ICCVAM was established to facilitate the review and adoption of scientifically valid safety testing methods designed to
protect human health, animal health and the environment while refining, reducing or replacing animal use where feasible.
Below is a list of alternative safety testing methods that are accepted by U.S. and international regulatory authorities.
Appropriate use of these test methods can significantly reduce animal use and improve animal welfare.
ICCVAM has also identified critical research, development, and validation efforts needed to
further advance numerous other alternative methods.
No. |
Alternative Test Method |
ICCVAM and ICCVAM Agency Contributions |
U.S. Regulatory Acceptance/
Endorsement and Applicable Regulations and Guidance |
OECD/Other Adoption |
EU Regulatory Acceptance/ Endorsement |
1 |
Murine local lymph
node assay (LLNA) for skin sensitization |
ICCVAM peer review and report; recommended in 1999 |
Accepted by U.S. agencies in 1999; EPA OPPTS 870.2600 (2003)
and FDA Guidance for Industry: Immunotoxicology Evaluation of Investigational New Drugs (2002) |
OECD TG 429 (2002)
ISO (2002) |
Via OECD |
2 |
Corrositex® in vitro membrane barrier skin corrosivity test |
ICCVAM peer review and report; recommended in 1999 |
Accepted by U.S. agencies in 1999; 49 CFR 173.137 (2011)
|
OECD TG 435 (2006) |
Via OECD |
3 |
Up-and-down procedure for acute oral toxicity |
ICCVAM peer review and report; recommended in 2001 |
Accepted by U.S. agencies in 2003; EPA OPPTS 870.1100 (2002)
|
OECD TG 425 (2001) |
Via OECD |
4 |
Fixed dose procedure for acute oral toxicity |
ICCVAM working group contributed to test guideline development |
Accepted by U.S. via OECD TG 420 |
OECD TG 420 (2001) |
Via OECD |
5 |
Acute toxic class method for acute oral toxicity |
ICCVAM working group contributed to test guideline development |
Accepted by U.S. via OECD TG 423 |
OECD TG 423 (2001) |
Via OECD |
6 |
ELISA test for batch potency testing of human
tetanus vaccines (refinement: antibody quantification) |
ICCVAM agency consideration |
21 CFR 610.10; use reviewed on a case-by-case basis
|
NA |
Published in European Pharmacopeia (2003) |
7 |
ToBI test for batch potency testing of human tetanus vaccines
(refinement: antibody quantification) |
ICCVAM agency consideration |
21 CFR 610.10; use reviewed on a case-by-case
basis |
NA |
Published in European Pharmacopeia (2003) |
8 |
EpiSkin™ in vitro human skin model skin corrosivity test |
ICCVAM review and report; recommended in 2002 |
Accepted by U.S. via OECD TG 431; 49 CFR 173.137 (2011) |
OECD TG 431 (2004) |
Via OECD |
9 |
EpiDerm™
in vitro human skin model skin corrosivity test |
ICCVAM review and report; recommended in 2002 |
Accepted by U.S. via OECD TG 431; 49 CFR 173.137 (2011) |
OECD TG 431 (2004) |
Via OECD |
10 |
SkinEthic™ in vitro human skin model skin corrosivity test |
ICCVAM contributed to U.S. OECD test guideline review |
Accepted by U.S. via OECD TG 431 (meets performance standards 2006) |
OECD TG 431 (2004) |
Via OECD |
11 |
Rat TER in vitro skin corrosivity test |
ICCVAM review and report; recommended in 2002 |
Accepted by U.S. via OECD TG 430 |
OECD TG 430 (2004) |
Via OECD |
12 |
3T3 NRU phototoxicity test for skin photo-irritation |
ICCVAM contributed to U.S. OECD test guideline review |
Accepted by U.S. via OECD TG 432 |
OECD TG 432 (2004) |
Via OECD |
13 |
3T3 NRU phototoxicity test: application to UV filter
chemicals |
ICCVAM contributed to U.S. OECD test guideline review |
Accepted by U.S. via OECD TG 432 |
OECD TG 432 (2004) |
Via OECD |
14 |
In vitro dermal absorption methods |
ICCVAM contributed to U.S. OECD test guideline review, expert consultation
meetings |
Accepted by U.S. via OECD TG 428 |
OECD TG 428 (2004) |
Via OECD |
15 |
Use of humane endpoints in animal testing of biological products |
ICCVAM agency initiative |
Addressed in 9 CFR 117.4e, Center for Veterinary Biologics (CVB) Notice No. 04-09 (2004) |
NA |
|
16 |
Rabies vaccine, humane endpoints |
ICCVAM agency initiative |
Addressed in 9 CFR 117.4e, CVB Notice No. 04-09 (2004) |
NA |
|
17 |
Relevance of the target animal safety test for batch safety
testing of vaccines for veterinary use |
ICCVAM agency consideration |
9 CFR 113.4 provides for authorizing exemptions from standard requirements |
NA |
Published in European Pharmacopeia (2004) |
18 |
Uterotrophic bioassay in rodents: a short-term screening
test for estrogenic properties |
ICCVAM contributed to U.S. OECD test guideline review |
Accepted by U.S. via OECD TG 440; EPA 890.1600 (2009) |
OECD TG 440 (2007) |
Via OECD |
19 |
Bovine corneal opacity and permeability in vitro test method to identify severe eye irritants/corrosives |
ICCVAM review and report; recommended in 2007 |
Accepted by U.S. agencies in 2008 |
OECD TG 437 (2009) |
Via OECD |
20 |
Isolated chicken eye in vitro test method to identify severe eye irritants/corrosives |
ICCVAM review and report; recommended in 2007 |
Accepted by U.S. agencies in 2008 |
OECD TG 438 (2009) |
Via OECD |
21 |
Acute toxicity in vitro starting dose procedure, 3T3 cells |
ICCVAM 2001 workshop report; ICCVAM 2006 peer review and report; recommended in 2008 |
Accepted by U.S. agencies in 2008 |
OECD GD 129 (2010) |
Via OECD |
22 |
Acute toxicity in vitro starting dose procedure, NHK cells |
ICCVAM 2001 workshop report; ICCVAM 2006 peer review and report;
recommended in 2008 |
Accepted by U.S. agencies in 2008 |
OECD GD 129 (2010) |
Via OECD |
23 |
ELISA test for batch potency testing of Leptospira interrogans serovar
pomona (replacement: antigen quantification) |
ICCVAM agency initiative |
USDA Supplemental Assay Method (SAM) 624 (2008) |
NA |
|
24 |
ELISA test for batch potency testing of Leptospira interrogans serovar
canicola (replacement: antigen quantification) |
ICCVAM agency initiative |
USDA SAM 625 (2008) |
NA |
|
25 |
ELISA test for batch potency testing of Leptospira interrogans serovar
icterohaemorrhagiae (replacement: antigen quantification) |
ICCVAM agency initiative |
USDA SAM 627 (2008) |
NA |
|
26 |
ELISA test for batch potency testing of erysipelas vaccines
(replacement: antigen quantification) |
ICCVAM agency initiative |
USDA SAM 613 (2008) |
NA |
Published in European Pharmacopeia |
27 |
ELISA test for batch potency testing of Leptospira kirschneri serovar
grippotyphosa (replacement: antigen quantification) |
ICCVAM agency initiative |
USDA SAM 626 (2009) |
NA |
|
28 |
Human whole blood/interleukin-1β in vitro pyrogen test |
ICCVAM peer review and report; recommended in 2008 |
Accepted by FDA in 2009; use addressed in June 2012 FDA guidance on pyrogen testing |
NA |
Published in European Pharmacopeia |
29 |
Human whole
blood/interleukin-1β in vitro pyrogen test:
application of cryopreserved human whole blood |
ICCVAM peer review and report; recommended in 2008 |
Accepted by FDA in 2009; use addressed in June 2012 FDA guidance on pyrogen testing |
NA |
Published in European Pharmacopeia |
30 |
Human whole blood/interleukin-6 in vitro pyrogen test |
ICCVAM peer review and report; recommended in 2008 |
Accepted by FDA in 2009; use addressed in June 2012 FDA guidance on pyrogen testing |
NA |
Published in European Pharmacopeia |
31 |
Human peripheral blood mononuclear cell/interleukin-6 in vitro pyrogen test |
ICCVAM peer review and report; recommended in 2008 |
Accepted by FDA in 2009; use addressed in June 2012 FDA guidance on pyrogen testing |
NA |
Published in European Pharmacopeia |
32 |
Monocytoid cell line Mono Mac 6/interleukin-6 in vitro pyrogen test |
ICCVAM peer review and report; recommended in 2008 |
Accepted by FDA in 2009; use addressed in June 2012 FDA guidance on pyrogen testing |
NA |
Published in European Pharmacopeia |
33 |
Inhalation toxicity - acute toxic class method |
ICCVAM contributed to U.S. OECD test guideline review |
Accepted by U.S. via OECD TG 436 |
OECD TG 436 (2009) |
Via OECD |
34 |
Hershberger bioassay in rats: a short-term screening assay for (anti) androgenic properties |
ICCVAM contributed to U.S. OECD test guideline review |
Accepted by U.S. via OECD TG 441; EPA OPPTS 890.1400 (2009) |
OECD TG 441 (2009) |
Via OECD |
35 |
Stably transfected human estrogen receptor-α transcriptional activation assay for the detection of estrogenic agonist-activity of chemicals |
ICCVAM contributed to U.S. OECD test guideline review, expert consultation meetings |
Accepted by U.S. via OECD TG 455; EPA OPPTS 890.1300 (2009) |
OECD TG 455 (2009); updated 2012 to include performance standards |
Via OECD |
36 |
EST-1000 in vitro test method for skin corrosivity testing |
ICCVAM contributed to U.S. OECD test guideline review |
Accepted by U.S. via OECD TG 431 (meets performance standards 2009) |
OECD TG 431 (2004) |
Via OECD |
37 |
Updated LLNA protocol (requires 20% fewer animals) |
ICCVAM peer review and report; recommended in 2009 |
Accepted by U.S. agencies in 2010; EPA updated policy on the use of the
LLNA for end-use pesticide products in 2011 |
OECD TG 429 (2010) |
Via OECD |
38 |
Reduced LLNA protocol (requires 40% fewer animals by using only the high dose group) |
ICCVAM peer review and report; recommended in 2009 |
Accepted by U.S. agencies in 2010; EPA adopted the reduced LLNA in 2011 |
OECD TG 429 (2010) |
Via OECD |
39 |
LLNA: DA for skin sensitization testing
(a nonradioisotopic LLNA test method) |
ICCVAM peer review and report; recommended in 2010 |
Accepted by U.S. agencies in 2010 |
OECD TG 442A (2010) |
Via OECD |
40 |
LLNA: BrdU-ELISA for skin sensitization testing
(a nonradioisotopic LLNA test method) |
ICCVAM peer review and report; recommended in 2010 |
Accepted by U.S. agencies in 2010 |
OECD TG 442B (2010) |
Via OECD |
41 |
EpiSkin™ in vitro human skin model skin irritation test |
ICCVAM contributed to U.S. OECD test guideline review |
Accepted by U.S. via OECD TG 439 |
OECD TG 439 (2010) |
Via OECD |
42 |
EpiDerm™
in vitro human skin model skin irritation test |
ICCVAM contributed to U.S. OECD test guideline review |
Accepted by U.S. via OECD TG 439 |
OECD TG 439 (2010) |
Via OECD |
43 |
SkinEthic™ in vitro human skin model skin irritation test |
ICCVAM contributed to U.S. OECD test guideline review |
Accepted by U.S. via OECD TG 439 |
OECD TG 439 (2010) |
Via OECD |
44 |
In vitro mammalian cell micronucleus test |
ICCVAM contributed to U.S. OECD test guideline review |
Accepted by U.S. via OECD TG 487 |
OECD TG 487 (2010); included in 2011 International Conference on
Harmonisation (ICH) harmonized guideline for testing human pharmaceuticals |
Via OECD |
45 |
Avian acute oral toxicity test (reduction of animal use) |
ICCVAM contributed to U.S. OECD test guideline review |
Accepted by U.S. via OECD TG 223 |
OECD TG 223 (2010) |
Via OECD |
46 |
Harmonized guidance for nonclinical safety studies for pharmaceuticals
(reduction of animal use) |
ICCVAM agency initiative |
Accepted by FDA via ICH in 2010 |
ICH Guidance Document M3(R2) |
|
47 |
Cytosensor microphysiometer in vitro test method for eye safety testing |
ICCVAM peer review and report; recommended in 2010 |
Accepted by U.S. agencies in 2011 |
New OECD test guideline under consideration |
|
48 |
Use of anesthetics, analgesics, and humane endpoints for in vivo eye safety testing
(refinement of animal use) |
ICCVAM peer review and report; recommended in 2010 |
Accepted by U.S. agencies in 2011 |
Updated OECD TG 405 (2012) |
Via OECD |
49 |
Cell-based potency assay for stability and potency of botulinum neurotoxin type A products
(replacement and reduction of animal use) |
ICCVAM workshop in 2006 |
Allergan, Inc., method accepted by FDA in 2011 |
NA |
|
50 |
USDA guidelines on master reference qualification and requalification for vaccine potency assays
(reduction of animal use) |
ICCVAM agency initiative |
Addressed in 9 CFR 113.8(d)(2), Veterinary Services Memorandum 800.211 (2011) |
NA |
|
51 |
In vitro H295R steroidogenesis assay |
ICCVAM contributed to U.S. OECD test guideline review |
Accepted by U.S. agencies via OECD TG 456 |
OECD TG 456 (2011) |
Via OECD |
52 |
In vitro post-column oxidation test method for paralytic shellfish toxin detection |
ICATM activity |
(may be used on an interim basis while awaiting implementation) |
Association of Analytical Communities official method (2011);
Canadian Food Inspection Agency (2011) |
|
53 |
Harmonized guidance for preclinical safety evaluation of
biotechnology-derived pharmaceuticals (reduction of animal use) |
ICCVAM agency initiative |
Accepted by FDA via ICH in 2012 |
ICH Guidance Document S6(R1) |
|
54 |
LLNA for potency categorization of skin sensitizers
(refinement and reduction of animal use) |
ICCVAM peer review and report; recommendations in 2011 |
Accepted by U.S. agencies in 2012 |
GHS (2009) |
Via GHS |
55 |
USDA guidelines on
use of humane endpoints and methods in animal testing of biological products (refinement of animal use) |
ICCVAM agency initiative |
Addressed in 9 CFR 117.4(e), CVB Notice No. 12-12 (2012) |
NA |
|
56 |
Serum
neutralization test for potency testing of inactivated veterinary rabies vaccines
(reduction and refinement of animal use) |
ICCVAM workshop in 2011 |
NA |
NA |
Published in European Pharmacopoeia Monograph 0451 (2012) |
57 |
Alternative test procedure for tuberculin, PPD Bovis, intradermic (reduces animal use by 65%) |
ICCVAM agency initiative |
Applies to 9 CFR 113.409(c), described in Veterinary
Services Memorandum 800.114 (2012) |
NA |
|
58 |
Histamine sensitization test for acellular pertussis vaccines based on
temperature measurement (refinement of animal use) |
ICCVAM workshop in 2010 |
NA |
WHO Technical Service Report 878 (update adopted 2012) |
|
59 |
Waiving or bridging of mammalian acute toxicity tests for pesticides and
pesticide products (reduction of animal use) |
ICCVAM agency initiative |
EPA guidance (2012) provides for authorizing exemptions from
standard requirements under 40 CFR 158.500, 40 CFR 161.340, other regulations and notices |
NA |
|
60 |
In vitro BG1Luc ER TA agonist assay to identify substances that induce human ER activity |
ICCVAM peer review and report; recommendations in 2012 |
Accepted by U.S. agencies in 2012 |
OECD TG 457 (2012) |
Via OECD |
61 |
In vitro BG1Luc ER TA antagonist assay to identify substances that inhibit human ER activity |
ICCVAM peer review and report; recommendations in 2012 |
Accepted by U.S. agencies in 2012 |
OECD TG 457 (2012) |
Via OECD |
62 |
In vitro fluorescein leakage test method for
identifying ocular corrosives and severe irritants |
ICCVAM contributed to OECD test guideline review |
Accepted by U.S. agencies via OECD TG 457 |
OECD TG 460 (2012) |
Via OECD |
63 |
Extended one-generation reproductive
toxicity study (reduction of animal use) |
ICCVAM agencies contributed to OECD test guideline review |
Accepted by U.S. agencies via OECD TG 443 |
OECD TG 443 (2012) |
Via OECD |
Totals |
63 |
60 |
41 |
47 |
