What is DART?
DART stands for ‘developmental and reproductive toxicology’ and is the acronym commonly used in place of the term reproductive toxicology. DART studies assess a chemical’s effect on several aspects of reproductive capacity in laboratory animals. These include mating performance and fertility, sexual maturation, and offspring growth and development, sometimes through to successive generations.
How are DART studies evolving?
In line with the 3Rs principles, the latest evolution in DART aims to reduce animal use. In addition, as scientific thinking and study designs evolve, so do regulatory endpoint requirements. For example, a recent focus has been the evaluation of endocrine disruptor potential which has been driven by many global regulations, including REACH.
How do DART studies compare to standard toxicology studies?
In general, DART studies are logistically more complex to conduct than standard toxicology studies such as acute oral toxicity studies (Test Guideline OECD 423) or repeat dose oral toxicity studies (Test Guideline OECD 407 or OECD 408).
This complexity has multiple causes:
- Often mating or littering is required as an inherent part of the study design. This complicates study logistics by introducing multiple start dates that span several days. This has a knock-on effect on scheduling tests, particularly those that may be specific to a day of gestation or lactation, and requires experienced management of resource availability.
- The inclusion of both adults and offspring in several study designs common to DART testing means additional welfare considerations. For example, the provision of environmental enrichment during late gestation and throughout lactation. This requires experience in routine procedures concerning care of both populations of animals.
- Test material calculations must be robust and consider multiple populations of animals at different stages of the appropriate study design. Some DART tests are of long duration to satisfy regulatory requirements so you must demonstrate sufficient GLP compliant stability, and expiry of test material, for each study.
- Sophisticated study designs are becoming more common with the goal to reduce the number of animals involved in DART testing and to optimize the information obtained from each animal. Some studies now include optional phases whose inclusion are driven by previous toxicity data. In certain cases, phases are triggered by specific endpoints within ongoing studies, such as the triggered breeding phase of the Extended One Generation Reproductive Study (EOGRTS Test Guideline OECD 443).
- Analytical assays should be robust and sensitive enough to detect analytes in a range of developmental stages, from adults to young offspring. For example, thyroid hormone testing for endocrine disruption endpoints is required in adults but also in offspring as young as 4 days old.
- DART studies include reproductive specific endpoints that require the support of appropriate Historical Control Data (HCD), particularly for assessments such as fetal pathology. It is vital that adequate HCD is available in order to put findings into context.
What test guidelines are applicable to DART?
The table below lists standard current OECD studies that fall under the DART umbrella.
Chemical guidelines applicable to DART studies
|OECD test number||Study name||Objectives|
|OECD 414||Prenatal developmental toxicity study||Provides general information concerning the effects of prenatal exposure on the pregnant animal and the developing fetus. These common studies are reasonably straightforward to conduct, and the usual test animal species is the rat. However, OECD 414 studies in rabbits may be triggered at higher tonnage bands (often when OECD 443 studies are requested).|
|OECD 421||Reproduction/developmental toxicity screening test||Aims to describe the effects of a test chemical on male and female reproductive performance, including endocrine disruptor endpoints.|
|OECD 422||Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test||This combines OECD 421 with a 4-week toxicology study (OECD 407). Provides limited information concerning the effects of a test chemical on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition.|
|OECD 416||Two-generation reproduction toxicity||Provides general information about the effects of a test substance on the integrity and performance of the male and female reproductive systems, and on the growth and development of the offspring. This is NOT the preferred longer-term study in the REACH framework but is still required for Crop Protection materials.|
|OECD 443||Extended one-generation reproductive toxicity study (EOGRTS)||This evaluates the reproductive and developmental effects resulting from pre- and postnatal chemical exposure as well as an evaluation of systemic toxicity in pregnant and lactating females and young and adult offspring. This study has replaced OECD 416 within the REACH Regulatory Framework and a major aim is to reduce animal usage. Reductions of 40% are possible if F1 breeding is not triggered.|
|OECD 426||Developmental neurotoxicity study||Developmental neurotoxicity studies are designed to provide data, including dose-response characterizations, on the potential functional and morphological effects on the developing nervous system of the offspring that may arise from exposure in utero and during early life.|
The Secrets of DART Success
Insightful study design
OECD 443 is the most challenging type of DART study as it is new and drives optimization of animal use. OECD 443 study designs are complex, involving mating and examination of a parental generation, evaluation of an F1 generation of offspring for multiple endpoints and, if triggered, breeding of the F1 generation to produce an F2 generation. Different regulators have different opinions regarding F1 breeding, so this also needs be considered up front.
Insight into the study design required by regulatory demands in the most cost-effective manner helps ensure success and optimizes spend on these expensive studies.
Specialist animal expertise
Expertise in animal handling and behavior is critical to the efficiency and success of DART studies.
- Effective strain selection optimizes the efficiency of animal usage. For some of the larger studies with multiple cohorts it is best to select a strain with a larger litter size and a more balanced offspring sex ratio.
- Managing pairing and resulting mating can support study scheduling and streamline resource usage, by effectively controlling the number of females mating at any one time. This also allows more control over littering numbers per day. This enables the prediction of dosing and sampling dates and prevents overloading resources such as necropsy. These are all critical for efficient study planning and excellent execution. Specialist animal technicians achieve this by monitoring estrous cycles, planning pairings, predicting the number of successful matings and when mating will happen. Such expertise also allows the estimation of the number of animals needed to fulfill specific endpoints with appropriate statistical power.
- Some DART studies, e.g., OECD 426, require behavioral endpoints. Comprehensive experience in handling and observing animals, in addition to experience in the required behavioral tests, is essential for successful behavioral assessments under test conditions.
- With multiple generations used in some studies, handling and health monitoring at all stages of development requires special care and attention.
Test dose estimation and demonstrating formulation stability
Demonstrating formulation stability is a key GLP regulatory requirement. Sufficient stability is required to be able to formulate dose at a frequency appropriate to study design. This helps manage resource requirements and ultimately cost.
Significant experience in DART study types imparts the knowledge required to accurately predict the amount of test material required for each study design.
Excellent pregnancy rates and healthy litter sizes allows group sizes to be kept to a minimum. This subsequently minimises test material requirements, particularly for larger models which, due to their bodyweight, need significantly more amounts of formulation and therefore test item per day than smaller rodents.
Excellent execution on logistics
DART studies that require conception may have multiple start dates due to different mating times, so application of test substance, sampling and other assessments regularly fall over several days, including weekends. Managing such logistics requires a dedicated team of specialists who can flex around these schedules. As well as a flexible workforce, flexible space is also necessary because complex studies can tie up multiple animal rooms. In addition, forward contingency planning for subsequent generations triggered in OECD 443 studies is essential. Capacity can often be an issue, especially for OECD 443 studies which can involve multiple arms and generations. Covance has the capacity to run several OECD 443 studies of varying designs each year.
Precision analytics and historic data controls
Most of the main OECD DART test guidelines now include endocrine disruptor endpoints. These include assessments of sexual maturation, ano-genital distance, thyroid histopathology and thyroid hormone assays (T3, T4 and TSH [thyroid stimulating hormone]).
Assessment of sexual maturation has been routine for many pharmaceutical study designs, and some chemical DART studies, for many years. It requires adequate interpretation relating to the age and bodyweight at attainment for meaningful evaluation.
Ano-genital distance requires skilled operators to avoid stretching of the skin which may artificially influence measurement of distance. These data also require evaluation relative to a measurement of growth, such as bodyweight, in order to provide meaningful interpretation of results.
Thyroid histopathology is a new addition to the OECD 414 guideline, which is a study design that historically included no histopathology except from fetal pathology evaluations. This has added another dimension of pathology reporting that could potentially influence overall report timings.
Thyroid hormones are hard to measure reliably at very low concentrations, such as those in studies involving fetuses and offspring. Assays for T3 and T4 must be highly sensitive, selective enough to differentiate between thyroid hormones, stable for samples stored until tests are triggered and reproducibly within and across laboratories. Furthermore, assays must be referenceable to historic data controls to allow the appropriate interpretation of any findings.
With enhanced regulatory standards driven by the 3Rs and the addition of endocrine disruptor endpoints, the complexity of DART studies outstrips those of standard toxicology studies. The most complex current test guideline is OECD 443 or EORGTS which has the potential to evaluate the potential impact of a chemical on the survival, growth, development, sexual function and fertility of multiple generations. Delivering successful OECD 443 studies requires scientific insight, dedicated and experienced teams as well as excellence in study logistics and execution.