Test whether the genotoxicity observed following administration of a commonly used herbal supplement (reference extract) to mice and rats was potentially related to folate deficiency induced by the supplement.

Use an in vitro micronucleus assay to investigate a possible mechanism of action for a genotoxic effect.

Then, test fifteen herbal extract preparations containing the active (or related) herbal ingredient of interest in TK6 lymphoblastoid cells under both physiological and supraphysiologic folate conditions, with and without metabolic activation.

Administer the reference extract concurrently at a single concentration previously shown to produce a positive response in the assay.


A set of optimization steps had to be solved in sequence before testing could begin:

Formulation of the reference herbal extract to the highest possible concentration

Determination of an optimal low-level folate concentration that cells would tolerate and that would not induce chromosomal damage

Determination of a non-cytotoxic dose range for testing the reference extract in TK6 cells

Determination of the optimal exposure time to allow for chemical-induced folate reduction

The herbal extracts were initially tested at concentrations selected based on a previous test of the reference material, but half of the extracts did not produce the level of cytotoxicity recommended by the OECD guideline; thus, further testing at higher concentrations was required.

The large number of micronucleus tests conducted during the optimization and testing phases, as well as the timing required for conditioning and exposing cells, necessitated careful and flexible scheduling to maintain satisfactory progress.

Some of the extracts were tested at concentrations resulting in formation of a precipitate in the test cultures, raising a concern that the particulate matter may interfere with the assay, artificially elevating the micronucleus counts and/or lowering the cytotoxicity.

TK6 cells needed to be conditioned to the defined medium containing specific folate concentrations for several days prior to performing the assays with the test herbal extracts.

One of the compounds to be tested was a tablet; its composition prevented preparation of formulations containing concentrations of the active ingredient adequate for testing.

A large number of cultures and conditioning media was required for each experiment.

The scientific, scheduling and logistical challenges required excellent teamwork and frequent communication between the study director, client and technical staff.


Adapted a conventional assay to tackle a mechanistic question related to a known genotoxic response, demonstrating agility and flexibility in re-directing routine lab capabilities to meet non-routine client needs.

Optimization steps were completed in a systematic manner to develop an exposure protocol suitable for use to meet the client’s goal.

The optimized protocol was used to test all of the herbal extract preparations (except for the tablet) under physiologic and supraphysiologic folate conditions, with and without metabolic activation; follow-up testing of several extracts at higher concentration ranges was necessary to meet test guideline recommendations.

The work has been presented in posters and talks at several internal, local and national meetings; a manuscript is being written for publication in a peer-reviewed journal.

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