UNIT 18.15 Developmental Immunotoxicity (DIT): Assays for Evaluating Effects of Exogenous Agents on Development of the Immune System

 

Introduction

Developmental immunotoxicity (DIT) is best defined as “the effects on the immune system resulting from pre- and/or postnatal exposure to physical factors (e.g., ionizing and ultraviolet radiation), chemicals (including drugs), biological materials, medical devices, and in certain instances physiological factors, collectively referred to as agents” (Luster et al., 2008). Immunotoxicity that arises from developmental exposure to agents may elicit suppression, hyperactivation, or misregulation of immune responses and may present clinically in the exposed organism as decreased resistance to pathogens, allergic and autoimmune diseases, and inflammatory diseases. Often, manifestation of the immune alteration induced by DIT is not detectable until the exposed organism reaches immunocompetence, or the time at which the immune system is capable of mounting a full immune response against pathogens. While it is possible to evaluate certain anatomical and functional components of the immune system prior to immunocompetence, evaluating the potential effects of DIT on an immature immune system fails to appreciate the effects of an agent on all critical windows of immune development. The main purpose of the protocols in this unit, therefore, is to describe the optimum exposure scenario that should be followed to ensure that exposure to the agent occurs during all critical windows of immune development in a rodent model.

The U.S. Environmental Protection Agency (US EPA) requires immunotoxicity testing of agents covered by the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA); however, DIT testing is not part of the harmonized test guidelines recommended for evaluating the immunotoxicity of agents under FIFRA (OPPTS 870.7800). In addition, the harmonized immunotoxicity test guidelines were designed to detect immune suppression in adult organisms. While the assays recommended in the test guidelines are nearly identical to those appropriate for evaluating DIT and will be described in this unit, they are not suitable for detecting DIT if the exposure scenario does not include all critical windows of immune development.

 

Strategic Planning

Currently, there are no consensus guidelines on the suite of assays most appropriate for evaluation of DIT, although several symposia and cross-organization workshops have discussed strategies for DIT testing. While large test batteries will fully evaluate all compartments of the immune system, the strategic use of selected assays can still provide a comprehensive overview of the potential for DIT to impact innate and adaptive immune function and the structural components of the immune system. In 2007, a comprehensive set of methodologies for evaluating DIT in rodent models was published by Dietert and Holsapple (2007), and this remains the best test guideline for DIT to date because it includes assays that are both observational and functional. This reference is the basis for the Basic Protocol.

In rodent models (rats and mice) commonly used for immunotoxicity studies, immune development occurs throughout gestation and lactation, and immunocompetence is not achieved until approximately 6 weeks after birth (Smialowicz et al., 2007). Therefore, the assays referenced throughout this manuscript are for young adult test organisms. The two exceptions are the first two assays referenced in the Support Protocol; these two protocols can be used to evaluate DIT in weaning-age offspring.

The Basic Protocol describes the scenario by which rodent models should be exposed to ensure that all critical windows of immune development are subjected to the potential actions of the agent. An Support Protocol is provided, which describes three other exposure scenarios that encompass fewer critical windows of immune development, but that are still sufficient for estimating the potential for DIT.

A Support Protocol for evaluation of DIT is included to direct investigators to corresponding protocols published in Current Protocols to give additional information, when necessary. Assays referenced in the Support Protocol include: immunophenotyping (Reference 1), histopathology (Reference 2), T cell-dependent antibody responses (Reference 3), delayed-type hypersensitivity responses (Reference 4), and natural killer cell activity and the cytotoxic T lymphocyte assay (Reference 5).

NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals.

NOTE: The investigator should consult the manufacturer's instruction manual for specific information regarding the operation of any and all specialized instrumentation, equipment, and software.

 

Basic Protocol: Exposure Scenario for Including All Critical Windows of Immune Development

As there is no guideline or consensus document for an exposure scenario most suitable for detecting DIT in rodent models, a method presented by Dietert and Holsapple (2007) will serve as the template for this protocol. This method (Dietert and Holsapple, 2007) emphasized the use of multifunctional assessments across different types of immune responses combined with histopathology and cell enumeration information. Multifunctional assessment is a core of the Basic Protocol, and the combination of different functional measures serves an important purpose. Because different immunotoxicants may target different portions of the developing immune system, it is important to collect information of sufficient breadth and diversity to cover the range of immune functions (i.e., innate immunity, humoral immunity, cell-mediated immunity). Single functional assay approaches do not reflect the entire risk for the immune system. For example, while humoral immune measures are prevalent in DIT risk assessment, Tonk et al. (2011) recently reported an example where cell-mediated immune function—e.g., the DTH (delayed-type hypersensitivity) response—was actually the critical functional endpoint in a DIT evaluation of di-n-octyltin dichloride (DOTC).

It is, therefore, important that initial DIT studies assess several endpoints, including structural and functional evaluations of the immune system. No single assay is sufficient for evaluating every aspect of the immune system, although host-resistance assays do allow for an examination of the overlapping functional capacities of the immune system. Host-resistance assays are one of the best measures of an agent's impact on an organism's overall ability to mount an effective immune response (Burleson and Burleson, 2008). If an agent prevents an organism from appropriately defending itself against infectious diseases, it is deemed immunotoxic. Some of the types of challenges used in host-resistance assays are likely to have future utility in DIT testing, as with the evaluation of cell-mediated immunity following a viral challenge (Burleson et al., 2010). However, these possible support applications are many, and each encompasses its own detailed protocol; as such, these auxiliary assays will not be described in the Basic Protocol. Please refer to the Support Protocol for additional information and relevant references to established protocols.

The Basic Protocol is appropriate for evaluating DIT in either rats or mice.

 Materials

• Rodents for testing: timed-pregnant dams or dams and sires ordered as breeding pairs
• Test agent in appropriate delivery vehicle, if necessary

• Apparatus for delivering test agent (gavage needles, water bottles, instillation apparatus, etc.)
• Apparatus for weighing dams and offspring
• Data sheets for recording weights and other data
• Additional reagents and equipment for evaluating DIT (Support Protocol)

 Expose dams to test agents

 1.

Obtain and house animals. Provide animals with nesting materials and food and water ad libitum unless agent is to be given via diet or drinking water.

p type = annotation

Dams may be ordered from suppliers as timed-pregnant dams, or dams and sires may be ordered as breeding pairs. As the rate of conception is likely to be less than 100%, it is recommended that 25% to 50% more dams be ordered than the minimal sample size needed.

p type = annotation

It is beyond the scope of this protocol to discuss the relative advantages and disadvantages of ordering timed-pregnant animals versus breeding pairs. This is a consideration that should be based on cost, experience with managing breeding pairs, and space for housing animals and their offspring.

p type = annotation

When ordering timed-pregnant females, the researcher may specify gestational day for delivery. It is possible to have pregnant dams delivered on GD1, which is considered the sperm-positive (sperm present in vaginas of mated females) or plug-positive (presence of a copulatory plug in the vagina) date by many animal suppliers. However, the stress of shipping and acclimatization to a new housing environment may increase pre-implantation losses. Implantation generally occurs between GD4 and GD5, so by GD6, implantation losses will be minimized. It is therefore recommended that delivery occur on GD6 and dosing occur on the day of delivery. This does eliminate an acclimatization period, but will reduce potential pre-implantation losses and ultimately reduce animal numbers needed.

p type = annotation

It also is beyond the scope of this protocol to thoroughly discuss housing options. Several housing options for dams or dam-sire pairs exist, depending on the strain of rodent, study design, and housing space available. Housing decisions should be based on strain of rodent (some strains are very social and do well with group housing, whereas others do not), the study design (i.e., if agent is to be delivered via food or water, group housing may not be the best option), and housing space. Current Protocols in Toxicology “Guidelines for Mating Rodents” (unit 16.2) and Current Protocols in Mouse Biology “Mouse Breeding and Colony Management” (Ayadi et al., 2011) provide detailed overviews of procedures for mating rodents.

 2.

Begin exposure of the dams no later than GD6 to ensure that the test agent is present in the dam when hematopoiesis begins at about GD7.

p type = annotation

If breeding in-house, exposure to the dams can begin as soon as they are paired with sires. If dosing timed-pregnant animals, beginning at GD6 is ideal due to reasons specified in step 1. The route, dose, methods, and timing and duration of exposure should be based on the situation being modeled by the study (Fig. 18.15.1).

p type = annotation

It is beyond the scope of this protocol to detail exposure routes. While daily dosing or exposure is recommended unless steady state of the agent can be maintained at a different dosing frequency, the investigator must determine the route most appropriate for the agent under study.

Figure 18.15.1 Schematic of protocol for developmental immunotoxicity as outlined by Dietert and Holsapple (2007), with inclusion of rat/mouse and human time frames for immunological development (Dietert et al., 2000). In this protocol, DIT is assessed at 6 weeks of age in the offspring. Exposure can occur during gestation only, during lactation only, or during gestation and lactation (stopping at weaning of offspring), or can continue past weaning at postnatal day (PND) 21 and through adulthood to encompass earlier measurements in the offspring. GD = gestation day, GW = gestation week. It is advised, except in specific cases where exposure to the agent of concern occurs only during certain developmental time points, that exposure occur from gestation (beginning at conception when using breeding pairs by starting exposure to the pairs when they are placed together or no later than GD6 when using time-pregnant animals) through PND42.

 3.

After dams have delivered litters (between GD20 and GD22 for most rodents), observe the F1 generation. Record the following:

1. Total number of offspring per dam as well as number of males and females per dam.
   
Numerous references are available for sexing of neonatal rodent offspring. One good reference is the American Association for Laboratory Animal Science Learning Library (see Internet Resources).
2. Weight of litter. Whole litter weight or weights of males and females separately.
   
3. Number of stillborn or cannibalized offspring.
   
4. General health observations.
   
5. Other.
   
   Many researchers choose to assess developmental milestones as part of a DIT study to distinguish developmental toxicity from DIT. Current Protocols in Neuroscience “Assessment of Developmental Milestones in Rodents” (Heyser, 2003) provides a detailed overview of what developmental endpoints to assess and how to assess them.
   
   

 4.

On PND2, standardize litters so that each dam has an equal number of male and female offspring. Number of offspring per litter will vary with the strain of rodent. It is recommended that the number of offspring not exceed the number of nipples (10 to 12).

p type = annotation

Some researchers comingle offspring from each dose and randomly reassign male and female offspring to each dam. Other researchers retain offspring with each dam and backfill litters with extra offspring from dams within the same dose if a particular dam does not have a sufficient number of offspring. Either method is acceptable, and most dams will accept offspring from other dams at PND2.

p type = annotation

If dams are born with very few offspring (i.e., 2 to 3), or if several offspring are stillborn or cannibalized for a particular dam, it is recommended that the dam be removed from the study unless it is absolutely necessary to retain such dams.

p type = annotation

Some dams will not deliver offspring between GD20 and GD22. Some of those dams may not have been pregnant, may not have been GD6 at the start of dosing, may have lost offspring during the pregnancy, or were not able to deliver offspring. At the start of the study, the researcher must decide how far past GD20 to allow dams to go before euthanizing them. For dams that are obviously not pregnant based on body weight, euthanasia can occur as early as GD13. For other dams, the decision will not be quite as obvious; humane euthanasia may be necessary to ensure that dams do not die of sepsis from in utero fetal death or from a pup that is blocking the vaginal canal. Veterinary staff can and should provide assistance in assessing the gestational stage and overall health of dams that have not delivered when expected.

p type = annotation

Uteri of dams that have not delivered can be assessed for implantation sites, and corpora lutea on ovaries may be counted as well.

p type = annotation

Current Protocols in Toxicology “In Vivo Assessment of Prenatal Developmental Toxicity in Rodents” (unit 13.5) provides a detailed overview of how to examine apparently nongravid uteri.

 5.

Continue to dose the dams through weaning of the offspring at postnatal day 21 (PND21) to ensure exposure of the offspring during gestation and lactation.

p type = annotation

For agents that are not known to be transferred via the lactational route, direct dosing of pre-weanling offspring may be necessary. See the following step for more details.

 Expose offspring

Gestational exposure of offspring will occur via dams for the gestational period. After birth, offspring exposure will be via lactation. Direct dosing of offspring may be necessary if human exposure considerations and the specific properties of the chemical being studied warrant it (Dietert and Holsapple, 2007). Direct dosing of offspring can begin as early as PND1 via specific routes of exposure. However, the appropriate selection of route, dose, methods, and timing and duration of exposure must be made with care (Moser et al., 2005). Guidelines for directly dosing pre-weaning rodents are available in Moser et al. (2005).

6.

Expose offspring via direct dosing after weaning until PND42 (6 weeks of age). p type = annotation The route, dose, methods, and timing and duration of exposure should be based on the situation being modeled by the study. p type = annotation At weaning, it is recommended that offspring be segregated by sex and housed in sibling groups. Male offspring from the same litters generally do not fight with one another. p type = annotation Numerous references are available for sexing of weaning-age rodent offspring. One good reference is the American Association for Laboratory Animal Science Learning Library (see Internet Resources).

7.

Begin evaluating offspring for DIT (Support Protocol) at or after PND42.

 

Alternate Protocol: Exposure Scenario for Evaluating Limited Windows of Exposure

The Basic Protocol ensures that offspring are exposed during all critical windows of immune system development. To meet situations being modeled by the study that may include a more limited window of exposure, alternative scenarios may be appropriate. As discussed in the Basic Protocol, the route, dose, methods, and timing and duration of exposure should be based on the situation being modeled by the study. Please refer to the Basic Protocol for annotations to the referenced steps of that protocol.

 Materials

• Pregnant dams or weaning-age offspring
• Test agent in appropriate delivery vehicle, if necessary

• Apparatus for delivering test agent (gavage needles, water bottles, instillation apparatus, etc.)
• Apparatus for weighing dams and offspring
• Data sheets for recording weights and other data

 For gestational exposure only

1a.	Follow step 1 of the Basic Protocol.
2a.	Dose the dams until they give birth (GD20 to GD22). p type = annotation This will ensure exposure of the offspring during gestation.
3a.	Follow steps 3 and 4 of the Basic Protocol.
4a.	Follow recommendations under step 6 of the Basic Protocol for weaning of offspring. Allow offspring to develop until PND42.
5a.	Begin evaluating offspring for DIT (Support Protocol) at or after PND42.

 For lactational exposure only

1b.	Follow steps 1 through 4 of the Basic Protocol, but do not dose dams until PND1.
2b.	Begin dosing the dams at PND1 and continue through weaning of the offspring at postnatal day 21 (PND21). p type = annotation This will ensure lactational exposure of the offspring.
3b.	Follow recommendations under step 6 of the Basic Protocol for weaning of offspring. Allow offspring to develop until PND42.
4b.	Begin evaluating offspring for DIT (Support Protocol) at or after PND42.

 For gestational and lactational exposure only

1c.	Follow steps 1 through 4 of the Basic Protocol.
2c.	Continue to dose the dams through weaning of the offspring at postnatal day 21 (PND21). p type = annotation This will ensure exposure to the offspring during gestation and lactation.
3c.	Follow recommendations under step 6 of the Basic Protocol for weaning of offspring. Allow offspring to develop until PND42.
4c.	Begin evaluating offspring for DIT (Support Protocol) at or after PND42.

 For direct dosing of offspring through weaning only

1d.	Follow steps 1 through 4 of the Basic Protocol but do not dose dams.
2d.	Begin directly dosing the offspring at PND1 and continue through weaning of the offspring at postnatal day 21 (PND21). p type = annotation This will ensure direct exposure to the offspring. The appropriate selection of route, dose, methods, and timing and duration of exposure must be made with care (Moser et al., 2005). Guidelines for directly dosing pre-weaning rodents are available in Moser et al. (2005).
3d.	Follow recommendations under step 6 of the Basic Protocol for weaning of offspring. Allow offspring to develop until PND42.
4d.	Begin evaluating offspring for DIT (Support Protocol) at or after PND42.

 For weaning to young adult exposure only

1e.	Follow steps 1 through 6 of the Basic Protocol; do not dose dams; do not dose offspring from PND1 to PND21.
2e.	Begin exposure to offspring at day of weaning (PND21) and continue through PND42. p type = annotation The route, dose, methods, and timing and duration of exposure should be based on the situation being modeled by the study. p type = annotation Unless offspring can be delivered on the day of weaning, it is recommended that offspring be generated in-house.
3e.	Begin evaluating offspring for DIT (Support Protocol) at or after PND42.

 

Support Protocol: Evaluation of DIT

As stated previously, currently there are no consensus guidelines on the suite of assays most appropriate for evaluation of DIT. The following assays provide a comprehensive overview of the potential for DIT to impact innate and adaptive immune function and the structural components of the immune system. In an ideal scenario, all of the assays would be evaluated in an initial DIT study. Minimally, a combination of observational (immunophenotyping, histopathology, or lymphoid organ weights), adaptive immune function (TDAR or DTH responses), and innate immune function (natural killer cell or cytotoxic T cell assays) should be performed.

Reference 1: Immunophenotyping

Immune cell phenotyping (immunophenotyping) may be performed on weaning-age offspring before they are fully immunocompetent. However, when evaluating the number and types of immune cells in an immature organism, it is critical that observations from the treated groups be compared only to the appropriate age-matched control group. It is not possible to make assumptions about the effects of an agent on “standard” values for immune cell populations in immature organisms. Further, the usefulness of immunophenotyping in immature organisms for detecting DIT is not known. See Current Protocols in Toxicology, unit 18.8, Immune Cell Phenotyping Using Flow Cytometry.

Reference 2: Histopathology

Histopathology of immune tissues may be performed on weaning age offspring before they are fully immunocompetent. As is true for immunophenotyping, it is critical that observations from the treated groups be compared only to the appropriate age-matched control group, as one cannot assume that the observations of an agent's effect on “standard” morphology will be applicable in the context of an immature organism. The value/utility of histopathology in immature organisms for detecting DIT also is not known. If histopathology cannot be evaluated, it is recommended that, at a minimum, the weights of lymphoid organs (spleen and thymus) be recorded.

See Current Protocols in Molecular Biology, unit 14.1, Fixation, Embedding, and Sectioning of Tissues, Embryos, and Single Cells (Zeller, 1989). Although it is recommended that sections be read by a veterinary pathologist, volume 34 (2006) of the journal Toxicologic Pathology has a special issue on immunopathology. This volume contains overviews of all of the major immune tissues.

Reference 3: T Cell–Dependent Antibody Responses

T cell-dependent antibody responses (TDAR) may be evaluated in weaning age offspring before they are fully immunocompetent. When evaluating TDAR in immature organisms, comparisons between treated groups must, again, be made according to an appropriate age-matched control group. In addition, TDARs to measure multiple isotypes of immunoglobulins are preferable to those measuring only one isotype, as isotype skewing, rather than changes in antibodies across all isotypes, is a relevant perinatal immunotoxic outcome (Dietert and Holsapple, 2007).

See Current Protocols in Toxicology, unit 18.11, Analysis of Immunotoxicity by Enumeration of Antibody-Producing B Cells for experimental details.

Reference 4: Delayed-Type Hypersensitivity Responses

Delayed-type hypersensitivity (DTH) responses may be evaluated in weaning-age offspring before they are fully immunocompetent; age matching between all groups is required here as well. Because DTH responses assess the robustness of Th1 cell–mediated immunity, which is often a target of prenatal or early postnatal exposures, it is a fairly valuable assay for evaluating DIT (Dietert and Holsapple, 2007).

See Current Protocols in Immunology, unit 4.5, Delayed-Type Hypersensitivity for experimental details (Luo and Dorf, 1993).

Reference 5: Natural Killer Cell Cytotoxicity and Cytotoxic T Cell Assays

See Current Protocols in Toxicology, unit 18.6, Measuring the Activity of Cytolytic Lymphocytes.

sect1 type = commentary

 

Commentary

 

Background Information

The immune system is a complex and compensatory system that protects the body from disease when working properly. Given recent increases in childhood diseases that are immune-based, such as recurrent otitis media, asthma, allergies, and type 1 diabetes, the role of environmental stressors during development of this system is an increasing concern (Dietert and DeWitt, 2010). For example, polychlorinated biphenyl (PCB) exposure in developmental animal models has been shown to alter immunological functions and increase susceptibility to infections (Dietert and DeWitt, 2010). This also has been confirmed in epidemiology studies indicating relationships between PCB exposure and rates of recurrent otitis media and recurrent respiratory infections (Dietert and DeWitt, 2010). Due to the vulnerability of the developing immune system, it is considered more sensitive to toxicant exposure than is the adult immune system (Luebke et al., 2006). Research related to DIT and its role in childhood disease is becoming a priority, as evidenced by several recent workshops and symposia (Burns-Naas et al., 2008). Notably, DIT testing has been included in regulatory developmental and reproductive toxicology (DART) protocols, along with developmental neurotoxicity testing (DNT) (Burns-Naas et al., 2008).

Important in DIT testing is the role of exposure during critical windows of susceptibility. Exposure during different times of development can result in differing effects on the immune system. Lead exposure is a prime example of this, with research indicating that exposure through gestation results in altered DTH responses, while exposure solely during early gestation has no effect (Dietert and DeWitt, 2010). The concept of effects related to critical developmental windows is integral to the US Environmental Protection Agency's (USEPA) Framework for Assessing Health Risks of Environmental Exposures to Children (Brown et al., 2008).

The full background, history, importance of exposure during critical windows, and potential links to immune-based disease states in relation to DIT testing is well reviewed (Holsapple et al., 2005; Dietert and Holsapple, 2007; Burns-Naas et al., 2008; Dietert and DeWitt, 2010; DeWitt et al., 2011), and is beyond the scope of this commentary section. However, the following points represent the general consensus:

1. For many agents, the developing immune system may be more sensitive than the adult immune system, and early-life exposures during critical windows of immune development may pose a greater risk for immune-based health concerns.
   
2. Initial assessment of DIT should be conducted in an exposure scenario that encompasses all critical developmental time points, unless a rationale exists for the agent of interest in which exposure only occurs in humans at a specific time point (e.g., a drug given to the mother during pregnancy or lactation).
   
3. Evaluation of DIT in both sexes of offspring is critical to understanding DIT risks.
   
4. Assessment of functional tests is critical and should include TDAR plus DTH, NK and/or CTL endpoints.
   
5. Immunophenotyping, histology, and cytokine measurements can support data obtained from functional tests, but should not be conducted in isolation or replace the functional tests.
   

 

Critical Parameters and Troubleshooting

 

Timed-pregnant dams

While it is beyond the scope of this protocol to discuss the relative advantages and disadvantages of ordering timed-pregnant animals or breeding pairs, one important consideration is necessary if ordering timed-pregnant dams. It is recommended that delivery occur on gestational day 6 (GD6), as embryos should be implanted into the uterine wall at this time. Delivery prior to GD6 increases the probability of preimplantation losses. This does eliminate an acclimatization period, but most Institutional Animal Care and Use Committee (IACUC) guidelines allow for exceptions if they are part of an approved animal use protocol.

The rate of conception will naturally be less than 100%. Be sure to order a sufficient number of dams or breeding pairs to account for a 25% to 50% shortfall in conception. For example, if a sample size of eight offspring per endpoint is desired, it is recommended that 12 to 16 dams be ordered.

 

Sample sizes

The US EPA Health Effects Test Guidelines for Immunotoxicity (OPPTS 870.7800) recommends at least eight animals in each dose and control group. For a developmental study, this translates to offspring from the litters of eight different dams in the control group and each dose group (Fig. 18.15.2). If, for example, TDAR, DTH, and NK cell cytotoxicity will be evaluated in offspring, at least three offspring of each sex per dam will be needed (depending on the antigen being used to elicit TDAR and DTH, as these responses may, sometimes, be evaluated in the same animals).

Figure 18.15.2 Potential experimental breakdown of pregnant dams and offspring for DIT assessment.

When performing developmental studies it is important to treat the litter as a unit. Two offspring from the same litter that are evaluated for the same immunological endpoint equal a sample size of one, not two. Two offspring from the same litter that are evaluated for different immunological endpoints may be treated as separate samples. However, when performing statistical analysis for gestational and/or lactational exposures, effects from the dam must be part of the analysis. Typically this is done with a nested ANOVA (unit 1.2), where the dam is a subgroup within each treatment group.

 

Replicates

It is recommended that at least two experimental replicates be part of any DIT study and that animals for positive control be included in the functional immunological assays performed on the offspring. Experimental replicates consist of two or more studies that treat separate groups of animals in the same manner. Myriad positive controls for detecting immunomodulation are available and are detailed in the protocols describing the functional assays recommended in this manuscript (see, principally, the Support Protocol).

 

Breeding/birth records

It is critical to a DIT study to maintain breeding and/or birth records for the dams and offspring. Example breeding and birth tables for recording such information are included in Figure 18.15.3. Offspring should be monitored daily until weaning. Dead offspring and the cause of death, if it can be determined, should be recorded.

Figure 18.15.3 Example form for recording birth from breeding pairs or time pregnant mice (in the case of time pregnant mice change breeding pair ID to the animal ID number for the time pregnant dam). The comments section can include information on survivability of the offspring. Not all offspring may survive to weaning age, due to various reasons related to the compound, or at early stages, to the dam's actions. Dead offspring and the date can be recorded here or may be recorded on a different form.

It also is important to include information on dams that did not complete the pregnancy but were pregnant (this can be confirmed by post-mortem examination of the uteri). A high number of dams that did not successfully deliver litters might indicate that the agent used in the study is at too high a dose and is contributing to offspring lethality. Or, the agent may be causing other deleterious effects during pregnancy beyond or in addition to alterations to the immune system. Body weights of dams and offspring should be assessed throughout the course of the study (Fig. 18.15.4A,B). Prior to weaning, litter weight is acceptable, especially when offspring are not given individual identification (ID) markers until weaning. Litter weights should be assessed at a minimum of once a week. Once weaned, weights of individual offspring should be monitored at least once a week.

Figure 18.15.4 (A) Example form for monitoring individual dam weights and dosing over the course of the study. (B) Example litter/individual offspring weight form. For ID marking, the correct information (i.e., ear or tail or litter box number) can be circled at the top of the column to indicate the ID information below in the column. Thus, these form examples can be used for litter weights, and then for individual weights once offspring are weaned, by simply changing which ID marker type is circled once the offspring are weaned. Additionally, during this time the dam ID can be circled as such while collecting litter weights, and the individual offspring ID can then be added and circled as such when individual offspring weights are collected.

 

Troubleshooting

See the Support Protocol for troubleshooting related to the recommended assays.

 

Anticipated Results

The objective of any DIT study is to determine the potential of an administered agent to alter the immune response in a way that renders the exposed organism incapable of mounting an effective immune response in one or more components of the immune system. The assays referenced within this unit, when used in combination, assess several endpoints, including structural and functional evaluations of the immune system. No single assay is sufficient for evaluating every aspect of the immune system; however, if used in the recommended combination, they are sufficient for detecting immunomodulation. For most of the functional assays mentioned in this protocol, immunomodulation will be in the form of immunosuppression. Although misregulated inflammation, allergic responses, and autoimmune responses are all types of immune dysfunction that occur in adults and that can be induced by DIT, currently, no functional assays exist to adequately detect these effects in young adult (developing) organisms. Therefore, suppression of the responses measured by the recommended assays in the treated groups relative to the untreated groups is generally regarded as an immunotoxic response.

Immunosuppression occurs when the responses of the treated group or groups are lower statistically and biologically compared to the responses of the untreated group. For the functional assays recommended in this protocol, when statistically significant changes are observed, they generally are considered to be biologically significant. In addition, when statistically significant changes are observed within multiple tests, the strength of the support for the agent as a developmental immunotoxicant is increased. Instances may arise when functional assays are not statistically significant but observational assays are, or the other way around. In these instances, it is recommended that the researcher put the results into the context of what is known about the agent in terms of inducing developmental toxicity, immunotoxicity, or DIT in other models or under other exposure scenarios. Such results should lead to additional functional assays to determine if the observational change is associated with a deficit in a specific immunological function.

Often, different immunological responses between male and female offspring will be observed. Therefore, responses of male and female offspring should be statistically compared before they are grouped together as one unit. If sex-based differences do not exist, it is statistically acceptable to group offspring responses together to increase the sample size and therefore the power of the statistical tests used to evaluate the experimental responses.

 

Time Considerations

The time required to evaluate DIT depends largely on the exposure protocol. Gestation in the mouse and rat is 21 days, and offspring generally are weaned at 21 days of age; therefore, it will take at least 42 days before data can be collected from weaning-age offspring. If all assays will be performed on immunocompetent offspring, it will take at least 63 days before data can be collected. Acclimatization periods, breeding periods, and length of DIT assays will all adjust the length of time to complete a particular study. In addition, if two experimental replicates are performed, it may be 4 to 6 months before data are appropriate for publication.

 

Acknowledgments

The authors would like to acknowledge Jackie EuDaly and Qing Hu for their valuable technical assistance during many DIT studies.