Direct Dosing of Preweaning Rodents in Toxicity Testing and Research: Deliberations of an ILSI RSI Expert Working Group

Abstract

Laboratory animal studies designed to assess the effects of exposure of a test substance during postnatal development are commonly utilized in basic research and to evaluate potential hazard to children for chemical and pharmaceutical regulation. Direct dosing, defined here as the administration of a test substance directly to a preweaning mammal, has been identified as a useful tool that can be used in the conduct of such studies for regulatory purposes. The International Life Sciences Institute Risk Science Institute (ILSI RSI) convened an Expert Working Group to develop guidance on the design and implementation of direct dosing regulatory studies on preweaning mammals, which was published as an ILSI monograph in 2003 (Zoetis and Walls, Principles and Practices for Direct Dosing of Pre-Weaning Mammals in Toxicity Testing and Research, Washington, DC: ILSI Press, 2003). A summary of the Working Group conclusions regarding direct dosing studies with laboratory rodents are presented here, although the ILSI monograph also includes rabbits, canines, swine and nonhuman primates. Issues to be considered when designing the protocol include selection of the test species, the route of administration, dose levels, and the timing of dosing. Knowledge of the maturational status of the test species and information on critical windows of development are important in creating a valid study design. Most common routes of administration (e.g., oral, inhalation, injection) are possible with typical laboratory species; however, adjustments may be necessary due to practical considerations. Information on the pharmacokinetic profile in young animals versus adults and in the test species versus humans is very useful for determining dosing parameters. The conduct of the study and the interpretation of the data will be improved by an understanding of confounding factors as well as statistical and biological issues specific for postnatal studies. Ultimately, the success of the study will depend upon careful preparation, including thorough training of the technical staff.

The use of animals to characterize the hazard of potentially toxic materials is broadly accepted as a critical aspect of safety and risk assessment to ensure the safety of humans exposed to these compounds. Many standardized protocols for toxicology testing recommend using adult animals, but it is widely recognized that children represent a unique segment of the human population, for which tests in adult animals may not be predictive (NRC 1993). Over the years, there has been extensive discussion on the need to evaluate potential toxicity in immature animals for risk assessment purposes (Guzelian, Henry, and Olin 1992; Kimmel and Makris 2001; NRC 1993; US EPA 1999; WHO 1986). Testing guidelines that include the use of immature animals have been developed for preclinical screening of pharmaceuticals (ICH 1994, 1996; US FDA 1966), and regulatory evaluations of pesticides and toxic substances (OECD 2001a, 2001b, 2003; US EPA 1998a, 1998b, 1998c) and food additives (US FDA 2001). These standard toxicity tests generally include the prenatal developmental toxicity study, the multigeneration reproduction study, and the developmental neurotoxicity study. In addition, studies designed to assess specific target organs or systems, toxicities, or pharmacological/toxicological effects for a particular developmental stage may also be required on a case-by-case basis. Such studies could include, for example, developmental immunotoxicity, developmental carcinogenicity, age-comparative biomarker, or juvenile animal toxicity studies. Although postnatal/preweaning direct dosing has been used for decades in several areas of basic research (e.g., Elkes, Eayrs, and Todrick 1955), its incorporation into these standard toxicity studies has not been as well established or accepted.

The exposure period used in developmental studies differs depending on the study (Manson and Kang 1994). In a prenatal developmental toxicity study, the test substance is administered to the mother during gestation, and the fetuses are removed from the uterus just prior to normal delivery. Fetal exposure occurs only in utero, via the placental blood supply; therefore, the prenatal developmental toxicity study protocol is not included in discussions of direct dosing. In a multigeneration reproduction study in rodents, first generation parental animals are administered the test substance for a defined period of time; treatment is typically maintained through the periods of mating, gestation, delivery, and lactation. After weaning, offspring are selected and administration of the test substance to these animals commences and is maintained throughout the growth and reproductive study phases. In a typical developmental neurotoxicity study (US EPA 1998c), maternal animals are administered the test substance from early gestation through mid-lactation or weaning. In these latter paradigms, the offspring are exposed to the test substance indirectly, via in utero exposure during gestation and through maternal milk during the lactation period. Depending on the route of administration used, there could also be some direct exposure of the pups prior to weaning, e.g., through intake of treated food or water.

In these standard study designs, there are usually no data collected or reported regarding the dose to the offspring, and the observation of an adverse treatment-related pre- or postnatal outcome is considered evidence of adequate exposure. Maternal dose levels are commonly used to express dose to the offspring, without any evidence of comparability between mother and offspring. This can lead to either underestimation or overestimation of exposure depending on the kinetics of the chemical in mother’s milk. One possible methodological approach to assure dose availability to the offspring is to directly dose preweaning mammals. Direct dosing techniques can be incorporated into studies such as the multigeneration reproduction study or the developmental neurotoxicity study. Some examples of situations in which direct dosing might be utilized to achieve these objectives include the following:

1. When direct dosing provides the best approximation to potential exposure of the compound of interest to infants and children, e.g., for pharmaceuticals or biologics, which are intended for pediatric use, or for pesticide residues in foods or water that will likely be consumed by infants and children.

2. When direct dosing enhances the delivery of the test substance to the animal model of concern. For instance, direct dosing may be particularly useful when it has been demonstrated that the test substance is not present in milk, or when widely varying quantities of the compound are present in the milk at different times during lactation, or when only an inactive metabolite is present in milk and the concern is over the toxicity of the parent compound.

3. When direct dosing facilitates interpretation of the study data, e.g., when it is suspected that an apparent age-related increase in susceptibility in pups during lactation on a dietary study is actually attributable to increased internal dose (dietary ingestion) to the offspring, or when there are indications that maternal toxicity during the lactation period may be compromising the well-being of the pups.

Direct dosing is defined in this report as the administration of a test substance directly to a preweaning mammal, as opposed to indirect dosing, for example, via maternal milk. This can be accomplished in rodent and nonrodent species using various routes of administration, e.g., oral intubation, inhalation, and subcutaneous, intraperitoneal, or intravenous injection. Weaning is considered to be the age at which mother’s milk (or infant formula) is substituted with other sources of nourishment appropriate to the maturation level of the offspring. Within developmental toxicity studies, weaning is most often accomplished by removing the maternal animal from her offspring or litter at an age or developmental stage when the young animal is able to feed itself within the laboratory environment.

An Expert Working Group was formed by the International Life Sciences Institute Risk Science Institute (ILSI RSI) to address issues unique to direct dosing of potentially toxic substances to preweaning (i.e., nursing) mammals in laboratory research or regulatory screening studies. The full monograph (Zoetis and Walls 2003) provides general guidance on how to undertake a direct-dosing laboratory study on preweaning mammals. The present report provides a summary of the conclusions of the Working Group, which were published in an ILSI RSI monograph (Zoetis and Walls 2003), focusing on studies conducted in common laboratory rodents.

EXPERIMENTAL CONSIDERATIONS AND METHODOLOGIES

Experimental Design

The study design is guided by the specific scientific question(s) to be addressed, whether the objective is to characterize the safety/risk of the test article or to provide basic scientific data. Historically, a number of different experimental designs have been used successfully to administer test articles directly to preweaning mammals. Depending on the hypothesis under investigation, the design can be incorporated as a modification of an existing study or as a separate study. When designing the protocol, several basic considerations must be addressed, including selection of the test species, the route of administration, dose levels, and the timing of doses. Assignment of animals to test groups should allow for meaningful statistical evaluation of the biological responses observed. The parameters that will be evaluated in the study will be based primarily upon the study objectives, but will also depend on other particulars of the study design. A comprehensive evaluation should be included to ensure that no new unexpected toxicity occurs in preweaning animals that might not have been noted or expected in the adult. Additionally, care should be taken to ensure that endpoints added to a standard developmental study do not compromise the integrity of the study design.

Test System/Species

Preweaning mammals may be used to investigate possible effects of a test article on postnatally developing systems or, conversely, to evaluate the influence of the postnatally developing system on the response to the compound. In many cases test animals undergo developmental changes in patterns similar to humans, with timeframes appropriate for their life span. The test system should include preweaning mammals undergoing similar developmental changes as those observed in the age group of children for which the research is directed. Other critical factors in the selection of the test species include existing toxicology data, technical feasibility, and likely route of human exposure, historical use of a particular species, comparative pharmaco/toxicokinetic data when available, and recommendations found in regulatory guidelines.

Timing and Duration of Dosing

The timing and duration of dosing preweaning mammals depends on the developmental stage of interest. Consideration of the differences between species in the time to achieve developmental milestones relative to birth is essential to the utility of the study design. Both structural and functional maturation are important considerations when selecting the appropriate age at which animals should be exposed to test materials.

The US Food and Drug Administration (US FDA 2000) has suggested a categorization of postnatal human development, referenced for clinical investigations of medicinal products in the pediatric population (Table 1). These very general categories provide a developmental basis for considering study designs for the pediatric population and may provide a developmental basis for preweaning direct dosing study designs. The human infant/toddler category corresponds roughly to the usual age at weaning for laboratory species. The preterm infant, neonate, and infant categories in humans will correspond to similar categories in laboratory animals during the preweaning period, and are the stages that this report addresses regarding direct dosing of preweaning mammals.

Cross-species comparison of the maturation profiles for various organ systems should be considered when designing studies for preweaning mammals. However, the process of defining comparable timeframes as they might apply to laboratory animal studies is a difficult task for at least two reasons. First, developmental events are compressed into a much shorter time frame for most laboratory animals compared to those in humans. Second, developmental schedules vary among species, strains within species, particular organ systems, as well as among various components within each organ system. Table 1 provides an example of a comparison of categorical reference information between rats and humans, based on combined developmental events occurring in the central nervous and neuroendocrine and reproductive systems (Kimmel and Buelke-Sam 2001). More detailed information may be obtained from the literature specifically focused on species-specific landmarks of development within selected structural and/or functional systems (Beckman and Feuston 2003; Dobbing and Sands 1973, 1979; Hew and Keller 2003; Holsapple, West, and Landreth 2003; Hurtt and Sandler 2003a, 2003b; Marty et al. 2003; Rice and Barone 2000; Wood, Beyer, and Cappon 2003; Zoetis and Hurtt 2003a, 2003b; Zoetis, Tassinari, and Hurtt 2003).

Developmental Pharmaco/Toxicokinetics

An understanding of the pharmacokinetic parameters (absorption, distribution, metabolism, and elimination) of the test chemical in the test species, as developed in physiologically based pharmacokinetic (PBPK) models, aids in the determination of the need for direct dosing studies (Andersen 2003; Nestorov 2003). For example, a direct dosing study may be indicated if there is no transfer of the test chemical through the milk, but children could be exposed early in life; in such a case, maternal dosing only would not provide sufficient data for evaluating potential effects in children. If direct dosing is selected, then ideally one needs to be able to assess the dose to the target tissue for comparison either with adult animals (to identify potential susceptibility of early life stages as compared to adults) or humans.

One of the difficulties in attempting to use information on pharmaco/toxicokinetics is that the developmental profiles of multiple factors must be considered and integrated, e.g., changes in absorption, distribution, and clearance (metabolic, urinary, exhalation) during development. Thus, PBPK models that describe the known changes in kinetic processes during postnatal development should be useful because they integrate all these factors together to evaluate the outcome (Ginsberg, Hattis, and Sonawane 2004). This requires knowledge of how the development of the key processes would impact the target tissue dose in early life stages.

For compounds that have simple absorption and clearance characteristics, it may be possible to anticipate the impact of alterations in these processes during the preweaning period in order to assist in setting doses or interpreting observed effects. With more complex patterns, it can be very difficult to evaluate correctly the quantitative impact without some kind of PBPK model. For example, birth initiates changes in metabolizing enzymes, in some cases resulting in the disappearance of fetal forms and the appearance of adult forms of the enzyme while in other cases enzymes increase towards adult levels from low fetal levels (Ginsberg et al. 2002; Ring et al. 1999). The rate of absorption via the oral, inhalation, and dermal routes also shows changes after birth (e.g., Shah et al. 1987). Changes in serum proteins that can alter distribution of certain compounds have also been observed (Dziegielewska et al. 1981). Preweaning mammals appear to have less fat and higher water content in lean tissue than adults, resulting in a larger volume of distribution for water-soluble compounds (Ginsberg et al. 2002).

Controls

As with studies in adult animals, at least one concurrent control group is necessary for studies using preweaning mammals. This negative-control group should be treated identically to the treated groups with the exception of receiving the test material (Wilson and Hayes 1994). Additional concurrent control groups may be necessary, including sham, nondosed, or pair-fed controls. Each type of control group serves as a comparison to the treated groups in different ways to improve interpretation of the data.

Route of Administration

The Working Group has used various routes of administration successfully in studies in preweaning mammals. The route of administration should mimic the likely and/or principle route and circumstances of known or potential human exposure, but there are clearly technical factors to be considered. For example, the administration of the test article by inhalation is technically feasible in preweaning rat litters, but this could introduce the confounding issue of multiple concurrent exposures, i.e., via the dermal and oral routes in addition to inhalation.

Physiological and anatomical characteristics of the preweaning mammal also play a role in species selection when considering route of administration. For example, intravenous administration in juvenile rat tail veins can only occur after the tail has developed to the extent to which the vein can be viewed and accessed by the technical personnel.

Dose Selection

In general, the dose selection process for studies using preweaning animals is similar to that for studies using adult animals (Foran 1997). The purpose of the study is the most important consideration in the dose selection process. For studies in preweaning mammals, there are four main sources of information that could assist in dose selection: (1) pilot studies in preweaning animals, (2) toxicity data from adult animal studies, (3) exposure estimates and toxicity data from other developmental studies, and (4) pharmaco/toxicokinetic data.

Assignment to Groups

Assigning animals in litters is an important and unique issue in the design and statistical analysis of studies involving mothers with multiple offspring. Researchers are advised to consult a statistician at the earliest stage of planning studies in preweaning mammals to ensure the ultimate interpretability and success of the study.

For direct dosing studies in rodents, several different methods of assigning offspring to dose groups have been used. All pups within a litter can be in the same dose group, each dose group can be represented within a litter, or pups can be cross-fostered such that the resultant litter represents several birth litters but all pups in the resultant litter are in one dose group. A fourth possibility is cross-fostered pups but with each dose group represented in the resultant litter. It is clear that the hypothesis under investigation should ultimately drive the study design. There are considerations for each method, and the reader is referred to Ruppert, Dean, and Reiter (1983) for a comparison of split- and whole-litter designs. Some procedures require reallocation of pups, and personal experiences of the Working Group members have shown that rat mothers of most strains rarely show rejection of pups they did not deliver when new litters are formed within a few days after birth.

SPECIES SPECIFIC METHODOLOGIES

The ILSI RSI monograph presents issues specific to the husbandry and dosing of rodents (rats/mice), rabbits, dogs, nonhuman primates, and swine. Only an overview of information on laboratory rats and mice is presented below.

Rats and Mice

Rodents, particularly rats, are commonly used species for studies of developing animals. Rats may be preferred over mice mainly due to their larger size. In addition, rats have historically been an accepted species by regulatory authorities for studies submitted to support the safety/risk assessment of a wide variety of compounds. This historical use of rats for testing provides three important pieces of information for the conduct and interpretation of studies in preweaning mammals. First, there exists a large database of information from adult rat studies of a large number of chemicals. Second, historical control information on parameters evaluated in pre-weaning rat pups are typically maintained by laboratories conducting developmental studies, and they are expanding these databases to include postnatal developmental information. These databases will prove useful in the understanding of effects of exposure over the animals’ life. Finally, there already exists technical expertise in the handling and husbandry of rat dams and pups.

Rats and mice have several reproductive characteristics that facilitate their use in preweaning mammal studies (Derelanko 2000). These include a relatively frequent estrus cycle, fecundity, and ample litter sizes. Rats and mice have a gestation period of approximately 21 days (19 to 21 days for mice), and produce an average of 9 to 12 pups per litter, depending on the strain. Parturition typically takes 1 to 3 h.

Carefully coordinated timing of mating and conception is essential to obtaining sufficient numbers of pups at the same age to conduct a study in preweaning rodents. This timing of mating and subsequent delivery is manageable for rodents and is accomplished by either breeding in-house or by purchasing timed-pregnant females from animal suppliers.

Rat and mouse pups are born hairless with closed eyes and ear canals, and virtually no motor or independent thermoregulatory abilities (Fox 1965). The only sensory ability that is well developed in the newborn is olfaction, which aids the pup in suckling and maintaining contact with the dam and littermates in the nest. The pink skin is somewhat transparent and it is very easy to see the milk in the pup’s stomach, sometimes referred to as the “milk band,” during the first few postnatal days. Although the anogenital distance is commonly used for distinguishing young males from females, caution is advised if using a test article with endocrine-mediating activity that may alter this parameter.

The pups are kept with their mothers except when separated for dosing and observation. The separation periods should be minimized; however, separation of rat pups for up to 6 h can be used without untoward effects (Pryce, Bettschen, and Feldon 2001; Stanton, Crofton, and Lau 1992). During separation for prolonged periods, all equipment supporting the pups should be kept warm to prevent hypothermia, e.g., by using a heating pad.

Tremendous growth and development takes place during the first postnatal days and weeks (Fox 1965). Within the first 2 weeks, the eyes have opened, pinna (outer ear) detachment has occurred, hair has emerged, and motor ability has improved tremendously. Weaning in the laboratory typically occurs at about 21 days of age for both rats and mice, and thus, preweaning direct dosing studies would last no longer than that.

Due to their small size and thin skin compared to other neonatal laboratory species, the method of identifying rat and mouse pups should be carefully considered. For individual identification, possible methods include the use of permanent marking pens to mark skin or tail and subdermal injection of tattoo ink on the footpad or tail (Allmann-Iselin 2000). Although these markings are not always permanent and may have to be repeated during the course of study, many members of the Working Group have used them with considerable success. Other identification procedures, e.g., toe clipping and ear notching, should be considered only with adequate justification, because these procedures breech the skin and could render the pup susceptible to maternal rejection.

Dosing Techniques

General recommendations for dosing methodologies are available in the literature but are generally focused on adult animals (e.g., Nebendahl 2000; Waynforth and Flecknell 1992). These procedures may be modified to accommodate the immature animal. For all routes of direct dosing in the preweaning mammal, restraint should be appropriate for the age and dexterity of the animal and species. For injections, the smallest needle diameter that allows reasonably swift administration should be used (e.g., 27 to 30 gauge). The length of the needle should only be long enough to reach the intended injection site. The smallest possible volume compatible with the solubility, concentration, pH, viscosity or other pertinent characteristics of the test compound should be used (e.g., 1 to 10 ml/kg). The syringes used for dosing should accurately measure the volume being administered (e.g., 0.5-cc syringes). Animals should be closely observed for any adverse effects of the dosing and handling procedures. The technical staff should be thoroughly trained in dosing preweaning animals.

The common route of direct oral administration in preweaning mammals is by oral gavage. In rodents, oral dosing in preweaning rats is possible from postnatal day (PND) 1 but more often begins at PND 4. Due to rapid growth rates, pups usually are weighed and dose calculations adjusted at the time of each dose. Dosing must be undertaken with care to avoid damage to the thin tissues, but with practice, this can be accomplished as a routine laboratory procedure. Successful studies have been conducted in rats using either flexible cannula tubing or a 1.5- or 3-inch intubation needle (27 gauge), curved or straight, with or without a ball tip. In mice, a cut and marked premature infant feeding tube can be used. The tip of the dosing apparatus should be smooth to avoid irritation or injury and can be lubricated with corn oil. Although the volume of administration should be as low as possible, 10 ml/kg is typically used (e.g., 0.15 ml for a 15-g pup). The Working Group felt that gavage dosing of preweaning rodents could be reliable and efficient with proper training and experience of the laboratory personnel.

Rodents can be dosed using the intravenous injection procedure from approximately PND 3. A magnifying lamp and/or wiping the tail with isopropyl alcohol or warm water can be used to visualize the veins of the tail as necessary (Rice et al. 1991; White et al. 1993). Both intraperitoneal and subcutaneous injections can be accomplished in preweaning rats using small (26 to 27 gauge) needles and small dose volumes; however, caution is advised. The limited space in the abdomen increases the likelihood of inadvertently puncturing an internal organ, as well as seepage from the injection site that can lead to maternal rejection.

Dermal exposures in preweaning rodents are generally not recommended. The architecture of rodent skin does not closely mimic human skin and species differences in absorption and metabolism may become important. In addition, dermal application of any test material can lead to maternal rejection due to changes in the smell of the pup.

Studies conducted in preweaning rodents via the inhalation route may be conducted using whole body exposure of the entire litter (Vitarella et al. 1998; Dorman et al. 2000). Depending on the chemical, however, this method may result in an inexact estimate of exposure. Pups in whole body inhalation exposure experiments may be exposed to additional compound through the mothers’ milk, dermally, or perhaps orally after the onset of grooming behavior.

Confounding Factors

Confounding factors can be easily and inadvertently introduced into studies using preweaning mammals. The experimental design should minimize confounds as much as possible. In addition to typical confounds (e.g., staggered testing, cage assignment, etc.), the maternal-offspring interaction is a critical factor in the success of studies using preweaning mammals. Nurturing behavior of the maternal mammal affects the ability of the offspring to survive and thrive, and therefore investigators should consider any factors that may interfere with this interaction as a potential confounder.

The maternal response to the pup is affected by various cues, some of which can be controlled during the preweaning rodent study. Olfactory cues can be controlled by using gloves when handling the pups, and by ensuring that the handled pups smell the same as the rest of the litter before reintroducing them to the dam. Rat dams may reject injured (e.g., bleeding) pups or those showing toxicity or failing to thrive. The cues for maternal rejection and nurturing are complex and should be normalized among dose groups to the greatest extent possible.

The chemical and physical properties as well as volume of the vehicle may also alter the outcome of the preweaning mammal study. For example, it is possible that a highly viscous vehicle may affect nursing behavior of the pups and resultant milk supply of the dams. Likewise, oral dosing with high volumes may satiate the pup and decrease nursing behavior. Dosing volumes should be considerably less than typical milk intake, which ranges from around 1 g/day to 2–2.5 g/day (around PND 10) (Maeda, Onkura, and Tsukamura 2000).

DATA INTERPRETATION

There are a few issues unique to the interpretation of studies using preweaning mammals, e.g., the maternal-offspring interaction. Specific methods of data interpretation may vary based on the purpose for which the study has been conducted (i.e., basic research, testing, regulatory requirements), the chemical and/or physical properties of the test article, and potential for human exposure. The ILSI RSI monograph describes these issues in more detail.

Statistical Issues

Appropriate statistical treatment of the data is required for any toxicological study, but this issue becomes more complex when analyzing data from developmental studies. Both maternal and genetic influences must be recognized and accounted for in any developmental study using a multiparous species, in particular, the laboratory rodent (Holson and Pearce 1992).

It is generally accepted that offspring within a litter (i.e., siblings) are more alike than nonsiblings. The specific approach used to consider within- and between-litter variances is dictated by the experimental design. The reader is encouraged to consult a statistician and/or the literature before designing any study in which more than one pup per litter is used (e.g., Abbey and Howard 1973; Cox 1994; Holson and Pearce 1992; Riley and Meyer 1984).

Biological Issues

Data interpretation of biological issues should be addressed in light of the purpose of the study. Specific guidance regarding data interpretation is beyond the scope of this report. Issues to consider when interpreting data from preweaning mammal studies include husbandry and handling, maternal-offspring interactions, anatomical features of the newborn, and pharmaco/toxicokinetic data.

SUMMARY

The ILSI Expert Working Group concluded that direct dosing of preweaning animals is a commonly used methodology that is applicable for use in regulatory toxicity testing that includes assessment of exposures to laboratory animals during postnatal development. Care must be taken to select the appropriate species, route of administration, dose levels, dosing methodologies, and timing and duration of exposure. Successful study design and implementation must also include consideration of pharmaco/toxicokinetic data and knowledge of critical windows of development for the specific organ systems or processes being evaluated. Good experimental design, minimizing confounding variables, and appropriate statistical analyses are required for interpretable studies. The ILSI RSI monograph contains details to help guide the researcher in making these decisions; however, experience and consultation with others are often crucial elements of designing and conducting a successful study that includes the application of direct dosing in preweaning mammals.