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
|
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. 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 |
EpiSkin™ in vitro human skin model skin corrosivity test |
ICCVAM review and report; recommended in 2002 |
Accepted by U.S. via OECD TG 431 |
OECD TG 431 (2004) |
Via OECD |
7 |
EpiDerm™
in vitro human skin model skin corrosivity test |
ICCVAM review and report; recommended in 2002 |
Accepted by U.S. via OECD TG 431 |
OECD TG 431 (2004) |
Via OECD |
8 |
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 |
9 |
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 |
10 |
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 |
11 |
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 |
12 |
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 |
13 |
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 |
|
14 |
Rabies vaccine, humane endpoints |
ICCVAM agency initiative |
Addressed in 9 CFR 117.4e, CVB Notice No. 04-09 (2004) |
NA |
|
15 |
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 |
16 |
Bovine corneal opacity and permeability 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 |
17 |
Isolated chicken eye 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 |
18 |
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 |
19 |
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 |
20 |
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 |
|
21 |
ELISA test for batch potency testing of Leptospira interrogans serovar
canicola (replacement: antigen quantification) |
ICCVAM agency initiative |
USDA SAM 625 (2008) |
NA |
|
22 |
ELISA test for batch potency testing of Leptospira interrogans serovar
icterohaemorrhagiae (replacement: antigen quantification) |
ICCVAM agency initiative |
USDA SAM 627 (2008) |
NA |
|
23 |
ELISA test for batch potency testing of erysipelas vaccines
(replacement: antigen quantification) |
ICCVAM agency initiative |
USDA SAM 613 (2008) |
NA |
Published in European Pharmacopeia |
24 |
ELISA test for batch potency testing of Leptospira kirschneri serovar
grippotyphosa (replacement: antigen quantification) |
ICCVAM agency initiative |
USDA SAM 626 (2009) |
NA |
|
25 |
Relevance of the target animal safety test for batch safety
testing of vaccines for veterinary use |
ICCVAM coordinated agency consideration |
9 CFR 113.4 |
NA |
Published in European Pharmacopeia |
26 |
ELISA test for batch potency testing of human
tetanus vaccines (refinement: antibody quantification) |
ICCVAM coordinated agency consideration |
21 CFR 610.10; use reviewed on a case-by-case basis
|
NA |
Published in European Pharmacopeia |
27 |
ToBI test for batch potency testing of human tetanus vaccines
(refinement: antibody quantification) |
ICCVAM coordinated agency consideration |
Per 21 CFR 610.10; use reviewed on a case-by-case
basis |
NA |
Published in European Pharmacopeia |
28 |
Human whole blood/interleukin-1β in vitro pyrogen test |
ICCVAM peer review and report; recommended in 2008 |
Accepted by FDA in 2009 |
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 |
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 |
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 |
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 |
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) |
Via OECD |
36 |
EST-1000 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 rLLNA 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; recommendations 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; recommendations 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 harmonized guideline for testing human pharmaceuticals |
Via OECD |
45 |
Avian acute oral toxicity test |
ICCVAM contributed to U.S. OECD test guideline review |
Accepted by U.S. via OECD TG 223 |
OECD TG 223 (2010) |
Via OECD |
46 |
Cytosensor Microphysiometer® test method for eye safety testing |
ICCVAM peer review and report; recommendations in 2010 |
Accepted by U.S. agencies in 2011 |
New OECD test guideline considered by Working Group of National Coordinators in 2011 |
|
47 |
Use of anesthetics, analgesics, and humane endpoints for in vivo ocular safety testing |
ICCVAM peer review and report; recommendations in 2010 |
Accepted by U.S. agencies in 2011 |
OECD TG 405 considered by Working Group of National Coordinators in 2011 |
|
48 |
Cell-based potency assay for stability and potency of botulinum neurotoxin type A products |
ICCVAM workshop in 2006 |
Allergan, Inc., method accepted by FDA in 2011 |
NA |
|
49 |
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 |
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 |
LLNA for potency categorization of skin sensitizers |
ICCVAM peer review and report; recommendations in 2011 |
Accepted by U.S. agencies in 2012 |
GHS (2009) |
Via GHS |
Totals |
51 |
51 |
32 |
41 |
