ICCVAM Advisory Committee Addresses In Vitro Cytotoxicity Methods

The Scientific Advisory Committee on Alternative Toxicological Methods recommends additional assays be identified for inclusion in a battery testing approach for determining in vivo acute toxicity starting doses.

A battery testing approach, which would comprise a series of assays including the 3T3 mouse fibroblast test and the normal human keratinocyte method, would be more "predictive" for estimating starting doses in acute oral in vivo tests than using the 3T3 and NHK tests alone, SACATM noted.

An advisory committee to the Interagency Coordinating Committee on the Validation of Alternative Methods, SACATM discussed the approach during an Aug. 3 teleconference, during which it reviewed a report of an ICCVAM peer review panel on 3T3 and NHK.

The peer review panel had concluded in its report that the in vitro cytotoxicity test methods are sufficient for use in a weight-of-evidence approach in determining starting doses of in vivo toxicity tests. The panel reached its conclusion after reviewing a validation test and draft recommendations of ICCVAM during a May 23 meeting (¹ (Also see "ICCVAM Peer Review Meeting Evaluates Two In Vitro Cytotoxicity Tests" - HBW Insight, 29 May, 2006.), p. 8).

3T3 and NHK set starting doses by estimating rodent acute oral toxicity LD50 values, therefore reducing the number of animals used in the tests by 10%-20%.

While noting 3T3 and NHK are a significant improvement on current practices, SACATM acknowledged that alone they do not take into account other critical aspects of in vivo acute toxicity.

Raymond Tice, deputy director of the National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods, concurred with the committee on that point, but noted NICEATM is looking for other sufficient assays.

"We really looked at [the 3T3 and NHK tests] as generating very good basal cytotoxicity data that can then be built upon," he said.

"I don't think there was any expectation that if all we did was focus on basal cytotoxicity and not take into account either target organ specificity or metabolism or some of the other components associated with in vivo acute toxicity that this assay would be highly predictive of that particular outcome."

"I think that we're looking at a particular component of cytotoxicity, and it by far doesn't measure all the things one needs to measure in order to accurately predict in vivo acute lethality," Tice added.

To that end, he noted, NICEATM is "looking for additional assays that can be added as a battery approach that ultimately perhaps will replace the acute toxicity assay."

NICETAM Director and ICCVAM Executive Director William Stokes, DVM, noted the scientific community is pushing for support of the ADME-Tox in vitro test and specific target organ cytotoxicity tests.

The ADME-Tox in vitro test provides information on the bioavailability of a substance within a particular organ, tissue or cell to indicate the concentration at which a substance becomes cytotoxic.

Specific target organ testing uses cultures created from highly-differentiated cells, such as blood or nerve cells, to study the effects of a toxic substance.

Although SACATM members present at the meeting reached a consensus in support of the peer review panel's recommendations, the committee's final approval of the report will be given at a later date because too few committee members participated in the conference call to constitute a quorum.

In addition to approving the report, SACATM provided suggestions on improving the process. Committee Chair Daniel Acosta, PhD, noted there is a lack of "immediate access" to toxicity data in the private sector. "In the literature there has to be a way of having better access and better information," he said.

Acosta added there is frustration in the scientific community due to delays in moving forward with improvements on in vivo methods. He questioned whether there is too much reliance on the LD50 data and whether it is time to explore new paradigms.

ICCVAM will take SACATM's comments, in addition to the peer review conclusions, into account as it develops its final draft recommendations, which will be distributed to federal agencies for consideration.

- Molly Laas