between each column and the mean of both
    columns is calculated and was used as a test acceptance criterion.
    If the difference was greater than 15%, then the test was rejected
    by the Study Director. This value is an indicator of reference
    substance volatility and potential cell seeding errors.

2)  A mean relative viability was determined for each concentration
    along with the standard deviation and % coefficient of variation
    (CV).

3)  Macros were included in the EXCEL^®^ template to perform an outlier
    test (Dixon and Massey 1981) on data in each well of the test plate.
    Extreme values at the 99% level were highlighted and could be
    removed to improve curve fit. The decision as to whether or not to
    remove outliers was made by the Study Director.

Other test-to-test measures of variability were considered for this
study.

-   Each set of assays include a PC plate. If the SLS PC data did not
    meet test acceptance criteria, then all tests associated with that
    PC were rejected. The SMT recommended testing a manageable number of
    definitive test plates (e.g., 4 -- 6) with each PC to avoid
    rejection of reference substance NRU assays that are unacceptable
    due only to a PC failure. In this validation study, 4.2% of all
    definitive tests performed were rejected only because the PC failed
    (i.e., the PC IC~50~ was outside the acceptable confidence limits).

-   Standard deviations and CVs were determined for mean IC~50~ values
    from replicate testing of the same substance. Replicate testing
    included three definitive tests per reference substance, each
    performed on a different day.

2.2.12 [Methods for Analyzing NRU Data]{.underline}

A calculation of cell viability expressed as NRU was made for each
concentration of the reference substance by using the mean NRU of the
six replicate values (minimum of four acceptable replicates wells) per
test concentration. This value was compared with the mean NRU of all VC
values (provided VC values have met the VC acceptance criteria).
Relative cell viability was expressed as percent of untreated VC. Raw OD
data from the microtiter plate reader was transferred to the EXCEL^®^
template for performance of these calculations. Where possible, the
eight concentrations selected for each reference substance tested ranged
from no effect up to 100% toxicity.

The IC~20~, IC~50~, and IC~80~ values were determined from the
concentration-response by using the PRISM^®^ template and applying a
Hill function to the data. The IC~20~ and IC~80~ values were calculated
for use in the development of a human prediction model resulting from
this study.

2.2.13 [Decision Criteria for Classification of Reference
Substances]{.underline}

The 3T3 and NHK NRU test methods were not used to classify reference
substances in hazard categories but rather to aid in setting the
starting dose for acute systemic toxicity assays (i.e., the Up and Down
Procedure \[UDP\], the Acute Toxic Class method \[ATC\], the Fixed Dose
Procedure \[FDP\]). The RC regression formula (i.e., the prediction
model) was used to predict an LD~50~ value from an NRU IC~50~ value. The
RC compilation (Halle 2003) contains *in vitro* cytotoxicity information
on 347 chemicals (i.e., one average IC~50x~ value/chemical based on
multiple reports in the literature) with corresponding *in vivo* acute
oral LD~50~ values (mmol/kg) for rats (282 values) or mice (65 values)
from RTECS (See Halle 2003 for the RC data). **Section 6** addresses the
potential of using the *in vitro* NRU cytotoxicity test methods for
predicting the GHS hazard category.

2.2.14 [Information and Data Included in the Test Report]{.underline}

*T**est and Control Substances*

(Laboratories in this study worked only with coded reference substances
and could not know the specific reference substance information.)

-   chemical name(s) such as the structural name used by the CASRN,
    followed by other names, if known

-   the CASRN, if known

-   formula weight, if known

-   purity and composition of the substance or preparation (in
    percentage(s) by weight)

-   physicochemical properties (e.g., physical state, volatility, pH,
    stability, chemical class, water solubility)

-   treatment of the test/control substances (solubility efforts) prior
    to testing, if applicable (e.g., vortexing, sonication, warming,
    grinding)

-   stability, if known

*Information Concerning the Sponsor and the Test Facility*

-   name and address of the sponsor, test facilities, study director,
    and laboratory technicians

-   justification of the test method and protocol used

*Test Method Integrity*

-   the procedure used to ensure the integrity (i.e., accuracy and
    reliability) of the test method over time (e.g., use of the PC data)

*Criteria for an Acceptable Test*

-   acceptable VC differences (between each column and the mean of both
    columns)

-   acceptable concurrent PC ranges based on historical data

-   number of cytotoxicity points on either side of the IC~50~ (i.e.,
    number of points \> 0 and ≤ 50% viability and \> 50 and \< 100%
    viability)

*Test Conditions*

-   experimental start and completion dates

-   details of test procedure used

-   test concentration(s) used

-   cell type used

-   description of any modifications of the test procedure

-   reference to historical data of the model (e.g., solvent and
    positive controls)

-   description of evaluation criteria used

*Results*

-   tabulation of data from individual test samples (e.g., IC~50~ values
    for the reference substance and the PC, reported in tabular form,
    including data from replicate repeat experiments as appropriate, and
    means and the standard deviation for each experiment)

*Description of Other Effects Observed*

-   for example, cell morphology*,* precipitate, NR crystals

*Discussion of the Results*

*Conclusion*

*Quality Assurance (QA) Statement for GLP-Compliant Studies*

-   This statement indicates all inspections made during the study, and
    the dates any results were reported to the Study Director. This
    statement also serves to confirm that the final report reflects the
    raw data.

During this study, testing at IIVS and ECBC, the GLP-compliant
laboratories, followed additional reporting requirements provided in the
relevant guidelines (e.g., OECD 1998; EPA 2003a, 2003b; FDA 2003).

Standard forms for data collection, EXCEL^®^ and PRISM^®^ templates,
were developed by the SMT and laboratories. The solubility test form was
derived from a standard form provided by IIVS. The EXCEL^®^ template was
an adaptation of a template format presented in the *Guidance Document*.

**2.3 Basis for Selection of the *In Vitro* NRU Cytotoxicity Test
Methods**

As stated in **Section 1**, Workshop 2000 participants recommended that
the approach proposed by ZEBET (Halle 1998; Spielmann et al. 1999) be
used for rapid adoption so that data could be generated to establish its
usefulness with a large number of chemicals (ICCVAM 2001a). To assist in
the adoption and implementation of the ZEBET approach, several workshop
participants wrote the *Guidance Document* (ICCVAM 2001b). NICEATM and
ECVAM used this document as the basis of test method protocol
development and designed the validation study to evaluate the
performance of the 3T3 and NHK NRU test methods.

### 

2.3.1 [*Guidance Document* Rationale for Selection of *In Vitro* NRU
Cytotoxicity Test Methods]{.underline}

The *Guidance Document* (ICCVAM 2001b) provides basic protocols for
using *in vitro* NRU basal cytotoxicity test methods as the means to
predict a starting dose for *in vivo* acute lethality assays. The
protocols take advantage of the relationship between *in vitro* IC~50x~
values and *in vivo* LD~50~ values derived from the RC for 347 chemicals
(Halle and Spielmann 1992; Halle 2003). The 3T3 NRU and NHK NRU test
method protocols used in the NICEATM/ECVAM validation study were derived
from the document. Guidance was also provided for qualifying these tests
for use with the RC regression to predict the starting dose.

The 3T3 NRU test method has been used most frequently in formal
validation programs, all of which were aimed at evaluation of
cytotoxicity in predicting eye irritancy. Large-scale studies include
Phases I, II, and III of the Cosmetic, Toiletry, and Fragrance
Association (CTFA) validation program (Gettings et al. 1991, 1992,
1994a, 1994b); the German eye irritation validation study (Spielmann et
al. 1991, 1993, 1996); the European Commission/British Home Office
(EC/HO) eye irritation validation study (Balls et al. 1995); and the
European Cosmetic Toiletry and Perfumery Association (COLIPA) eye
irritation study (Brantom et al. 1997). The 3T3 NRU Phototoxicity Test,
a modification of the 3T3 NRU test, has been fully validated (Spielmann
et al. 1998a,b), and has gained regulatory acceptance. See **Section 9**
for comparison of these studies to this validation study.

2.3.2 [*Guidance Document* Rationale for Selection of Cell
Types]{.underline}

The Workshop (ICCVAM 2001a) concluded that there are no significant
differences between the basal cytotoxicity results obtained using
permanent mammalian cell lines, primary human cells, or using the
IC~50x~ approach of Halle and Spielmann (Halle 2003; Spielmann et al.
1999; Halle and Spielmann 1992). The Workshop recommended that near-term
*in vitro* studies designed to reduce and refine animal testing in acute
lethality tests should follow the ZEBET approach of using basal
cytotoxicity assays in conjunction with the RC database. This can be one
of the factors used to identify appropriate starting doses for *in vivo*
acute lethality studies, as described by Spielmann et al. (1999).

*Cell Types for Basal Cytotoxicity Testing*

Established rodent (rat and mouse) cell lines were recommended because:

-   it was assumed that such cells would give the best prediction of
    rodent (rat and mouse) acute lethality

-   the use of an immortalized standard cell line that is easy to grow
    and readily available for *in vitro* cytotoxicity testing would
    hasten the generation of a database that can be used to analyze the
    usefulness of this approach

Human cells also offer potential advantages. An analysis of the RC
rodent (rat and mouse) acute lethality data relative to cytotoxicity
data generated using human cell lines in the MEIC program showed that
both human and rodent cells were highly correlative (R^2^=0.90) (ICCVAM
2001). A long-term advantage of using human cells is that the human cell
cytotoxicity data derived from *in vitro* cytotoxicity testing can be
added to human toxicity databases to facilitate the development of test
methods that may later better predict acute human lethality.

*Differentiated Cells for Metabolic Capabilities*

The *Guidance Document* explained why highly differentiated cells were
not used in the basal cytotoxicity assays. Such cells may not give the
best prediction of acute lethality for the large variety of chemicals
likely to be tested for acute toxicity (Ekwall et al., 1998). For
example, to eliminate the possibility of metabolic activation or
inactivation of chemicals, neither hepatocyte nor hepatoma cytotoxicity
data were included in the RC database. This does not preclude the use of
hepatocytes in future studies, however, either to estimate cytotoxicity
or to investigate the effect of metabolism or cell-specific toxicity
(Seibert et al., 1996). Hepatocytes are essential to investigations of
metabolism-mediated toxicity (Seibert et al., 1996).

The Workshop participants agreed that the current *in vitro* basal
cytotoxicity tests do not take into account metabolism-mediated
toxicity. Simple predictive systems (*in vitro* or *in silico*) must be
developed for early identification of those substances likely to be
metabolized to more toxic or less toxic species than the parent chemical
(e.g., Fentem et al., 1993; Seibert et al., 1996; Curren et al., 1998;
Ekwall et al., 1999). Participants concluded that the available *in
vitro* assays require further development to accurately predict acute
lethality (i.e., LD~50~). See **Section 3.3.4 -- Metabolism** for
metabolic information on the NICEATM/ECVAM reference substances.

*Historical Testing*

Historical data exists for 3T3 cells including data from controlled and
blinded validation studies (Gettings et al. 1991, 1992, 1994a, 1994b;
Spielmann et al. 1991, 1993, 1996; Balls et al. 1995; Brantom et al.
1997). Human NHK or fibroblasts have also been used in validation
studies for basal cytotoxicity test methods with good results (Willshaw
et al. 1994; Sina et al. 1995; Gettings et al. 1996; Harbell et al.
1997). See **Sections 5**, **6**, **7**, **8**, and **9** for data
generated for the NICEATM/ECVAM validation study.

**2.4 Proprietary Components of the *In Vitro* NRU Cytotoxicity Test
Methods**

The only proprietary components used in these test methods are the NHK
cells and the NHK basal culture medium obtained from CAMBREX
Clonetics^®^. All other components are readily available through various
scientific product suppliers. The NHK cells consisted of pooled donor
primary neo-natal foreskin keratinocytes from an unidentified source.
The use of this specific supplier ensured that the laboratories would
have access to the same source of keratinocytes throughout the entire
validation study. Keratinocytes from other sources are acceptable if
they meet the growth requirements identified in the protocols.

The contents of the NHK basal culture medium are proprietary, but the
formulation is based on a commercially available basal medium (MCDB 153
formulation). This medium was chosen since it was recommended by the
laboratories for use with the CAMBREX Clonetics^®^ NHK cells and would
be available for the laboratories throughout the study. Other media are
acceptable for the NRU test methods if they meet the performance
standards prescribed in the media prequalification protocol and achieve
parity with the CAMBREX Clonetics^®^ products (see **Appendix B-4** and
**Section 2.6.3** -- *Inadequate Cell Growth in NHK Medium*).

**2.5 Basis for Number of Replicate and Repeat Experiments for the 3T3
and NHK NRU Test Methods**

The NICEATM/ECVAM study protocols required each laboratory to test the
reference substances in at least one range finding test using a log
dilution factor and in at least three definitive tests on three
different days using a smaller dilution factor than used in the range
finding test. Assays were performed over a number of days to assess
day-to-day variability.

Laboratories tested each coded reference substance until three
definitive tests met the test acceptance criteria. Additional testing
was often dictated by:

-   chemical issues (low toxicity, volatility, insolubility, and
    precipitation)

-   PC failure

-   technical difficulties such as NR crystal formation

A stopping rule for insoluble reference substances was incorporated into
the protocols to prevent infinite retesting:

> "If the most rigorous solubility procedures have been performed and
> the assay cannot achieve adequate toxicity to meet the test acceptance
> criteria after three definitive tests, then the Study Director may end
> all testing for that particular chemical."

**2.6 Basis for Modifications to the 3T3 and NHK NRU Test Method
Protocols**

2.6.1 [Phase Ia: Laboratory Evaluation Phase]{.underline}

All protocol revisions were implemented [during]{.underline} Phase Ia
unless otherwise stated.

*NR Dye Crystals*

NR dye crystals formed in the 96-well test plates in both NRU test
methods when used at 50 μg/mL (OD values measured in the blanks
increased from \~ 0.05 to 0.10). Troubleshooting efforts explored
incubating the NR medium overnight, centrifuging, filtering, and
reducing the concentration of NR dye. The laboratories performed tests
using a reduced NR concentration of 33 μg/mL. Since there were no
differences in results between tests with 50 μg/mL and tests with 33
μg/mL NR, the SMT accepted tests with both concentrations.

> *Protocol Revision:* The NR dye concentration was reduced to 33 μg/mL
> for both cell types.

#### 

*3T3 Cell Growth*

Cell growth for 3T3 cells was slower than expected in that the cells
required more time in culture after seeding cells from the
cryogenically-preserved pool into culture vessels to obtain the proper
density.

> *Protocol Revision:* 3T3 cells must be passaged 2-3 times after
> thawing before reference substance application/toxicity evaluation.
> The protocol also emphasized attainment of the percent cell confluency
> required for both cell types prior to reference substance application
> rather than the amount of time in culture.

*NHK Cell Growth*

The NHK cells also had an additional growth problem that manifested as a
ring of dead/dying cells around the center of the wells. Troubleshooting
efforts included evaluating various brands of 96-well plates and
eliminating the change of medium prior to reference substance treatment.
All laboratories participated in evaluating the effect of changing
(i.e., refeeding) or not changing (i.e., no refeeding) the medium by
performing a small study with SLS, the PC. Tests were performed 1) after
refeeding the cells with fresh medium, and 2) by adding SLS to the
medium already on the cells. Control ODs were generally higher in the
tests in which the medium was not replenished, but SLS sensitivity was
unchanged (see **Table 2-2**). The SMT accepted both tests with
refeeding and those without refeeding as long as they met the test
acceptance criteria.

> *Protocol Revision:* Step 2 of the NHK NRU test method was eliminated
> (change of medium prior to addition of reference substance). The
> volume of medium with cells placed into the 96-well plates was changed
> from 250 µL/well to 125 µL/well.

# Table 2-2 Refeeding/No Refeeding Data

  ------------ ------------- ----------- ------------- ----------- ------------ -----------
  ** **        **ECBC^1^**               **IIVS^2^**               **FAL^3^**   

               **Refeed**    **No        **Refeed**    **No        **Refeed**   **No
                             Refeed**                  Refeed**                 Refeed**

  Number of    4             4           6             6           2            4
  Test Plates                                                                   

  Mean Abs. OD 0.265         0.621       0.885         1.12        1.41         1.24
  (VC)                                                                          

  Standard     0.151         0.322       0.057         0.033       0.127        0.430
  Deviation                                                                     
  (SD)                                                                          

  SLS IC~50~   3.33          3.23        3.41          3.49        6.21         8.14
  (µg/mL)                                                                       

  SLS IC~50~   0.47          0.61        0.58          0.39        0.88         0.40
  SD                                                                            
  ------------ ------------- ----------- ------------- ----------- ------------ -----------

#### ^1^Edgewood Chemical Biological Center

> ^2^Institute for In Vitro Sciences
>
> ^3^FRAME Alternatives Laboratory

The FAL laboratory could not get satisfactory levels of NHK cell
adherence to the 80-cm^2^ culture flasks when seeded with thawed cells
(one ampule) from the cryogenically-preserved pool of cells.

> *Protocol Revision (FAL only):* Culture flasks were to be coated with
> fibronectin-collagen to promote adherence.

#### 

*OD Limits*

VC control OD limits (OD value must be ≥ 0.3 and ≤ 1.1 as related in the
protocols) were frequently unattainable in both test methods. Study
Directors reported that the cells were adequately responsive and were
neither senescent nor 100% confluent. The SMT withdrew the VC control OD
limits as a test acceptance criterion.

> *Protocol Revision for Phase Ib:* OD data from all laboratories, a
> review of cell responsiveness (i.e., dose response data), and the
> ability of each test to pass the other acceptance criteria were
> analyzed for both cell types and new OD ranges were calculated as
> guidelines for each cell type.

*Precipitate Formation*

During solubility testing, precipitates were occasionally observed in
the 3T3 medium but not in the NHK medium at the same reference substance
concentrations. Some liquid reference substances (e.g., 2-propanol)
caused precipitation in the 3T3 medium only. The precipitates were
attributed to the serum in the 3T3 medium rather than insoluble
reference substance.

> *Protocol Revision:* The reference substance was dissolved in 3T3
> medium without NCS. Then, for reference substance exposure, the
> dissolved 2X reference substance was added to medium containing 10%
> NCS to reach the final 5% NCS and 1X reference substance
> concentrations.

*Dilution Factor*

Once a range finder test had been performed, the definitive test assays
were to be performed using a ^6^√10 = 1.47 dilution scheme centered on
the IC~50~. The laboratories sometimes deviated from the protocols and
used dilution factors other than the required one. The SMT accepted data
generated using dilution factors other than the recommended 1.47 for
definitive tests if all other test acceptance criteria were met. The use
of smaller dilution factors generally increased the number of points
between 10 - 90% viability and the precision of the IC~50~ calculation
was improved.

> *Protocol Revision:* The ^6^√10 = 1.47 dilution scheme was presented
> as a suggestion and was not a criterion for test acceptance after
> Phase Ia.

*Test Acceptance Criteria*

*The test acceptance criteria for Phase Ia were:*

-   the IC~50~ for SLS was within the 95% CI of the historical PC mean
    established by the Test Facility (*not applicable to Phase Ia*)

-   mean OD values of the left and right VCs (columns 2 and 11 in the
    96-well test plate) did not differ by more than 15% from the mean of
    all VC OD values

-   at least two calculated cytotoxicity values, one on either side of
    the IC~50~, between 10 and 90% viability (*added after commencement
    of Phase Ia*)

-   Hill function coefficient of determination R^2^ \> 0.9
    [or]{.underline} 0.8 \< R^2^ \< 0.9 and curve fit was evaluated on a
    case by case basis for acceptability by the SMT (*added after
    commencement of Phase Ia*); (note: this determination would be made
    by the Study Director in non-validation studies)

-   OD~540~ of VCs (with blank subtracted) was ≥ 0.3 and ≤ 1.1
    (*rescinded after commencement of Phase Ia*)

2.6.2 [Phase Ib: Laboratory Evaluation Phase]{.underline}

*NR Crystal Formation*

FAL and ECBC routinely observed NR crystals forming in the 96-well test
plates in 3T3 assays at 33 μg/mL NR. All laboratories tested 25 and 33
µg/mL NR concentrations and 2- and 3-hour exposure durations to
determine which exposure duration would provide optimal NRU without
crystal formation. In addition to determining whether NRU had reached a
plateau at these concentrations and durations, the laboratories also
tested SLS to determine whether sensitivity to SLS differed under these
conditions. Crystals were observed only at 33 µg/mL NR when present for
3 hours. **Figure 2-2** shows that the average OD results were very
similar for the concentrations and durations tested. **Figure 2-3**
shows that the SLS IC~50~ was approximately the same at these
concentrations and durations. To minimize changes for the Phase III
protocol, the SMT and laboratories agreed to use 25 µg/mL NR for three
hours in the subsequent protocol revisions for the 3T3 test method. The
NR concentration for the NHK NRU test method remained at 33 µg/mL.

> *Protocol Revision for Phase II*: The NR concentration for the 3T3 NRU
> test methods was changed to 25 µg/mL NR for the three-hour incubation.
> Revised methods for preparation of the NR dye solution included
> filtration of the solution, maintenance of the solution at 37ºC, and
> application of the NR dye solution to the cells within 15 minutes
> after removing from 37ºC. Cells should be observed during the NR
> incubation period of the 3T3 and/or NHK NRU test method assays to
> monitor possible crystal formation.

**Figure 2-2 Optical Density with NR Concentration and Duration**

![](media/image2.pct)

**Figure 2-3 SLS IC~50~ for Each NR Concentration and Duration**

![](media/image4.pct)

*Heating Reference Substance Solutions*

The laboratories had difficulty with the solubility of arsenic trioxide.
Mechanical applications for solubilizing reference substances into
culture medium were reviewed and revised.

> *Protocol Revision for Phase II*: The range for duration of heating
> the reference substance solution was increased from 5 -- 10 minutes to
> 5 -- 60 minutes.

#### 

*OD Readings*

OD readings were frequently lower than acceptance criteria for the VC
wells.

> *Protocol Revision for Phase II:* The OD range was eliminated as a
> test acceptance criterion. The OD data from the VCs in the
> laboratories for both cell types was used to calculate OD ranges to
> serve as guidelines (see **Section 2.2.9**).

To adjust for potential reference substance interference with NR dye,
the reference substance was added to the blank wells that were used to
generate the background OD at 540 nm that was subtracted from the
reference substance concentration ODs. Each reference substance
concentration was applied to six wells containing cells and to two blank
wells without cells.

*Laboratory Error Rates*

The SMT suggested that FAL needed additional guidance to become more
GLP-like (e.g., improve documentation) and to improve performance (i.e.,
fewer test failures and errors) throughout Phases Ib and II. The SMT
compiled a list of the errors (e.g., transcriptional and typographical
errors in the data sheets) and error rates (number of tests with
errors/number of tests) for the existing Phase Ib data and provided the
information to each laboratory (see **Table 2-3**). IIVS management
sponsored a weeklong laboratory training exercise at the IIVS facilities
so that FAL technicians would have exposure to a GLP laboratory
environment. ECBC was invited to participate and all three testing
laboratories shared information and thereby harmonized procedures during
the training exercise. Harmonization of the laboratory procedures
illustrated the need to make additional protocol revisions.

**Table 2-3 Error Rates^a^** in Phase Ib by Laboratory and Test Method

  ---------------- ------------------------- ----------------------------
  **Laboratory**   **NRU Test Method**       

                   **3T3**                   **NHK**

  ECBC             1/9 (10%)                 4/17 (23%)

  FAL              42/45 (93%)               12/29 (41%)

  IIVS             1/20 (5%)                 1/20 (5%)
  ---------------- ------------------------- ----------------------------

> ^a^Number of tests with errors/total number of tests (some data files
> had more than one error)

*Resultant protocol changes for Phase II*

*The protocol changes include:*

-   use multi-channel repeater pipettes for plating cells in the 96-well
    plates, dispensing plate rinse solutions, NR medium, and desorb
    solution, but not for dispensing reference substances to the cells;
    repeater pipettes are not accurate enough to deliver equal
    quantities of the reference substance solution to the wells

-   use 8-channel reservoirs for applying dosing solutions to the wells
    so multi-channel single delivery pipettes could be used

-   use a standardized length of time that HBSS rinses remain on the
    cell monolayers in flasks during the cell subculturing step

-   protect plates from high light levels during the shaking step for NR
    extraction; all laboratories will cover plates (e.g., with aluminum
    foil) during this step

-   allow plates to stand for at least five minutes after the shaking
    step is complete and break any bubbles observed in the wells before
    measuring OD

-   change the seeding density range for 3T3 NRU test method from
    2.5x10^3^ cells/well to 2 -- 3x10^3^ cells/well

-   change NHK culture flask size (at FAL) from 80-cm^2^ (for start-up
    of cryopreserved cells) to 25-cm^2^ (same as other laboratories) and
    discontinue using a fibronectin-collagen coating

*Test Acceptance Criteria*

*Criteria were modified as follows:*

-   the IC~50~ for SLS (PC) is within 2 SDs (approximately 95%) of the
    historical mean established by each laboratory in Phase Ia
    (originally used the 95% confidence interval)

-   mean OD values of the left and right VCs (columns 2 and 11 in the
    96-well test plate) do not differ by more than 15% from the mean of
    all VC OD values

-   at least one calculated cytotoxicity value is between 10 and 50%
    viability and one calculated cytotoxicity value between 50 and 90%
    viability

-   Hill function R^2^ \> 0.9 [or]{.underline} 0.8 \< R^2^ \< 0.9 and
    curve fit is evaluated on a case by case basis for acceptability by
    the SMT (note: this determination would be made by the Study
    Director in non-validation studies)

-   VC OD criteria are based on Phase Ia data (mean ± two SDs): 0.3-0.8
    for the 3T3 test method, and 0.6-1.7 for the NHK NRU test method
    (*rescinded after commencement of Phase Ib*)

2.6.3 [Phase II: Laboratory Qualification Phase]{.underline}

All revisions were implemented [during]{.underline} Phase II unless
otherwise stated.

*Testing Volatile Reference Substances*

When 2-propanol was tested according to the protocol, vapors from the
highest concentration wells contaminated the adjacent VC and appeared to
affect some lower concentration wells (i.e., the wells exhibited
unexpectedly reduced levels of NRU). An example dose-response curve is
shown in **Figure 2-4**. The tests for which such contamination was
present failed the VC criterion. When lower concentrations were used to
avoid contaminating the VC adjacent to the highest concentration,
toxicity was inadequate to produce an IC~50~. To address this problem,
IIVS repeated their tests using film plate sealers, which isolated all
wells from each other, and obtained acceptable results. Based on these
data, the SMT recommended the use of film plate sealers to the other
laboratories to test 2-propanol.

FAL had previous experience using mineral oil as a cell culture cover to
keep volatile reference substances from escaping and provided 2-propanol
test data where mineral oil had been added to each well. The FAL showed
that the average oil vs. film IC~50~ values were not significantly
different. However, there was less variability in the film sealer data
than the mineral oil data so the SMT decided on the use of plate
sealers.

A general indicator of volatility issues in the NRU test methods is the
percent difference in the mean OD values for the two VC columns on the
test plate. If the difference is greater than 15%, then reference
substance volatility is suspected, especially if the VC adjacent to the
highest test concentration had a significantly reduced OD value.
Volatility may be an issue for reference substances with a specific
gravity of less than 1. **Table 5-11** lists the study reference
substances that had volatility issues in the NRU test methods.

> *Protocol Revision*: The SMT included the use of film sealers to test
> suspected volatile compounds in the Phase III protocols.

**Figure 2-4 Representative Dose-Response for 2-propanol in a 3T3 Range
Finder Test**

![](media/image5.pct)

> %Difference of the two VC columns from the average VC was 63%. Mean
> corrected OD for VC1, adjacent to the highest 2-propanol concentration
> was 0.070, while that for VC2, adjacent to the lowest 2-propanol
> concentration, was 0.310. The 100% viability of the mean VCs shifted
> the toxicity curve such that lower concentrations of 2-propanol seem
> to have viability percentages much greater than the VCs.

*Unusual Dose-Response Curves*

Some laboratories observed unusual dose-response curves for aminopterin
and colchicine. When the range finder tests produced a biphasic response
(see **Figure 2-5** for an example), the SMT advised the laboratories to
focus the definitive tests on the lowest concentrations that produced
responses around 50% viability. In the definitive tests, they noted that
no matter how much reference substance was used, viability was not
reduced to 0% (see **Figure 2-6**). This effect with colchicine was very
reproducible across laboratories in the NHK NRU test method, but only
FAL achieved this type of response with colchicine in the 3T3 NRU test
method. Aminopterin produced a similar dose response in the NHK NRU test
method at ECBC and FAL, but not at IIVS. In the 3T3 NRU test method,
only FAL obtained an unusual response with aminopterin.

The SMT assumed the unusual dose-responses with these reference
substances were due to their mechanisms of action. Colchicine binds to
microtubular protein and interferes with function of mitotic spindles,
which arrests cell division (NLM 2003). Aminopterin blocks the use of
folic acid by the cells, which kills cells during the S phase of the
cell cycle by inhibiting metabolism, RNA production, and protein
synthesis (NLM 2002). The variability of results among the laboratories
may be due to cells in the culture populations being in different cell
cycle phases when reference substance was applied to the cultures.
Application of reference substance to the cell systems is based on the
cells being at a certain monolayer confluency that assures the cells are
in exponential growth phase. A subjective visual observation of the cell
cultures determines time point 0 for the reference substance exposure
period for the NRU test method.

**Figure 2-5 Representative Dose-Response for Aminopterin in a NHK Range
Finder Test**

![](media/image6.pct)

> Representative dose-response for aminopterin in a NHK range finder
> test. Laboratories were instructed to focus definitive tests
> (concentration-response assays) on the lowest doses that produced 50%
> viability.

**Figure 2-6 Representative Dose-Response for Aminopterin in a NHK
Definitive Test**

![](media/image7.pct)

> Representative dose-response for aminopterin in a NHK definitive test
> (concentration-response assay). %Viability did not reach 0%.

*Hill Function*

The Hill function used in the previous phases of this study was defined
as follows:

$$\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, logIC~50~ is
logarithm of X at the response midway between Top and Bottom, and
HillSlope describes the steepness of the curve.

$X = \text{log}\text{EC}_{\text{50}} - \frac{\text{log}\left( \frac{\text{Top} - \text{Bottom}}{Y - \text{Bottom}} - 1 \right)}{\text{HillSlope}}$Since
the unusual dose-responses did not fit the Hill function well, R^2^
values often failed the acceptance criterion. To obtain a better model
fit, the Bottom parameter was estimated without constraints (the
previous practice was to use Bottom = 0). However, when Bottom ≠ 0, the
EC~50~ reported by the Hill function was not the same as the IC~50~
since the Hill function relies on EC~50~ defined as the point midway
between Top and Bottom. Thus, the Hill function calculation using the
Prism^®^ software was rearranged to calculate the concentration
corresponding to the IC~50~ as follows:

where X is the logarithm of concentration at 50% response, logEC~50~ is
logarithm of concentration at the response midway between Top and
Bottom, Top is the maximum response, Bottom is the minimum response, Y =
50 (i.e., 50% response), and HillSlope describes the steepness of the
curve.

IIVS performed the recalculations for their colchicine tests in the NHK
NRU test method, but the SMT performed the necessary recalculations for
the other laboratories. Tests that were recalculated by the SMT are
noted in the data summaries.

> *Protocol Revision*: The protocol was revised to state that if a range
> finding test produces a biphasic curve, then the concentrations
> selected for the subsequent tests should cover the most toxic
> dose-response range.

#### Insoluble Reference Substances

Lithium carbonate was insoluble in 3T3 medium. Only ECBC was able to
expose 3T3 cells to sufficient lithium carbonate to produce three tests
that passed the test acceptance criteria. Precipitate was reported for
two of those tests in the wells at the three highest concentrations.
Since the third highest concentration, 510.2 μg/mL, was approximately
the IC~50~ (average was 564 μg/mL), the true IC~50~ for lithium
carbonate may actually be lower than that calculated and therefore the
LD~50~ value would be underestimated. The data were not discarded.

> *Protocol Revision for Phase III*: The protocol was revised to allow
> an increase in the solubility stirring/rocking duration in an
> incubator from 1 to 3 hours if cytotoxicity in the range finder test
> was limited by solubility. Also, a **Stopping Rule for Insoluble
> Chemicals** was added (see **Section 2.5**)

*Inadequate Cell Growth in NHK Medium*

IIVS and FAL had several NHK NRU test method assay failures that were
attributed to poor cell growth. FAL found that medium/supplement lot
combinations that performed poorly in the NHK NRU test method performed
well for the laboratory's research on corneal cell cultures. The SMT
compiled information from the laboratories on the KBM and SingleQuot
lot numbers that the laboratories were using along with their assessment
of NHK cell growth. The information was distributed to the laboratories
to identify the lots that produced adequate growth. The SMT also
obtained quality assurance and quality control test results from CAMBREX
Clonetics on the lots of KBM, but the information provided was
inadequate for determining how the medium would perform in the NHK NRU
test method.

> *Resolution*: A protocol for prequalifying the medium was developed
> (see **Appendix B-4**). For Phase III, the SMT asked IIVS to
> prequalify new lots of KBM and SingleQuots for use by all
> laboratories.

*Performance Standards for Media to Support NHK Growth*

A prequalification-of-medium protocol (**Appendix B-4**) was developed
and IIVS performed several tests of different lots of medium and
supplements to find various combinations that maintained the typical
growth characteristics of cells in this study. The laboratories then
reserved samples of these acceptable lots at CAMBREX so that the supply
of media would not be interrupted due to unavailability of the
materials.

*Test Acceptance Criteria* *for Prequalifying Media*

-   R^2^ (coefficient of determination) value calculated for the Hill
    model fit (i.e., from PRISM^®^ software) is ≥ 0.85

-   Difference between the mean of all VCs and (a) the left mean VC,
    and (b) the right mean VC is ≤ 15%

-   At least one point \> 0 % and ≤ 50.0 % viability and at least one
    point \> 50.0 % and \< 100 % viability

-   After meeting all other acceptability criteria, the SLS IC~50~ must
    be within the historical range established by the laboratory (i.e.,
    mean SLS IC~50~ ± 2.5 standard deviations)

*Other Criteria for Prequalifying Media (for consideration by a Study
Director)*

-   General culture observations: rate of proliferation; percent
    confluence; number of mitotic figures per field; colony formation;
    distribution of cells; absence or presence of contamination

-   Cell morphology observations should include overall appearance
    (e.g., good, fair, poor), and presence of abnormal cells

-   Mean corrected OD~540-550~ of the VCs

-   Cell morphology and confluence of the VCs at the end of the 48-hour
    treatment.

-   Cell doubling time (determined by the laboratory for first time use
    of the NRU test method \[prior to testing with SLS\])

*Test Acceptance Criteria for Phase II*

-   IC~50~ for SLS (PC) is within 2.5 SDs of the historical mean
    established by the Test Facility (*Phases Ia and Ib*)

-   Mean OD values of the left and right VCs (columns 2 and 11 in the
    96-well test plate) do not differ by more than 15.0 % from the mean
    of all VC OD values (*change in decimal point only*)

-   At least one calculated cytotoxicity value ≥ 10.0 % and ≤ 50.0 %
    viability and at least one calculated cytotoxicity value ≥ 50.1 %
    and ≤ 90.0 % viability (*change in decimal point only*)

-   R^2^ ≥ 0.90. Test fails if R^2^ \< 0.80. If the R^2^ ≥ 0.80 and \<
    0.90, the SMT evaluates the model fit (note: this determination is
    made by the Study Director in non-validation studies)

2.6.4 [Phase III: Laboratory Testing Phase]{.underline}

The changes below were made in the Phase III protocols as a result of
the experience in Phase II.

*Cytotoxicity Values Around the IC~50~*

Obtaining at least one calculated cytotoxicity value \> 0 % and ≤ 50.0 %
viability and at least one calculated cytotoxicity value \> 50.0 % and
\< 100 % viability may be difficult or unattainable for reference
substances with a steep dose response.

> *Protocol Revision*: The test acceptance criterion was qualified so
> that tests with only one point between 0 and 100 % were acceptable if
> the smallest practical dilution factor (i.e., 1.21) was used
> **[and]{.underline}** all other test acceptance criteria were met.

*Data Analysis Revisions*

> *Protocol Revision*: If the lowest toxic concentration calculates to
> be less than 0%, then the bottom values for IC calculations are set at
> zero (0) for the Hill function analysis.
>
> *Protocol Revision*: If a biphasic toxicity curve was obtained, the
> IC~80~ and IC~50~ were calculated from the initial toxicity part of
> the curve (the IC~20~ was not determined).
>
> *Protocol Revision*: The requirement for test articles to fit the Hill
> equation with R^2^ ≥ 0.90 was rescinded. The Hill equation was used to
> characterize the reference substance response curve shape rather than
> establish acceptance criterion. The PC acceptance criterion was
> modified to R^2^ ≥ 0.85.

**2.7 Differences in 3T3 and NHK NRU Test Method Protocols and the
*Guidance Document* Standard Protocols**

2.7.1 [Optimization of the *Guidance Document* Protocols Prior to
Initiation of the Study]{.underline}

As the NICEATM/ECVAM validation study progressed through Phases I and
II, the protocols provided in the *Guidance Document* (ICCVAM 2001b)
were optimized to address problems that were encountered. Changes to the
*Guidance Document* protocols are described below.

-   3T3 cell seeding density for 96-well plates was increased from
    1x10^4^ cells/well to 2.0 -- 3.0x10^4^ cells/well to achieve
    adequate cell growth.

-   The calcium concentration in NHK medium was changed from 0.15 mM to
    0.10 mM. The test laboratories had expressed concern that cell
    differentiation would occur at the higher concentration and
    requested a lower concentration. CAMBREX Clonetics^®^, the supplier
    of the NHK cells and NHK medium used in this study, normally grows
    NHK cells in 0.15 mM calcium without differentiation issues. The
    supplier agreed that the cells would grow well at 0.10 mM but should
    not be cultured at concentrations \< 0.10 mM in order to avoid
    morphology and growth changes (CAMBREX technical division, personal
    communication).

-   NHK cells were subcultured once (rather than the three passages
    suggested in the *Guidance Document*). The laboratories expressed
    concern about differentiation occurring in the cells if kept in
    culture too long.

-   The highest final concentrations of DMSO and ETOH in the culture
    media were reduced from 1% to 0.5%. IIVS performed experiments with
    both cell types to determine the appropriate solvent concentration
    to avoid toxicity. 3T3 cells were tested with ETOH at 0.5, 1, and 2%
    concentrations and DMSO at 0.1, 0.2, 0.3, 0.4, 0.5, 1, and 2%
    concentrations. The 0.5% concentrations of both solvents were chosen
    as optimal since that concentration of ETOH produced no toxicity.
    Although 0.5% DMSO produced slight toxicity (i.e., cells were 91%
    viable as compared to the control cells -- See **Appendix E**), it
    was chosen by the SMT and laboratories as an acceptable trade off
    between slight toxicity and the ability to reference substances at
    higher doses and was used throughout the study (see Curren et al.
    2003). However, ETOH was not used as a solvent in the NICEATM/ECVAM
    validation study.

-   The pH of reference substance solutions was not adjusted with NaOH
    or HCl regardless if solutions became acidic or basic (optimum
    mammalian cell culture pH is \~ 7.4 \[Freshney, 2000\]) since some
    of the basal cytotoxicity produced by these reference substances may
    be due to pH extremes. See **Appendix F** for pH values of reference
    substances in culture medium.

-   The CO~2~ concentration in the incubator was reduced from 7.5%
    *(Guidance Document*) to 5.0% since the laboratories were already
    set up to use 5% CO~2~ (a typical optimum CO~2~ concentration for
    mammalian cell culture).

-   Washing and fixing the cells with a formaldehyde solution prior to
    NR elution from the cells was eliminated. FAL's regulatory waste
    disposal requirements concerning formaldehyde were an issue and the
    NR desorb solution (1% glacial acetic acid, 50% ETOH, 49% H~2~O)
    adequately fixed the cells to the test plate (INVITTOX 1991). The
    SMT and laboratories agreed that the use of formaldehyde was
    unnecessary.

-   Reference substance exposure time for 3T3 cells was extended from 24
    hours *(Guidance Document*) to 48 hours (see **Section 2.2.6** and
    **Appendix E**).

-   Cell culture seeding densities for subculture were provided as
    guidelines and the laboratories were given liberty to determine
    adequate cell densities (see **Table 2-4**).

Table 2-4 Cell Culture Seeding Densities

+--------------+-------------+-------------+-------------+-------------+
| **Protocol** | **3T3       | **3T3       | **NHK       | **NHK       |
|              | cells/cm^2^ | c           | cells/cm^2^ | c           |
|              | subculture  | ells/well** | subculture  | ells/well** |
|              | to flasks** |             | to flasks** |             |
|              |             | **96-well   |             | **96-well   |
|              |             | Plate**     |             | Plate**     |
+--------------+-------------+-------------+-------------+-------------+
| *Guidance    | 1.25x10^4^  | 2.5x10^3^   | 3.5x10^3^   | 2 --        |
| Document*    |             |             |             | 2.5x10^3^   |
+--------------+-------------+-------------+-------------+-------------+
| Phase Ia     | 0.42 --     | 2.5x10^3^   | 2.5 --      | 2 --        |
|              | 1.68x10^4^  |             | 9x10^3^     | 2.5x10^3^   |
+--------------+-------------+-------------+-------------+-------------+
| Phase Ib     | 0.42 --     | 2.5x10^3^   | 2.5 --      | 2 --        |
|              | 1.68x10^4^  |             | 9x10^3^     | 2.5x10^3^   |
+--------------+-------------+-------------+-------------+-------------+
| Phase II     | 0.42 --     | 2 --        | 2.5 --      | 2 --        |
|              | 1.68x10^4^  | 3x10^3^     | 9x10^3^     | 2.5x10^3^   |
+--------------+-------------+-------------+-------------+-------------+
| Phase III    | 0.42 --     | 2 --        | 2.5 --      | 2 --        |
|              | 1.68x10^4^  | 3x10^3^     | 9x10^3^     | 2.5x10^3^   |
+--------------+-------------+-------------+-------------+-------------+

2.7.2 [Optimization of the *Guidance Document* Protocols During the
Study]{.underline}

*Changes in Phase Ia*

-   To avoid precipitation of serum components, reference substances
    were dissolved in treatment medium without NCS for the 3T3 NRU test
    method (*Guidance Document* recommended 10% NCS). The final 5% NCS
    on cells in the test plate came from the 50:50 dilution of the
    treatment medium with the 10% NCS in the routine culture medium (see
    **Section 2.6.1** -- *Precipitate Formation*).

-   The volume of NHK medium was reduced from 250 µL per well to 125 µL
    well for cell seeding. Culture medium was not removed prior to
    reference substance application. Cell death occurred during the
    refeeding step (see **Section 2.6.1** -- *Cell Growth*).

-   To avoid NR crystal formation, NR dye concentrations were reduced
    from 50 µg/mL to 33 µg/mL (3T3) and 25 µg/mL (NHK) (see **Section
    2.6.1** -- NR Dye Crystals).

-   The PC test acceptance criterion for the IC~50~ was changed for 3T3
    and NHK cells to historical mean ± 2.5 standard deviations instead
    of within the recommended 95% confidence interval of historical mean
    for 3T3 cells and 2 standard deviations for NHK cells.

-   The test acceptance criterion for the mean OD~540~ (\> 0.3) of the
    VC was eliminated. The study protocols provided an OD~540~ range as
    a guideline (see **Table 2-1** and **Section 2.2.9.**).

*Changes in Phase Ib*

-   NHK cells were deemed ready for reference substance application when
    they reached 20+% confluency rather than the range of 30 -- 50%
    confluency. Laboratory experience in Phase Ia dictated this change.

-   A recommendation for obtaining three cytotoxicity points between 10
    and 90% inhibition of NRU for use as a quality check of the dose
    responses was changed to become a test acceptance criterion. The
    dose response curve had to have at least one calculated cytotoxicity
    value ≥ 10.0 % and ≤ 50.0 % viability and at least one calculated
    cytotoxicity value ≥ 50.1 % and ≤ 90.0 % viability (see **Section
    2.6.2** -- *Test Acceptance Criteria*).

-   Instructions for using plate sealers were added to the protocols for
    testing volatile reference substances (see **Section 2.6.3** --
    *Testing Volatile Reference Substances*).

**2.8 Overview of the Solubility Protocol**

The SMT, with assistance from the laboratories, developed a solubility
protocol to provide information to the laboratories to optimize the
determination of the most appropriate solvent to use among three
solvents: culture medium, DMSO, and ETOH. Each laboratory tested the
solubility of each reference substance using this protocol and provided
the data to the SMT prior to initiating the cytotoxicity testing of each
reference substance. The SMT analyzed the solubility data provided by
BioReliance and each testing laboratory, then assigned the solvents for
each test article for this study. This eliminated potential variability
in the NRU test methods that may have been produced if different
solvents had been used for testing the same substance between
laboratories.

#### 

The solubility protocol is based on an EPA guideline (EPA 1998) that
involves testing for solubility in a particular solvent, beginning at a
relatively high concentration and proceeding to successively lower
concentrations by adding more solvent as necessary for dissolution.
Testing stops when, upon visual observation, the procedure produces a
clear solution with no cloudiness or precipitate. The solubility
protocol used by the *in vitro* laboratories during Phase III required
testing reference substances in the various solvents at equivalent
reference substance concentrations applied to the cultures. The
solubility flow chart in **Figure 2-7** shows, for example, that 2 mg/mL
medium and 200 mg/mL DMSO or ETOH were equivalent concentrations since
they yielded 1 mg/mL in cell culture. When applied to cultures, medium
was diluted by one-half. The 0.5% \[v/v\] final concentrations were
achieved by diluting DMSO and ETOH by 200. At each concentration, the
following mixing procedures were employed, as necessary, to completely
dissolve the reference substance in this order: vortex (1--2 minutes);
sonication (up to 5 minutes); warming to 37°C (5 -- 60 minutes \[NRU
protocols allow warming to be extended to three hours if cytotoxicity in
the range finder test was limited by solubility\]). If the reference
substance was still undissolved, the next concentration/solvent was
tested.

**Figure 2-7 Flow Chart for Determination of Reference Substance
Solubility in Medium, DMSO, or** **ETOH**

![](media/image8.pct){width="6.249305555555556in"
height="3.470138888888889in"}

Notes: 3T3 Medium - DMEM (Dulbecco's Modification of Eagle's Medium)
with supplements; NHK medium - KBM (Keratinocyte Basal Medium) with
supplements (from CAMBREX Clonetics).

[]{.mark}

**2.9 Components of the Solubility Protocol**

2.9.1 [Medium, Supplies, and Equipment Required]{.underline}

*Medium and Chemical Supplies*

-   [3T3 Cell Medium]{.underline}: DMEM without L-Glutamine and
    containing Hanks' salts and high glucose \[4.5gm/l\]; L-Glutamine
    200 mM; NCS

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-   [NHK Cell Medium]{.underline}: Keratinocyte Basal Medium without
    Ca^++^ (KBM^®^, Clonetics^®^ CC-3104); KBM^®^ SingleQuots^®^ medium
    supplements (Clonetics^®^ CC-4131) \-- epidermal growth factor,
    insulin, hydrocortisone, antimicrobial agents, bovine pituitary
    extract; Calcium SingleQuots^®^ (Clonetics^®^ CC-4202);
    penicillin/streptomycin solution

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-   U.S.P. analytical grade DMSO

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-   U.S.P. analytical grade (100%, non-denatured) ETOH

*Equipment*

-   waterbath (37°C)

-   sonication unit

-   vortex unit

-   pippettors (micropipettors)

-   balance

-   pH meter

*Procedures*

The first Phase III solubility protocol procedure was the dissolving of
\~10 mg of a reference substance in \~ 0.5 mL medium (both 3T3 and NHK
media were tested) for a concentration of 20 mg/mL (see **Appendices B-1
and B-2**). In order, the mixture was vortexed for 1-2 minutes,
sonicated for up to 5 minutes, and warmed to 37°C for 5-60 minutes as
necessary to dissolve the reference substance. The endpoint for
dissolution was that a clear, not cloudy solution with no noticeable
precipitate. If the reference substance was not soluble in medium at 20
mg/mL, then more medium was added to a concentration of 2 mg/mL (i.e., a
total volume of \~5 mL) (Step 2). The mixing procedures were repeated as
necessary to dissolve the reference substance. If the reference
substance was not dissolved, \~10 mg reference substance in \~0.5 mL
DMSO was added in an attempt to dissolve it at 200 mg/mL DMSO (Step 3).
If the reference substance was not dissolved, the same concentration was
attempted in ETOH (Step 4). Step 5 began in the same way with 0.2 mg/mL
medium and then to 20 mg/mL DMSO and then 20 mg/mL ETOH.

Determination of solubility of reference substances was limited to
visual observation of the reference substance in solution. If a solution
appeared clear, then solubility testing ceased. If particles were
visible or the solution appeared cloudy, then more stringent mixing
procedures were employed. If necessary, the solubility procedure
proceeded to the next solvent/concentration tier. The duration of the
solubility test was dependent on mechanical procedures used to achieve
solubility. Some reference substances were immediately solubilized
(e.g., liquids) and others required up to 60 minutes of heating and
other mechanical procedures.

### 

2.9.2 [Data Collection]{.underline}

All laboratories (including the reference substance distribution
laboratory \[BioReliance\]) used a worksheet designed to capture the
solubility information for the reference substances. The protocol's
tiered approach to determining solubility of each reference substance
was followed. The endpoint for each step was a visual observation of the
solution and a documented comment of soluble or insoluble. Each
worksheet contained:

-   reference substance code and physical description

-   solvent (3T3 medium, NHK medium, DMSO, ETOH)

-   amount of reference substance (mg)

-   volume of solvent added and total volume (mL)

-   concentration (µg/mL)

-   pH and solvent color

-   mechanical procedures (vortexing, sonication, heating)

-   comments (soluble/insoluble at the particular concentration; visual
    observations)

The solubility test data from the laboratories were transferred via
email to the SMT and stored on the NICEATM server and as hard-copy
printouts. Each laboratory also maintained electronic and hard-copy
files of the data.

### 

2.9.3 [Variability in Solubility Measurement]{.underline}

Solubility analyses were not replicated since within-laboratory results
were not expected to vary. Comparison of the laboratory results to
determine laboratory concordance for the 72 reference substances (see
**Section 4** for results) provided a measure of variability among the
laboratories (see **Section 7**).

### 

2.9.4 [Solubility and the 3T3 and NHK NRU Test Methods]{.underline}

Reference substance solutions were monitored throughout all aspects of
the *in vitro* NRU cytotoxicity test methods and observations were
documented. The 2X and 1X solutions for the range finder tests were
permitted to contain precipitates. The lowest concentration of reference
substance in a 2X solution that contained observable precipitates,
particles, globules, or oily droplets was noted in the EXCEL^®^
template. After reference substance exposure, all wells of the 96-well
test plates were observed microscopically and scored using a visual
observation code as per the NRU protocol. The code addressed growth
characteristics and the presence or absence of precipitates. The Study
Directors made determinations of test acceptance based on the effect
that precipitates had on the NRU results.

2.9.5 [Methods for Analyzing Solubility Data]{.underline}

During Phase III, the SMT used the solubility data from all the
laboratories to determine the solvent that would be used for
cytotoxicity testing (see **Section 5** for solubility results and SMT
selections). If the solubility of an individual reference substance in
3T3 medium and NHK medium was different, the SMT chose the same solvent
for both test methods, rather than choosing one for the 3T3 NRU test
method and one for the NHK NRU test method. For example, if solubility
in one medium was ≥ 2 mg/mL and solubility in the other medium was \< 2
mg/mL, and the reference substance was soluble in DMSO at 200 mg/mL,
then the SMT selected DMSO as the solvent for cytotoxicity testing.
Where possible, the SMT chose a solvent such that all cytotoxicity
laboratories could obtain solubility at some concentration. For example,
if a reference substance had low solubility in medium (i.e., 2 mg/mL) at
one laboratory and high solubility in DMSO at the other laboratories,
the SMT chose DMSO.

Solubilizing enough reference substance to produce cytotoxicity was
challenging for relatively insoluble low toxicity reference substances
such as lithium carbonate (in the 3T3 NRU test method) but generally was
not a problem for toxic reference substances. Some insoluble and highly
toxic reference substances were problematic, however, because the amount
of powdered reference substance added to solvent was very small, so it
was difficult to determine the absence of solute particles in solution
(i.e., if the solution was visibly clear). Any undissolved reference
substance remaining might have been too little to see. Arsenic trioxide
is an example of such a solute.

**2.10 Basis of the Solubility Protocol**

The solubility protocol used by BioReliance, which tested solubility of
the reference substances prior to testing by the *in vitro*
laboratories, is provided in **Appendix G**. The protocol is based
largely on information from the literature and Internet searches for
solubility procedures, the experience of the SMT and IIVS, and the
solubility and IC~50~ information for the RC chemicals (Halle 2003). The
only formal solubility protocol discovered was the EPA Product
Properties Test Guideline, OPPTS 830.7840 Water Solubility Column
Elution Method; Shake Flask Method (EPA 1998).

2.10.1 [Initial Solubility Protocol Development]{.underline}

BioReliance tested reference substances in cell culture media at 2000
mg/mL, 400 mg/mL, and 200 mg/mL, and if not soluble, in DMSO, and then
ETOH at the same concentrations (initial protocol). It was apparent that
these concentrations were not low enough when the laboratory was unable
to achieve solubility for arsenic trioxide. The solubility protocol was
revised twice to lower the concentrations tested (see **Table 2-5**). An
extra tier of concentrations ≤ 1 mg/mL was added for insoluble reference
substances. Because of this experience, this solubility protocol for the
cytotoxicity laboratories was revised to reduce the number of steps
required (by testing in log units) and to test in tiers in which the
reference substance concentrations reflected the same concentrations in
cell cultures.

In Phases Ib and II, the SMT used the data from BioReliance to determine
the solvent for the *in vitro* laboratories to use for NRU testing. When
it became apparent that the laboratories sometimes obtained different
results than those reported by BioReliance, the SMT used the
cytotoxicity results from all the laboratories to determine the solvents
for Phase III reference substances.

Table 2-5 Comparison of Concentrations Tested in Various Solubility
Protocols

+-------------+-------+--------+---------+-------+--------+---------+
| *           | *     |        |         |       |        |         |
| *Solubility | *Conc |        |         |       |        |         |
| Protocol    | entra |        |         |       |        |         |
| Version**   | tions |        |         |       |        |         |
|             | T     |        |         |       |        |         |
|             | ested |        |         |       |        |         |
|             | (mg/  |        |         |       |        |         |
|             | mL)** |        |         |       |        |         |
+-------------+-------+--------+---------+-------+--------+---------+
|             | *     | **Step | **Step  | *     | **Step | **Steps |
|             | *Step | 2**    | 3**     | *Step | 5**    | 6-10**  |
|             | 1**   |        |         | 4**   |        |         |
+-------------+-------+--------+---------+-------+--------+---------+
| BioReliance | 2,000 | 400    | 200     |       |        |         |
| 1 (4/26/02) |       |        |         |       |        |         |
| and Phase   |       |        |         |       |        |         |
| Ia for      |       |        |         |       |        |         |
| c           |       |        |         |       |        |         |
| ytotoxicity |       |        |         |       |        |         |
| l           |       |        |         |       |        |         |
| aboratories |       |        |         |       |        |         |
+-------------+-------+--------+---------+-------+--------+---------+
| BioReliance | 200   | 40     | 20      | 10    | 2      |         |
| 2 (9/17/02) |       |        |         |       |        |         |
+-------------+-------+--------+---------+-------+--------+---------+
| BioReliance | 200   | 40     | 20      | 10    | 2      | 1, 0.5, |
| 3           |       |        |         |       |        | 0.25,   |
| (10/11/02)  |       |        |         |       |        | 0.125,  |
|             |       |        |         |       |        | 0.05    |
+-------------+-------+--------+---------+-------+--------+---------+
| Phases Ib,  | 20    | 2      | 0.2     | 2     | 0.2    |         |
| II, III for | M     | Medium | Medium  | DMSO  | DMSO   |         |
| c           | edium |        |         |       | 0.2    |         |
| ytotoxicity |       | 200    | 20 DMSO | 2     | ETOH   |         |
| l           |       | DMSO   |         | ETOH  |        |         |
| aboratories |       | 200    | 20 ETOH |       |        |         |
|             |       | ETOH   |         |       |        |         |
+-------------+-------+--------+---------+-------+--------+---------+

DMSO -- dimethyl sulfoxide

ETOH -- ethanol

Medium -- cell culture medium

### 

The protocol provided a tiered approach for determining the 2X stock
concentration for each reference substance, based on the solvent and
solubility of the reference substance (see **Figure 2-7**). The
solubility protocol was developed to reduce the number of steps for
testing (compared to that used by BioReliance) so that solubility
testing was less time consuming (see **Appendix B-3**).

#### 

2.10.2 [Basis for Modification of the Phase II Protocol]{.underline}

All three cytotoxicity laboratories found arsenic trioxide (tested in
Phase Ib) less soluble than that reported by BioReliance (0.25 mg/mL in
3T3 medium and 0.05 mg/mL in NHK medium). Use of the solubility
procedures in the protocol did not dissolve arsenic trioxide. IIVS
warmed the stock solution (at least 200 μg/mL for 2X) for longer than
the protocol specified (i.e., 30-50 min) but still had small,
undissolved particles persist in the non-homogeneous stocks (i.e.,
particles readily fell out of suspension). ECBC obtained a clear
solution (highest 2X concentration was 30-50 μg/mL), but found
precipitated particles after the solution stood at room temperature.
Sonication time was increased to 15-30 min, and heating time to \~ 30
min to get a finer suspension. This procedure achieved a more
homogeneous mixture, resulting in better series dilutions and more
uniform application of reference substance to the cells. FAL stirred the
suspension (\~ 20-90 μg/mL) in the CO~2~ incubator for 1.5 to 2 hours to
get clear medium.

> *Protocol Revision for Phase II*: The duration of the solution heating
> range was increased from 5-20 minutes to 5-60 minutes.

11. **Summary**

-   The *Guidance Document* NRU protocols were the basis of the
    NICEATM/ECVAM study protocols. The SMT and laboratories made initial
    modifications to the protocols prior to implementation of the study.
    Other protocol modifications were made after commencement of testing
    and were the result of comments and recommendations from the
    laboratories and the SMT. The resulting optimized protocols were
    used in the main testing phase (Phase III) and were the final
    protocols for the NICEATM/ECVAM study.

-   The solubility protocol was developed to provide specific guidance
    to laboratories to assure that solubility issues could be
    satisfactorily addressed and reference substances from a specific
    study set could be adequately prepared and evaluated for *in vitro*
    cytotoxicity effects.

***\[This Page Intentionally Left Blank\]***

[^1]: IC~50~ values are used for estimating the LD50 value of a
    reference substance. The IC~20~ and IC~80~ values were collected (as
    per request in the validation study's Statement of Work \[SOW\]) for
    possible use in estimating human lethal concentrations in blood.

[^2]: Vehicle control wells are considered to have 100% cell viability
    (i.e., all cells are alive). Cell viability in other test wells is
    referenced to the vehicle control value.