For the risk assessment and labelling of pharmaceuticals, food additives, crop protection products and chemicals, multi-generation reproduction and developmental neurotoxicity studies provide important information of specific relevance to infants and small children. Most regulatory bodies require data from one or two species and the rat is the rodent species of choice to perform such studies.
Historically, toxicokinetic monitoring in developmental toxicity studies has been focused largely on the detection of test item in the dams, based on a general assumption that the offspring are automatically exposed in utero and via the milk. In the light of the forthcoming OECD Guideline, this approach is unlikely to find regulatory acceptance in the future. In an exploratory study, the authors investigated the milk transfer of a xenobiotic compound in rats with the help of 14C-labelled test compound.
According to the relevant EPA guideline and the draft OECD Guideline No 426, exposure of the dams and/or the offspring has to be accomplished for the entire period from the day of implantation (day six after gestation) to weaning (day 21 after delivery). In the first two postnatal weeks, exposure of the offspring is ideally accomplished through the milk, although direct dosing of the offspring may be required in some cases. OECD Guideline no. 426 emphasizes the importance of sound toxicokinetic data to support instances where direct dosing of the test compound to the pups is not performed. "In general, it is assumed that exposure of the pups will occur through the maternal milk. However, direct dosing of pups should be considered in those cases where there is a lack of evidence of continued exposure to offspring. Evidence of continuous exposure can be retrieved from for example, pharmacokinetic information, offspring toxicity or changes in biomarkers."
Although toxicokinetic data for the dams may be obtained by measurement of plasma levels of the parent test compound, proof of exposure for the offspring is more demanding. Whole-Body Autoradiography (QWBA) is a well-established method to monitor the exposure of the offspring in utero. Demonstration of postnatal exposure via the milk can be more challenging, as not only the parent compound but also maternal metabolites may be passed on to the offspring. If toxicokinetic monitoring of the suckling pups is to include the parent compound and metabolites, the use of standard HPLC-MS techniques require reference compounds and expensive validation procedures.
An alternative approach is the investigation of fetal and postnatal exposure in rats in a separate toxicokinetic study using radiolabelled test compound. We present data from an exploratory study on the milk transfer of a xenobiotic in rats with 14C-labelled test compound. Milk, blood and plasma of lactating females were sampled on four occasions during the lactation period to investigate the concentration of radioactivity in the collected specimen and to calculate the milk to plasma ratio (M/P). Sampling times after dosing were chosen based on the pharmacokinetics of the test compound in rats, at tmax and t(Cmax/2).
Direct dosing
In toxicological studies the route of administration that best mimics the most likely route of human exposure to the product should be used. For human pharmaceuticals, this is defined by the proposed route of therapeutic administration. For agrochemicals, as for veterinary pharmaceuticals, adventitious consumer exposure generally occurs through the food, while dermal and/or lung exposure is of relevance for agricultural workers and for those involved in the manufacture of these products.
Dosing of the maternal animals in multi-generation reproduction or developmental neurotoxicity studies does not necessarily expose the fetuses and neonates to the test compound and metabolites. However, in the past, toxicokinetic data were rarely reported in dossiers, which were based on the standard EPA guideline. As this is likely to change with the new OECD Guideline No 426, which is expected to be finalized in 2007, more attention to this question will be required in the future.
The first question to be answered in the design of a multigeneration study is whether to rely on passive exposure via the milk or dose the test compound directly to the pups. For those pharmaceuticals with pediatric applications, direct dosing is the most reliable way to ensure quantitative and reproducible exposure of the offspring, while for pharmaceuticals intended for adult use, the only relevant exposure for the offspring is through the milk.
For pesticide residues in foods or water, both routes of exposure are relevant to infants and children: uptake of residues through the milk and direct consumption with the food. Although direct dosing of preweaning animals is feasible at least by the oral, intravenous and intraperitoneal routes, it requires high technical skills and may be difficult to perform in a routine testing environment. A further consideration is the possibility of additional stress to the juvenile animals interfering with the toxicological end points.
Therefore, exposure of the pups through milk has both practical and scientific advantages, if exposure of the offspring to the test compound can be demonstrated unequivocally.
Transfer of xenobiotics into milk
The exposure of the offspring to a xenobiotic compound through the milk after administration to the dam depends on a variety of factors like the molecular properties of the test compound, as well as on the physiology of both dam and offspring. Maternal metabolism, overall milk yield, fat and protein content, pH of the milk as well as metabolism in the offspring have a marked influence on the toxicokinetic behavior of a test compound. Furthermore, molecular properties such as lipophilicity, protein binding, and pKA value of a xenobiotic compound determine its uptake and retention in milk.
Milk composition, especially fat content, depends on food intake of the dam and additionally shows changes during feeding (hind vs. fore milk), diurnal changes and changes during the lactation period. These effects have been studied in humans, but relatively little is known about dynamic changes in milk composition in common laboratory animal species.
A robust paradigm for a toxicokinetic study supporting multi-generation reproduction and developmental toxicity studies should therefore include milk and blood sampling at several time points post dosing and on several occasions during the lactation period.
Milk transfer study in rats
Method: All experimental animal work was carried out under a project specific licence issued by Swiss animal welfare authorities responsible for the canton of Baselland.
Lactating rats were treated with oral doses of test compounds at two dose levels, 50 mg/kg body weight (Group 1) or 500 mg/kg body weight (Group 2). Dose selection would be expected to bracket the dose levels employed in the subsequent developmental study. Twelve lactating dams (6 for each dose group) were dosed, by gavage, on days 2, 4, 8, and 12 of lactation (day 1 of lactation = the day after completion of parturition). Each dose group was further divided into subgroups of 3 lactating dams each. The concentrations in maternal milk, whole blood and plasma were determined at two time points (one per subgroup) after each oral dose, at both dose levels. The time points were selected based on a previously conducted pharmacokinetics study, which showed tmax values in blood of 0.5 and 2.0 hours and t (Cmax/2) values of 1.5 and 4.0 hours for the low and the high dose groups, respectively.
Three hours prior to milking, the dams were separated from the pups and milk injection into the mammary glands was stimulated by an intraperitoneal injection of oxytocin (4IU/kg) about 5 minutes prior to milking. The milk specimen was obtained by an in-house built vacuum driven milking pump in conscious animals (see Figure 2). Milk samples were frozen for determination of total radioactivity by liquid scintillation counting. After milking, a blood sample of about 0.5 ml was withdrawn from the sublingual vein from each animal at the selected time point. Aliquots were separated and used for determination of total radioactivity in plasma by liquid scintillation counting. After milk and blood sampling the dams were placed back with their pups.
Results and conclusion
When the test compound was administered to the lactating rat as single oral doses of 50 or 500 mg/kg, the parent compound and/or metabolites were absorbed from the GI tract and could be found in the plasma. In the low dose group, the concentration of the test substance in plasma reached a level of 32-35 µg (parent equivalent)/ml 0.5 hours after administration. Test compound and/or metabolites rapidly partitioned into maternal milk at concentrations up to approximately 1.5 to 2-fold higher than those of whole blood or plasma. At the second sampling time point (1.5 h), the concentrations in plasma and milk had decreased by approximately 50 per cent, but milk concentrations remained consistently higher than those in plasma on all lactation days evaluated.
In the high dose group, the kinetic profile closely mirrored that of the low dose, the concentrations in blood and plasma were 140 µg/ml, those in milk 180 µg/ml, 2 hours after administration. At 4 hours after administration the test item concentration in milk decreased to half the maximum level.
The levels of radioactivity in maternal plasma were not markedly influenced by the day of lactation within the range examined (day 2-day 12), but there was a trend towards increasing concentration of radiolabel in milk later in the lactation period.
The concentrations determined in plasma and milk after administration at the high dose level (500 mg/kg) were only 4 times higher compared to the 10-fold higher dose level.
During the first half of the lactation period, when neonatal nutrition is almost exclusively derived from maternal milk, the data strongly suggested that pups would be exposed to test compound and/or metabolites from birth to at least day 12 of the lactation period. Since milk concentrations of test compound-derived radioactivity showed no evidence of decline as lactation progressed from day 2 to day 12, the data also suggest continued exposure of suckling pups via the maternal milk beyond day 12 of lactation.
In conclusion, the study provided evidence of neonatal exposure of suckling pups to test compound and/or its metabolites via the maternal milk.
Discussion
The milk transfer study shows an approach to generate supporting toxicokinetic information that demonstrates exposure of rat pups to radio labelled test compound and/or maternal metabolites. Depending on the nature of the test compound and the extent of maternal metabolism, the measurement of total radioactivity may be complemented by metabolite profiling in the milk.
Another important issue in the experimental design of a toxicokinetic study in lactating rats is the specification of sampling occasions during the lactation period and the number of sampling time points at each occasion. Several sampling occasions will help to detect fluctuations of milk transfer during the lactation period. More time points per occasion can provide a more robust estimate of the total quantities of test compound and metabolites excreted through the milk and thus of the overall exposure of the pups.
An alternative approach to measure radioactivity in milk samples is the determination of radiolabel in dams and pups by QWBA. This method has the potential not only to demonstrate the exposure of the offspring to the test compound and metabolites, but also the systemic uptake and distribution into potential target tissues such as the CNS.
Driven by increasing regulatory demands, these technologies will become more important. However, there will be no standard design for generation toxicokinetic studies as prior information about the pharmacokinetics and metabolism, as well as its intended future use will determine the best way to proceed.
(The authors are with Environmental Safety and Metabolism)