The US Food and Drug Administration (FDA) has premarket review authority over food additives, but a food manufacturer may, according to the legislation, intentionally add a substance to human food or animal food without their
premarket review or approval if the substance is generally recognized, among qualified experts, to be safe under the conditions of its intended use. Generally recognized as safe (GRAS) implies that the current scientific community
agrees on the adequacy of how data is generated. This system has come under public pressure because of doubts as to its efficiency and the FDA’s recent GRAS rule is part of the response. The FDA guidance for testing food additives,
known as the “Redbook”, is about two decades old. Work toward a new “Redbook” is on the way, but the US Grocery Manufacturer Association (GMA) also has initiated the development of an independent standard on how to perform
GRAS determinations.
This review of the current guidance shows a very rigorous system for higher concern levels, but also many waiving options. Opportunities and challenges for safety evaluations of food additives are discussed. Where scientific progress
has allowed improving existing and adapting new methods, these should be adopted to improve product safety and animal welfare. The continuous adaptation of such improved methods is therefore needed. Especially, there are opportunities
to embrace developments within the toxicity testing for the 21st century movement and evidence-based toxicology approaches. Also, the growing understanding of the limitations of traditional tests needs to be considered.

Private consumers and professionals may experience acute inhalation toxicity after inhaling aerosolized impregnation products. The distinction between toxic and non-toxic products is difficult to make for producers and product users alike, as there is no clearly described relationship between the chemical composition of the products and induction of toxicity. The currently accepted method for determination of acute inhalation toxicity is based on experiments on animals; it is time-consuming, expensive and causes stress for the animals. Impregnation products are present on the market in large numbers and amounts and exhibit great variety. Therefore, an alternative method to screen for acute inhalation toxicity is needed. The aim of our study was to determine if inhibition of lung surfactant by impregnation products in vitro could accurately predict toxicity in vivo in mice. We tested 21 impregnation products using the constant flow through set-up of the constrained drop surfactometer to determine if the products inhibited surfactant function or not. The same products were tested in a mouse inhalation bioassay to determine their toxicity in vivo. The sensitivity was 100%, i.e., the in vitro method predicted all the products that were toxic for mice to inhale. The specificity of the in vitro test was 63%, i.e., the in vitro method found three false positives in the 21 tested products. Six of the products had been involved in accidental human inhalation where they caused acute inhalation toxicity. All of these six products inhibited lung surfactant function in vitro and were toxic to mice.

The maximal chemical concentration that causes an acceptably small or no effect in an organism or isolated cells is an often-sought-after value in toxicology. Existing approaches to derive this value have raised several concerns; thus, it is often chosen case-by-case based on personal experience. To overcome this ambiguity, we propose an approach for choosing the non-toxic concentration (NtC) of a chemical in a rational, tractable way. We developed an algorithm that identifies the highest chemical concentration that causes no more than 10% effect (≤ EC10) including the modeled 95% confidence intervals and considering each of the measured biological replicates; and whose toxicity is not significantly different from no effect. The developed algorithm was validated in two steps: by comparing its results with measured and modeled data for 91 dose-response experiments with fish cell lines and/or zebrafish embryos; and by measuring actual effects caused by NtCs in a separate set of experiments using a fish cell line and zebrafish embryos. The algorithm provided an NtC that is more protective than NOEC (no-observed-effect-concentration), NEC (modeled no-effect concentration), EC10 and BMD (benchmark dose). Despite focusing on small-scale bioassays here, this study indicates that the NtC algorithm could be used in various systems. Its application to the survival of zebrafish embryos and to metabolic activity in cell lines showed that NtCs can be applied to different effect measure­ments, time points, and levels of biological organization. The algorithm is available as Matlab and R source code, and as a free, user-friendly online application.

Evidence regarding carcinogenic mechanisms serves a critical role in International Agency for Research on Cancer (IARC) Monograph evaluations. Three recent IARC Working Groups pioneered inclusion of the US Environmental Protection Agency (EPA) ToxCast program high-throughput screening (HTS) data to supplement other mechanistic evidence. In Mono­graph V110, HTS profiles were compared between perfluorooctanoic acid (PFOA) and prototypical activators across multiple nuclear receptors. For Monograph V112-113, HTS assays were mapped to 10 key characteristics of carcinogens identified by an IARC expert group, and systematically considered as an additional mechanistic data stream. Both indi­vidual assay results and ToxPi-based rankings informed mechanistic evaluations. Activation of multiple nuclear receptors in HTS assays showed that PFOA targets not only peroxisome proliferator activated receptors, but also other receptors. ToxCast assays substantially covered 5 of 10 key characteristics, corroborating literature evidence of “induces oxidative stress” and “alters cell proliferation, cell death or nutrient supply” and filling gaps for “modulates receptor-mediated effects.” Thus, ToxCast HTS data were useful both in evaluating specific mechanistic hypotheses and in contributing to the overall evaluation of mechanistic evidence. However, additional HTS assays are needed to provide more comprehensive coverage of the 10 key characteristics of carcinogens that form the basis of current IARC mechanistic evaluations.

The implantation of chondrocyte-laden hydrogels is a promising cartilage repair strategy. Chondrocytes can be spa­tially positioned in hydrogels and thus in defects, while current clinical cell therapies introduce chondrocytes in the defect depth. The main aim of this study was to evaluate the effect of spatial chondrocyte distribution on the reparative process. To reduce animal experiments, an ex vivo osteochondral plug model was used and evaluated. The role of the delivered and endogenous cells in the repair process was investigated.
Full thickness cartilage defects were created in equine osteochondral plugs. Defects were filled with (A) chondrocytes at the bottom of the defect, covered with a cell-free hydrogel, (B) chondrocytes homogeneously encapsulated in a hydrogel, and (C, D) combinations of A and B with different cell densities. Plugs were cultured for up to 57 days, after which the cartilage and repair tissues were characterized and compared to baseline samples. Additionally, at day 21, the origin of cells in the repair tissue was evaluated.
Best outcomes were obtained with conditions C and D, which resulted in well-integrated cartilage-like tissue that com­pletely filled the defect, regardless of the initial cell density. A critical role of the spatial chondrocyte distribution in the repair process was observed. Moreover, the osteochondral plugs stimulated cartilage formation in the hydrogels when cultured in the defects. The resulting repair tissue originated from the delivered cells.
These findings confirm the potential of the osteochondral plug model for the optimization of the composition of cartilage implants and for studying repair mechanisms.

Since the early 20^th century, extracorporeal perfusion of large animal organs has been used to investigate a broad variety of research questions, thereby overcoming common drawbacks of in vitro studies without suffering from ethical concerns associated with live animal research. The technique is in accordance with the 3R principles and represents an excellent opportunity to investigate the physiology of organs in detail under standardized conditions. It is also suitable for the translation of basic pre-clinical research into a more relevant arena prior to or avoiding live animal research altogether. Furthermore, organ perfusion has also been an important tool in developing methods of organ preservation for transplantation surgery. Yet, due to the nature of the experiments, only short-term observations can be made and while cells are still exposed to their regional secretome, the whole organ itself is isolated from the body and correlations between organ systems cannot be taken into consideration. This review gives an overview over the history of extracorporeal perfusion of large animal organs and limbs, highlighting major achievements in the field and discussing different experimental set-ups. Advantages and limitations of the technique are presented. Prospective future research perspectives, which may include tracking of specific cell types and study of their distinct behavior towards different stimuli, are given to illustrate the relevance of this method.

The supplementation of culture medium with fetal bovine serum (FBS, also referred to as “fetal calf serum”) is still common practice in cell culture applications. Due to a number of disadvantages in terms of quality and reproducibility of in vitro data, animal welfare concerns, and in light of recent cases of fraudulent marketing, the search for alternatives and the development of serum-free medium formulations has gained global attention. Here, we report on the 3^rd Workshop on FBS, Serum Alternatives and Serum-free Media, where regulatory aspects, the serum dilemma, alternatives to FBS, case-studies of serum-free in vitro applications, and the establishment of serum-free databases were discussed.
The whole process of obtaining blood from a living calf fetus to using the FBS produced from it for scientific purposes is de facto not yet legally regulated despite the existing EU-Directive 2010/63/EU on the use of animals for scientific purposes. Together with the above-mentioned challenges, several strategies have been developed to reduce or replace FBS in cell culture media in terms of the 3Rs (Refinement, Reduction, Replacement). Most recently, releasates of acti­vated human donor thrombocytes (human platelet lysates) have been shown to be one of the most promising serum alternatives when chemically-defined media are not yet an option. Additionally, new developments in cell-based assay techniques, advanced organ-on-chip and microphysiological systems are covered in this report. Chemically-defined serum-free media are shown to be the ultimate goal for the majority of culture systems, and examples are discussed.