In Vitro Methods for Assessing Acute Systemic Toxicity
Evaluation of In Vitro Cytotoxicity Test Methods
Validation Study of In Vitro Cytotoxicity Test Methods
In vitro methods that use mammalian cell cultures and various cytotoxicity endpoints have been proposed
as alternatives to in vivo acute oral toxicity tests that use rodents. In vitro cytotoxicity test methods
that measure basal cytotoxicity (general cytotoxicity that affects structures or processes intrinsic to all cell types)
are not currently regarded as suitable replacements for rodent
acute oral toxicity tests. However, some methods have been validated for establishing the starting dose for acute
oral toxicity tests so as to reduce and refine the use of animals for such testing.
The use of in vitro cytotoxicity test methods to reduce animal use in acute toxicity testing
was evaluated at an October 2000 Workshop on In Vitro Methods for Assessing Acute Systemic Toxicity.
A Guidance Document on Using
In Vitro Data to Estimate In Vivo Starting Doses for Acute Toxicity was prepared by ICCVAM with the assistance
of the workshop participants. ICCVAM recommended that near-term validation studies should focus on two
standard basal cytotoxicity assays: one using a human cell system and one using a rodent cell system. The results of the
validation study can be found in the Test Method Evaluation Report.
In February 2008, ICCVAM forwarded recommendations on the use of these two in vitro test methods for estimating
starting doses for acute oral systemic toxicity tests. ICCVAM recommended that these test methods be considered before
using animals for acute oral toxicity testing, and that the methods should be used where determined appropriate. Data
from the test methods should be used in a weight-of-evidence approach for determining starting doses for in vivo
studies. Using these in vitro methods where appropriate is expected to reduce the number of animals required
for each toxicity test. To follow up on this recommendation, NICEATM and ICCVAM will conduct a study to determine
how the two recommended cell culture test methods can be used to set the starting doses for acute toxicity tests of mixtures.
NICEATM and ICCVAM, in partnership with ECVAM (European Centre for the Validation of Alternative Methods) and JaCVAM (Japanese
Center for the Validation of Alternative Methods) sponsored a workshop to explore alternative methods for acute chemical safety testing.
The goals of this February 2008 meeting of international experts in the fields of in vitro and in vivo toxicology and human and veterinary
medicine were to:
- Review the state-of-the-science and identify knowledge gaps (at the whole organism, organ
system, cellular, and/or molecular levels) regarding the key in vivo pathways involved in acute systemic toxicity
- Recommend how these knowledge gaps can be addressed by collecting mechanistic biomarker
data during currently required in vivo safety testing
- Recommend how in vivo key pathway information can be used to develop more predictive
mechanism-based in vitro test systems and to identify biomarkers that may serve as
earlier, more humane endpoints for in vivo test methods
- Recommend how mechanism-based in vitro test systems and earlier, more humane endpoints
can be used to further reduce, refine, and eventually replace animal use for acute systemic
toxicity testing while ensuring the protection of human and animal health
Up-and-Down Procedure
The Up-and-Down Procedure (UDP) is an acute oral toxicity test alternative to the traditional oral
median lethal dose (LD50) test.
For the UDP, one animal is tested at a time. The response of the animal to a test substance determines whether the
next animal receives a higher or lower dose. The Organisation for Economic Co-operation and Development (OECD) UDP
test guideline adopted in 1998 was revised to conform with a new globally harmonized hazard classification scheme and
to include a limit test for nontoxic substances and a primary test for the remaining substances. A peer review panel
sponsored by NICEATM and ICCVAM evaluated the revised UDP in July 2000 and in August 2001.
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