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

Regulatory Toxicology and Pharmacology 43 (2005) 35–44 www.elsevier.com/locate/yrtph

Immune assessments in developmental and juvenile toxicology: Practical considerations for the regulatory safety testing of pharmaceuticals 夽 Paul C. Barrow ¤, Guillaume Ravel MDS Pharma Services, Les Oncins, 69210 Saint-Germain sur l’Arbresle, France Received 14 March 2005 Available online 15 August 2005

Abstract 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 eVective 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 eVects resulting from post-natal exposure to the drug. Adequate testing methods are available to screen for developmental eVects 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.  2005 Elsevier Inc. All rights reserved. Keywords: Developmental immunotoxicology; Juvenile toxicology; Risk assessment

1. Introduction Interest in developmental immunotoxicology has been stimulated following suggestions over recent years that xenobiotics, including environmental contaminants, pesticides, food additives, and drugs, may be capable of inXuencing the human immune system in such a way as to render the population more vulnerable to diseases. It has been shown that the developing human (i.e., embryo, 夽 All of the results presented in this paper were generated in the Sprague–Dawley rat (OFA.SD.(IOPS Caw), supplied by Charles River Laboratories, Saint Germain sur l’Arbresle, France). * Corresponding author. Fax: +33 4 74 01 63 99. E-mail address: [email protected] (P.C. Barrow).

0273-2300/$ - see front matter  2005 Elsevier Inc. All rights reserved. doi:10.1016/j.yrtph.2005.06.001

fetus, new-born, infant, or child) is more sensitive to the eVects of several immunotoxicants than the adult (Holladay and Luster, 1994; Landrigan et al., 2004). The pharmaceutical, chemical, and agrochemical industries, therefore, have every interest in developing methods with which to assess the safety of their products with respect to adverse eVects on the developing immune system. The possible manifestations of immunotoxicity include the following: reduced resistance to bacterial, viral, or parasitic infectious diseases; more frequent virus-associated malignancies; increased susceptibility to allergy or idiopathic autoimmune diseases; drug-induced hypersensitivity; and drug-induced autoimmunity (Descotes, 2004b). The methods and protocols currently proposed

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for the immunotoxicology screening of pharmaceuticals and chemicals are broadly based on the recommendations of the National Institute of Environmental Health Sciences National Toxicology Program (Luster et al., 1992). The U.S. Food and Drug Administration (US FDA) has issued a guidance document on the immunotoxicology evaluation of investigational new drugs (US FDA, 2002). Also, concern for a lack of agreed regulatory approach to immunotoxicity testing per se led to the recent publication of a draft guideline from the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) on the topic (ICH, 2004). Both these documents, however, cover only immunotoxicology testing in adults. The majority of developmental immunotoxicity studies to date have concentrated on immunosuppression (Holsapple, 2002). It has also been suggested that some medicines may play a role in the induction of autoimmune diseases. Although childhood vaccines are generally considered safe based on the large clinical experience, there have been case reports of autoimmune diseases following vaccination, such as multiple sclerosis, diabetes, or Guillian–Barre syndrome (Classen and Classen, 2001; JeVerson and Heijbel, 2001; Ropper and Victor, 1998). No causal links have been demonstrated to date (Vial and Descotes, 2004), however. An involvement of medicines has also been suggested in the aetiology of childhood asthma (Landrigan et al., 2004; Richter-Reichhelm et al., 2002); though nutrition, environmental chemicals and increased standards of hygiene all appear to play a role (Armstrong et al., 2005). It has been postulated that the lack of exposure to potential pathogens during immune development may render the child more prone to allergy due to faulty T-cell programming (Rook and Stanford, 1998).

2. Manifestations of immunotoxicity in the immature organism Pre-clinical immunotoxicity studies are designed to detect adverse eVects of xenobiotics on the immune system of the adult animal. By far the most reported and best understood manifestation of immunotoxicity in animal studies is immune depression (Descotes, 2005). The fetus, neonate and juvenile are often more sensitive than adults to the adverse eVects of toxic agents, including immunotoxicity (Schwenk et al., 2003). The range of possible toxic eVects in the developing organism is much wider than in the adult, in view of the complex and varied ontogenic processes underway at the various stages of development. Furthermore, immune eVects that are transient in the adult may prove to be permanent following developmental exposure (Dietert et al., 2002). This has been demonstrated in rats following in utero exposure versus adult exposure with dexamethasone (Dietert

et al., 2003), but was not conWrmed with cyclosporine (Hussain et al., 2005). The nature and degree of immune eVects also vary markedly depending on the pre- or postnatal stage of development at the time of exposure. For example, fetal exposure of rodents to cyclosporine blocks thymocyte development and delays immune development, whereas adult exposure predominately aVects the size of the thymus, corticomedullary ratios and the delayed-type hypersensitivity response (Dietert et al., 2002; Hussain et al., 2005).

3. Applications of immune assessments in immature animals Immune assessments in immature animals have two major applications in regulatory safety testing: (1) developmental immunotoxicology and (2) screening for immunotoxicity in juvenile toxicology studies. Both of these topics are of high current concern to the regulatory authorities involved in the safety assessment of medicines. 3.1. Developmental immunotoxicology Developmental immunotoxicology is a relatively new Weld of research. Reports that the immune system of the immature organism may be more sensitive to toxic insult than that of the adult started to appear in the mid- to late 1970s (reviewed by Roberts and Chapman, 1981). Many xenobiotics have now been shown to cause developmental immunotoxicology in animals. The chemicals involved include pesticides, environmental pollutants, industrial reagents, and medicines (Descotes, 2004a; Holladay and Smialowicz, 2000). Most of the developmental immunotoxicants identiWed to date are known to be immunotoxic in the adult (Descotes, 2004a). The induction of persistent immune deWciency in the rat following pre-natal exposure to low doses of diazepam (Schlumpf et al., 1994), however, raised the worrying prospect that the developing immune system may be a preferential target for drug-induced immunosuppression. Similar results were obtained with 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) (Neubert et al., 1994). At present, there are no requirements to test new drugs for developmental immunotoxicology, but the regulatory agencies have expressed an interest in this area (Holsapple, 2002). In fact, the current harmonised guidelines for the reproductive toxicity testing of medicinal agents (ICH, 1994) make no provision for the post-natal exposure of the developing animal other than via the milk following treatment of the mother. This means that adverse drug eVects on the later stages of immune development are unlikely to be detected in routine safety testing studies. Such eVects

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can, however, be detected in an appropriately designed juvenile toxicity study, provided that animals are retained for an immune assessment following a treatment-free follow-up period (see below) to distinguish between direct immune modulation and developmental defects. Developmental toxicants have three major common properties (Schmidt and Johnson, 1997). (1) They provoke persistent alterations, which result from a defect in development. (2) The manifestations of toxicity are phase-speciWc and vary according to the timing of exposure. (3) The observed eVects are more severe or occur at lower doses following developmental exposure than the eVects seen following exposure of the adult. These characteristics are used to distinguish between general and developmental toxicity. In the few animal studies performed to date to compare the sensitivity to immunotoxicants across life stages, the embryo, fetus, and neonate have proved to be more sensitive to immunotoxic insult than the adult (Dietert et al., 2003). The study designs used for the detection of developmental immune eVects will inevitably be more complicated than those used today for the detection of other types of developmental eVects, owing to the extremely long and complex ontogenic mechanisms involved in immune development. The potential period of vulnerability of the developing immune system starts during early in utero development, with the initiation of haemopoiesis (Kimmel et al., 2005; Leibnitz, 2005). Immune development then continues throughout preand post-natal life, with a series of closely orchestrated events involving sequential waves of haematopoietic cell production and cell migration, resulting in Wnal maturation at about the time of puberty (Dietert et al., 2000; Holladay and Smialowicz, 2000). This situation is unusual in developmental biology, since most other organ systems have much shorter and well-deWned windows of development (Schardein, 2000). Two other organ systems with comparable complexity and length of development are the central nervous system (CNS) and reproductive system. Indeed, there are many interactions between the immune organs and neuroendocrine systems during the course of development (Basta, 2005; Spinedi et al., 2005). All regulatory guidelines for the testing of drugs for developmental toxicity were written with the principal aim of detecting adverse reproductive outcomes following exposure during organogenesis (Francis, 1994; Palmer, 1981). The vast majority of morphological birth defects can only be induced during the embryonic period (Hoar and Monie, 1981). The regulatory guidelines for the testing of pharmaceuticals do make some provision for the detection of functional eVects induced later in development, such as those aVecting reproduction or behaviour, but there is much less insistence on these examinations than on embryotoxicity.

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3.2. Juvenile immunotoxicology Pre-clinical toxicity studies in juvenile animals have become more popular recently as a possible pre-requisite to clinical trials in children following the US FDA recommendations (1998) and guidance from the European Medicines Agency EMEA (2001). Protocols for the assessment of toxicity of drugs in neonate and juvenile rats have been in existence for many years (Barrow, 1990). These studies were rarely performed in the past, except in the case of a drug speciWcally intended for paediatric use. Regulatory guidance documents on juvenile toxicity studies in animals are presently in preparation (US FDA, 2003). The species of choice is the rat, but other species such as the dog, minipig, or primates can also be used. The immune system is one of the principal targets of toxicity in this type of study in view of its late development and increased vulnerability in the developing animal. Immune responses may vary considerably in both nature and degree between animals of diVerent ages because of the prolonged evolving state of the immune system. A juvenile toxicity study may be considered superXuous if adequate exposure throughout post-natal development can be demonstrated in other studies. For this reason, there is every interest in obtaining pharmacokinetic data from the pups during the course of the preand post-natal developmental toxicity study (see above). Also, there may be an advantage in using relatively young animals for one of the sub-chronic or chronic toxicity studies, to cover exposure during the post-weaning maturation period.

4. Proposed study designs Despite the diVerent study objectives, the same basic battery of immune tests can be incorporated into the protocols used for both developmental and juvenile toxicology studies. There are, however, major diVerences in the experimental designs used for the two types of study, particularly with regard to the timing of the various immune tests and to the time of exposure to the test substance. In juvenile studies, the immune assessments are usually performed during the treatment period, in order to detect immune modulation and evaluate whether the drug is likely to render the young animal more sensitive to infectious disease, for instance. Developmental immunotoxicity screening requires exposure of the animal during one or more critical periods of development (e.g., organogenesis) and then assessing the integrity of the immune system at a more mature age. It is important to perform at least part of the immune assessments after cessation of treatment, otherwise it is not possible to distinguish between the direct immunomodulatory eVects of the test substance and functional defects arising from a defect of development.

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4.1. Characteristics and timing of immune tests The battery of immune assessments included in regulatory developmental or juvenile toxicity studies should be comprised of the following three types of test: (1) Assessment of the immune system using similar parameters to those used in the adult to detect potential immunotoxic eVects of xenobiotics (i.e., haematology parameters, immune system organ weights and histology); (2) milestones of immune development (e.g., the time of appearance of the various lymphocyte subsets in the blood, spleen and/or thymus); and (3) functional evaluations of humoral and cellular immunity. The comparison of the results from these three types of test can be expected to provide a robust screen for the detection of immune eVects. In addition, it should be possible to diVerentiate between the possible manifestations of immunotoxicity in the immature organism, i.e., delayed or advanced immune development versus permanent functional defects arising from abnormal development. The test battery should also be capable of detecting a shift in the type 1 T helper (Th1) and type 2 T helper (Th2) balance, such as the skewing towards Th2 activity that was noted in rats as a result of pre-natal exposure to lead (Bunn et al., 2001). The studies with lead also demonstrated gender diVerences in eVects on interleukin-10 (IL-10) secretion, illustrating the need to evaluate both male and female oVspring. It should be possible to integrate the new immune tests into the existing regulatory protocols; hopefully, without requiring treating additional animals. To this end, it will be necessary to develop and validate standard procedures which give reliable and reproducible results. An extensive control data bank will have to be compiled for each test in the selected strain of rat at each appropriate age. It is therefore very important to carefully select and deWne all of the experimental methods, parameters and conditions in advance, since it will be laborious and expensive to rederive historical control data at a later date. Within the context of a developmental immunotoxicology study, there is a considerable advantage in performing immune assessments as early as possible. This allows monitoring of immune development during the course of the study. Any observed variation of the normal course of development could then trigger a more indepth functional assessment at the end of the study (or a prolongation of the study, if necessary). Also, some toxic agents have been shown to induce a transient suppression of immune functions in post-natal mice, which is no longer detectable in the unchallenged adult later in life (Landreth and Dodson, 2005). 4.2. Species and strain selection To correctly interpret the results of animal studies and extrapolate to humans, it is necessary to understand

the anatomic and physiological diVerences between species. When investigating developmental eVects, it is also necessary to comprehend any intraspecies diVerences in the chronology and relative rates of immune development. One example of such a diVerence is the variable degree of immunocompetence between species at the time of birth. The mouse is the most studied species with respect to immune development (Holladay and Smialowicz, 2000). Less is known for other laboratory species (Holsapple et al., 2003). The rat, however, is the species used in almost all post-natal developmental toxicity studies on pharmaceuticals. More research is therefore required to develop a testing battery that can be easily integrated into the existing regulatory protocols. DiVerent strains of rat have a diVerent resting balance between Th1 and Th2 activity, which may inXuence their relative sensitivity to immunotoxic agents or susceptibility to antigens (Bunn et al., 2001). Such considerations need to be taken into account when selecting a strain of rat for use in developmental or juvenile immunotoxicology studies. In the same way, the Fischer 334 strain, for example, shows a more robust response to keyhole limpet haemocyanin (KLH) antigen than the Sprague–Dawley (SD) strain and may be chosen for such studies. However, the ICH testing guideline speciWcally states that strains of low fecundity, such as Fischer 344, should be avoided for reproductive toxicology studies (ICH, 1994). The Fischer 344 also diVers from other strains in its post-natal period of development (Francis, 1994). It would seem reasonable, therefore, to select immune testing methods which are best adapted for use with the strains of rat most often employed in reproductive regulatory safety testing, i.e., Sprague–Dawley and/or Han– Wistar. As stated above, non-rodent species are sometimes used for juvenile toxicology studies. The basic immune evaluations, such as clinical pathology, histopathology and lymphoid organ cellularity, can also be performed in non-human primates, dogs, rabbits and minipigs (Hendrickx et al., 2005; Rothkötter et al., 2005). Some development work may be necessary, however, to adapt the more specialised examinations and functional tests for use in juvenile animals. Many human diagnostic kits can be adapted for use in monkeys (Hendrickx et al., 2005) but it is more diYcult to Wnd suitable reagents for use in other species. The Wnding that exposure to a varied range of potential pathogens is necessary to assure normal T-cell programming could have wide-ranging implications on the use of pathogen-free animals in developmental toxicity testing. These consequences have yet to be explored, even though germ-free animals have been shown to have low levels of immunoglobulins A (IgA) and M (IgM) (Thorbecke, 1959).

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4.3. Testing occasions during a routine ICH pre- and postnatal developmental toxicity study All regulatory pre-clinical programmes for medicinal agents now follow the harmonised ICH guidelines for reproduction toxicity evaluation (ICH, 1994). There is some Xexibility in the testing strategy used, but practically all safety testing programs include a pre- and postnatal study Fig. 1. This study design provides several opportunities for performing immune tests on the excess rat pups before and after weaning. It may, however, be necessary to extend the study design, or to perform a separate additional study, to ensure exposure of the pup during post-natal development (see above). The available opportunities for immune testing at various ages are listed in Table 1 and discussed below. 4.3.1. Day 4 post-partum Many laboratories cull excess pups at 4 days of age to standardise the size of the litters to four or Wve pups of each sex. The aim of the procedure is to standardise the pre-weaning litter responses, but the scientiWc merit of this practice is debatable (Agnish and Keller, 1997; Chapin and Heck, 1997; Palmer and Ulbrich, 1997). This practice does have the advantage of providing excess pups at 4 days of age that can be used for pharmacokinetic analyses, immune tests, or terminal examinations. One disadvantage is that the number of pups available depends on the number of pups born to each dam, which cannot be predicted in advance. The nested statistical analysis of these results can be challenging, since the litter origin of each pup has to be taken into account and unequal numbers of pups are obtained from each dam. 4.3.2. Weaning One male and one female pup per litter are selected at weaning to form the second generation which is not

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treated, but is followed through to maturity. The excess pups (i.e., three or four of each sex) are necropsied or retained for supplementary examinations, e.g., behavioural tests. Immune parameters, such as organ weights or haematology evaluations, can be determined in the pups necropsied at this time. 4.3.3. Post-weaning Some of the above non-selected pups may be retained as satellite groups for testing at a more advanced age. Sub-groups may be used for various functional tests and may even be directly exposed to the drug. Note that the main study animals of the Wrst generation (F1) will be used for a fertility assessment, so the females will normally not be useful for the determination of terminal immune parameters (unless results in pregnant females are of interest). Terminal assays may, on the other hand, be performed at necropsy of the F1 males. 4.4. Proposed immune assessments for incorporation in regulatory pre-clinical safety studies 4.4.1. Clinical pathology determinations Blood samples can be withdrawn for rats of any age and used for routine haematology and serum clinical chemistry determinations. The total and diVerential white blood cell counts, for example, may give a Wrst indication of abnormal immune development. 4.4.2. Pathology Any immunotoxicity investigation should begin with a thorough review of the available pathology data in association with the haematology results. These examinations alone, without speciWc immune tests, may be suYcient to reveal developmental immunotoxicity. For instance, exposure of the new-born rat to low doses of TCDD results in a severe immunosuppression persisting

Fig. 1. ICH pre- and post-natal toxicity study design.  Academic Press (Barrow, 2000). G D day of gestation. L D day of lactation.

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Table 1 Possible occasions for routine immune assessments in an ICH pre- and post-natal developmental toxicology study Age

Origin of pups

Proposed tests

4 days

Excess pups after culling

Routine clinical pathology Spleen and thymus cellularity and lymphocyte subset analysis

Weaning (3 weeks)

Excess pups after selection of F1 generation

Routine clinical pathology Spleen and thymus cellularity and lymphocyte subset analysis Primary response to SRBC Delayed-type hypersensitivity Serum cytokine levels

Post-weaning (3–9 weeks)

Excess pups retained after selection of F1 generation Pups retained for behavioural tests

Routine clinical pathology Spleen and thymus cellularity and lymphocyte subset analysis Humoral response to KLH Serum cytokine, immunoglobulin, autoantibody levels

Adult (10 weeks)

Excess pups retained after selection of F1 generation Pups retained for behavioural tests F1 males after mating

Routine clinical pathology Spleen and thymus cellularity and lymphocyte subset analysis Histopathology of immune organs and bone marrow Serum cytokine, immunoglobulin, autoantibody levels

into adulthood. The Wrst discernible sign of this eVect is an involution of the thymus (Gehrs et al., 1997), which is visible in the weaning rat and is easily detected in a conventional pre- and post-natal study (even following treatment only of the mother). This lesion is very easily overlooked in the adult, in which thymic involution is normal. All pups killed or dying during the study should be given a necropsy examination, paying particular attention to lymphoid organs (e.g., lymph nodes, thymus, spleen, Peyer’s patches). Organ weights and histopathological examination of these organs, plus those organs involved in the development of haemopoiesis (e.g., liver and bone marrow) or potential sites of deposition of immune complexes (e.g., kidneys), can also give useful information. Routine toxicological histopathology examinations may not be suYcient, however, to detect the consequences of a developmental toxic insult. Tissue abnormalities resulting from defects in development tend to be morphological in nature and are not necessarily manifest as those types of lesion most easily detected using routine histological methods employed in toxicology studies (e.g. necrosis, apoptosis, inXammation, hypertrophy). Quantitative morphometric examinations, Table 2 Spleen weight and cellularity in the SD rat Sex

Age

Splenocytes 106/g of spleen

Spleen weight (g)

Male

4 days 19 days 6 weeks 8 weeks

596 § 96 603 § 228 704 § 95 681 § 221

0.049 § 0.006 0.189 § 0.043 0.773 § 0.101 0.735 § 0.116

Female

4 days 19 days 6 weeks 8 weeks

629 § 130 662 § 89 702 § 109 718 § 134

0.045 § 0.010 0.216 § 0.055 0.534 § 0.120 0.511 § 0.058

Values given are mean § SD, n D 10.

such as thymus medulla:lobe ratios (Dietert et al., 2003) or cellularity measurements (Gehrs et al., 1997), are perhaps more likely to reveal developmental defects than detailed microscopic examinations. Typical values for spleen cellularity at various ages in our laboratory are given in Table 2. 4.4.3. Lymphocyte subset analysis in the blood, spleen, and thymus This evaluation can be used to monitor the development of the thymus and the seeding of the spleen with lymphocytes. A sample of spleen is taken at necropsy. A splenocyte suspension is then analysed in a FACScan Xow cytometer to study the various surface markers of lymphocytes. Mature subset types Wrst appear in signiWcant numbers in the rat from about 4 weeks of age. Typical values at various ages obtained in our laboratory are given in Fig. 2. It may also be useful to monitor immature subset types, such as CD4+CD8+ double positive T cells, that are prevalent in the developing rat, but only represent a minor subpopulation of T lymphocytes in the peripheral circulation of the adult. An increase in the relative double positive T cell counts, for instance, may be indicative of immunotoxicity (Parel and Chizzolini, 2004). The lymphocyte subset counts in the spleen may be compared with the counts in peripheral blood. Lymphocyte subset counts may also be performed in the thymus. As for immunotoxicity studies following exposure of the adult, immunophenotyping of lymphocyte subsets in lymphoid organs and tissues can be analysed retrospectively by immunohistochemistry. Immunohistochemistry also allows the characterisation of immune changes within speciWc compartments of the organ. 4.4.4. Primary antibody response to SRBC In our experience, a direct plaque forming cell assay can be performed in rats from about 15 days age, when

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% B cells % CD3 T cells

Relative lymphocyte counts (%)

60

% CD4 T cells % CD 8 T cells

50 40 30 20 10 0 4 days

6 weeks Age of males

8 weeks

4 days

6 weeks Age of females

8 weeks

Fig. 2. Lymphocyte subset analysis in the blood of 4-day, 6-week, and 8-week old SD rats mean § SD. n D 10 for all data points.

the intravenous administration of 100
L of a suspension of 109 sheep red blood cells (SRBC) becomes feasible. The pups are necropsied 4 days after sensitisation. A spleen cell suspension is prepared and plaques are counted in a Cunningham slide following mixing with SRBC and guinea-pig complement. Each plaque is equivalent to one antibody-producing cell. Typical values in pre-weaning rats obtained in our laboratory are given in Table 3. Sensitisation is also possible via the intraperitoneal route (Holsapple, 1995). Alternatively, SRBC-speciWc IgM antibodies can be assayed in serum using an enzyme-linked immunoTable 3 Primary response to SRBC in the SD rat following sensitisation at 19 days of age Sex

Log PFC/105 cells

PFC/105 cells

PFC/g of spleen

Male Female

1.26 § 0.35 1.18 § 0.44

24.9 § 23.1 21.1 § 13.9

154.5 § 136.9 136.4 § 89.9

Values given are mean § SD, n D 10.

sorbant assay (ELISA), but this assay has so far proven to be relatively insensitive in the young rat due to inherently high background IgM levels (Ladics et al., 2000). 4.4.5. Humoral response following KLH administration The T-cell dependant antibody response can be evaluated using the measurement of the antibody response induced by the administration of the KLH antigen (Ulrich et al., 2004). In our experience, this test may be performed in rats as young as 4 weeks of age. KLH is given by subcutaneous injection on two occasions seven days apart. Serum samples taken before each injection and seven days after the second injection (i.e., at 4, 6, and 7 weeks of age) are analysed for anti-KLH IgG and IgM antibodies. Typical results obtained in our laboratory are given in Fig. 3. The intravenous route of sensitisation is sometimes used in the adult and may also be applicable to developmental and juvenile immunotoxicology studies.

Fig. 3. Humoral response following KLH administration at 4 weeks of age mean § SD. n D 10 for all data points.

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Table 4 Serum IgM, IgG, and anti-dsDNA levels in the SD rat at 8 week of age Sex

Total IgM (
g/mL)

Total IgG (
g/mL)

Anti-dsDNA antibody (mUI/mL)

Male Female

219 § 68 207 § 37

1536 § 624 2023 § 725

9502 § 4615 11300 § 4766

Values given are mean § SD, n D 10.

4.4.6. DTH response The depression of the delayed-type hypersensitivity (DTH) response is reported to be one of the most sensitive indicators for the detection of developmental immunotoxicity (Gehrs and Smialowicz, 1999). The DTH response can Wrst be detected in the rat from about the time of weaning (Bunn et al., 2001). The pups are given a sensitising injection of bovine serum albumin (BSA) mixed with Freund’s complete adjuvant by injection into the subcutaneous tissue at the base of the tail. Six days later, a challenge injection of heat-aggregated BSA is given into the left rear footpad. The right footpad is given a control injection of buVer solution. The thickness of each footpad is measured before and 24 h after injection. Swelling of the right footpad only following challenge is indicative of a DTH response. The test has been successfully performed in our laboratory in the SD rat with a BSA sensitisation at three weeks of age. In recent years, the KLH antigen has become more popular as the sensitising agent, in which case the test may be conveniently combined with an assessment of the humoral response to KLH (Bunn et al., 2001; Dietert et al., 2003). 4.4.7. Analysis of serum levels of cytokines, immunoglobulins, and autoantibodies Serum samples may also be used for the determination of the concentrations of cytokines such as IL-2, IL-4, IL5, interferon-gamma (IFN-) and tumour necrosis factor alpha (TNF-), immunoglobulins (e.g., total IgG and IgM) and anti-double strand (anti-ds) DNA antibodies. The development of FACScan Xow cytometry techniques permits cytokine assays with very small sample volumes (approximately 100 mL of serum), but the necessary cell markers are not yet available in the rat. Typical values of total IgG, total IgM and anti-ds DNA serum concentrations in rats at 8 weeks of age obtained in our laboratory using an ELISA technique are given in Table 4. Other classes and subclasses of immunoglobulins (e.g., IgE, IgG1, IgG2) and other types of autoantibody (e.g. organ-speciWc autoantibodies) could also be evaluated.

detection of immune defects following developmental exposure. The severe technical constraints necessary to prevent the spread of infection within the animal unit and the inXuence of the infection on the interpretation of the clinical and pathological Wndings in the experiment dictate that these assays be performed as separate dedicated experiments. Host resistance assays, on the other hand, may be considered for follow-up experiments when potential eVects have been identiWed in the routine studies. Probably the most promising infectious agent for this purpose is the nematode Trichinella spiralis, which was used to demonstrate a lasting immune depression in rats following pre-natal exposure to diazepam (Schlumpf et al., 1994) or TCDD, (Luebke et al., 1999). Other common host resistance assays include the use of bacteria such as Listeria monocytogenes or Streptococcus pneumoniae or subcutaneous tumourigenesis induced by inoculation with PYB6 tumour cells (Karrow et al., 2003).

5. Future directions for pre-clinical testing: hypersensitivity and autoimmunity The majority of the immune assessments described above are intended for the detection of immune depression. At the present time, there is little scope to screen for xenobiotics which promote hypersensitivity and/or autoimmunity. A few animal models of autoimmune diseases have been developed, such as the non-obese diabetic (NOD) mouse (Atkinson and Leiter, 1999; Ravel et al., 2004) or the Bio-Breeding (BB) rat (Kroemer et al., 1992) for type 1 diabetes, and the (NZB £ NZW) F1 mouse (Burnett et al., 2004; Ravel et al., 2002), for systemic lupus erythematosus. Their use in developmental or juvenile studies would be extremely problematic, owing to the compromised reproductive capacity of the animal strains used. A model of induced autoimmune encephalomyelitis in the rat has nonetheless been used to demonstrate the promotion of autoimmunity in adults as a consequence of pre-natal exposure to dexamethasone, even though this chemical is not known to induce autoimmunity in adult rats or humans (Bakker et al., 2000). There is an urgent need for research in the area of hypersensitivity and autoimmunity to develop reliable and practical methods for use in the safety assessment of new drugs. Another type of adverse immune eVect in need of research, particularly for large molecules, is immunogenicity. As yet, no robust animal models have been validated to predict immunogenicity of pharmaceuticals in either mature or immature humans.

4.5. Follow-up tests: host resistance assays Some immune deWcits may only become evident when the immune system is placed under the stress of infection or infestation (Meade et al., 1998). Host resistance assays are, therefore, likely to be particularly sensitive for the

6. Conclusion The developing immune system has been shown to be uniquely susceptible to the detrimental eVects of toxic

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