Juvenile Nonclinical Safety Studies in Support of Pediatric Drug Development.

A pediatric assessment is now a required component of every drug marketing application in North America, Europe, and Japan, unless a waiver has been granted previously. Nonclinical juvenile toxicity studies are often required as part of this assessment. The protocols for juvenile toxicity studies are best devised in consultation with the regulatory authorities. It is important to submit the pediatric investigation plan (PIP) or pediatric study plan (PSP) early, in order not to delay the marketing authorization of the drug in adults. The choice of species and the design of juvenile toxicity studies are based on a series of complex considerations, including the therapeutic use of the drug, age at which children will be treated, duration of treatment, and potential age- or species-specific differences in efficacy, pharmacokinetics, or toxicity.

The teratology testing of food additives.

The developmental and reproductive toxicity testing (including teratogenicity) of new foods and food additives is performed worldwide according to the guidelines given in the FDA Redbook. These studies are not required for substances that are generally recognized as safe, according to the FDA inventory. The anticipated cumulated human exposure level above which developmental or reproduction studies are required depends on the structure-alert category. For food additives of concern, both developmental (prenatal) and reproduction (multigeneration) studies are required. The developmental studies are performed in two species, usually the rat and the rabbit. The reproduction study is generally performed in the rat. The two rat studies are preferably combined into a single experimental design, if possible. The test methods described in the FDA Redbook are similar to those specified by the OECD for the reproductive toxicity testing of chemicals.

Developmental toxicity testing of vaccines.

Preventative and therapeutic vaccines are increasingly used during pregnancy and present special considerations for developmental toxicity testing. The various components of the vaccine formulation (i.e., protein or polysaccharide antigen, adjuvants, and excipients) need to be assessed for direct effects on the developing conceptus. In addition, possible adverse influences of the induced antibodies on fetal and/or postnatal development need to be evaluated. A guidance document on the preclinical testing of preventative and therapeutic vaccines for developmental toxicity was issued by the FDA in 2006. Preclinical studies are designed to assess possible influences of vaccines on pre- and postnatal development. The choice of model animal for these experiments is influenced by species differences in the timing and extent of the transfer of the induced maternal antibodies to the fetus. The cross-placental transport of maternal immunoglobulins generally only occurs in late gestation and tends to be greater in humans and monkeys than in non-primate species. For many vaccines, the rabbit shows a greater rate of prenatal transfer of the induced antibodies than rodents. For biotechnology-derived vaccines that are not immunogenic in lower species, nonhuman primates may be the only appropriate models. It may be advisable to test new adjuvants using the ICH study designs for conventional pharmaceuticals in addition to the developmental toxicity study with the final vaccine formulation.

The teratology testing of cosmetics.

In Europe, the developmental toxicity testing (including teratogenicity) of new cosmetic ingredients is performed according to the Cosmetics Directive 76/768/EEC: only alternatives leading to full replacement of animal experiments should be used. This chapter presents the three scientifically validated animal alternative methods for the assessment of embryotoxicity: the embryonic stem cell test (EST), the micromass (MM) assay, and the whole embryo culture (WEC) assay.

Reporting of teratology studies.

The regulatory toxicology report is an unusual document that requires a particular skill to write. The report must be clear, accurate, concise, and focused. A clear and direct writing style is required. The end-users of the report will hope to find the information they seek with as little effort as possible. Few, or none, will read the entire document. The author should aim to appease the user by obliging him to read as little text and turn as few pages as possible. This chapter gives tips and guidance on how to present the experimental data and write the narrative text in the final study report for a teratology study.

Preclinical evaluation of juvenile toxicity.

A pediatric assessment is now a required component of every New Drug Application in North America or Marketing Authorization Application in Europe, unless a waiver has been granted previously. Nonclinical juvenile toxicity studies are usually required as part of this assessment. The protocols for juvenile toxicity studies are devised in consultation with the FDA or EMEA. It is important to approach the regulatory authority well in advance in order not to delay the marketing authorization of the drug and to confirm the need or not to perform a preclinical evaluation in juvenile animals. The choice of species and the design of juvenile studies are based on a series of complex considerations, including: the therapeutic use of the drug, the age at which children will be treated, the duration of treatment, and potential age- or species-specific differences in pharmacokinetics or toxicity.

Reproductive toxicity testing for pharmaceuticals under ICH.

Reproductive toxicity testing of drugs is performed as a 2- or 3-segment package. The choice of species for routine studies with small molecule drugs is essentially limited to the rat, rabbit, mouse and minipig. The lack of alternative species is a threat to the successful screening of drugs for teratogenicity. A proposed revision of the ICH M3 guideline addresses contradictions concerning the timing of the various reproductive and juvenile studies. This M3 draft, however, raises questions concerning the confidence that can be attributed to preliminary embryotoxicity studies with as few as six females per group. The detection of reproductive hazards could be improved by implementing methods in routine use for the testing of chemicals, such as double staining of fetuses and primordial follicle counts. Modern imaging techniques hold promise for application in the morphological examination of fetuses. An assessment of developmental immunotoxicity should be included in any future revision of the reproductive toxicity guidelines.

Juvenile toxicity of cyclosporin in the rat.

The pups from 32 litters of SD rats were given 0, 1, 3 or 10mg/kg-d of cyclosporin by oral gavage from 4 to 28 days of age. 10mg/kg-d resulted in a persistent impairment of the primary antibody response at 10 weeks of age. Indications of systemic toxicity, including the death of 10/64 pups and severely depressed weight gain, were also observed at this dose level. Arteriopathy of the heart and tubular basophilia and edema in the cortico-medullary region of the kidney were observed at 3 and 10mg/kg-d. In conclusion, while pharmacological effects were seen at all dose levels, the adverse effects of cyclosporin on the development of the immune system in the rat only occurred at a dose level that also induced systemic toxicity.

Developmental immunotoxicity investigations in the SD rat following pre- and post-natal exposure to cyclosporin.

BACKGROUND: The development and function of the immune system was assessed in juvenile SD rats following pre- or post-natal exposure to cyclosporin. The main objective was to assess the feasibility of the methods available for the detection of adverse effects on the development of the immune system for use in the safety assessment of medicines. METHODS: In a pre-natal experiment, 15 pregnant rats were given 10 mg/kg/day of cyclosporin by gavage from day 6 of gestation until 4 days after parturition. A control group received olive oil. In a post-natal experiment, the pups from 35 litters were given 10 mg/kg/day of cyclosporin by gavage from 4 to 28 days of age. Half of the pups in each litter were given water and acted as controls. Immune endpoints were determined in the pups in both experiments from two to 10 weeks of age, including: lymphocyte subsets, serum immunoglobulin titres, serum autoantibodies, primary antibody response to sheep red blood cells (SRBC), delayed-type hypersensitivity response, humoral response to keyhole limpet haemocyanin, spleen cellularity, immune organ weights, and histopathology. RESULTS: Pre-natal exposure caused no effects on immune function. Post-natal exposure caused immune depression during the treatment period and a persistent impairment of the immune system characterised by lymphoid hyperplasia in the spleen and a reduced primary antibody response to SRBC at 10 weeks of age. CONCLUSIONS: These results demonstrate the importance of a post-treatment follow-up period in developmental immunotoxicity studies, in order to distinguish between the transient effects of immune modulation and the persistent consequences of developmental toxicity.

Immune assessments in developmental and juvenile toxicology: practical considerations for the regulatory safety testing of pharmaceuticals.

The developing organism is considered to be more sensitive than the adult to immunotoxic agents. There is every reason, therefore, to include immune assessments in the regulatory testing for developmental toxicity of drugs that are intended to be used in young patients or pregnant woman. An effective strategy would be to incorporate immune assessments in the existing recommendations on pre- and post-natal toxicity study in the rat from the International Conference on Harmonisation. Immune assessments could also be included in juvenile toxicity studies to screen for effects resulting from post-natal exposure to the drug. Adequate testing methods are available to screen for developmental effects that result in immune depression. Routine immune assessments may comprise histopathological examination of the lymphoid organs/tissues and immunophenotyping of lymphocyte subsets in the blood, spleen, or thymus. These tests should be performed in rodents at various ages and at various stages of pre- and post-weaning development. Immunoglobulin and cytokine measurements, assessment of the T-cell dependent antigen response to sheep red blood cells or keyhole limpet haemocyanin antigens, and host resistance studies may be performed as apical tests at maturity. More research is required to develop methods for the detection of drugs that may render the developing organism more susceptible to hypersensitivity or autoimmunity.

Reproductive toxicology studies and immunotherapeutics.

There are many ways in which immunotherapeutic agents could potentially cause developmental toxicity. According to the published data in humans and animals, however, this class of drugs does not appear to present any increased risk of reproductive toxicity by comparison with other therapeutic classes. The basic testing strategy outlined in the ICH guidelines is suitable for the preclinical reproductive toxicity assessment of these drugs (except vaccines). Particular consideration needs to be given to the choice of species when testing antibodies or human molecules. Immune-active drugs may have the potential to interfere with the development of the immune system. For this reason, it may be advisable to assess the integrity of the immune system in animals previously exposed to the test substance during development. Such tests can easily be appended to the study design of the pre- and post-natal study. In view of the lack of any provision for post-weaning exposure in the guidelines and the late maturation of the immune system, a separate post-natal study may be considered for drugs that are likely to be prescribed to children.

Reproductive toxicity testing of vaccines.

Vaccines play a major role in the prevention of human birth defects by protecting the pregnant woman from teratogenic or otherwise harmful infections. Until now, it has not been common practice to perform preclinical developmental toxicity tests for new vaccines. Despite the excellent safety record of vaccines, increased attention is now being given to the feasibility of screening new vaccines for developmental hazards in animals before their use in humans. Contrary to previous assumptions, many vaccines are now given to potentially pregnant women. Any new components of the vaccine formulation (adjuvants, excipients, stabilisers, preservatives, etc.) could also be tested for influences on development, although based on past experience the risks are limited by the very low dosages used. The conferred immunity following vaccination lasts for several years. Therefore, the developing conceptus may theoretically be exposed to the induced antibodies and/or sensitised T-cells, even if the pregnant woman was last vaccinated during childhood (particularly if she encounters the antigen during pregnancy through exposure to infection). However, it should be kept in mind that viral or bacterial infections represent a higher risk for a pregnant woman than the potential adverse effects related to vaccination or the associated immune response. Non-clinical safety studies may be employed as an aid for hazard identification. In these studies interactions of the vaccine with the maternal immune system or with the developmental systems of the offspring are considered. Post-natal examinations are necessary to detect all possible manifestations of developmental toxicity, such as effects on the immune system. Species selection for the preclinical studies is based on immunogenicity to the vaccine and the relative timing and rate of transfer of maternal antibodies to the offspring. A single study design is proposed for the pre- and post-natal developmental assessments of vaccines in rodents and rabbits.