PREDART

Background

Developmental and reproductive toxicity studies in rats and rabbits form the main basis for regulatory assessment of the potential effects of chemicals on the developing foetus. These studies use large numbers of animals, are expensive and time consuming. In addition, the relevance to humans of effects seen in these studies is not always clear and significant species differences exist. In addition, there remains a dearth of information about the fundamental pathways of embryogenesis and the effects of toxicity on them.

If a compound is found to be toxic to reproduction and/or development, this has significant consequences for potential use of the product, including restriction from occupational and/or consumer use or prohibition of authorisation for any use under regulatory schemes, including REACH and the EU’s plant protection products regulation. It is therefore important to obtain an indicator of developmental toxicity early in the product development pipeline.

In the past the use of alternative models in lower species or in vitro systems has been hampered by the drive for a 1:1 predictive screen for mammalian outcomes. However, in the absence of an understanding of the complex underlying developmental pathways and species differences this has proved to be unachievable.

In recent years numerous alternative developmental biology model systems have been explored, ranging from whole organism systems including zebrafish, Caenorhabditis elegans and slime moulds, to cell based systems using mouse or human embryonic stem (ES) cells. Groups working with these systems are embracing and exploiting the explosion in molecular biology and genetics to better understand the underlying pathways driving normal embryogenesis and the effects of perturbations to these pathways.

The key to unlocking the potential of these alternative test systems is to understand the conservation of key pathways across different species, including mammals, and to use these non-mammalian (surrogate) systems to inform mammalian toxicity potential. The issue of exposure concentrations in the test systems versus those in rats/rabbits and humans will also need to be assessed. There has been progress in this direction in recent years, but there is now a need for a focused effort towards practical, commercial solutions, which will include an integration of the available systems.

3Rs benefits

A standard two-generation reproductive and developmental toxicity study typically uses around 2,500 test animals. An early indication of developmental toxicity potential during compound development (such as relevant pathway perturbations) would be of benefit as a screening or ranking tool, helping to inform compound selection and direct testing strategies. The information can also be used to predict structure activity relationships (SAR) and steer chemistry design away from these key alerts.

This could impact on animal use in a number of ways including.

• Reducing the number of regulatory toxicity studies carried out in animals (e.g. developmental and reproductive toxicity, teratogenicity, carcinogenicity) on compounds with potential for developmental toxicity that would not therefore be progressed into product development.
• Supporting weight of evidence and/or read across arguments for registration, consequently reducing and replacing the use of animals for developmental toxicity testing. In the longer term as our understanding of underlying pathway perturbations improves and confidence is gained in these models, the opportunities to replace the use of rodents and rabbits will increase.

Phase 1 winners

Project teams led by:

• Professor R Wayne Glasse-Davies, Brainwave‐Discovery Ltd, £96,600.
• Dr Nils Klüver, Helmholtz Centre for Environmental Research GmbH ‐ UFZ, £99,957.
• Professor Raymond Pieters, Institute for Risk Assessment Sciences/ Toxicology, £100,000.

Phase 2 winner

Project team led by:

• Professor Raymond Pieters, Institute for Risk Assessment Sciences/ Toxicology, £750,000.

Full Challenge information

Through the PREDART Challenge, the team developed a test battery of assays combining three alternative non-mammalian test systems (the zebrafish Danio rerio, the nematode C.elegans and the social amoeba Dictyostelium discoideum) to pre-screen compounds for developmental and reproductive toxicity (DART) and identify mechanisms of action, reducing the number of regulatory animal studies carried out.

The advantages of the test systems include rapid high throughput screening, short turnover time enabling the entire developmental and reproductive cycle to be examined and easy identification of adverse outcomes pathways and molecular imitating events. The assays have been validated using 42 well characterised DART positive and negative compounds. D. Discoideum, C.elegans and D.rerio detected DART compounds with assay sensitivity values of 87%, 87% and 74%, respectively (Table 1.). Combining all three test systems increases the sensitivity to 92%, highlighting the value of the combinatorial approach.

The team has formed a company, Vivaltes B.V. which offers the alternative non-mammalian test system assays as a screening service. If you are interested in using alternative non-mammalian models as a screen or would like further information, please contact Dr Marjolein Wildwater.

Table 1. Sensitivity of the three alternative non-mammalian test systems in detecting DART.