Requirement of second species testing for evaluating developmental toxicity potential
Over the last decade, there has been ongoing debate on the regulatory requirement of a second-species testing requirement to identify developmental toxicity. A number of workshops and reviews have been undertaken across the EU and the US to understand the added value of using rabbit as the second species. More recently, an analysis of more than 1000 compound data by Braakhuis et al. (2019) has questioned the reproducibility of developmental toxicity results and the comparison approach using NOAELs. The current insight attempts to present the state-of-the-art information on the ongoing discussion.
It is a standard information requirement under different regulatory programmes to test chemical substances or active pharmaceutical ingredients for embryo-foetal and pre-and post-natal developmental toxicity in two different species, one rodent (preferred species: rat) and one non-rodent (preferred species: rabbit) species. In recent years, with the advent and implementation of the 3Rs strategy, tiered testing approaches have been adopted and the decision for conducting the second species testing is now taken based on the results of the first study.
The need and additional benefit of using a second species in developmental toxicity studies has been debated over years and evaluated in a number of publications. The evaluation by Janer et al. (2008) indicated that the rat and the rabbit developmental toxicity studies were similarly sensitive, the average ratio of the no observed adverse effect levels (NOAELs) between the two species was about one, and for most compounds there were no differences between rat and rabbit studies in terms of classification for developmental toxicity. The discrimination between direct and indirect (i.e., as a consequence of maternal toxicity) developmental effects was often doubtful and is one of the factors that could explain the apparent differences between the two species (Janer et al. (2008)). On the other hand, the analysis by Hurtt et al. (2003) and Knudsen et al. (2009) showed that rat studies alone predicted teratogenicity in 61% of chemicals that showed teratogenicity in rat, mouse or rabbit, whereas a rat study and a rabbit study together identified teratogenicity in 100% of these chemicals.
The same authors noted that, higher frequency of growth retardation and skeletal variations observed in rat studies might be explained by differences in the dosing schedule. The post-treatment period from the end of exposure to terminal evaluation in the evaluated dataset was different between rat and rabbit. In other words, the longer period between exposure and evaluation that can allow rabbit foetuses more time to recover from transitory delays than might be detected in the rat foetus. Similar findings were reported by Julien et al. (2004) and Theunissen et al. (2016).
In a retrospective analysis specifically conducted with 379 marketed and non-marketed pharmaceuticals, (Theunissen et al. (2014); Theunissen et al. (2016); Theunissen et al. (2017)) indicated that both rat and rabbit are similarly sensitive, and the average ratio of the NOAELs between the two species were similar to one although species differences were observed in the frequency of different EFDT manifestations. Additionally, the relative extent of embryo-foetal toxicity in the presence of maternal toxicity was not different between species. For 31% of compounds in the database, EFDT was observed in one species only, and 74% of compounds detected developmental toxicity in at least one species when two species were used. Therefore, on the basis of an overall EFDT LOAEL comparison, both species are equally sensitive, but selective EFDT toxicity in one species is not uncommon. Based on systemic exposure and after human equivalent dose conversion (HED), the rat and rabbit show similar sensitivity (within 10-fold difference between systemic or HED LOAEL in both species) to prenatal developmental exposure for roughly 80% of the tested substances. Based on the power of detection and given differences in the nature of developmental effects between rat and rabbit study outcomes for individual compounds, EFDT studies in two species have added value over single studies. This suggests that the use of both species has a higher degree of detecting developmental toxicants than either one alone.
In a more recent analysis of over 1000 compounds from chemicals, drugs and pesticides, Braakhuis et al. (2019) confirmed that overall rat and rabbit do not differ in sensitivity to developmental effects, however substantial difference in NOAELs resulted when a same compound was tested twice in the same species. This reproducibility error could arise due to random sampling errors, other experimental errors, differences in study design and in experimental conditions and seem to overwhelm interspecies difference in developmental effects. Much of these study replication errors can be assigned to the NOAEL approach which could be reduced by the application of the Benchmark dose (BMD) analysis for deriving a point of departure (POD).
Overall, the above-mentioned analyses and workshops indicated that both the species exhibit similar sensitivity to developmental effects, although differences exist in terms of nature of the effects. None of the analysis clearly indicated that the evaluation of developmental toxicity, as opposed to other types of toxicity, would specifically require the rabbit as an additional test species. It would be interesting to see if the ongoing debate would trigger further refinement of the regulatory guidelines to adopt a tiered approach based on chemical nature, previous study results and species-specific attributes and shift from the existing two-species requirements.
