In Vitro Toxicity Screens May Aid 30% Reduction In Test Animals - ICCVAM

Use of in vitro cytotoxicity assays in conjunction with in vivo acute oral toxicity tests may allow for a 30% reduction in the number of test animals needed, according to a report by the Interagency Coordinating Committee on the Validation of Alternative Methods.

ICCVAM is an advisory group to the National Institute of Environmental Health Sciences.

Released Oct. 3, the report expresses ICCVAM's recommendations on in vitro testing for acute toxicity based on the conclusions of experts attending an October 2000 workshop on the topic.

The workshop was organized by ICCVAM and the National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM).

ICCVAM states it "agrees with the workshop report that data from in vitro cytotoxicity assays can be useful as one of the tools...in setting a starting dose for the in vivo assessment of acute oral toxicity."

The report includes data evaluating the influence of the starting dose in the Up-and-Down Procedure (UDP) of evaluating oral toxicity in vivo. "The percentage of animals saved when the starting dose equals the true LD50 value is about 30%...and independent of the dose mortality slope," it notes.

ICCVAM recommends that near-term validation studies should focus on standard cytotoxicity assays involving rodent and human cell systems.

The committee identified the murine BALB/c 3T3 neutral red uptake assay involving mouse cell lines as one "for which data for a number of chemicals support its potential utility for estimating the starting dose."

ICCVAM also suggests federal agencies consider making information about the in vitro approach available.

The murine BALB/c assay is described in a guidance document released by ICCVAM along with the report. The guidance addresses the use of in vitro data to estimate in vivo starting doses. Public comment on the report and guidance will be accepted through Nov. 13.

The report further recommends consideration of validation studies on rat cell lines as "most acute oral toxicity testing is conducted in this species."

In addition, since "one of the aims of toxicity testing is to make predictions of potential toxicity in humans," ICCVAM states, "future validation studies should therefore compare rodent and human in vitro data with one another, with rodent in vivo data and with human in vivo data."

The Environmental Protection Agency and the National Institutes of Environmental Health Sciences are working together to "further characterize the usefulness of in vitro methods for acute toxicity testing," ICCVAM notes.

To assist the effort, the report advocates establishment of an "interagency expert group under ICCVAM to advise on near-term activities such as assay selection, study design and chemical selection."

Use of in vitro screens prior to in vivo testing would further reduce the number of animals used in acute toxicity tests now being supported as alternatives to the traditional LD50. For example, the UDP requires use of as few as six to nine rats, while older LD50 tests typically require 50-200 animals.

At an Aug. 21 public meeting held by NICEATM, a scientific panel agreed on final adjustments to the UDP, clearing the way for its adoption in the U.S. as a replacement for old LD50 tests. A report on the adjustments is scheduled to be released late this month.

The Organization for Economic Cooperation & Development said in late 2000 it would remove old LD50 procedures from its guidelines in favor of tests like the UDP that use fewer animals. Other alternatives, each involving eight to 14 animals, include the Fixed Dose Procedure and the Acute Toxic Class Method, both developed in Europe.

OECD is an international trade group that includes several European countries, Japan and the U.S. Within a year of final OECD approval, the older LD50 method can be replaced by the regulatory agencies of the member countries with less animal-intensive tests. An official, international switch to the new tests is expected in late 2002, according to NIEHS.