The inhibition of adipogenesis via an in vitro assay can reduce animal use by more precisely estimating the starting dose for the acute toxic class method.

In the present work, we established an adipogenesis inhibition assay as an adequate and sensitive in vitro model for reducing animal use by estimating the starting dose for the acute toxic class (ATC) method. First, human adipose-derived stem cells (ADSCs) underwent adipogenic differentiation induction for 14 days. Then, by high-content imaging analysis, we determined the percentage and area of cell differentiation that we considered suitable for negative and positive internal control according to the quality control criteria strictly standardized mean difference (SSMD) and robust SSMD. Moreover, we established sodium dodecyl sulfate (SDS) as an external positive control in this assay. To measure reduction in animal use to estimate the starting dose for the ATC method, we evaluated 10 chemicals representing Globally Harmonized System of Classification and Labeling of Chemicals (GHS) toxicity categories 1-5 and unclassified toxicity and determined the dose-response curves for percentage and area of cell differentiation by using the Hill function with an R2 ≥ 0.85. The resulting IC50 values were used for LD50 prediction and for estimating the starting dose for the ATC method. Our results indicated that use of the inhibition of adipogenesis assay to estimate the starting dose for the ATC method would decrease animal use for 7 out of 10 tested substances, possibly all substances if we consider the more toxic test substances in GHS categories 1, 2, and 3. We can conclude that the present assay is a suitable alternative to reduce animal testing in the first steps of predicting highly toxic substances. Moreover, this method also presents internal and external controls as differentials, which guarantee the quality of the assay as well as the results. These features are important for suggesting a methodology for regulatory purposes.

The use of human adipose-derived stem cells based cytotoxicity assay for acute toxicity test.

Human adipose-derived stem cells (ADSC) were evaluated as cell culture model for cytotoxicity assay and toxicity prediction by using the neutral red uptake assay (NRU). In this study, we compared ADSC and the murine cell line BALB/c 3T3 clone A31 to predict the toxicity of 12 reference substances as recommended by the Interagency Coordinating Committee on the Validation of Alternative Methods. We predicted the LD50 for RC-rat-only weight and RC-rat-only millimole regressions for both cell culture models. For RC rat-only weight regression, both cells had the same accordance (50%), while for RC rat-only millimole regression, the accordance was 50% for ADSC and 42% for 3T3s. Thus, ADSC have similar capability for GHS class prediction as the 3T3 cell line for the evaluated reference substances. Therefore, ADSCs showed the potential to be considered a novel model for use in evaluating cytotoxicity in drug development and industry as well as for regulatory purposes to reduce or replace the use of laboratory animals with acceptable sensitivity for toxicity prediction in humans. These cells can be used to complete the results from other models, mainly because of its human origin. Moreover, it is less expensive in comparison with other existing models.