We have funded the creation of phenotypic screens using two model organisms to assess chemicals for developmental and reproductive toxicity (DART).
Challenge Contractor: Professor Raymond Pieters
Organisation: University of Applied Sciences Utrecht, The Netherlands
Start date: 2012
Duration: 3 years
Sponsors: Shell | Syngenta
Chemicals and pharmaceuticals are tested for potential reproductive and/or developmental toxicity, usually in two species as specified in OECD and ICH test guidelines for chemicals and pharmaceuticals respectively. The studies use large numbers of rats and rabbits with the standard multi-generation reproductive and developmental toxicity study involving around 2,500 animals per substance. The tests are also resource intensive and expensive because of their long duration and the associated animal housing and husbandry costs.
In academic research, many of the biological phenomena associated with development and reproduction are studied in model organisms such as the nematode Caenorhabditis elegans and zebrafish embryos. Nematodes have highly reproducible developmental timings and in zebrafish most of the organs are formed within three days post-fertilisation. These models have 3Rs potential since they are not covered by regulations on the use of animals in scientific procedures*, however, despite the evolutionary conservation of the molecular pathways involved in development and reproduction, the use of C. elegans and zebrafish embryos by industry for DART studies had not been fully explored. Shell and Syngenta posed the PREDART Challenge to develop the use of these model organisms for the early and rapid prediction of DART.
The PREDART Challenge was awarded to a consortium led by Professor Raymond Pieters at the Institute of Risk Assessment at the University of Applied Sciences Utrecht, that included collaborators from four Dutch institutions and the University of Oxford.
The consortium developed a phenotypic screen for DART hazard assessment using C. elegans and zebrafish embryos (up to five days post-fertilisation).
3Rs and scientific benefits
The phenotypic screen employs assays for various developmental and reproductive effects, including for the nervous, intestinal and reproductive systems. The consortium tested the assays using a range of concentrations of well-characterised DART chemicals identified by Shell and Syngenta. This demonstrated that 28/32 (87%) and 26/35 (74%) of the chemicals tested were correctly identified as having a DART-phenotype by the nematode and fish embryos respectively. Those that were missed by one of the two models were picked up as DART chemicals by the other, such that all chemicals were scored correctly by combinatorial testing using nematodes and zebrafish. Testing in these two model organisms is cheap and fast, taking one week to complete compared to mammalian studies conducted to comply with OECD test guidelines which can take up to 30 weeks.
Achieving full 3Rs impact requires regulatory acceptance and considerably more development and validation work, likely to take many years. But the study shows that the models could be utilised as part of a weight-of-evidence approach to avoid testing in rodents and rabbits for DART purposes and this has generated further interest and investment. A spin-out company, Vivaltes, has been formed as a result of the Challenge to provide DART screening using C. elegans (with the sub-contracting of testing in zebrafish embryos). It has conducted assignments from Shell including testing water samples, expanding the application of the screen to ecotoxicology as well as human health.
Vivaltes has received two Dutch development grants totalling €80k to support the commercialisation of the screens and to further validate the C. elegans test system against a broader range of chemicals to build confidence in its utility for mechanism of action studies. Vivaltes and the University of Applied Sciences Utrecht are also part of a consortium with Shell working on the €4.28M 3R TOXFLOW project, applying DART testing using C. elegans and zebrafish embryos to complex petrochemical mixtures to address the REACH regulation requirements.
During the Challenge, the consortium incorporated high content assays into the nematode and fish embryo phenotypic screens as well as conducting some analyses in the social amoeba, Dictyostelium discoideum, a model commonly used to study cell signalling pathways. For eight DART chemicals, RNAseq analyses were carried out on the three model organisms. Although different phenotypic outcomes were observed, toxicogenomic profiles were common across the test species identifying potential molecular mechanisms with human relevance. This type of mechanistic information supports the development of Adverse Outcome Pathways (AOPs) relevant to DART. A team led by Raymond was subsequently awarded £1M in the DARTpaths CRACK IT Challenge sponsored by Shell and Syngenta to integrate information on genes involved in DART in model organisms and humans into functional AOPs that can be used to predict DART and select the best non-mammalian model for testing.
Members of the consortium from the University of Oxford have been awarded an NC3Rs grant of £0.31M to develop fluorescent reporter constructs for different developmental events in C. elegans and D. discoideum, further expanding their utility for mechanism of action data for DART. Through CRACK IT Solutions, the Oxford team have partnered with Syngenta to conduct validation work on the reporter constructs.
Sponsor in-kind contributions
Shell and Syngenta provided a range of in-kind contributions. This included DART compounds for testing, developmental toxicity data from rats and zebrafish for validation purposes and toxicogenomic data. Each company provided 0.36 of a full-time equivalent (scientific staff) over the three-year period.
This case study was published in our 2019 CRACK IT Review.
* Zebrafish are protected, by EU legislation on the use of animals in scientific procedures, five days after fertilisation.