139 - P - BALBc 3T3 Neutral Red Uptake Cytotoxicity Assay

© ECVAM DB-ALM: Protocol
DB-ALM Protocol n° 139 : BALB/c 3T3 Neutral Red Uptake Cytotoxicity Assay (3T3 NRU)
Systemic Toxicity
The cytotoxic effect of substances upon cells in culture is measured by a cell survival/viability assay, the Neutral Red
Uptake assay (NRU), which is based on the ability of the cells to incorporate and bind the Neutral Red (NR). The i n
vitro data are used to estimate an IC50 value which in turn is used to predict a LD50 value that can serve as the starting
dose for the acute oral toxicity test in vivo. (NICEATM/ECVAM validation study protocol, method evaluated in the EU
Intergrated Project "ACuteTox").
Objective & Application
TYPE OF TESTING : Screening, Adjunct, Part of a test battery, Reduction,

Refinement
LEVEL OF ASSESSMENT : Toxic potential, Toxic potency, Risk assessment approaches
PURPOSE OF TESTING : Substance-related mechanistic studies, Screen or priority
setting
Context of Use:
Regulatory and/or non-regulatory purpose.The purpose of this test method is to evaluate the capacity of test
substances to cause injury to structures and functions universal to all cells. To this end the BALB/c 3T3 NRU
cytotoxicity assay is used, which measures the lysosome integrity. OECD Guidance Document No 129 (OECD, 2010)
describes the concept of using the in vitro cytotoxicity test methods to estimate the starting doses for in vivo acute
oral systemic toxicity tests. Furthermore, this method is considered as a suitable candidate for use in the potentially
forthcoming integrated testing strategies for human acute oral toxicity testing (http://www.acutetox.eu/).
Applicability Domain:
The test method is suitable for water-soluble, non-volatile pure substances and formulations which exert their effects
via mechanisms present in BALB/c 3T3 cells. In this regard, the test method will not detect the augmented toxic effect
due to specific organ toxicity. For example neurotoxic or cardiotoxic substances or those which acquire toxicity after
metabolic activation might be under-predicted with this test method.The method may be used for volatile chemicals,
as long as they do not pose a danger to the operator as a result of their toxicity and volatility and all acceptance
criteria are fulfilled. The test method is not suitable for non-stable emulsions/suspensions.
Additional Information:
NR can be phototoxic at concentrations, exceeding those attained in the cells in the assay. However, the high light
exposure of the cells during the dye uptake step and the prior to the endpoint measurement should be avoided.
Certain substances which affect lysosomal function can also alter the retention capacity of the cells, leading to false
data (e.g. Antihelminth drugs).
Résumé
Please refer to a general Method Summary for "Neutral Red Uptake”, available in DB-ALM which describes the key
principle and main applications of the assay. The preceding studies which led to the protocol in its current form are
outlined in the Status section.
Experimental Description
Endpoint and Endpoint Measurement:
The assay measures the Neutral Red Uptake (NRU), quantified spectrophotometerically and performed in a
concentration-response format.
CELL VIABILITY: is expressed as a vital dye uptake ratio and plotted against the test substance concentration
Endpoint Value:
IC50: The concentration of substance that reduces the NRU by 50% compared with the vehicle treated control set as
100% (in some studies also referred to as EC50 or NRU 50)
Experimental System(s):
BALB/c 3T3 mouse fibroblast cell line
Basic Procedure
BALB/c 3T3 cells are routinely grown as a monolayer in tissue culture grade flasks (e.g., 75 - 80 cm2) at 37ºC ± 1ºC, 90
% ± 5 % humidity, and 5.0 % ± 1 % CO 2/air. The cell density should be examined daily so that the cells are
https://ecvam-dbalm.jrc.ec.europa.eu/methods-and-protocols/protocols page 1 / 27
sub-cultured at post 50% confluence and before reaching 80%.

Prior to the cytotoxicity assay, the solubility of the test substances should be determined. If not yet known from
literature or laboratory practice, a step-wise testing procedure is recommended. If the test substance does not
dissolve at the concentration of min. 20mg/mL in aqueous Chemical Dilluton Medium (CDM), dilutions are made in
10-fold increments, using one of the following solvents: CDM, DMSO or ethanol, until a substance is dissolved into a
clear solution or has been judged incompatible with the test.
The assay consists of two subsequent runs: the range-finding experiment to estimate the IC 50 and the main
experiment over a narrower range of concentrations above and below of the expected IC50 to facilitate an accurate
measurement.
For performing the cytotoxicity assay, BALB/c 3T3 cells are seeded in 96-well plates. Once well attached and
proliferating, the cells are treated with test substances for 48 hours. Afterwards, NR stain is added to the wells. After 3
hours incubation, the staining solution is removed and a desorbing fixative (ethanol / acetic acid / water) is added for
20-45 minutes. NR taken up by the viable cells is extracted and the absorption is measured in a spectrophotometer.
Cytotoxicity is expressed as a concentration-dependent reduction of the uptake of NR, compared to the untreated or
vehicle-treated controls.
For each set of plates, used in an assay for test substances, a separate plate of positive control concentrations (i.e.
sodium lauryl sulphate, SLS) needs to be set up in parallel. The positive control IC50 must be within ± 2.5 standard
deviations of the historical mean established by the laboratory. This value should be used as an acceptance criterion
for the assay.
Data Analysis/Prediction Model
Rat acute oral toxicity data available from the Registry of Cytotoxicity (RC) were used to develop a regression model in
the NICEATM/ECVAM validation study (Halle, 2003). Up-to-date RC list is available from ZEBET on individual
request, adressed to: zebet@bfr.bund.de, and can be obtained as paper copy (Willi Halle: "Registry of Cytotoxicity") or
MS Access database (RC_5DB.mdb).
The following formulas were used to predict LD50 value from the NRU IC50 values obtained with 3T3 NRU test
method. For more details, please consult section 4 of the Background Review Document (NIH, 2006):
Validated millimole regression model:

Log LD50 (mmol/kg) = 0.439 log IC50 (mM) + 0.621
Validated weight regression model:

Log LD50 (mg/kg) = 0.372 log IC50 (µg/mL) + 2.024
The millimole regression model is especially suitable for substances of known molecular weight that are relatively
pure.
The weight regression model is applicable to mixtures, substances whose structures or molecular weights are
unknown, and substances with high impurity ratio.
The validation study concluded that the weight regression model significantly overestimated the toxicity of the high
molecular weight substances (≥400 g/mole vs. <400 g/mole) while the millimole regression did not.
Predictions of the Globally Harmonised System (GHS) acute oral toxicity categories (UN, 2009; NIH, 2006) by the
3T3 NRU using the RC rat-only regression models:
GHS acute oral toxicity categories % of correct predictions*

Millimole regression Weight regression
Category 1 (LD 50 ≤ 5 mg/kg) 0% (0/6) 0% (0/6)
Category 2 (5 < LD 50 ≤ 50 mg/kg) 9% (1/11) 9% (1/11)
Category 3 (50 < LD50 ≤ 300 mg/kg) 42% (5/12) 33% (4/12)
Category 4 (300 < LD50 ≤ 2 000 mg/kg) 81% (13/16) 75% (12/16)
Category 5 (2 000 < LD50 ≤ 5000 mg/kg) 0% (0/10) 40% (4/10)
Unclassified (LD 50 > 5 000 mg/kg) 17% (2/12) 0% (0/12)
* data from NICEATM/ECVAM validation study (NIH, 2006)
Test Compounds and Results Summary
In the NICEATM/ECVAM validation study (See also the Status section below), the ability of 3T3 NRU test method to
estimate rodent oral LD50 values across the GHS categories was addressed. For the study, a total of 72 chemicals were
selected, with 12 chemicals representing each GHS category, including “Not Classified”. These chemicals included
pharmaceuticals (35%), pesticides (22%), solvents (10%) and food additives (6%). In addition, selected chemicals,
used for manufacturing and consumer products were included.
The results of this study showed that the overall accuracy of the 3T3 NRU test method in correctly predicting each of
the GHS acute oral toxicity classification categories was low (31 %), being highest (75%) in GHS category 4, i.e. 300 <
LD 50 < 2 000 mg/kg. For correct category prediction ± 1 category the accuracy of the 3T3 NRU method was 69%
(47/67) with millimole regression model and 75% (50/67) with weight regression model. The validation study affirmed
the potential application of the 3T3 NRU method in a weight-of-evidence approach to determine the starting dose for
acute oral in vivo toxicity protocols.
In this study, two laboratories, IIVS and ECBC, had GLP certification.
Modifications of the Method
As the cells are fixed it is possible to quantify their total protein content using, for example, the FRAME Kenacid Blue
method (DB-ALM Protocol No. 15). It is also possible to adapt the test to moderately volatile chemicals by using a
plate sealer film, i.e. prevent well to well contamination by the test substance.
Discussion
3R impact
The test method is based on a commercially available cell line and thus does not involve use of animals. The 3T3
NRU test method is not accurate enough to be used as a stand alone method for the classification purpose.
However the test method may be included in a testing strategy or a weight-of-evidence approach and potentially
contribute to the reduction and refinement of animal use in the current toxicological in vivo methods e.g. by
providing the starting dose information. The NICEATM/ECVAM validation study (NIH, 2006) estimated that
5-10% animals required for in vivo testing could be saved when the staring dose for acute oral toxicity testing
would be based on the result of 3T3 NRU assay rather than a default dose prescribed in OECD TG 423: Acute Oral
Toxicity – Acute Toxic Class Method (OECD, 2001).
Toxicity – Acute Toxic Class Method (OECD, 2001).

The estimates, however, may vary depending on the set of substances used in the simulations. A recent study by
Schrage et al. (2011) used 203 substances with in vivo data (187 of which where BASF in-house substances
including 70% of agrochemical active ingredients and formulations) and showed that the number of animals
could be saved only for a small subset of substances tested. In fact, a more accurate estimate of the starting dose
was achieved by the expert judgment considering all available information on the test substance: structural
information, physicochemical properties, data in biochemistry, physiology and toxicology of the test substance
and related compounds (Schrage et al., 2011).
Practical aspects
Special equipment
No.
Required Training and Expertise
Personnel performing in vitro testing should have training in basic cell culture techniques including
cryostorage, liquid handling, adherent cell handling and maintenance and operation of standard laboratory
equipment. Laboratory personnel should be trained in the application of GLP requirements and in the safe
storage, handling, and disposal of toxic substances.
Adaptation for high-throughput testing
The method can be tailored to a high-throughput testing, and has proven to function well (Norlén et al .
2007; Bouhifd et al. , 2012).
Costs
The method uses an immortalized cell line that can be propagated indefinitely by passaging cells and
periodically cryopreserving batches of cells, and only basic cell culture medium (DMEM supplemented with
Newborne Calf Serum and glutamine) is required. A fresh batch of frozen cells from the stock lot of cells
should be thawed approximately every two months. The cells should be used below passage nr 60 from the
frozen stock, as over this they can change significantly in their response. Equipment for cell culture
experiments constitutes the highest cost, but no new investment in method-specific equipment is needed.
For 10 chemicals approximately 2 person-months are required for a complete testing cycle: culture
establishment and maintenance, solubility testing, range finding experiment and a completion of three
acceptable runs of the main experiment.
Advantages and limitations
Advantages
The NRU cytotoxicity assay is a quantitative and relatively quick assay to perform. Compared to other
cytotoxicity tests, such as tetrazolium salts, enzyme leakage or protein content, it is considered more
sensitive, cheaper, of lower interference, and with more stable reagents.
Limitations
Some substances with low toxicity and low solubility might not be compatible with the in vitro NRU
cytotoxicity assays. In particular, for substances where the fully soluble concentration is lower than the
concentration range inducing toxic effects, no definitive toxicity prediction is possible. Thus, the
amount of dissolved substance may be too low to obtain a measurable IC 50 value.
The 3T3 NRU test method is better at predicting the toxicity of substances with 50 < LD 50 ≤ 300 mg/kg
and 300 < LD 50 ≤ 2 000 mg/kg ranges than predicting the toxicity of substances with higher or lower LD
50 values (NIH, 2006).
The method is unsuitable for chemicals exerting their toxic effects through mechanisms absent in 3T3
cells, such as tissue-specific receptor toxicity (e.g. neuro- or cardiotoxicity) or through metabolic
pathways that the cells do not contain (both toxifying or detoxifying).
There are indications that certain chemicals that affect lysosomal pH can modulate the NRU storage so
that the test outcome might not directly correspond to membrane damage and cell viability.
The only other major problem is in connection with the precipitation of the NR. If this occurs then the
results will be inaccurate since these crystals are very difficult to wash off. The unincorporated NR will
be included in the overall OD, i.e. the OD will be significantly higher than it should be. However, the
crystals are easily noticeable during the inspection of the cells for the morphological scoring.
Status
Known Laboratory Use:
- Institute for In Vitro Sciences (IIVS), US

- U.S. Army Edgewood Chemical Biological Center (ECBC), US
- FRAME Alternatives Laboratory (FAL), University of Nottingham, Nottingham, UK

- Advanced In Vitro Cell Technologies, S.L.,C/ Baldiri i Reixac,Barcelona, ES
- GAIKER Technology Centre, Zamudio, ES
- Institute for Health and Consumer Protection, Joint Research Centre, Ispra, IT
Participation in Evaluation Studies:

The method has been evaluated in ACuteTox (http://www.acutetox.eu/), an integrated EU FP6 project on the
optimisation and prevalidation of an in-vitro test strategy for predicting human acute oral toxicity (January 2005-June
2010) (Prieto et al. 2012a, Clothier et al. 2008).
Participation in Validation Studies:

1. NICEATM/ECVAM validation study (2002-2006), " In vitro Cytotoxicity Test Methods for Estimating Acute Oral
Systemic Toxicity", addressed the ability of two NRU test method to estimate rodent oral LD 50 values across the GHS
categories and to identify starting doses for in vivo acute oral toxicity test. The study confirmed that none of the test
methods evaluated (3T3 NRU and NHK NRU) was sufficiently accurate as “stand-alone” assay to predict each of the
GHS acute oral toxicity hazard categories. However both methods may be used to determine the starting dose for
acute oral in vivo methods. When compare to NHK NRU, the 3T3 NRU method was judged as less labour intensive
and cheaper to conduct and hence recommended for general use.
A complete documentation of this validation study can be found under:

http://iccvam.niehs.nih.gov/methods/acutetox/inv_nru_announce.htm
2. The EURL ECVAM follow-up validation study (2007 -2011) was conducted to evaluate the predictive capacity of the
3T3 Neutral Red Uptake cytotoxicity assay to correctly identify substances not requiring classification for acute oral
toxicity under the EU CLP system (LD 50 > 2 000 mg/kg) (Prieto et al. , 2012b).
The outcome of the study has been peer-reviewed by ESAC (March 2012). Subsequent EURL ECVAM
recommendation ( http://ihcp.jrc.ec.europa.eu/ ; EURL ECVAM, 2013) concluded that the 3T3 NRU test method
shows a high sensitivity (ca. 95%) and, consequently, a low false negative rate (ca. 5%) when employed in conjunction
with a prediction model to distinguish potentially toxic versus non-toxic (i.e. classified versus non-classified)
substances. Therefore, substances found to be negative in this test would most likely not require classification for
acute oral toxicity under the EU CLP Regulation.
Regulatory Acceptance:
Today the NRU cytotoxicity assay is recognised only as additional test that can be used for estimating the initial doses
for acute oral systemic toxicity tests in vivo.
Based on the results of the NICEATM/ECVAM validation study OECD has adopted a Guidance Document (GD No.
129) describing methods to determine the in vitro basal cytotoxicity of test substances using NRU assays and the use
of the in vitro data to determine starting doses for in vivo acute oral systemic toxicity tests (OECD, 2010).
Health and Safety Issues

General Precautions
When culturing the cells, gloves (non-allergenic latex or vinyl) and laboratory coat must be worn by the operators.
Only sterile equipment must be used in cell handling. Discard all used materials according to the procedure for
special biological waste. During handling of cryogenic vials in liquid nitrogen, a full-face mask and gloves must be
worn. All solutions (except NR stock solution, NR medium and NR desorb), glassware, pipettes, etc., shall be sterile
and all procedures should be carried out under aseptic conditions and in the sterile environment of a laminar flow
cabinet (biological hazard standard). Appropriate environmental controls and safety procedures should be used when
handling toxic or chemical hazardous substances. All methods and procedures must be adequately documented.
MSDS Information
SLS (positive control) CAS#151-21-3; e.g. : http://www.sigmaaldrich.com/catalog/product/fluka/71727
Abbreviations and Definitions
Abbreviations
CDM: Chemical Dilution Medium
DB-ALM: EURL ECVAM's DataBase service on ALternative Methods to animal experimentation
DB-ALM: EURL ECVAM's DataBase service on ALternative Methods to animal experimentation

DMEM: Dulbecco's Modified Eagle Medium
D-PBS: Dulbecco’s Phosphate Buffered Saline
DMSO: Dimethyl sulphoxide
ECVAM: European Centre for the Validation of Alternative Methods. As of 2011, ECVAM has been
established as the European Union Reference Laboratory for the Validation of Alternative Methods.
EURL ECVAM: European Union Reference Laboratory for the Validation of Alternative Methods
(formerly referred to as ECVAM)
HBSS: Hanks’ Balanced Salt Solution
GHS: Globally Harmonised System
GLP: Good Laboratory Practice
IC50: Concentration of the test substance which causes 50% inhibition of the endpoint measured
ICx : geometric average of IC50 determined in several experiments
LD50: median lethal dose of a test substance required to kill half of the animals in a toxicological test
N(B)CS: New Born Calf Serum

NICEATM: National Toxicology Program Interagency Center for the Evaluation of Alternative
Toxicological Methods
NR: Neutral Red dye
NRU: Neutral Red Uptake, equivalent of cell viability in the context of this document.
OD: Optical density
OECD: Organisation for Economic Cooperation and Development
PBS: Phosphate Buffered Saline
PC: positive control
RC: the Registry of Cytotoxicity
SLS: Sodium Lauryl Sulphate
VC: vehicle control
Definitions
Hill function: a four parameter logistic mathematical model relating the concentration of test substance
to the response being measured in a sigmoidal shape.
Y = Bottom +(Top-Bottom)/(1+10(logIC50 X)HillSlope )
Where:
Y = response
X = the logarithm of dose (or concentration)
Bottom = the minimum response
Top = the maximum response
logIC50 = logarithm of X at the response midway between Top and Bottom
Hill Slope describes the steepness of the curve
Soluble: Substance exists in a clear solution without visible cloudiness or precipitate.

Highest soluble concentration: the highest concentration that can be dissolved in one of the defined
solvents, with the mild heating or shaking as defined in the Mechanical Procedures, and that will stay
visibly in a clear solution without visible cloudiness or precipitate for at least 1 hour.
Last update: 4 February 2013
PROCEDURE DETAILS, 28 January 2013

BALB/c 3T3 Neutral Red Uptake Cytotoxicity Assay (3T3 NRU)
DB-ALM Protocol n° 139
The protocol presents the standard operating procedure used in the EURL ECVAM follow-up validation study
(2007-2011): "Assessment of the predictive capacity of the 3T3 Neutral Red Uptake cytotoxicity test method to identify
substances not classified for acute oral toxicity under the EU CLP system CLP system(LD50 > 2000 mg/kg)"
Contact Details
Dr. Richard Clothier
Reader in Cell toxicology and Trustee of FRAME
Fund for the Replacement of Animals in Medical Experiments
FRAME
Clifton Boulevard
Nottingham NG7 2UH
United Kingdom
email: richard.clothier@btinternet.com
Mr. Hans Raabe

Institute for In Vitro Sciences, Inc.
30 W.Watkins Mill Road Suite 100
Gaithersburg 20878
United States
email: hraabe@iivs.org
telephone: +1 (301) 947 6523
General Instructions
Required Training and Expertise

Personnel performing 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, cell growth and measurement of growth curves, viability assays, and storage and
freezing/thawing of cryo-stored cells. Additionally, training is encouraged for special culture procedures such as
primary cell and tissue cultures, toxicity testing, and viability assays. Laboratory personnel should be trained in
the application of GLP requirements, and in the safe storage, handling, and disposal of toxic substances.
Specific Training and Expertise Needed
Personnel performing the in vitro 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; and document observations of cell cultures throughout
all aspects of the procedure.
Perform the in vitro assays by following the protocols to grow the cells, count, transfer, and feed the
cells, conduct techniques in 96-well microtiter plate formats, treat the cells with test substances,
perform application of adhesive plate sealers to culture plates for control of volatile substances,
perform the NRU assay, perform optical density measurements, transfer data to electronic
templates.
Operate standard equipment necessary for maintaining cell culture laboratories (e.g., water baths,
incubators, biohazard hoods, spectrophotometric microtiter plate readers).
General Laboratory Expertise Needed

Personnel should also be able to understand and perform 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 substances for application to cell cultures, follow solubility protocols to adequately
prepare test substances in solution, recognize solubility issues (e.g., insolubility nature of
substance, precipitation), and implement procedures for dissolving the test substances.
Monitor and control laboratory environment (e.g., temperature, humidity, lighting, traffic),
maintain equipment to support cell cultures (e.g., correct CO 2 levels, temperature, humidity, gas
flow, calibrations) and microtiter plate spectrophotometers.
Study Documentation
All methods and procedures should be noted in a Study Workbook; logs should be maintained for general
laboratory procedures and equipment (e.g., media preparation, test substance preparation, solubility testing,
laboratory balance calibration, incubator function); solubility reports should be in electronic and paper format;
all optical density data obtained from the spectrophotometer plate reader should be saved in electronic and
paper formats; all calculations of IC 50 values and other derived data should be in electronic and paper format; all
data need to be archived.
Materials and Preparations
Cells or Test System

BALB/3T3 clone A31, murine fibroblast cell line
CCL-163, LGC Reference Materials, Customer Service, Queens Road, Teddington, Middlesex, TW110LY,
UK
CCL-163, American Type Culture Collection [ATCC], Manassas, VA, USA
Fixed Equipment
1. Incubator: 37ºC ± 1ºC, 90 % ± 5 % humidity, 5.0 % ± 1 % CO 2 /air

2. Laminar flow clean bench/cabinet (standard: "biological hazard")
3. Water bath: 37ºC ± 1ºC
4. Inverse phase contrast microscope
5. Centrifuge (optionally: equipped with microtiter plate rotor)
6. Laboratory balance
7. 96-well plate spectrophotometer (i.e., plate reader) equipped with 540 nm ± 10 nm filter
8. Shaker for microtiter plates
9. Cell counter or haemocytometer
10. Pipetting aid
11. Pipettes, pipettors (multi-channel and single channel; repeater (multichannel) pipette), dilution
block
12. Multichannel reagent reservoir
13. Waterbath sonicator
14. Magnetic stirrer
15. Antistatic bar ionizer/antistatic gun (optional for neutralizing static charge on 96-well plates)
16. Dry heat block (optional)
Consumables
1. Sterile glass tubes with caps (e.g.: 5 mL)

2. Cryotubes
3. Tissue culture flasks (e.g., 75 - 80 cm 2 , 25 cm 2 )
4. 96-well flat bottom tissue culture microtiter plates (e.g., Nunc # 167 008; Falcon tissue
culture-treated)
5. pH paper (wide and narrow range)
6. Adhesive film plate sealers (e.g., Excel Scientific SealPlate™, Cat # STR-SEAL-PLT or equivalent)
7. Filters/filtration devices
NOTE: Tissue culture flasks and microtiter plates should be prescreened to ensure that they
adequately support the growth of 3T3 cells. Multi-channel repeater pipettes may be
used for plating cells in the 96-well plates, dispensing plate rinse solutions, NR
medium, and desorb solution. Do not use the repeater pipette for dispensing test
substances to the cells.
Media, Reagents, Sera, others
1. Dulbecco’s Modification of Eagle’s Medium (DMEM) without L-Glutamine; should have high
glucose [4.5 g/L] and sodium
2. pyruvate (e.g., ICN-Flow Cat. No. 12-332-54)
3. L-Glutamine 200 mM (e.g., ICN-Flow # 16-801-49)
4. New Born Calf Serum (NBCS or NCS) (e.g., Biochrom # SO 125)
NOTE: Due to lot variability of NBCS/NCS, first check a lot for growth stimulating
properties with 3T3 cells (approximately 20-24 h doubling time) and then
reserve a sufficient amount of NBCS/NCS.
5. 0.05 % Trypsin/0.02 % EDTA solution (e.g., SIGMA T 3924, ICN-Flow, # 16891-49)

6. Phosphate buffered saline (PBS) without Ca 2+ and Mg 2+ (for trypsinization)
7. Hanks’ Balanced Salt Solution (HBSS) without Ca 2+ and Mg 2+ (CMF-HBSS)
8. Dulbecco’s Phosphate Buffered Saline (D-PBS) [formulation containing calcium and magnesium
cations; glucose optional] (for rinsing)
9. Penicillin/streptomycin solution (e.g. ICN-Flow # 16-700-49)
10. Neutral Red (NR) Dye – tissue culture-grade; liquid form (e.g., SIGMA N 2889)
11. Dimethyl sulphoxide (DMSO), U.S.P. analytical grade (Store under nitrogen @ -20ºC)
12. Ethanol (ETOH), U.S.P. analytical grade (100 %, non-denatured for test substance preparation; 95 %
can be used for the desorb solution)
13. Glacial acetic acid, analytical grade
14. Distilled H 2 O or any purified water suitable for cell culture and NR desorb solution (sterile)
15. Sterile/non-sterile paper towels (for blotting 96-well plates)
Media and Endpoint Assay Solutions
NOTE: all solutions (except NR stock solution, NR medium and NR desorb), glassware,
pipettes, etc., shall be sterile and all procedures should be carried out under aseptic
conditions and in the sterile environment of a laminar flow cabinet (biological hazard
standard). All methods and procedures must be adequately documented.
1. Media
1. DMEM (buffered with sodium bicarbonate) supplemented with (final concentrations in DMEM are
quoted):
2. Freeze Medium , for cell freezing; contains 2X concentration of NBCS/NCS and DMSO of final
freezing solution.
40 % NBCS/NCS
20 % DMSO
3. Routine Culture Medium - for routine cell culture
10 % NBCS/NCS
4 mM Glutamine
4. Chemical Dilution Medium (CDM) - for test substance dilution
NOTE: The CDM will dilute the serum concentration of the Routine Culture Medium
in the test plate to 5 %. Serum proteins may mask the toxicity of the test
substance, but serum cannot be totally excluded because cell growth is
markedly reduced in its absence
4 mM Glutamine
200 IU/mL Penicillin
200 µg/mL Streptomycin
5. NR Dilution Medium - for dilution of NR stock solution
5 % NBCS/NCS
4 mM Glutamine
100 IU/mL Penicillin
100 µg/mL Streptomycin
Completed media formulations should be stored at 2-8°C for no longer than two weeks.
2. Endpoint Assay Solutions
NR Stock Solution
The liquid tissue culture-grade stock NR Solution will be the first choice for performing
the assay (e.g., SIGMA #N2889, 3.3 mg/mL). Store liquid tissue culture-grade NR Stock
Solution at the storage conditions and respect the shelf-life period recommended by the
manufacturer.
NR Medium ( to be prepared fresh directly before use)

The final concentration of the NR Medium is 25 µg NR dye/mL and aliquots will be
prepared on the day of application.
Example:
0.758 mL (3.3 mg NR dye/mL solution) NR Stock Solution

99.242 mL NR Dilution Medium (pre-warmed to 37° C)
The NR Medium should be filtered (e.g., Millipore filtering, 0.2 – 0.45 µm pore size) to
reduce NR crystals. Keep the aliquots of the NR Medium at 37°C (e.g., in a waterbath)
before adding to the cells and use within 30 min of preparation and no more then 15 min
after removing from 37°C.
Ethanol/Acetic Acid Solution (NR Desorb)
1 % Glacial acetic acid solution
50 % Ethanol
49 % H2O
Test Compounds
NOTE: Preparation under red or yellow light is recommended to preserve substances that
degrade upon exposure to light.
The solvent to be used for each test substance needs to be selected by the testing facility using good
scientific judgment and all ready available information. The preference of solvent for dissolving test
substances is CDM, then DMSO, followed by ethanol. The final concentration of DMSO or ethanol in
the medium in which the cells are incubated, cannot exceed 0.5%.
An example of a routine solubilisation test is described below (see Figure 1 ). Note that starting
concentration here is 20mg/mL. For substances with high molecular weight and/or suspected low
toxicity, higher starting concentration might be needed, if feasible. Other solubility schemes can be
found in OECD Guidance Document (p. 45; OECD, 2010) and in Stokes e t al . (2008).
1. Solubility scheme
If the test substance solubility is not known in advance from the technical documentation, literature or
laboratory practice, it can be determined in a step-wise procedure. The solubility testing starts with an
attempt to dissolve a test substance at high concentration in CDM (e.g. 200mg/mL), followed by a
sequence of mechanical procedures: vortexing, sonicating, stirring and heating, see also instructions in
the subsequent section: 2. Mechanical Procedures. If the substance does not dissolve, the volume of
CDM is increased so as to decrease the concentration by a factor of 10. The sequence of mechanical
procedures is then repeated in an attempt to solubilise the substance at the lower concentration. If this
fails, the same procedure is repeated with an organic solvent (first DMSO and then ethanol). If the test
substance does not dissolve, then solubility at a concentration likely to cause toxicity is not achievable.
1. Tier 1 begins with testing 20 mg/mL of test substance in CDM. Weigh approximately
10 mg (10,000 mg) of the test substance into a glass tube. Document the weight. Add
approximately 0.5 mL of CDM into the respective tube so that the final concentration
is precisely 20,000 mg/mL (20.0 mg/mL). Mix the solution as specified in the
Mechanical Procedures .If complete solubility is achieved, then additional solubility
procedures are not needed.
2. If the test substance is NOT soluble, add CDM up to 5mL and mix the solution as
specified in the Mechanical Procedures. If complete solubility is achieved, then
additional solubility procedures are not needed.
3. If the test substance is NOT soluble in CMD, or in DMSO or ethanol at Tier 2
concentration (20mg/mL), progress to Tier 3 by adding enough solvent to increase the
volume by 10-fold and attempt to solubilise again using the sequence of mixing
procedures as specified in the Mechanical Procedures . If the test substance
dissolves, no additional solubility procedures are necessary.
4. If the test substance does NOT dissolve, then the solvent volume can be increased up
to 50mL and the solution mixed as specified in the Mechanical Procedures .
However, note that at this volume of solvent the concentration likely to cause toxicity
might not be achievable.
Figure 1: Example of a solubilisation scheme for substances tested in the 3T3 NRU assay (used in the
NICEATM/ECVAM validation study)
* organic solvent in the stock solution cannot exceed 0.5% in the medium in which the cells will be
incubated.
** same solvent as in the preceding step
CDM: Chemical Dilution Medium
2. Mechanical Procedures
The following sequence of mixing procedures is recommended to dissolve the test substance:
1. Add test substance to the solvent. Both test substance and solvent should be at room
temperature.
2. Gently mix at room temperature. Vortex the tube (1 –2 minutes).
3. If test substance has not dissolved, use water bath sonication for up to 5 minutes.
4. If test substance is not dissolved after sonication, then warm solution to 37°C for 5 -
60 min. This can be performed by warming tubes in a 37°C water bath or in a CO 2
incubator at 37°C. The solution may be stirred while heated (stirring in a CO 2
incubator will help maintain proper pH).
5. If the substance has not dissolved, proceed to the subsequent Tier.
3. Preparation of test substances
Allow test substances to equilibrate to room temperature before dissolving and diluting.
Prepare test substance solutions immediately prior to use. Avoid bulk preparations for use in
subsequent tests. Ideally, the solutions must not be cloudy nor have noticeable precipitate. Each
stock dilution should have at least 1-2 mL total volume to ensure adequate solution for the test
wells in a single 96-well plate.
For substances dissolved in DMSO or ethanol, the final organic solvent concentration for
application to the cells cannot exceed 0.5% (v/v) in the vehicle controls and in all of the eight test
concentrations (1% in 50 µL of test substance dosing solution and 0% in 50 µL medium on the cells).
The stock solution for each test substance should be prepared at the recommended highest
concentration. The highest test concentration applied to the cells in each range finding experiment
is:
0.5 times the highest soluble concentration in CDM, defined as the concentration that
can be dissolved in one of the defined solvents, with the mild heating or shaking as
defined in the Mechanical Procedures, and that will stay visibly in solution for at least
1 hour.
or
1/200 the highest concentration found to be soluble in the solubility test if the
chemical was soluble only in DMSO or ethanol.
The seven lower concentrations in the range finding experiment are prepared by successive
dilutions that decrease by one log unit each. The following example illustrates the preparation of
test substance in solvent and the dilution of dissolved test substance in CDM before application to
3T3 cells.
Example:
1. Preparation of Test Substance using a Log Dilution Scheme. DMSO was determined to be the
optimal solvent.Dissolve the test substance in DMSO at 2,000 µg/mL to obtain 1mL stock solution
2. Label eight glass tubes #1 – #8. Add 0.9 mL DMSO (solvent) to tubes #2 - #8.
3. Transfer 0.5mL of stock solution of 2,000 µg test substance/mL solvent in tube #1.
4. Add 0.1 mL of 2,000 µg/mL dilution from tube #1 to tube #2 to make a 1:10 dilution in solvent
(i.e., 200 µg/mL).
5. Add 0.1 mL of 200 µg/mL dilution from tube #2 to tube #3 to make another 1:10 dilution (i.e.,
1:100 dilution from stock solution) in solvent (i.e., 20 µg/mL).
6. Continue making serial 1:10 dilutions in the remaining solvent tubes. Each concentration is now
200 fold greater than the concentration to be tested.
7. Make a Dosing Solution by diluting 1 part of dissolved substance in each tube with 99 parts of
CDM (e.g.: 0.1 mL test test substance in DMSO + 9.9 mL CDM), to obtain eight 2X concentrations
ready for application to 3T3 cells. The 3T3 cells will have 0.05 or 0.1 mL Routine Culture Medium in
the wells prior to application of the Dosing Solution. Using 2X concentration enables the solvent to
be diluted to 0.5% v/v.
NOTE: A test substance prepared in DMSO or ethanol may precipitate upon transfer
into the CDM. The 2X Dosing Solutions should be evaluated for precipitates
and the results recorded in the workbook. It will be permissible to test all of
the dosing solutions in the range finding assay and main experiments.
However, concentrations containing test substance precipitates should be
avoided in the IC 50 determinations for the main tests. Precipitates in 2X
dosing solutions are permissible for range finder tests but not acceptable in
definitive tests.
8. Document all test substance preparations in the Study Workbook.

9. The conditions of testing (cell preparation, exposure and endpoint measurement) are the same
as in the definitive test, see Method section for details.
4. Concentrations of Test Substance

4.1 Range Finder Experiment
NOTE: To avoid e.g. the need to repeat a range finder experiment, because no cytotoxicity was
detected, the highest soluble concentration should be considered as a starting point in
the preparation of 2X dosing solutions
Select eight concentrations of the test substance, starting from the highest soluble from the solubility
testing. The initial dilution range should be log dilution series (e.g., 1:1, 1:10, 1:100, 1:1000, etc.).
If a range finding test produces a biphasic curve (e.g. in Figure 2 ), then the concentrations selected for
the subsequent definite experiments should cover the most responsive concentration range (e.g. in
Figure 2, the most sensitive range is 0.001 – 0.1 µg/mL), where evidence of cytotoxicity at the lowest
concentrations tested is observed.
Figure 2. Biphasic curve - Example
4.2. Main Experiment
NOTE: After the range finding assay is completed, the definitive concentration-response
experiment shall be performed at least twice on two different days for each substance
(i.e., one plate per day per substance). During the NICETAM/ECVAM validation study
three acceptable definitive runs were requested in order to evaluate the reproducibility
of the test method
Depending on the slope of the concentration-response curve estimated from the range finder, the
dilution/progression factor in the concentration series of the main experiment should be respectively
smaller that in range finding experiment (e.g., dilution factor of 6 √10 = 1.47 i). A progression factor of 12
√10 =1.21 is regarded the smallest increment achievable.
Cover the relevant concentration range around the IC 50, estimated in the range finding experiment,
(between 0 %< and < 100 % effect) with several points of a graded effect, with a minimum of two points,
one on each side of the estimated IC 50 value, avoiding too many non-cytotoxic and/or 100%-cytotoxic
concentrations. Experiments revealing less than one cytotoxic concentration on each side of the IC 50
value shall be repeated, where possible, with a smaller dilution factor. Each experiment should result
with at least one cytotoxicity value 0 %< and ≤ 50.0 % viability and at least one cytotoxicity value 50.0 %<
and < 100 % viability.
Example :
DMSO was determined to be the optimal solvent. Dose-response curve in the range-finding
experiment had gentle slope and IC 50 estimate is 5 µg/mL,selected dilution factor is 1.47 :
1. Dissolve the test substance in DMSO at 2,000 µg/mL to obtain 2mL stock solution.
2. Label eight glass tubes #1 – #8.
3. Transfer 1.5mL stock solution of 2,000 µg test substance/mL solvent in tube #1.
4. Transfer 1 volume from tube # 1 to tube#2 and add 0.47 volumes of DMSO.
5. After equilibration, transfer 1 volume of this solution to tube #3 and add 0.47 volumes of the
DMSO (e.g.: 1 mL test substance in DMSO + 0.47 mL DMSO).
6. Repeat this 6 more times, until all tubes are full.
7. Make a 2x Dosing Solution by diluting 1 part of dissolved substance in each tube with 99 parts of
CDM (e.g.: 0.1 mL test test substance in DMSO + 9.9 mL CDM).
8. Document all test substance preparations in the Study Workbook.
4.3 Maximum Concentrations to be Tested in the Main Experiments

If minimal or no cytotoxicity was measured in the range-finding assay, a maximum concentration for
the main experiments will be established as follows:
For test substances prepared in CDM, the highest test substance concentration that may be applied
to the cells in the main experiments will be 100 mg/mL. Test substance will be weighed into a glass
tube and the weight will be documented. More stringent solubility procedures may be employed if
needed based on results from the range finder experiment. The highest soluble stock solution will
be used to prepare the 7 additional serial stock dosing solutions.
For test substances prepared in either DMSO or ethanol, the highest test substance concentration
that may be applied to the cells in the main experiments will be either 2.5 mg/mL, or less,
depending upon the maximum solubility in solvent. Weigh the test substance into a glass tube and
document the weight. Add the DMSO and mix the solution using the sequence of mechanical
procedures. If complete solubility is achieved in the solvent, then 7 additional serial stock dosing
solutions may be prepared from the 200X stock. The highest soluble stock solution will be used to
prepare the 7 additional serial stock dosing solutions.
If precipitates are observed in the 2X dilutions, continue with the experiment; make the appropriate
observations and documentation.
4.4. pH of Test Substance Solutions

Prior to or immediately after the application of the test substance to the 96-well plate, measure the pH of
the highest 2X dosing concentration of the test substance (i.e., C1 in the test plate, see Figure 3 ) in
culture medium. Use pH paper (e.g., pH 0 - 14 to estimate and pH 5 – 10 to determine more precise
value) for measurements. The pH paper should be in contact with the solution for approximately one
minute. Document the pH and note the colour of the 2X concentration medium (i.e., in the Excel ®
Study template, available in Downloads section of this protocol). Medium colour for all dosing dilutions
should be noted in the workbooks. Do not adjust the pH.
4.5. Volatility of Test Substances
Highly volatile test substances may generate vapours from the treatment medium during the long
treatment with test substance. These vapours may become re-absorbed into the treatment medium in
adjacent wells. If the test substance is particularly toxic at the concentrations tested, the cross
contamination may be evident as a significant reduction in viability in the vehicle control cultures (i.e.,
Figure 3 - VC1) adjacent to the highest test substance concentrations.
If potential test article volatility is suspected (e.g., for low density liquids) or if the initial range finder
test (non-sealed plate) results show evidence of toxic effects in the control cultures (i.e., > 15 %
difference in viability between VC1 [column 2] and VC2 [column 11]), then use the Plate Sealer Method
with the subsequent test plates:
Plate Sealer Method
1. Prepare test plates and substances as usual according to the Test Material Exposure Procedures.
2. Immediately after the 96-well culture plate has been treated with the suspected volatile substance,
apply the adhesive plate sealer (e.g., using a hand, microplate roller, etc.) directly over the culture
wells. Assure that the sealer adheres to each culture well (well tops should be dry). Place the 96-well
wells. Assure that the sealer adheres to each culture well (well tops should be dry). Place the 96-well
plate cover over the sealed plate and incubate the plate under specified conditions.
NOTE: Do not jam the plate lid over the film. This can cause the sealer to detach from
culture wells. Loose fit of the plate lid is acceptable.
3. At the end of the treatment period, the plate sealer should be carefully removed to avoid spillage.
Continue with the NRU assay.
Positive control
The positive control substance, sodium lauryl sulphate (SLS), is prepared in the same manner as test
substances (See Section Test Compounds).
Negative control
Vehicle control: NRU dilution medium (DMEM containing 5% NBCS, 4 mML Glutamine, 100 IU/mL
Penicillin, 100 µg/mL Streptomycin).
Method
Test System Procurement

BALB/3T3 commercially available cell line (CCL-163) is used as a test system. BALB/C 3T3 cells are
derived from 14 to 17 day old BALB/C mouse embryo. The cells possess properties similar to 3T3
fibroblasts and they exhibit contact inhibition, grow to a high dilution and have a low saturation
density. They are readily transformed by SV40 and murine sarcoma virus.
Receipt of Cryopreserved Cells

Upon receipt of cryopreserved BALB/c 3T3 cells, the vial(s) of cells shall be stored in a liquid nitrogen
freezer until needed.
Thawing Cells
Thaw cells by dipping the ampoules in a water bath at 37°C ± 1ºC. Immediately after thawing remove
the ampoules from the water bath.
1. Resuspend the cells in pre-warmed Routine Culture Medium and transfer into
prewarmed Routine Culture Medium in a tissue-culture flask.
2. Incubate at 37ºC ± 1ºC, 90 % ± 5 % humidity, and 5.0 % ± 1 % CO 2 /air.
3. When the cells have attached to the bottom of the flask (within 4 to 24 h), decant the
supernatant and replace with fresh pre-warmed (37ºC) Routine Culture medium.
Culture as described above.
4. Passage at least three times before using the cells in a cytotoxicity test.
A fresh batch of frozen cells from the stock lot of cells should be thawed out and cultured approximately
every two months or if approaching the 60 subcultures.
Freezing Cells
Procedure required only if current stock of frozen cells is depleted.
1. Stocks of BALB/c 3T3 cells can be stored in sterile, freezing tubes in a liquid nitrogen freezer. DMSO
is used as a cryoprotective agent.
2. Centrifuge trypsinized cells at approximately 200 x g. Ensure there is a cell pellet.
3. Resuspend the cells in cold Routine Culture Medium (half the final freezing volume) so a final
concentration of 1-5x10 6 cells/mL can be attained.
4. Slowly add cold Freeze Medium to the cells so that the solvent will equilibrate across the cell
membranes. Bring the cell suspension to the final freezing volume. The final cell suspension will be
10% DMSO. Aliquot the cell suspension into freezing tubes and fill to 1.8 mL.
5. Place the tubes into an insulated container (e.g., styrofoam trays) which gives a freezing rate of
approximately 1°C/min and place in a freezer (-70 to -80°C) for 24 h.The Test Facility needs to
ensure that the freezing protocol is applicable to the 3T3 cells and that the cells are viable when
removed from cryopreservation.
Place the frozen tubes into liquid nitrogen for storage.
Routine Procedures
Routine Culture
BALB/c 3T3 cells are routinely grown as a monolayer in tissue culture grade flasks (e.g., 75 - 80 cm 2 ) at
37ºC ± 1ºC, 90 % ± 5 % humidity, and 5.0 % ± 1 % CO 2 /air. The cells should be examined on a daily (i.e.,
on workdays) basis under a phase contrast microscope, and any changes in morphology or their
adhesive properties noted in a Study Workbook.
When cells exceed 50% confluence (but less than 80%) they should be removed from the flask by
trypsinization:
1. Decant medium, briefly rinse cultures with 5 mL PBS or Hanks’ BSS (without Ca 2+ , Mg 2+ ) per 25
cm 2 flask (15 mL per 75 cm 2 flask). Wash cells by gentle agitation to remove any remaining serum
that might inhibit the action of the trypsin.
2. Discard the washing solution. Repeat the rinsing procedure and discard the washing solution.
3. Add 1-2 mL trypsin-EDTA solution per 25 cm 2 to the monolayer for a few seconds (e.g., 15-30
seconds).
4. Remove excess trypsin-EDTA solution and incubate the cells at room temperature.
5. After 2-3 minutes, lightly tap the flask to detach the cells into a single cell suspension.
Cell Counting
After detaching the cells, add 0.1-0.2 mL of pre-warmed (37ºC) Routine Culture Medium/cm 2 to the
flask (e.g., 2.5 mL for a 25 cm 2 flask). Disperse the monolayer by gentle trituration. It is important to
obtain a single cell suspension for exact counting. Count a sample of the cell suspension obtained using
a haemocytometer or cell counter (e.g., Coulter counter).
Sub-culture of Cells
After determination of cell number, the culture can be sub-cultured into other flasks or seeded into
96-well microtiter plates. BALB/c 3T3 cells are routinely passaged at suggested cell densities as listed in
Table 2 (approximate doubling time is 20-24 h). The individual laboratories will need to determine and
adjust the final density to achieve appropriate growth rate.
Table 1. Cell density guidelines for sub-culturing
Days in Culture Seeding Density (cells/cm 2 ) Total Cells seeded Total Cells seeded
per 25 cm 2 flask per 75 cm 2 flask
2 16 800 4.2 x 10 5 1.26 x 10 6
3 8 400 2.1 x 10 5 6.3 x 10 5
4 4 200 1.05 x 10 5 3.15 x 10 5
NOTE: It is important that cells have overcome the lag growth phase when they are used for
the test.
Determination of Doubling Times

A cell doubling time procedure to be performed on the initial lot of cells that are used. The doubling
time only needs to be determined if a new lot of cells is used.
1. Establish cells in culture and trypsinize for subculture. Resuspend cells in NR Dilution Medium (5 %
NBCS/NCS). Seed cells at 4200 cells/cm 2 .
2. Seed five sets of cell culture vessels in triplicate (e.g., 15 tissue culture dishes [60mm x 15mm]). Use
appropriate volume of Routine Culture Medium for the culture vessels. Note number of cells placed
into each culture dish. Place dishes into the incubators (37ºC ± 1ºC, 90 % ± 5 % humidity, 5.0 % ± 1
% CO 2 /air).
3. After 4 - 6 hours (use the same initial measurement time for each subsequent doubling time
experiment), remove three culture dishes and trypsinize cells. Count cells using a cell counter or
haemocytometer. Cell viability may be optionally determined by dye exclusion (e.g., Nigrosin). Use
appropriate size exclusion limits if using a Coulter counter. Determine the total number of cells and
document. Repeat sampling at 24 h, 48 h, 72 h, and 96 h post inoculation. Change culture medium
at 72 h or sooner in remaining dishes if indicated by pH drop.
4. Plot cell concentration (per mL of medium) on a log scale against time on a linear scale. Determine
lag time and population doubling time. Additional dishes and time are needed if the entire growth
curve is to be determined (lag phase, log phase, plateau phase).
Test Material Exposure Procedures

Cell preparation
1. Cultured cells that are going to be used in seeding the 96-well plates should be fed fresh medium the day before
subculturing to the plates. On the day of plate seeding, prepare a cell suspension of 2.0 – 3.0x10 4 cells/mL in
Routine Culture Medium. Using a multichannel pipette, dispense 100 µL Routine Culture Medium only into the
peripheral wells of a 96-well tissue culture microtiter plate (see Figure 3: Blanks are in columns 1 and 12, rows A
and H). In the remaining wells, dispense 100 µL of a cell suspension of 2.0 – 3.0x10 4 cells/mL (= 2.0 – 3.0x10 3
cells/well). The seeding density should be noted to ensure that the cells in the control wells are not overgrown
after three days (i.e., 24 h incubation in step b and 48 h exposure to test substances). Prepare one plate per each
substance tested. The order of concentrations given in the Figure 3 matches the Excel Study Template, provided
in the Downloads section of this protocol. The layout of the plate can also be reversed so that lowest
concentrations are on the left and highest on the right side (i.e. C8 to C1 from left to right). In such case the
calculation sheets must be adopted accordingly.
1 2 3 4 5 6 7 8 9 10 11 12
A VCb VCb C 1b C 2b C 3b C 4b C 5b C 6b C 7b C 8b VCb VCb
B VCb VC1 C1 C2 C3 C4 C5 C6 C7 C8 VC2 VCb
C VCb VC1 C1 C2 C3 C4 C5 C6 C7 C8 VC2 VCb
D VCb VC1 C1 C2 C3 C4 C5 C6 C7 C8 VC2 VCb
E VCb VC1 C1 C2 C3 C4 C5 C6 C7 C8 VC2 VCb
F VCb VC1 C1 C2 C3 C4 C5 C6 C7 C8 VC2 VCb
G VCb VC1 C1 C2 C3 C4 C5 C6 C7 C8 VC2 VCb
H VCb VCb C 1b C 2b C 3b C 4b C 5b C 6b C 7b C 8b VCb VCb
Figure 3. 96-well plate configuration for positive control (PC) and test substance assays
VC1 and VC2 = VEHICLE CONTROLS (VC1 is adjacent to the highest and VC2 to the lowest test substance
concentration)
C1 – C8 = Test Substances or PC (SLS) at eight concentrations (C1 = highest, C8 = lowest)
C x b = BLANKS (Test substance or PC, but contain no cells)
VCb = VEHICLE CONTROL BLANK (contain no cells)
2. Incubate cells for 24 ± 2 h (37ºC ± 1ºC, 90 % ± 5 % humidity, 5.0 % ± 1 % CO 2 /air) so that cells form a less
than half (< 50%) confluent monolayer. This incubation period assures cell recovery and adherence and
progression to exponential growth phase.
3. Examine each plate under a phase contrast microscope to assure that cell growth is relatively even across
the microtiter plate. This check is performed to identify experimental and systemic cell seeding errors.
Record observations in the Study Workbook.
Test Procedure
1. 96-Well Plate Configuration
The 3T3 NRU assay for test substances will use the 96-well plate configuration as shown in Figure 3,
which matches the layout of the attached EXCEL® Study Template provided in the Downloads section
of this protocol.
2. Application of Test Substance
For instructions on preparation of dosing solutions, refer to section Test compounds , Paragraph: 3.
Preparation of test substances (p. 16) . One of two optional methods for rapidly applying the 2X dosing
solutions onto the 96-well plates may be utilized.
The first method is to add each of the 2X dosing solutions into labelled, sterile
reservoirs (e.g., Corning/Costar model 4870 sterile polystyrene 50 mL reagent
reservoirs; or Corning/Transtar model 4878 disposable reservoir liners, 8-channel; or
other multichannel reservoirs).
The second method utilizes a “dummy” plate (i.e., an empty sterile 96-well plate)
prepared to hold the dosing solutions immediately prior to treatment of the test plate
(with cells). The test substance and control dosing solutions should be dispensed into
the dummy plate in the same pattern/order as will be applied to the plate containing
cells. More volume than needed for the test plate (i.e. greater than 100 µL/well) should
be in the wells of the dummy plate.
At the time of treatment initiation, a multi-channel micropipettor is used to transfer the 2X dosing
solutions, from the reservoirs or dummy plate, to the appropriate wells on the treatment plate. These
methods will ensure that the dosing solutions can be transferred rapidly to the appropriate wells of the
test plate to initiate treatment times and to minimize the range of treatment initiation times across a
large number of treatment plates, and to prevent “out of order” dosing. Do not use a repeater pipette for
dispensing test substance to the plates.
1. After 24 h ± 2 h incubation of the cells, remove the Routine Culture Medium from the
pre-incubated cells by careful inversion of the plate (i.e., “dump”) over an appropriate
receptacle. Gently blot the plate on a sterile paper towel so that the monolayer is
minimally disrupted. Do not use automatic plate washers or vacuum aspiration.
2. Immediately add 50 or 100 µL of fresh pre-warmed Routine Culture Medium to all of
the wells, including the blanks. Transfer 50 or 100 µL respectively of dosing solution
from the 8-channel reservoir (or dummy plate) to the appropriate wells of the test
plate using a single delivery multi-channel pipettor.
Example:
In the plate layout shown in Figure 3 the VC may be transferred first
(into columns 1, 2, 11, and 12), followed by the test substance dosing
solutions from lowest to highest concentration, so that the same pipette
tips on the multi-channel pipette can be used for the whole plate. The
Vehicle Control blank (VCb) wells (in column 1, column 12, wells A2,
A11, H2 and H11) will receive the Vehicle Control dosing solutions (with
any solvents used). Blanks for wells A3 – A10 and H3 – H10 shall receive
the appropriate test substance solutions for each concentration (e.g.,
wells A3 and H3 receive C1 solution).
3. Incubate cells for 48 h ± 0.5 h (37ºC ± 1ºC, 90 % ± 5 % humidity, and 5.0 % ± 1 % CO 2

/air).
4. Positive Control: For each set of test substance plates used in an assay, a separate
plate of positive control concentration range has to be tested (e.g. in the
NICEATM/ECVAM validation study, the exposure concentration ranged from 9.49
µg/ml to 100 µg/ml). If multiple sets of test substance plates are set up, then clearly
designate the positive control plates for each set. Each set will be an individual entity.
The mean ICx ± two and a half standard deviations (SD) for the SLS acceptable tests
(after the removal of outliers) are the values that will be used as an acceptance
criterion for test sensitivity for the 3T3 NRU assay. This plate will follow the same
schedule and procedures as used for the test substance plates (including appropriate
substance concentrations in the appropriate wells and meeting test acceptance
criteria).
3. Microscopic Evaluation
After at least 46 h treatment, examine each plate under a phase contrast microscope to identify
systematic cell seeding errors and growth characteristics of control and treated cells. Record any
changes in morphology of the cells due to the cytotoxic effects of the test substance. These records form
circumstantial evidence, but are not to be used for any quantitative measure of cytotoxicity .
Undesirable growth characteristics of control cells may indicate experimental error and may be cause
for rejection of the assay. Use the following Visual Observations Codes, Table 3, in the description of cell
culture conditions. Numerical scoring of the cells (see below) should be determined and documented in
the Study Workbook and in the appropriate section of Addendum II of theEXCEL® Study Template
provided in the Downloads section of this protocol.
Table 2. Visual observations codes
Note Code Note Text

1 Normal Cell Morphology
2 Low Level of Cell Toxicity
3 Moderate Level of Cell Toxicity
4 High level of Cell Toxicity
1P Normal Cell Morphology with Precipitate
2P Low Level of Cell Toxicity with Precipitate
3P Moderate Level of Cell Toxicity with
Precipitate
4P High level of Cell Toxicity with Precipitate
5P Unable to View Cells Due to Precipitate
Endpoint Measurement
1. Carefully remove (i.e., “dump”) the medium with test substance and rinse the cells gently with 250
µL pre-warmed D-PBS. Dump the rinsing solution and gently blot excess on paper towels. Add 250
µL NR medium to all wells including the blanks and incubate (37ºC ± 1ºC, 90 % ± 5 % humidity, and
5.0 % ± 1 % CO 2 /air) for 3±0.1 h. Examine the cells briefly during the NR incubation (e.g., between
2 and 3 h) for NR crystal formation. Record observations in the Study Workbook. If excessive NR
crystallization has occurred, the experiment results might have to be rejected.
NOTE: It is vital that the cells are not allowed to dry out as this will give rise to the so-called
“ring of death” appearance at the end of the NRU exposure period. This is where the
cells in a ring around the centre of the well do not appear to take up NR whilst the
cells in the centre and around the periphery do.
2. After incubation, remove the NR medium, and carefully rinse cells with 250 µl prewarmed D-PBS.
3. Decant and gently blot D-PBS from the plate.
4. Add exactly 100 µL NR Desorb (ETOH/acetic acid) solution to all wells, including blanks.
5. Shake microtiter plate rapidly on a microtiter plate shaker for 20 – 45 min to extract NR from the
cells and form a homogeneous solution. Plates should be protected from light by using a cover
during shaking.
6. Plates should be still for at least five minutes after removal from the plate shaker (or orbital mixer).
If any bubbles are observed, make sure that they are ruptured prior to reading the plate. Measure
the absorption (within 60 minutes of adding NR Desorb solution) of the resulting coloured solution
at 540 nm ± 10 nm in a microtiter plate reader (spectrophotometer), using the blanks as a reference.
NOTE: The mean OD value for the plate blanks in the NICEATM/ECVAM validation study
(NIH, 2006) was 0.057 ± 0.043 for 3T3 cells (± 2.5 standard deviations; data from 3 labs;
N = 189). Use this range as a guide for assessment of the blank values.
7. Save raw data in the Excel® Study Template provided.
Acceptance Criteria
Test Acceptance Criteria

All acceptance criteria (i.e., criteria 1, 2, and 3) must be met for a test result to be valid.
1. The PC (SLS) IC x must be within ± two and a half (2.5) standard deviations of the historical mean
established by the test facility, and must meet criteria 2 and 3, and must have an r 2 (coefficient of
determination) value calculated for the Hill model fit (i.e., from PRISM® software) ≥ 0.85.
2. The left and right mean of the VCs do not differ by more than 15% from the mean of all VCs.
3. At least one calculated cytotoxicity value at 0 %< and ≤ 50.0 % viability and at least one calculated
cytotoxicity value at 50.0 %< and < 100 % viability must be present.
Exception: If a test has only one point between 0 and 100 % and the smallest dilution factor (i.e., 1.21)
was used and all other test acceptance criteria were met, then the test will be considered valid.
was used and all other test acceptance criteria were met, then the test will be considered valid.
Stopping Rule for Insoluble Substances: 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 trials, then the substance can be judged as incompatible with the test
NOTE: A corrected mean OD 540 ± 10nm of 0.103 - 0.813 for the VCs (mean ± 2.5 standard
deviations, N = 98, from the NICEATM/ECVAM validation study, (NIH, 2006)) is a
target range but not a test acceptance criterion. However, any VC should be within
range determined from previous VC OD values.
Checks for Systematic Cell Seeding Errors

To check for systematic cell seeding errors, untreated VCs are placed both at the left side (row 2) and the
right side (row 11 for the test plates) of the 96-well plate. Aberrations in the cell monolayer for the VCs
may reflect a volatile and toxic test article present in the assay. If volatility is suspected, then proceed to
section Plate Sealer Method.
Checks for cell seeding errors may also be performed by examining each plate under a phase contrast
microscope to assure that cell quantity is consistent.
Data Analysis
Good scientific judgment should be used for determining “unusable” wells that will be excluded from the data
analysis and explanations for the exclusion of any data from the analysis must be recorded on in the study workbook.
A calculation of cell viability expressed as NRU is made for each concentration of the test substance by using the
mean NRU of the six replicate values (minimum of four acceptable replicate well) per test concentration (blanks will
be subtracted). This value is compared with the mean NRU of all VC values. Relative cell viability is then expressed as
percent of untreated VC. Ideally, the eight concentrations of each chemical tested will span the range of no effect up
to total inhibition of cell viability.
Raw data from the microtiter plate reader can be transferred to the Excel® spreadsheet template provided. The
template will automatically determine cell viability, IC 50 values by linear interpolation, and perform statistical
analyses (including statistical identification of outliers).
The Hill function analysis shall be performed using statistical software (e.g., GraphPad PRISM® 3.0 or greater) and a
template specified to calculate IC20, IC 50, and IC80 values (and the associated confidence limits) for each test substance.
If the Hill function analysis includes IC20,IC 50 and IC80 values, all three can be transferred to the EXCEL® Study
Template provided in the Downloads section of this protocol, which will calculate the concentrations associated
with 50 % viability.
Prediction Model
The following formulas were used to predict LD50 value from the NRU-generated IC 50 values obtained with 3T3 NRU
test method, and are included in the Excel Study Template (for details, please consult section 4 of the BRD (NIH,
2006)):
Validated millimole regression model:

Log LD50 (mmol/kg) = 0.439 log IC 50 (mM) + 0.621
Validated weight regression model:

Log LD50 (mg/kg) = 0.372 log IC50 (µg/mL) + 2.024
The millimole regression model is especially suitable for substances of known molecular weight that are relatively
pure.
The weight regression model is applicable to mixtures, substances whose structures or molecular weights are
unknown, and substances with high impurity ratio.
The validation study concluded that the weight regression model significantly overestimated the toxicity of the high
molecular weight substances (≥400 g/mole vs. <400 g/mole) while the millimole regression did not.
Bibliography
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Automation of an in vitro cytotoxicity assay used to estimate starting doses in acute oral systemic toxicity tests.
Food and Chemical Toxicology 50, 2084-2096
Clemedson, C. (2008)
The European ACuteTox project: a modern integrative in vitro approach to better prediction of acute toxicity.
Clinical Pharmacology and Therapeutics 84, 200-202
Clothier, R., Dierickx, P., Lakhanisky, T., Fabre, M., Betanzos, M., Curren, R., Sjöström, M., Raabe, H., Bourne, N.,
Hernandez, V., et al. (2008)
A database of IC50 values and principal component analysis of results from six basal cytotoxicity assays, for use in
the modelling of the in vivo and in vitro data of the EU ACuteTox project.
Alternatives to Laboratory Animals (ATLA) 36, 503-519
Clothier, R., Gómez-Lechón, M.J., Kinsner-Ovaskainen, A., Kopp-Schneider, A., O’Connor, J.E., Prieto, P., and
Stanzel, S. (2013)
Comparative analysis of eight cytotoxicity assays evaluated within the ACuteTox Project.
Toxicology In Vitro 27(4), 1347-1356
EURL ECVAM (2013)
EURL ECVAM Recommendation on the 3T3 NRU Assay for Supporting the Identification of Substances Not
Requiring Classification for Acute Oral Toxicity. Link to document (last access 20.06.2013)
European Union Reference Laboratory for Alternatives to Animal Testing
NIH (2006)
NIH NICEATM-ICCVAM - Validation Study of In Vitro Cytotoxicity Test Methods - Overview. NIH Publication Nr
07–4518. Link to document (last access 17.01.2013)
National Institutes of Health
Norlén H., Bouhifd M., Bories G., Whelan M., Casado J., Parissis N., Fernando F. and Coecke S. (2007)
Report on 3T3/NRU assay results for the testing period January – April 2007. JRC internal publication.
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OECD Series on Testing and Assessment No. 129: Guidance Document on Using Cytotoxicity Tests To Estimate
Starting Doses for Acute Oral Systemic Toxicity Tests. Link to document (last access 17.01.2013)
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OECD Test Guideline No. 423: Acute Oral toxicity - Acute Toxic Class Method. Link to document (last access
17.01.2013)
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Assessment of the predictive capacity of the 3T3 Neutral Red Uptake cytotoxicity test method to identify substances
not classified for acute oral toxicity (LD50>2000mg/kg): Results of an ECVAM validation study.
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L., Di Consiglio, E., et al. (2013)
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139 - P - BALBc 3T3 Neutral Red Uptake Cytotoxicity Assay

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© ECVAM DB-ALM: Protocol

Objective & Application

TYPE OF TESTING : Screening, Adjunct, Part of a test battery, Reduction,

BALB/c 3T3 mouse fibroblast cell line

sub-cultured at post 50% confluence and before reaching 80%.

Data Analysis/Prediction Model

Validated millimole regression model:

Validated weight regression model:

GHS acute oral toxicity categories % of correct predictions*

* data from NICEATM/ECVAM validation study (NIH, 2006)

Test Compounds and Results Summary

Modifications of the Method

Toxicity – Acute Toxic Class Method (OECD, 2001).

- Institute for In Vitro Sciences (IIVS), US

- FRAME Alternatives Laboratory (FAL), University of Nottingham, Nottingham, UK

Participation in Evaluation Studies:

Participation in Validation Studies:

A complete documentation of this validation study can be found under:

Health and Safety Issues

Abbreviations and Definitions

DB-ALM: EURL ECVAM's DataBase service on ALternative Methods to animal experimentation

ICx : geometric average of IC50 determined in several experiments

N(B)CS: New Born Calf Serum

Y = Bottom +(Top-Bottom)/(1+10(logIC50 X)HillSlope )

Soluble: Substance exists in a clear solution without visible cloudiness or precipitate.

Last update: 4 February 2013

PROCEDURE DETAILS, 28 January 2013

Mr. Hans Raabe

Required Training and Expertise

Work with cryogenic freezing apparatus.

General Laboratory Expertise Needed

flow, calibrations) and microtiter plate spectrophotometers.

Materials and Preparations

Cells or Test System

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

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

Media, Reagents, Sera, others

5. 0.05 % Trypsin/0.02 % EDTA solution (e.g., SIGMA T 3924, ICN-Flow, # 16891-49)

Media and Endpoint Assay Solutions

2. Endpoint Assay Solutions

NR Medium ( to be prepared fresh directly before use)

0.758 mL (3.3 mg NR dye/mL solution) NR Stock Solution

5. If the substance has not dissolved, proceed to the subsequent Tier.

3. Preparation of test substances

8. Document all test substance preparations in the Study Workbook.

4. Concentrations of Test Substance

Figure 2. Biphasic curve - Example

4.2. Main Experiment

4.3 Maximum Concentrations to be Tested in the Main Experiments

4.4. pH of Test Substance Solutions

Test System Procurement

Receipt of Cryopreserved Cells

Place the frozen tubes into liquid nitrogen for storage.

Table 1. Cell density guidelines for sub-culturing

Determination of Doubling Times

curve is to be determined (lag phase, log phase, plateau phase).

Test Material Exposure Procedures

3. Incubate cells for 48 h ± 0.5 h (37ºC ± 1ºC, 90 % ± 5 % humidity, and 5.0 % ± 1 % CO 2

Table 2. Visual observations codes

Note Code Note Text

7. Save raw data in the Excel® Study Template provided.

Test Acceptance Criteria

Checks for Systematic Cell Seeding Errors