# High-Throughput Testing for Unknown Mutagens and Cytotoxica via Duplex Planar Ames–Cytotoxicity Bioassay Including Metabolic S9 Activation

## TL;DR

A new high-throughput bioassay detects unknown mutagens and cytotoxic compounds in complex samples, improving safety testing for products like cosmetics and teas.

## Contribution

A novel duplex planar Ames–cytotoxicity bioassay with metabolic activation for sensitive, selective, and high-throughput detection of unknown mutagens.

## Key findings

- The new method reduces time to results by 5-fold and manual work by 330-fold compared to traditional methods.
- Previously unknown mutagens were detected in complex samples like skin care products at levels exceeding safe thresholds.
- The method uses open-source tools and sustainable materials for global application in safety and risk assessments.

## Abstract

Current nontarget effect-directed analysis of complex
samples for
mutagens is hampered by matrix effects, associated cytotoxicity, diffusion
effects, insufficient sensitivity, and a lack of selectivity. Non-target
analysis may overlook highly potent, unknown mutagens at trace levels.
To overcome these limitations, a duplex planar Ames mutagenicity–cytotoxicity
bioassay was developed to sensitively and selectively detect individual
mutagens and cytotoxic compounds with or without metabolic activation.
Key innovations included high-throughput testing of samples, either
directly or as raw extracts, separated in parallel by planar chromatography,
substance zone fixation to prevent diffusion during long incubation
times, integration of the human versus rat liver S9 enzyme systems
for metabolic de/activation, and use of a tetrazolium salt substrate
that provides a dual end-point read-out. Compared to the state of
the art, time to result was reduced 5-fold, manual work was reduced
330-fold, and costs for nearly plastic-free consumables were reduced
651-fold. The selective, sensitive, and quantitative unmasking of
previously unknown mutagens and cytotoxica was shown for highly complex
samples, such as teas, cosmetics, skin care creams, and perfumes.
An exemplary daily exposure to 11.5 g of skin care products exceeded
the half-maximal effective mutagenicity dose by at least 4 orders
of magnitude. Using the open-source and sustainable 2LabsToGo-Eco,
the new duplex planar bioassay method can be applied worldwide to
serve as a valuable tool for hazard minimization and to support regulatory
safety and risk assessments, industrial quality control, and drug
development.