11.0 PRACTICAL CONSIDERATIONS 11-3

11.1 Transferability of the 3T3 and NHK NRU Test Methods 11-3

> 11.1.1 Facilities and Major Fixed Equipment 11-4
>
> 11.1.2 Availability of Other Necessary Equipment and Supplies 11-6

11.2 3T3 and NHK NRU Test Method Training Considerations 11-8

> 11.2.1 Required Training and Expertise 11-8
>
> 11.2.2 Training Requirements to Demonstrate Proficiency 11-10
>
> 11.3 Test Method Cost Considerations 11-11
>
> 11.3.1 3T3 and NHK NRU Test Methods 11-11
>
> 11.3.2 *In Vivo* Rodent Acute Oral Toxicity Testing 11-12

11.4 Time Considerations for the 3T3 and NHK NRU Test Methods 11-14

11.5 Summary 11-15

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**11.0 PRACTICAL CONSIDERATIONS**

The 3T3 and NHK NRU test methods are proposed as adjuncts, rather than
replacements for, the *in vivo* acute oral toxicity assays. Data from
these *in vitro* basal cytotoxicity test methods are used with a
prediction model to estimate the rodent oral LD~50~ of the test
chemical. This LD~50~ value is then used to determine the starting dose
for subsequent *in vivo* acute oral toxicity assays. This section
discusses practical issues involved in applying these two *in vitro* NRU
cytotoxicity test methods to the prediction of starting doses for rodent
acute systemic toxicity assays. Practical issues to consider for
implementation of these cell culture test methods include the need for
and availability of specialized equipment, training and expertise
requirements, cost considerations, and time expenditure. Good Cell
Culture Practice: ECVAM Good Cell Culture Practice Task Force Report 1
(Hartung et al. 2002) encourages the establishment of practices and
principles that will reduce uncertainty in the development and
application of *in vitro* test methods.

Good cell culture practices (in conjunction with good laboratory
practices) are essential for all *in vitro* cytotoxicity testing and
should be employed to ensure that data produced from the 3T3 and NHK NRU
test methods are reproducible, reliable, credible, and acceptable.

**11.1 Transferability of the 3T3 and NHK NRU Test Methods**

Transferability of a test method is defined as the ability of a test
method or procedure to be accurately and reliably performed in
different, competent laboratories (ICCVAM 2003). Accuracy and
reliability of these test methods are discussed in **Sections 6** and
**7**, respectively.

Protocols for the 3T3 and NHK NRU test methods, solubility testing, and
prequalification of keratinocyte growth medium have been optimized and
are available on the ICCVAM/NICEATM website
(http://iccvam.niehs.nih.gov/methods/invitro.htm). The protocols were
designed with GLP-compliance in mind and can be easily implemented or
adapted by scientists with the appropriate technical experience.

While *in vitro* and *in vivo* methods require some similar skills
(e.g., preparation of solutions and test chemical doses, documentation),
*in vitro* testing requires skills specific to cell culture systems
(e.g., aseptic techniques, microscopic evaluation of cell cultures,
propagation of cells in medium) but not to the maintenance, handling, or
treatment of rodents.

11.1.1 [Facilities and Major Fixed Equipment]{.underline}

The following lists of facility requirements, equipment and supplies,
and training and expertise are common to most *in vitro* mammalian cell
culture laboratories. Required equipment and supplies are also described
in the NICEATM/ECVAM validation study 3T3 and NHK NRU test method
protocols (**Appendices B and C**), the *Guidance Document* (ICCVAM
2001b, **Appendix D**) and Hartung et al. 2002.

*Facility Requirements*

The testing facility should provide structures and infrastructures
necessary for operating a scientific laboratory (e.g., laboratory space,
access to utilities, shipping/receiving department \[for appropriate
receipt and handling of cell culture materials\], etc.). Each facility
should provide:

-   personnel that are competent in performing *in vitro* cytotoxicity
    assays under aseptic laboratory conditions

-   adequate facilities, equipment, and supplies

-   proper health and safety guidelines

-   satisfactory quality assurance procedures

Each facility should conform to all appropriate statutes (i.e., local,
state, provincial, federal, national, international) concerning safety
guidelines (e.g., general workplace safety guidelines, chemical handling
and disposal guidelines, biohazard guidelines, etc.). Hartung et al.
2002 provides recommended safety guidelines for working with potentially
infectious materials (e.g., HIV, hepatitis B, hepatitis C) and human
materials (e.g., cells, tissues, fluids).

The facility management should establish scientific guidelines and
procedures, train and supervise professional and technical staff, and
evaluate results and performance within their discipline area relative
to the testing requirements. Personnel should have mandatory training in
basic cell culture practice, in specific procedures for specialized
culture procedures, and in specific safety practices appropriate to the
types of materials that may be used in the laboratory (Hartung et al.
2002). The management should maintain records of the qualifications,
training and experience, and job descriptions for each professional and
technical individual involved in the testing.

*Cell Culture Laboratory*

The testing facility should have a designated cell culture laboratory to
ensure that *in vitro* cytotoxicity assays are performed under clean and
proper aseptic conditions. The laboratory should be located such that
through traffic is minimal to reduce possible disturbances that may
compromise the cell culture assays. Room temperature of the laboratory
should be regulated, monitored, and documented. Access to the laboratory
and test chemicals should be restricted to appropriate personnel.

*Major Equipment*

Each testing facility should have at a minimum the following equipment:

-   incubator (37ºC ± 1ºC, 90% ± 10% humidity, 5.0% ± 1% CO~2~/air)

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

-   inverse phase contrast microscope

-   96-well plate spectrophotometric plate reader equipped with 540 nm ±
    10 nm filter (if testing in 96-well plates)

-   autoclave

-   refrigerator

-   freezer (-70ºC)

-   liquid nitrogen

-   cryogenic freezer/storage unit

-   computer

Equipment maintenance and calibration should be routinely performed and
documented as per GLP guidelines and testing facility procedures.

11.1.2 [Availability of Other Necessary Equipment and
Supplies]{.underline}

*General Equipment*

Each testing facility should have at a minimum the following equipment:

-   centrifuge

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-   waterbath

-   pipettors

-   balance

-   pH meter

-   cell counting system

-   water bath sonicator

-   magnetic stirrer

-   vortex mixer

-   antistatic bar ionizer

Equipment maintenance and calibration should be routinely performed and
documented as per GLP guidelines and testing facility procedures. These
types of equipment are available from scientific and laboratory supply
companies (e.g., Fisher Scientific, Thomas Scientific, etc.).

*General Cell Culture Materials and Supplies*

The following supplies are needed for the NRU test methods:

-   tissue culture plasticware

-   glassware

-   sterile filtration systems

-   culture medium and supplements

-   serum

-   balanced salt solutions

-   NRU assay chemicals

Cell culture supplies are generally available through the major
scientific and laboratory supply companies and through specialty
companies (e.g., GIBCO, SIGMA-Aldrich, CAMBREX/Biowhittaker, Becton
Dickinson, etc.). Compositions of culture media, supplements/additives,
salt solutions, NRU assay chemicals and the volumes needed for the test
methods should be defined. All culture vessels needed to assure proper
cell propagation should be defined.

During this study, obtaining an adequate supply of NHK medium was
problematic for FAL. Communication between the UK distributor and the
laboratory was uneven and the SMT intervened on several occasions in an
attempt to resolve the supply issue. This illustrates the need for
additional sources of keratinocyte cell culture medium. Periodically, it
was also difficult to obtain NHK medium and supplements that adequately
supported keratinocyte growth similarly in all the laboratories.
Although the purchased medium met the manufacturer's QA/QC standards,
certain lots of the medium and supplements did not support the growth of
NHK cells to the extent needed to meet the growth characteristics
required by the test method protocol. This necessitated the need to
incorporate an NHK medium prequalification protocol into the study.
Prequalification of medium is recommended to avoid unnecessarily
repeating studies.

*Cell Cultures*

3T3 Mouse Fibroblasts: BALB/c 3T3 cells, clone 31, can be obtained from
national/international cell culture repositories (e.g., CCL-163,
American Type Culture Collection \[ATCC\], Manassas, VA).

Normal Human Epidermal Keratinocytes (NHK): non-transformed keratinocyte
cells from cryopreserved primary or secondary cells can be obtained from
national/international cell culture repositories (e.g., CAMBREX Bio
Science, 8830 Biggs Ford Road, Walkersville, MD) or isolated from
donated tissue (using proper collection, preparation, and propagation
techniques).

Obtaining adequate supplies of keratinocytes may be difficult since
preparing a pool of cells depends on the availability of tissue donors.
Procurement of a commercially available stock pool of cells and storing
them indefinitely in a cryogenics freezer is recommended.

2.  **3T3 and NHK NRU Test Method Training Considerations**

    1.  [Required Training and Expertise]{.underline}

Hartung et al. 2002 recommends that scientists involved in *in vitro*
testing should have training in basic cell culture aspects such as:
sterile technique, handling culture media, feeding cultures, cell
counting, subculture (trypsinization), detection and elimination of
contamination, growth parameters, growth curves, viability assays,
storage and freezing/thawing of cells. Additionally, training is
encouraged for special culture procedures such as: primary cell and
tissue cultures, toxicity testing, viability assays, cloning,
transfection, expression cloning, cell transformation and
immortalization, and virus propagation and isolation. Laboratory
personnel should be trained in the application of GLP requirements (see
**Section 8.1.1**).

*Training and Expertise*

*In vitro* NRU cytotoxicity test methods require personnel trained
specifically in sterile tissue/cell culture techniques and general
laboratory procedures. Performance of the test methods requires a
relatively moderate degree of technical capability and a high degree of
skill in monitoring and maintaining appropriate cell growth conditions,
troubleshooting potential and real problems in culture systems, and
interpreting and analyzing cytotoxicity data. Each individual engaged in
the conduct of or responsible for the supervision of a study shall have
education, training, and experience, or combination thereof, to enable
that individual to perform the assigned duties. The NRU test methods do
not require that personnel be trained to perform *in vivo* testing.

*Specific Training and Expertise Needed* *for the In Vitro NRU
Cytotoxicity Test Methods*

Personnel involved in performing the *in vitro* NRU cytotoxicity test
methods should be well experienced in general cell culture techniques
and should be able to:

-   work with cryogenic freezing apparatus

-   pipette solutions with large volume pipettors and multi-channel
    pipettors

-   establish cells in culture vessels under aseptic conditions and
    monitor growth; recognize normal and abnormal cell growth
    characteristics; document observations of cell cultures throughout
    all aspects of the cultures

-   perform the *in vitro* assays by following the protocols to: grow
    the cells, treat the cells with test chemicals, perform the NRU
    assay, measure endpoints (i.e., optical density measurements),
    transfer data to electronic templates

-   operate equipment necessary for maintaining cell culture
    laboratories (e.g., incubators, biohazard hoods, spectrophotometric
    microtiter plate readers)

*General Laboratory Expertise Needed for the In Vitro NRU Cytotoxicity
Test Methods*

Personnel should also be able to perform and understand basic laboratory
techniques and laboratory management:

-   prepare cell culture solutions (e.g., culture medium, NRU
    solutions); measure pH; know proper storage conditions and maintain
    proper documentation

-   prepare test chemicals for application to cell culture test plates;
    follow solubility protocols to adequately prepare test chemicals in
    solution; recognize solubility issues (e.g., insolubility nature of
    chemical, precipitation) and implement mechanical procedures for
    solubilizing the test chemicals

-   monitor and control laboratory room conditions (e.g., temperature,
    humidity, lighting, traffic); maintain equipment at conditions
    essential to cell cultures (e.g., temperature, humidity, gas flow,
    calibrations)

*Personnel Needed to Perform the In Vitro NRU Cytotoxicity Test Methods*

-   [Study Director]{.underline}: the single point of study control; has
    the overall responsibility for the technical conduct of the testing
    (e.g., GLP adherence); determines test acceptance, provides SOPs,
    interprets and analyzes the data, documents testing aspects, and
    produces all written reports.

-   [Quality Assurance Officer]{.underline}: monitors the testing to
    assure conformance with GLP requirements; must be independent of the
    Study Director.

-   [Laboratory Technician(s)]{.underline}: individuals trained in
    sterile tissue/cell culture techniques and general laboratory
    procedures and capable of performing the *in* *vitro* NRU
    cytotoxicity test methods in a GLP-manner.

11.2.2 [Training Requirements to Demonstrate Proficiency]{.underline}

Laboratories set their own criteria for proficiency, but in general,
personnel should be able to understand the protocol, carry out the
protocol with guidance from an experienced supervisor/trainer, and then
carry out the protocol with no supervision. An experienced supervisor
determines when a technician is adequately trained since there is no
precise level of training that can be measured. Once the technician
demonstrates competence in executing all the aspects of the *in vitro*
NRU cytotoxicity test method(s), it is appropriate to initiate routine
assessments of observations among personnel using a benchmark control
test substance (SLS for these two NRU test methods) to ensure
consistency.

The laboratories in this study were experienced in performing *in vitro*
cytotoxicity assays but were required to train and develop additional
skills through Phases I and II (e.g., data collection and transfer to
Excel^^ and PRISM^^ templates). Inexperienced laboratory personnel
were trained by completion of "training" NRU assays using SLS. In the
early phases of the ICCVAM/ECVAM validation study, the laboratories
continued training by the testing of coded reference chemicals of
various toxicities and performing solubility testing on the chemicals.
This training improved proficiency among the staff of the laboratories
for the final phase of the validation study.

### GLP-Compliance Proficiency Criteria

ECBC and IIVS conducted this study in compliance with GLP Standards (see
**Section 8.1.1**). The appropriate QA unit (as per GLPs) reviewed the
various aspects of the study and issued a QA statement that identified
whether the methods and the results described in the Final Report
accurately followed the test method protocol and reflected the raw data
produced during the study, respectively, and provided assurance that all
testing was done under the principles of GLP. FAL (non GLP-adherent)
followed GLP standards referenced in **Section 8.1.1** as guidelines for
conducting this study. FAL had no QA unit to judge their compliance with
GLP guidelines.

**11.3 Test Method Cost Considerations**

11.3.1 [3T3 and NHK NRU Test Methods]{.underline}

*Laboratory Costs*

Supplies such as cell culture chemicals, the reagents used to measure
NRU, and cell culture plasticware are available from numerous suppliers
and are not cost prohibitive. Major instruments and equipment that *in
vitro* cytotoxicity laboratories need to implement the *in vitro* NRU
cytotoxicity test methods are described in **Section 11.1.1**.

The 3T3 NRU test method is generally less expensive to use than the NHK
NRU test method. One vial of the immortalized 3T3 cells (\$180) can be
propagated indefinitely by passaging cells and periodically
cryopreserving pools (i.e., numerous vials of cells). NHK cells require
a fresh sample of primary cells for each test run (\$380 per vial).
Since primary NHK cells are only passaged once after initiating into
culture, there are no cells available to cryopreserve a stock pool of
cells. The D-MEM medium used for the 3T3 cells is less expensive, more
"generic", and more readily available than keratinocyte-specific medium.
(See **Table 11-1**)

**Table 11-1 Costs for Cell Culture Materials and Commercial Laboratory
*In Vitro* Cytotoxicity Testing**

+----------------+-----------------+------------+----------------------+
| **Item**       | **Cost**        | **Number   | **Other**            |
|                |                 | of Tests   |                      |
|                | **              | Possible** |                      |
|                | (approximate)** |            |                      |
+----------------+-----------------+------------+----------------------+
| 3T3 Cells      | \$180/vial^1^   | indefinite | One vial can produce |
|                |                 |            | an indefinite supply |
|                |                 |            | of cells by          |
|                |                 |            | propagating the      |
|                |                 |            | cells in culture and |
|                |                 |            | periodically         |
|                |                 |            | freezing a pool of   |
|                |                 |            | cells.               |
+----------------+-----------------+------------+----------------------+
| NHK Cells      | \$380/vial^1^   | \~5        | Since cells are      |
|                |                 | (96-well   | passaged only once   |
|                |                 | plates)    | beyond               |
|                |                 |            | cryopreservation,    |
|                |                 |            | new ampules should   |
|                |                 |            | be thawed frequently |
|                |                 |            | to maintain          |
|                |                 |            | continuous testing.  |
+----------------+-----------------+------------+----------------------+
| Dulbeccos'     | \$20/500mL^1^   | \~15       | Establish cells in   |
| Minimum        |                 | (96-well   | culture (\~20        |
| Essential      |                 | plates)    | mL/vial of cells; 60 |
| Medium (D-MEM) |                 |            | mL/3 vials), seed    |
| with           |                 |            | cells in 96-well     |
| supplements    |                 |            | plates (12 mL/plate; |
|                |                 |            | 180 mL/15 plates);   |
|                |                 |            | prepare stock        |
|                |                 |            | solution and eight   |
|                |                 |            | concentration        |
|                |                 |            | dilutions (\~20      |
|                |                 |            | mL/chemical; 300     |
|                |                 |            | mL/15 plates).       |
+----------------+-----------------+------------+----------------------+
| NHK Medium     | \$80/500 mL^1^  | \~15       | Same as DMEM (above) |
| with           |                 | (96-well   |                      |
| supplements    |                 | plates)    |                      |
+----------------+-----------------+------------+----------------------+
| Commercial     | \$1050/\$1950   | 1          | *in vitro* NRU       |
| Laboratory     | (USP/ISO) per 3 | tes        | cytotoxicity test    |
| Testing (MB    | test            | t/material | (24-hour test        |
| Research       | materials^2^    |            | period)              |
| Laboratories)  |                 |            |                      |
+----------------+-----------------+------------+----------------------+
| Commercial     | \$1120 (GLP)    | 1 range    | *in vitro* NRU       |
| Laboratory     | per test        | finder, 2  | cytotoxicity test    |
| Testing        | material        | definitive | (48-hour test        |
| (Institute for | (minimum of 5   | tests per  | period)              |
| In Vitro       | materials)^2^   | test       |                      |
| Sciences))     |                 | material   |                      |
+----------------+-----------------+------------+----------------------+
| Commercial     | \$1850 (GLP)    | 1 range    | *in vitro* NRU       |
| Laboratory     | per single test | finder, 2  | cytotoxicity test    |
| Testing        | material^2^     | definitive | (48-hour test        |
| (Institute for |                 | tests per  | period)              |
| In Vitro       |                 | test       |                      |
| Sciences))     |                 | material   |                      |
+----------------+-----------------+------------+----------------------+

^1^catalogue price

^2^personal communication

*Commercial Testing Laboratories*

A representative of MB Research Laboratories (Spinnerstown, PA,
<http://www.mbresearch.com/>) provided a quote (personal communication
2005) for an *in vitro* NRU cytotoxicity test (24-hour \[and not a
48-hour\] test period) of \$1050/\$1950 (USP/ISO) per set of three test
chemicals. The lead laboratory for the NICEATM/ECVAM study, IIVS
(Gaithersburg, MD, <http://www.iivs.org/>) provides commercial
laboratory GLP-compliant testing using this study's protocols (48-hour
test period) at a cost of \$1120 - \$1850 per chemical/sample (personal
communication with Hans Raabe \[IIVS\] 2005).

11.3.2 [*In Vivo* Rodent Acute Oral Toxicity Testing]{.underline}

**Table 11-2** provides commercial prices for acute oral systemic
toxicity testing.

MB Research Laboratories performs the UDP test at a cost of \$750 for
three rats and charges \$250 for each additional rat needed. In the
best-case scenario, the UDP test needs only three rats (\$750). In the
worst-case scenario, this test would need an additional 12 rats (15
maximum for the test); the total cost of the test would be \$3750. In
this costing strategy, \$250 is saved from the total cost of the UDP for
each rat saved by using the 3T3 or NHK NRU test method to predict the
starting dose. Considering that adding the *in vitro* NRU cytotoxicity
test costs from \$350 to \$1850 per chemical, the NRU test does not
provide cost savings if fewer than two to six animals are saved.

The President of Product Safety Laboratories (Dayton, NJ,
<http://www.productsafetylabs.com/>), Gary Wnorowski, provided a cost
quote of \$2700 for determination of an LD~50~ value using the UDP test;
the cost is independent of the number of rats that are needed. Each
testing dose is administered \~24-48 hours after the previous dose and
each animal test generally does not exceed four days. Time involved in
providing the LD~50~ value is approximately three months (initiation of
the test to provision of the final report). Knowing the estimated LD~50~
value does not affect the cost of the *in vivo* test in this case but
could reduce the number of animals needed for the test.

Bio Research Laboratories (BRL) performs the Acute Oral Rat Toxicity
Test bioassay to determine the relative acute toxicity of an unknown
substance. The method determines lethality and signs of acute toxicity
from a waste sample administered in a single dose by gavage to a limited
number of rats. The bioassay determines if the test sample exhibits a
median lethal dose (LD~50~) either greater than or less than a
regulatory threshold corresponding to a hazardous waste designation
(i.e., 5000, 500, 50 mg/kg). A minimum of ten rats is used at the tested
dosage for the pertinent regulatory threshold value that is relevant to
the test sponsor. Knowledge of the estimated LD~50~ does not reduce
animal use or test costs if a single predetermined dose is tested.

###### Table 11-2 Commercial Prices for Conducting *In Vivo* Acute Toxicity Testing 

  --------------------- ------------------- ----------------- -------------------
  **Test**              **GLP-Compliant**   **Non             **Company**
                                            GLP-Compliant**   

  Acute Oral Toxicity   \$1200              \$1000            Product Safety
  UDP: Limit Test -                                           Laboratories (PSL)
  2000 mg/kg                                                  

  Acute Oral Toxicity   \$800               \$650             PSL
  UDP: Limit Test -                                           
  5000 mg/kg                                                  

  Acute Oral Toxicity   \$2700              \$2200            PSL^a^
  UDP: LD~50~                                                 

  Acute Oral Rat        \$950               NA                Bio Research
  Toxicity: single                                            Laboratories (BRL)
  dose^b^                                                     

  Acute Oral Rat        \$1500              NA                BRL
  Toxicity: two                                               
  doses^b^                                                    

  Acute Oral Rat        \$3000              NA                BRL
  Toxicity: LD~50~                                            

  Acute Oral Toxicity   \$730 for the first NA                MB Research
  -- UDP                3 animals; \$250                      Laboratories^a^
                        each additional                       
                        animal                                
  --------------------- ------------------- ----------------- -------------------

^a^provided to NICEATM through personal communication

^b^Washington State Biological Testing Methods #80-12 For the
Designation of Dangerous Waste; Part B: Acute Oral Rat Toxicity Test
\[http://www.ecy.wa.gov/pubs/80012.pdf\] The method is an adaptation of
the EPA Health Affects Test Guidelines OPPTS 870.110 Acute Oral Toxicity
and American Society for Testing and Materials (ASTM) methods E 1163-90
(Standard test method for estimating acute oral toxicity in rats) and E
1372-90 (Standard test method for conducting a 90-day oral toxicity
study in rats).

**11.4 Time Considerations for the 3T3 and NHK NRU Test Methods**

*The 3T3 NRU Test Method*

Approximately one week is needed to thaw cryopreserved 3T3 cells,
propagate the cells in flasks, and passage/subculture the cells at least
two times before subculturing to the 96-well test plate. After
subculture into 96-well plates, the cells are incubated another 24 hours
to reach the proper percentage of confluency, and then exposed to test
chemical for 48 hours. The entire 3T3 NRU assay process takes
approximately 10 days. However, once the cells are established in
culture, they can be passaged for approximately two months before
starting the initial propagation from frozen stock. Multiple chemicals
can be tested at the same time, and different tests can overlap each
other; thus, many chemicals can be tested in a relatively short time.

*The NHK NRU Test Method*

Approximately one week is needed to thaw cryopreserved NHK cells,
propagate the cells in flasks, and passage/subculture the cells
([once]{.underline}) directly to the 96-well test plate. After
subculture into 96-well plates, the cells are incubated another 48-72
hours to reach the proper percentage of confluence and then exposed to
test chemical for 48 hours. The entire NHK NRU assay process (range
finder or definitive test) requires approximately 11-12 days. Cells can
be seeded at different densities in the culture flasks so that passaging
the cultures can take place on different days. Once the cells are
established in culture, they are passaged once to the 96-well test
plates. Multiple chemicals can be tested at the same time, and different
tests can overlap each other; thus, many chemicals can be tested in a
relatively short time.

### In Vivo Testing

According to guidelines for acute oral toxicity testing for the main
test and limit dose test, single animals or groups of animals are dosed
in sequence, usually at 2-4 day intervals, and observations are
generally made for up to 14 days (for animals that are not moribund)
(EPA 2002a; OECD 2001a; OECD 2001b, OECD 2001c). The addition of NRU
testing to estimate a starting dose prior to the implementation of the
UDP main test or limit dose test will take 10-12 days, but could save up
to 14 days of observation for every animal saved.

5.  **Summary**

-   All equipment and supplies are readily available. Direct
    communication with the NHK medium supplier assured that specific
    lots of medium were available to the laboratories. The test methods
    should be easily transferable to laboratories experienced with
    mammalian cell culture methods.

-   Much of the training and expertise needed to perform the 3T3 and NHK
    NRU test methods are common to all mammalian cell culturists.
    Additional technical training would not be intensive since there are
    no extraordinary techniques needed and these test methods are
    similar in general performance to other *in vitro* mammalian cell
    culture assays. GLP training should be provided to technicians to
    ensure proper adherence to protocols and documentation procedures.

-   Price levels for commercial testing for one chemical are \$1120 to
    \$1850 (**Table 11-2**) for *in vitro* NRU cytotoxicity testing to
    determine the IC~50~ (IIVS, personal communication) versus \$750 -
    \$3750 (**Table 11-2**) for *in vivo* rat acute oral testing for
    LD~50~ determination. Comparison of costs of the *in vitro* testing
    to *in vivo* testing is difficult since the *in vitro* NRU
    cytotoxicity test methods are not replacements for the animal
    testing. Use of these test methods may not necessarily reduce the
    overall cost of the *in vivo* rat acute oral toxicity test but can
    reduce the number of animals needed for a study.