Neutral Red Uptake Cytotoxicity Tests for Estimating Starting Doses for Acute Oral Toxicity Tests

William S. Stokes1, Silvia Casati2, Judy Strickland3, Michael Paris3

1 The National Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM), Research Triangle Park, North Carolina, 2 European Centre for the Validation of Alternative Methods (ECVAM), Ispra, Italy, 3 Integrated Laboratory Systems (ILS), Research Triangle Park, North Carolina
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 20.4
DOI:  10.1002/0471140856.tx2004s36
Online Posting Date:  May, 2008
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Abstract

In vitro cytotoxicity assays can be used as alternative toxicity tests to reduce the total number of animals needed for acute oral toxicity tests. This unit describes two methods for determining the in vitro cytotoxicity of test substances using neutral red uptake (NRU) and using the in vitro data to determine starting doses for in vivo acute oral systemic toxicity tests, e.g., the up‐and‐down procedure or the acute toxic class method. The use of the NRU methods to determine starting doses for acute oral toxicity tests may reduce the number of animals required, and for relatively toxic substances, this approach may also reduce the number of animals that die or require humane euthanasia due to severe toxicity. An interlaboratory validation study has demonstrated that the methods are useful and reproducible for these purposes. Two standardized protocols provide details for performing NRU tests with rodent and human cells. Curr. Protoc. Toxicol. 36:20.4.1‐20.4.20. © 2008 by John Wiley & Sons, Inc.

Keywords: in vitro; cytotoxicity; alternative; neutral red; 3T3; keratinocyte

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: BALB/C 3T3 Neutral Red Uptake (NRU) Cytotoxicity Test
  • Alternate Protocol 1: Normal Human Epidermal Keratinocyte (NHK) Neutral Red Uptake (NRU) Cytotoxicity Test
  • Support Protocol 1: Preparation of Test Substances
  • Support Protocol 2: Solubility Determination of Test Substances
  • Support Protocol 3: Prequalification of Normal Human Epidermal Keratinocyte (NHK) Growth Medium
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: BALB/C 3T3 Neutral Red Uptake (NRU) Cytotoxicity Test

  Materials
  • BALB/c 3T3 mouse fibroblast cells, clone A31 (e.g., ATCC #CCL‐163; passage ∼64; see ICCVAM; )
  • 3T3 routine culture medium (see recipe), prewarmed to 37°C ± 1°C
  • Phosphate‐buffered saline (without Ca2+ and Mg2+; PBS; appendix 2A) or Hanks' balanced salt solution without Ca2+ and Mg2+(CMF‐HBSS; appendix 2A; also commercially available)
  • 0.05% trypsin/0.02% EDTA solution (e.g., Sigma or ICN‐Flow)
  • Solutions (see protocol 3) for test substances (toxicants) and SDS positive control (e.g., >99% purity, Sigma‐Aldrich)
  • 3T3 chemical dilution medium (see recipe)
  • Dulbecco's phosphate‐buffered saline (DPBS; commercially available), glucose optional, prewarmed to 37°C ± 1°C
  • 3T3 NR medium (see recipe)
  • NR desorb solution (see recipe)
  • 75‐ to 80‐cm2 and 25‐cm2 tissue culture flasks
  • Inverted phase‐contrast microscope
  • Coulter counter or hemacytometer
  • Multichannel pipettors
  • Multichannel repeat pipettor
  • 96‐well flat bottom tissue culture microtiter plates (noncoated, tissue culture treated)
  • Adhesive film plate sealers (e.g., Excel Scientific SealPlate), optional
  • Paper towels, sterile
  • Plate shaker/mixer
  • Microtiter plate reader (spectrophotometer): 540 nm ± 10 nm filter with maximum absorbance of 3.0

Alternate Protocol 1: Normal Human Epidermal Keratinocyte (NHK) Neutral Red Uptake (NRU) Cytotoxicity Test

  • NHK cells: nontransformed cells from cryopreserved primary or secondary pooled neonatal foreskin cells (e.g., Clonetics CC‐2507)
  • NHK routine culture medium (see recipe)
  • HEPES‐buffered saline solution (HEPES‐BSS; e.g., Clonetics)
  • 0.025% trypsin/0.02% EDTA solution (e.g., Clonetics)
  • Trypsin neutralizing solution (TNS; e.g., Clonetics # CC‐5002), room temperature
  • Hanks’ balanced salt solution without Ca2+ and Mg2+ (CMF‐HBSS; appendix 2A; also commercially available)
  • NHK NR medium (see recipe)

Support Protocol 1: Preparation of Test Substances

  Materials
  • Test substances (toxicants) and SDS positive control (e.g., >99% purity, Sigma‐Aldrich)
  • 3T3 chemical dilution medium or NHK routine culture medium (depending on cells used; also see protocol 4)
  • Dimethyl sulfoxide (DMSO; USP analytical grade; store under nitrogen at −20°C) or 100% nondenatured ethanol (USP analytical grade): also see protocol 4
  • 5‐ml glass tubes with caps, sterile

Support Protocol 2: Solubility Determination of Test Substances

  Materials
  • Test substance
  • Chemical dilution medium or NHK routine culture medium (see recipes)
  • Dimethyl sulfoxide (DMSO; USP analytical grade; store under nitrogen at −20°C)
  • 100% nondenatured ethanol (USP analytical grade)
  • 5‐ml glass tubes with caps, sterile
  • 50‐ml tubes (glass or plastic) with caps, sterile
  • Bath sonicator
  • 37°C water bath or cell culture incubator
  • Inverted phase contrast microscope

Support Protocol 3: Prequalification of Normal Human Epidermal Keratinocyte (NHK) Growth Medium

  • NHK cells: nontransformed cells from cryopreserved primary or secondary pooled neonatal foreskin cells (e.g., Clonetics CC‐2507)
  • NHK routine culture medium (see recipe)
  • HEPES‐buffered saline solution (HEPES‐BSS; e.g., Clonetics)
  • 0.025% trypsin/0.02% EDTA solution (e.g., Clonetics)
  • Trypsin neutralizing solution (TNS; e.g., Clonetics), room temperature
  • Hanks' balanced salt solution without Ca2+ and Mg2+ (CMF‐HBSS; appendix 2A; also commercially available)
  • NHK NR medium (see recipe)
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Figures

Videos

Literature Cited

   Borenfreund, E. and Puerner, J.A. 1985. Toxicity determination in vitro by morphological alterations and neutral red absorption. Toxicol. Lett. 24: 119‐124.
   Halle, W. 1998. Toxizitätsprüfungen in Zellkulturen für eine Vorhersage der akuten Toxizität (LD50) zur Einsparung von Tierversuchen. Life Sciences/Lebenswissenschaften. Forschungszentrum Jülich, Jülich, Germany.
   Halle, W. 2003. The Registry of Cytotoxicity: Toxicity testing in cell cultures to predict acute toxicity (LD50) and to reduce testing in animals. Altern. Lab. Anim. 31: 89‐198. [translation from Halle, ].
   ICCVAM. 2001. Report of the International Workshop on In Vitro Methods for Assessing Acute Systemic Toxicity. NIH Publication No. 01‐4499. National Institute for Environmental Health Sciences, Research Triangle Park, N.C.
   ICCVAM. 2006a. Background Review Document: In Vitro Basal Cytotoxicity Test Methods for Estimating Acute Oral Systemic Toxicity. National Institute for Environmental Health Sciences, Research Triangle Park, N.C.
   ICCVAM. 2006b. ICCVAM Test Method Evaluation Report: In Vitro Cytotoxicity Test Methods for Estimating Starting Doses for Acute Oral Systemic Toxicity Tests. National Institute for Environmental Health Sciences, Research Triangle Park, N.C.
   OECD. 2001a. Guideline for Testing of Chemicals, 425. Acute Oral Toxicity—Up‐and‐Down Procedure. Organisation for Economic Co‐operation and Development, Paris.
   OECD. 2001b. Guideline For Testing of Chemicals, 423. Acute Oral Toxicity—Acute Toxic Class Method. Organisation for Economic Co‐operation and Development, Paris.
   Seibert, H., Balls, M., Fentem, J.H., Bianchi, V., Clothier, R.H., Dierickx, P.J., Ekwall, B., Garle, J.J., Gómez‐Lechón, M.J., Gribaldo, L., Gülden, M., Liebsch, M., Rasmussen, E., Roguet, R., Shrivastava, R., and Walum, E. 1996. Acute toxicity testing in vitro and the classification and labelling of chemicals. The report and recommendations of ECVAM Workshop 16. Altern. Lab. Anim. 24: 499‐510.
   Spielmann, H., Genschow, E., Liebsch, M., and Halle, W. 1999. Determination of the starting dose for acute oral toxicity (LD50) testing in the up‐and‐down procedure (UDP) from cytotoxicity data. Altern. Lab Anim. 27: 957‐966.
   UNECE 2005. Globally Harmonized System of Classification and Labelling of Chemicals (GHS), First Revised Edition. United Nations Economic Commission for Europe, New York and Geneva [http://www.unece.org/trans/danger/publi/ghs/ghs_rev01/01files_e.html].
Key Reference
   ICCVAM. 2006b. See above.
  This reference provides performance characteristics and recommended uses for the 3T3 and NHK NRU test methods.
Internet Resource
   http://iccvam.niehs.nih.gov/methods/acutetox/acutetox.htm
  This Web site has the most recent information about the use of the NRU methods for determining starting doses for acute oral toxicity test methods, as well as templates for NRU data collection and analysis.
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