Validation of a mouse 3D gastruloid-based embryotoxicity assay in reference to the ICH S5(R3) guideline chemical exposure list

Abstract

There is growing interest in establishing alternative methods in place of conventional animal tests to assess the developmental and reproductive toxicity (DART) of chemicals. Gastruloids are 3D aggregates of pluripotent stem cells that spontaneously exhibit axial elongation morphogenesis similar to gastrulation. They have been explored as in vitro embryogenesis models for developmental and toxicological studies. Here, a mouse gastruloid-based assay was validated for DART assessment in accordance with the ICH S5(R3) guideline, which provides the plasma concentration data of various reference drugs in rodents, specifically C_max and AUC for NOAEL and LOAEL. First, adverse effect concentrations of the reference drugs and their known metabolites on gastruloid development were determined based on morphological impact, namely reduced growth or aberrant elongation. Then, the NOAEL to LOAEL concentration range obtained from the gastruloid assay was compared with that in rodents to examine similarities in sensitivity between the in vitro and in vivo assays for each chemical. For 18 out of the 24 reference drugs that have both NOAEL and LOAEL information in rodents, the sensitivity of the gastruloid assay was comparable to the in vivo assay within an 8-fold concentration margin. For 7 out of the 8 additional reference drugs that have only NOAEL or LOAEL information in rodents, the gastruloid assay was in line with the in vivo data. Altogether, these results support the effectiveness of the gastruloid assay, which may be exploited as a non-animal alternative method for DART assessment.

Keywords: Embryotoxicity; Gastruloid; ICH S5(R3) guideline; New approach methodology (NAM); P19C5; Pluripotent stem cell.

Figure 1.

A. Schematic diagram depicting the experimental procedure to examine the effects of test chemicals on P19C5 3D gastruloids. See text (2.2, 2.3) for details. B. Representative images of gastruloids after 4 days of exposure to cytarabine and fluorouracil at varying concentrations. Scale bar = 500 μm. Note that representative images of gastruloids exposed to other test chemicals are shown in Supplementary Figure S1. C. Schematic diagram describing how the in vitro NOAEL to LOAEL range is compared with the in vivo data and how their similarities are classified as the extent of the match. See text (2.6) for details.

Figure 2.

Morphological impact of test chemicals on P19C5 3D gastruloids. Bar graphs show the relative values of the three morphometric parameters: area, elongation distortion index (EDI), and aspect ratio (AR). For each treatment condition, a total of 45 to 48 gastruloids from three independent sets of experiments are examined. The compiled data are presented as mean ± 95% CI. The keys (top box) describe different shades of bars and symbols in the graphs. An adverse effect (asterisk) is defined as a reduction in the relative area by >20% or a change in the relative EDI or AR by >40%, compared to the corresponding control. The highest concentration with no adverse effect is the no-observed-adverse-effect-level (NOAEL; open triangle), and the lowest concentration with an adverse effect is the lowest-observed-adverseeffect-level (LOAEL; closed triangle).

Figure 2.

Morphological impact of test chemicals on P19C5 3D gastruloids. Bar graphs show the relative values of the three morphometric parameters: area, elongation distortion index (EDI), and aspect ratio (AR). For each treatment condition, a total of 45 to 48 gastruloids from three independent sets of experiments are examined. The compiled data are presented as mean ± 95% CI. The keys (top box) describe different shades of bars and symbols in the graphs. An adverse effect (asterisk) is defined as a reduction in the relative area by >20% or a change in the relative EDI or AR by >40%, compared to the corresponding control. The highest concentration with no adverse effect is the no-observed-adverse-effect-level (NOAEL; open triangle), and the lowest concentration with an adverse effect is the lowest-observed-adverseeffect-level (LOAEL; closed triangle).

Figure 3.

Comparison of the adverse effect concentrations between the gastruloid assay and the in vivo rodent data for the ICH reference drugs that have both NOAEL and LOAEL information. The keys in the top box describe symbols in the graphs. The extent of the match between the in vitro and in vivo values are classified by the number of plus symbols (++++, +++, ++, +), namely, the more plus symbols are the closer match. See text (2.6) for details.

Figure 4.

Comparison of the adverse effect concentrations between the gastruloid assay and the in vivo rodent data for the ICH reference drugs that have only LOAEL or NOAEL information. The keys (top box) describe symbols in the graphs. See text (2.6) for details.