Regulatory Forum Opinion Piece *

Abstract

The National Toxicology Program (NTP) has developed a new flexible study design, termed the modified one generation (MOG) reproduction study. The MOG study will encompass measurements of developmental and reproductive toxicity parameters as well as enable the setting of appropriate dose levels for a cancer bioassay through evaluation of target organ toxicity that is based on test article exposure that starts during gestation. This study design is compared and contrasted with the new Organization for Economic Co-operation and Development (OECD) 443 test guideline, the extended one generation reproduction study. The MOG study has a number of advantages, with a focus on F ₁ animals, the generation of adequately powered, robust data sets that include both pre and postnatal developmental toxicity information, and the measurement of effects on reproductive structure and function in the same animals. This new study design does not employ the use of internal triggers in the design structure for the use of animals already on test and is also consistent with the principles of the 3R’s.

One of the major roles of the National Toxicology Program (NTP) has been in the development of new toxicology test methods. Following 2 workshops (King-Herbert and Thayer 2006; Thayer and Foster 2007) that focused on the NTP selection of a new rat strain for all of its toxicological studies and that there would be a greater emphasis on early life test article exposures in the conduct of its cancer bioassays, it became apparent that there was a need for some dose range finding studies that involved early life test article exposure (gestation, lactation, and continuing exposure through adulthood). At the same time, NTP also showed that they could markedly increase the power to detect postnatal developmental effects (including those consequent to in utero exposure) in their developmental and reproductive toxicity (DART) studies by simply retaining more of the offspring from each litter post weaning (on most DART littering studies, only 1 male and female from each litter is retained) that would normally be culled or only given a cursory examination (Blystone et al. 2010).

Taken together, the program realized that in performing the necessary setting of dose levels and identification of target organ toxicity in order to undertake a perinatal cancer bioassay, it was possible at the same time to use animals already produced following exposure during gestation and lactation to develop additional, high-quality DART information in a single design, which we have termed the modified one generation (MOG) study (http://ntp.niehs.nih.gov/ntp/About_NTP/BSC/2011/April/MOGDesign.pdf). The design basically commences with time mated rats (although this could easily be adapted to mice) with exposure from gestation day (GD) 6 (implantation) continuously through pregnancy and lactation (with at least 20 pregnant dams per dose group). At weaning, the offspring (normally 4 males and 4 females per litter) are then assigned to different “testing cohorts.” The first group (10 males and females from different litters) will be continuously dosed for 90 days post weaning for assessment of target organ toxicity and clinical pathology—analogous to a standard 90-day study. In a second group, 1 male and female from each litter (i.e., 20 males and females) will be continuously dosed until sexual maturity and bred (nonsiblings) and then at GD 21, a routine evaluation of fetuses for external, visceral, and skeletal effects would occur (analogous to a teratology study). A third group (again with 1 male and female per litter) would be treated similarly to the second group, but after breeding, the females would be allowed to litter and raise their offspring to weaning (and potentially beyond if required). This leaves one final group of approximately 30 male and female offspring that could be used for any other assessment of developmental toxicity (e.g., developmental neurotoxicity or immunotoxicity). Importantly, this approach allows a 10-week exposure period before mating (i.e., covers the whole period of rat spermatogenesis) such that any changes in reproductive organ structure can be directly correlated with functional outcome (i.e., fertility and fecundity) in at least 40 breeding pairs with necropsies on all the F ₁ offspring for evaluation of developmental effects.

There have been a number of other international efforts to try to improve DART study designs. One effort from ILSI-HESI on agrochemical testing attempted to improve life stage testing, in what would be a very rich toxicology data set produced, for example, with a food use pesticide (Cooper et al. 2006) and again endeavored to make the maximum use of the animals already produced within the study. This change in DART design was then taken up and amended by OECD, with the aim that it could replace the multigeneration reproduction study and also be used for all chemicals (not just agrochemicals) where the toxicology data portfolio was much poorer. One of the major drivers for this change was the effort to reduce animal use under REACH in Europe and the realization that approximately 60% of the animals used or produced in toxicity testing on a given agent come from DART studies (van der Jagt et al. 2004). This change in DART study design became the OECD 443, extended one generation test guideline (OECD 2012). This design commences with adult male and female rodents (usually rats), which are exposed to test article for 2 weeks prior to mating with sufficient numbers to achieve 20 litters per dose group. This exposure duration would equate to the period of sperm maturation in the epididymis of male rats. Following continuous exposure of both parents through the period of gestation and lactation, the pups from different litters are allocated to groups (10 males and 10 females per group) and continuously dosed. The groups are evaluated for developmental immunotoxicity, and neurotoxicity at weaning and a third group of 10 males and 10 females from different litters for evaluation of neurotoxicity at sexual maturity. The remaining 40 male and female offspring would be exposed and taken to sexual maturity for necropsy, or if “triggered,” half of these animals would be bred (nonsiblings) to produce F ₂ litters. The developmental neurotoxicity and immunotoxicity arms of the study, originally a requirement in the draft guideline, are now optional.

Offspring that have been exposed in utero and taken to adulthood represent a unique exposure population in toxicology, and, if these animals are not bred, it represents a missed major opportunity to garner toxicity information throughout the reproductive cycle. Moreover, a laboratory will have to plan and cost the use of rooms, equipment, and personnel irrespective of whether the internal triggers will be activated. It is the triggers to breed the F ₁ animals that will also produce some significant issues for toxicology laboratories. The guideline states that one of the triggers for breeding will be an effect on parental fertility (implantations, pregnancy rate, and gestational interval) in the absence of a corresponding treatment- related reproductive organ histopathology. This implies that all the pathology on reproductive organs of the F ₀ parents must be complete and appropriately analyzed before the animals of the F ₁ generation reach 90 days of age. The F ₀ parents will be necropsied at weaning of the F ₁ pups (e.g., 21 days of age). So when will these data be available to make such a decision to move forward with breeding? One might anticipate some study stagger (not all the animals will get pregnant at the same time), the necropsy data will need to be collected, tissues fixed, trimmed, embedded, cut, stained, evaluated by a pathologist, statistics conducted, plus any potential discussions with a sponsor or regulatory authority before triggering the breeding—all to be performed in less than 10 weeks. This may be technically possible, but perhaps unlikely, under normal conditions and will certainly be at a cost premium.

A second trigger for breeding the F ₁ animals in OECD 443 is if a finding is made on F ₁ developmental landmarks (e.g., anogenital distance, nipple retention, and puberty onset). The Guideline indicates that these changes must be dose related and in the absence of bodyweight-mediated changes in these animals. For example, vaginal opening in rats usually occurs around 32 days after birth, when the animals are in a rapidly growing phase. If one sees an advancement in this parameter in treated groups of say, 4 days, will their body weights be lower? The answer is yes. But is the bodyweight “effect” due to toxicity or because they were younger? I would suggest it is because of their age and that such a finding should not be easily dismissed. Similar arguments about study stagger and data collection and statistical analysis (now post weaning for some of these parameters) would also apply as for the trigger based on fertility and fecundity changes.

I believe the major advantages of the MOG over OECD 443 include the following:

In the MOG, there is a focus on the F ₁ generation—the animals exposed from “womb to tomb.” The critical periods of the reproductive life cycle are not assessed in the OECD 443 due to the lack of F1 mating and delivery of the F ₂ routinely.

The MOG has no internal triggers—a decision on how to use the animals is made ahead of study start; the use of triggers in the OECD 443 has never been successfully accomplished.

The MOG is adequately powered and generates robust data sets, whereas the use of only 10 males and females for immuno- and neurotoxicity assessment in OECD 443 is insufficient, except for the most profound effects.

The MOG maintains the 10-week pre-breed exposure period—this is based on sound biology of spermatogenesis and would be even more important when a 90-day toxicity study is not available (for many chemicals).

The MOG includes measuring reproductive structure and function in the same animals. These designs are supposed to provide critical information on reproduction for risk assessment and classification and labeling and, therefore, should assess reproductive function in a comprehensive manner.

The MOG incorporates the provision of other developmental outcome data (prenatal developmental toxicity/teratology).

The design of the MOG has the ability to set dose levels for a perinatal cancer bioassay through the generation of target organ toxicity data in offspring following early life exposure.

The MOG is also not without some issues, especially if the test article happens to have very significant developmental toxicity that would preclude having a suitable number of offspring to assign to the various testing cohorts. NTP has tried several options to ameliorate such effects, including lowering dose levels during gestation/lactation versus post weaning, commencing the study at a later time in gestation (e.g., GD 15 rather than GD 6 to overcome early embryonic loss) or deciding that separate studies would be more amenable to interpretation.

In conclusion, the MOG offers a number of very positive advantages over conducting individual DART and range finding studies. Our current experience is with 3 studies that have now completed their in-life portions and are in the reporting phase. One of the test agents studied in the MOG was also studied in a direct comparison with the segmented ICH DART designs (using the same stock of rat, diet, and dose levels) and in evaluation of the preliminary information (before QA and peer review) has produced completely equivalent data. Thus, we continue to support the “3Rs” through refining our toxicity study designs, replacing certain other standard toxicity studies by folding them into the MOG design and reducing overall animal use compared to conducting individual DART and 90-day toxicity studies.