Alternatives Approved by Regulators

Alternative methods endorsed by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM), the U.S. Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and/or the Organisation for Economic Co-operation and Development (OECD)

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Toxicity endpoint Replacement or refinement methods Regulatory acceptance of method in the U.S.
ICCVAM / ECVAM / Other OECD
Skin corrosion Integrated approach on testing and assessment (IATA) Via OECD OECD guidance document (GD) 203 published in 2014
In vitro membrane barrier test, Corrositex ICCVAM report in 1999, recommended to U.S. agencies in 1999; ECVAM ESAC statement in 2000 OECD test guideline (TG) 435 published in 2006
Reconstructed human epidermis (RHE) test, EpiSkin ICCVAM report in 2002; ECVAM ESAC statement in 1998 OECD TG 431 published in 2004
RHE test, EpiDerm ICCVAM report in 2002; ECVAM ESAC statement in 2000 OECD TG 431 published in 2004
RHE test, SkinEthic ECVAM ESAC statement in 2006 OECD TG 431 published in 2004
RHE test, epiCS/EST1000 ECVAM ESAC statement in 2009 OECD TG 431 published in 2004
Rat Skin TER ICCVAM report in 2002; ECVAM ESAC statement in 1998 OECD TG 430 published in 2004
Skin irritation Integrated approach on testing and assessment (IATA) Via OECD OECD GD 203 published in 2014
RHE test, EpiSkin ECVAM ESAC statement in 2007 OECD TG 439 published in 2010
RHE test, EpiDerm ECVAM ESAC statements in 2007 and in 2008 OECD TG 439 published in 2010
RHE test, SkinEthic ECVAM ESAC statement in 2008 OECD TG 439 published in 2010
Skin absorption / penetration In vitro diffusion method Via OECD OECD TG 428 published in 2004
Skin sensitization KeratinoSens™ Assay EURL ECVAM recommendation in 2014 OECD DRAFT TG published in 2014
Direct Peptide Reactivity Assay (DPRA) EURL ECVAM recommendation in 2013 OECD DRAFT TG published in 2014
Skin sensitization Human Cell Line Activation Test (h-CLAT) OECD DRAFT TG published in 2014
Adverse Outcome Pathway (AOP) for skin sensitization Via OECD OECD Series on Testing and Assessment 168 published in 2012
Reduced LLNA (rLLNA) ICCVAM report in 2008, recommended to U.S. agencies in 2010; ECVAM ESAC statement in 2007 OECD TG 429 published in 2010 (revised TG)
Murine Local Lymph Node Assay (LLNA) ICCVAM report and recommended to U.S. agencies in 1999; ECVAM ESAC statement in 2000 OECD TG 429 published in 2002
LLNA: BrdU–ELISA, non-radioactive Recommended to U.S. agencies in 2010 OECD TG 442B published in 2010
LLNA: DA, non-radioactive Recommended to U.S. agencies in 2010 OECD TG 442A published in 2010
Eye corrosion Fluorescein leakage test method Via OECD OECD TG 460Published in 2012
Cytosensor microphysiometer (CM) assay Recommended to U.S. agencies in 2010
Reconstituted human cornea-like epithelium (RhCE) test method OECD DRAFT TG published in 2014
Short Time Exposure (STE) in vitro test method OECD DRAFT TG published in 2014
Bovine Corneal  Opacity and Permeability (BCOP) test method ICCVAM report in 2007; recommended to U.S. agencies in 2008 OECD TG 437 published in 2009
Isolated Chicken Eye (ICE) test method ICCVAM report in 2007; recommended to U.S. agencies  in 2008 OECD TG 438 published in 2009
Eye irritation Fluorescein leakage test method Via OECD OECD TG 460Published in 2012
Cytosensor microphysiometer (CM) assay* ECVAM ESAC statement in 2009; ICCVAM report in 2010, recommended to U.S. agencies as a screening test in 2010
Eye irritation RhCE test method OECD DRAFT TG published in 2014
STE in vitro test method OECD DRAFT TG published in 2014
Bovine Corneal  Opacity and Permeability (BCOP) test method* ECVAM ESAC statement in 2007 OECD TG 437 published in 2009
Isolated Chicken Eye (ICE) test method ECVAM ESAC statement in 2007 OECD TG 438 published in 2009
Always use anesthetics, analgesics and humane endpoints in the Draize eye test Recommended to U.S. agencies in 2010
*EPA has proposed an in vitro irritation testing approach for antimicrobial cleaning products employing CM, BCOP and the EpiOcular model. More information is available at the link at the end of this table.
Miscellaneous ocular toxicity Always use anesthetics, analgesics and humane endpoints in the Draize eye test Recommended to U.S. agencies in 2010
Acute toxicity 3T3 NRU cytotoxicity test to identify substances not requiring classification EURL ECVAM recommendation in 2013
3T3 NRU cytotoxicity test to estimate starting doses for oral acute systemic toxicity ICCVAM report in 2008; recommended to US agencies in 2008 OECD GD 129 published in 2010
NHK NRU cytotoxicity test to estimate starting doses for oral acute systemic toxicity ICCVAM report in 2008; recommended to U.S. agencies in 2008 OECD GD 129 published in 2010
Up and Down Procedure (UDP) ICCVAM report in 2001, recommended to U.S. agencies in 2002; ECVAM ESAC statement in 2007 OECD TG 425 published in 1998, revised in 2002 and 2006. TG 401 has been deleted.
Fixed Dose Procedure (FDP) Via OECD; ECVAM ESAC statement in 2007 OECD TG 420 published in 1992 and revised in 2002
Acute toxicity Acute Toxic Class (ATC) method Via OECD; ECVAM ESAC statement in 2007 OECD TG 423 published in 1996, revised in 2002
Inhalation toxicity acute toxic class method Via OECD OECD TG 436 published in 2009
Genotoxicity / mutagenicity In vitro micronucleus test ECVAM ESAC statement in 2006 OECD TG 487 published in 2010
Bacterial reverse mutation test Via OECD OECD TG 471 published in 1997
In vitro mammalian chromosome aberration test Via OECD OECD TG 473 published in 1997
In vitro mammalian cell gene mutation test Via OECD OECD TG 476 published in 1997
Carcinogenicity Cell transformation assay EURL ECVAM recommendation in 2013
In vitro cell transformation assays (CTA) EURL ECVAM recommendation in 2012
Combined chronic toxicity and carcinogenicity studies Via OECD OECD TG 453 published in 2009
Pyrogenicity In vitro monocyte activation tests (MAT) ICCVAM report in 2008; ECVAM ESAC statement in 2006, adopted into European Pharmacopoeia in 2009
Limulus amoebocyte lysate (LAL) test US Pharmacopeia general chapter 85; European Pharmacopoeia general chapter 2.6.14; ICH Annex 14
Hematotoxicity CFU-GM assay ECVAM ESAC statement in 2006
Acute phototoxicity 3T3 NRU Phototoxicity Test ECVAM ESAC statement in 1997 OECD TG 432 published in 2004
Reproductive toxicity Embryonic stem cell test ECVAM ESAC statement in 2001
Micromass embryotoxicity assay ECVAM ESAC statement in 2001
Whole rat embryotoxicity assay ECVAM ESAC statement in 2001
Extended one-generation study Via OECD OECD TG 443 published in 2012
Endocrine disruptor screening Estrogen receptor-alpha transcriptional activation assay Via OECD OECD TG 455 published in 2009
H295R steroidogenesis assay Via OECD OECD TG 456 published in 2011
BG1Luc estrogen receptor method Via OECD OECD TG 457 published in 2012
Aquatic toxicity Fish embryo acute toxicity test Via OECD OECD TG 236 published in 2013
Threshold approach, tiered testing strategy ECVAM ESAC statement in 2006 OECD GD 126 published in 2010
Freshwater alga and cyanobacteria growth inhibition test Via OECD OECD TG 201 published in 2011
Daphnia sp. acute immobilization test Via OECD OECD TG 202 published in 2004
All endpoints Guidance on the grouping of chemicals Via OECD OECD GD 194 published in 2014
Clinical signs as humane endpoints Various ICCVAM and ECVAM recommended methods OECD GD 19 published in 2000

Detailed information on the guidance documents and test methods described in this table can be found at the following sites:

Toxicity endpoint

Replacement or refinement methods

Regulatory acceptance

Biologics testing

In vitro leptospirosis potency assay USDA Supplemental Assay Methods (SAM) 624, 625, 626 and 627
In vitro erysipelas potency assay USDA SAM 612 and 613
In vitro clostridial potency assay USDA draft SAM 220
In vitro tetanus toxoid potency assay USDA SAM 217
In vitro recombinant and monoclonal antibody production methods USDA, NIH and ECVAM endorsement (expanded recombinant antibody information presented on the following pages)
Humane endpoints for vaccine-challenge methods USDA CVB Notice 12-12
Anesthesia for rabies vaccine challenge test
Veterinary TABST Can be waived following demonstration of compliance

Advantages of Recombinant Antibody Use

Non-animal

technology

As outlined in a 1998 ESAC statement, scientifically acceptable in vitro methods are now practicably available for all levels of monoclonal antibody production. Recombinant antibodies derived from synthetic or human antibody libraries are an entirely non-animal technology. This alleviates animal welfare concerns associated with traditional monoclonal antibody production (animal immunizations, euthanasia and ascites hybridoma expansion).

Speed

Once an antibody library is established, a researcher skilled in the art of recombinant antibody production can furnish an antigen-specific antibody suitable for research purposes in as little as 8 weeks.1 This is significantly shorter than the 4 or more months required for “in vitro” hybridoma technology.

Control

Recombinant antibody production gives researchers control over the state of the antigen to which antibodies bind.

Isotype

conversion

Once a desirable antibody fragment is found it can be easily converted into any antibody isotype (e.g. IgA, IgM, IgG, etc.) from any species by adding the appropriate constant domain.2,3

Applications

and other

advantages

Recombinant antibodies obtained from antibody gene libraries can be used in all applications in which traditional mAbs are used (e.g., western blotting, immunohistochemistry, fluorescence-activated cell sorting (FACS), and immunofluorescence).4
Recombinant antibodies from antibody gene libraries require less purified antigen to produce5 and eliminate constraints on the types of antigens that can be used. Antibodies to highly toxic or non-immunogenic antigens can be created using library methods, unlike animal immunization technologies.6 Furthermore, the affinity of antibodies derived from libraries can be increased to levels unobtainable by an animal’s natural immune system.
Recombinant antibodies from antibody gene libraries are also amenable to high-throughput production,7 a fact that makes them attractive to the field of proteomics. Because they are derived from human or synthetic genes, recombinant antibodies do not trigger the intense immunogenic reactions in patients that animal-derived antibodies do,8 making them ideal for clinical use. As one researcher from Tufts University pointed out, “[T]he number of applications in which rAb technology has advantages over mAb technology is becoming too numerous, and the power of these applications too great for many labs to resist.9­”

References

  1. Gawrylewski, A. (2007). Antibodies go recombinant. The Scientist. 21(5), 74-76.
  2. Gao, J., Sidhu, S.S. & Wells J.A. (2009). Two-state selection of conformation-specific antibodies. Proc. Natl. Acad. Sci. U.S.A. 106(9), 3071-3076.
  3. Moutel, S., El Marjou, A., Vielemeyer, O., et al. (2009). A multi-Fc-species system for recombinant antibody production. BMC Biotechnol. 9, 14.
  4. Bradbury, A.R.M. & Marks, J.D. (2004). Antibodies from phage antibody libraries. J. Immunol. Methods. 290(1-2), 29-49.
  5. Hacker, G.W., Goetschel, A.F. & Schwamberger, G. (2005). Conscious production and purchase of reagents for molecular morphology: methodogical, ethical, and legal considerations. In: Hacker GW, Tubbs RR, eds. Molecular morphology in human tissues: techniques and applications. Boca Raton, FL: CRC Press; 253-263.
  6. Maynard, J. & Georgiou, G. (2000). ANTIBODY ENGINEERING. Annu. Rev. Biomed. Eng. 2(1), 339-376.
  7. Schofield, D.J, Pope, A.R., Clementel, V., et al. (2007). Application of phage display to high throughput antibody generation and characterization. Genome Biol. 8(11), R254.
  8. Weiner, L.M. (2006). Fully human therapeutic monoclonal antibodies. J. Immunother. 29(1), 1-9.
  9. Shoemaker, C.B. (2005). When will rAbs replace mAbs in labs? Vet. J. 170(2), 151-152.