Five critical success factors for navigating EOGRTS complexity and smoothing the progress of your chemical registration

The EOGRTS or OECD 443 is a scientifically and logistically complex study. Five critical success factors can help you overcome the complexities to achieve DART success.

What makes EOGRTS so challenging?

The EOGRTS or extended one-generation reproductive toxicity study evaluates the reproductive and developmental effects resulting from pre- and post-natal exposure to a chemical as well as the systemic toxicity in pregnant and lactating females and young and adult offspring. The test guideline for EOGRTS – OECD TG 443¹ – was adopted by the OECD in 2011, replacing the previous two-generation reproductive toxicity study – OECD TG 416 – as the required study for REACH (Registration, Evaluation, Authorisation and restriction of Chemicals) registration by the European Chemicals Agency (ECHA). OECD 416 remains a valid test guideline, however. The advantages of OECD TG 443 over OECD TG 416 are more targeted breeding of the F1 generation to evaluate suspected reproductive toxicity, the ability to examine multiple endpoints in the F1 generation, if required, and a reduction in the use of test animals. In fact, the EOGRTS can reduce animal usage by ~40% compared with OECD TG 416.

As a result, OECD TG 443 study designs are complicated. They involve the mating and examination of an F0 parental generation (P), evaluation of an F1 generation of offspring for multiple endpoints and, if triggered, breeding of the F1 generation to produce an F2 generation.¹ The studies need robust study design, careful planning for triggered breeding, specialist animal management and robust sampling and analytical approaches. The bottom line is they need more resources and time than the previous OECD TG 416, meaning they are a substantial investment and one you need to get right.

Critical success factor 1: use a preliminary study to optimize your EOGRTS design

Ironically, it is the flexibility at the heart of EOGRTS that makes it so complex. The hallmark of EOGRTS is the ability to build in triggers based on specific criteria which, when observed, initiate breeding of the F2 generation and/or the evaluation of multiple endpoints in the F1 generation. Multiple protocols for OECD 443 exist but in summary, after weaning, the F1 generation is assigned to cohorts as follows:

• Cohort 1A: the integral test cohort, with both males and females examined for toxicology and reproductive endpoints
• Cohort 1B: the ‘spare’ set of animals used for triggered breeding to produce the F2 generation, if required
• Cohort 2A: the cohort tested for developmental neurotoxicity (DNT) as adults, if triggered
• Cohort 2B: assessed for DNT at weaning, if triggered
• Cohort 3: assessed for developmental immunotoxicity (DIT), if triggered

To yield the highest levels of success from your EOGRTS, you need to have the right study design and consider how best to avoid possible downstream challenges or delays.

Example study designs based on the OECD TG 443 protocols

Figures 1–3 show some of the study protocol variants developed and used routinely. Figure 3 follows the recommended ECHA approach.

Using a preliminary study to enhance EOGRTS success

To maximize EOGRTS success it is usually advisable to first run a robust dose range-finding study. This identifies any unpredicted issues that may jeopardize your study’s viability, such as previously undetermined reduced litter size or increased offspring mortality. As many chemicals may have limited existing developmental and reproductive toxicology (DART) data, this preliminary study also helps you evaluate the endpoints critical to the EOGRTS before you embark on the main study. In addition, it enables appropriate dose selection and flags any possible assessments that should be included.

For example, if dose-related and test item-related reductions in thymus weight are recorded in the F0 animals, it may be prudent to include the Cohort 3 (DIT) in the EOGRTS study, even if not initially required by regulators such as the ECHA. Similarly, if unexpected signs of neurotoxicity, such as test item-related tremors or convulsions, are observed in the F1 offspring on the preliminary study, it may be prudent to include the F1 Cohort 2 (DNT). The registrant is free to expand an EOGRTS to address concerns identified after the ECHA decision has been issued.

Endpoints recommended for the dose range-finding study include mating success, fertility, litter production and pup survival, including evaluation of pup exposure during gestation and lactation and, importantly, effects of direct treatment of the offspring post weaning.

Critical success factor 2: select the right test model and enhance study scheduling by using the expertise of specialist animal technicians

Your study design will influence the rodent model you select for an EOGRTS. For a standard EOGRTS design based on REACH, i.e., without DNT and DIT cohorts, then Han Wistar or Sprague Dawley strains of rat are appropriate. The Sprague Dawley rat is recommended for the full OECD TG 443 study, which includes DIT and DNT cohorts, as it is has a larger litter size and balanced sex ratio offspring.

As well as selecting the right rodent strain, you can also benefit from having specialist animal technicians who can predict and manage mating. This can support study scheduling, streamline resource usage and increase the efficiency of study planning and execution. In addition, because multiple generations may be used, dams, fetuses and pups must be handled to the highest welfare standards; this means consideration of litter and individual animal health.

Critical success factor 3: ensure you have the right test dose and route of administration

Preliminary work is needed to establish suitable dose levels in littering females. Toxicokinetic data collection is recommended to assess potential exposure in fetuses and offspring during lactation and at puberty. The highest dose level will normally be expected to induce some systemic toxicity or be at the level at which toxicokinetic exposure becomes non-linear, with two or more lower doses selected at intervals of less than 10x to determine no observed adverse effect level (NOAEL) values.

When considering the route of test dose administration, you should take into account the most relevant route for human exposure. For example, if the main exposure route in humans is via inhalation, inhalation dosing may be appropriate. This needs special consideration; especially as whole-body inhalation requires the simultaneous exposure of both parents and pups to avoid separation of family groups, which can result in exposure via dermal and oral routes.

There are other considerations too, including whether oral administration (by gavage, via food or drinking water) to dams can be continued through birth and in the neonatal period and if offspring exposure to test substance occurs via maternal milk. If this is unclear, it may be appropriate to consider direct dosing of pups during the pre-weaning stage. Dosing of pups can be technically and logistically complicated.

Critical success factor 4: understand the triggers and have robust assessment techniques and resources ready to measure them

There are a range of possible scenarios that can pre-determine inclusion of cohorts 2A, 2B or 3 in study design (based on previous Toxicity information) or that can trigger breeding of the F1 generation based on observations within-study in both the F0 and F1 adults or F1 offspring (Table 1). Within-study Triggers broadly fall into two categories: effects that may predict toxicity in the offspring; and effects in adults that raise concerns for offspring development.

It is essential to understand the triggers and have the necessary assessment approaches in place to ensure they are effectively evaluated.

Histopathology timelines for specific EOGRTS cohorts

With the variability in number of animals and cohorts, resourcing necropsy and histopathology to support your study is important. Estimated histopathology timings for individual cohorts are given in Table 2. Remember also to allow lead in time of approximately 12 weeks, at Covance it’s 8 weeks for litter mates.

Critical success factor 5: leverage regulatory insight to plan and design an EOGRTS to satisfy your global registration goals

EOGRTS evaluates reproductive endpoints that need the sexual interaction of males and females and which are not assessed by other reproductive toxicology studies. In general, these may be required if results from studies such as repeat dose studies (OECD TG 422) or short-term endocrine disruptor screening studies indicate that there is a possible reproductive toxicity concern. Different regulators have different opinions regarding F1 breeding, so you need to factor in your longer-term global launch strategy up front to maximize the value of your EOGRTS.

For EU registrations, the design of your EGORTS is stipulated by ECHA based on all available toxicity data. Full and robust justification for the addition, or perhaps more importantly the omission, of additional cohorts 2 (neurotoxicity) and 3 (immunotoxicity) is an extremely important aspect when planning your study for this jurisdiction. For subsequent registrations in the U.S. and Canada, it is important to have comprehensive justification to support the inclusion or exclusion of breeding F1 generation animals, and to build in within-study triggers to support this; these are clearly identified in OECD Guidance document 117.³

EOGRTS timescales vary, so be prepared

The overall length of an EOGRTS depends on the triggers as well as extent of pre-treatment (Table 3). As you can see, some study durations can take almost a year to complete, so you need to plan appropriately.

Conclusions

OECD TG 443 or EORGTS evaluates the potential impact of a chemical on the sexual function and fertility of several generations of test animals. To achieve OECD TG 443 study success, you need to follow the five critical success factors outlined here:

1. Use a preliminary study to optimize your EOGRTS design
2. Select the right test model and enhance study scheduling by using the expertise of specialist animal technicians
3. Ensure you have the right test dose and route of administration
4. Understand the triggers and have robust assessment techniques and resources ready to measure them
5. Leverage regulatory insight to plan and design an EOGRTS to satisfy your global registration goals

This can be accomplished if you partner with a provider who has scientific insight, dedicated and experienced teams as well as excellence in study logistics and execution.

To learn more, read our latest eBook on the subject, EOGRTS – What You Need to Know: Understand the Complexities and Overcome the Challenges for Your Substances.

Animal Welfare considerations must be included in study design as well as study conduct. Animal care and studies must be conducted in alignment of all applicable animal welfare regulations.