SOWandAppendices.pdf

STATEMENT OF WORK

STUDIES TO EVALUATE THE POTENTIAL FOR TEST AGENTS SELECTED

BY THE NATIONAL TOXICOLOGY PROGRAM TO INDUCE

IMMUNOTOXICITY

October 9, 2012

TABLE OF CONTENTS

Page

I. BACKGROUND, HISTORY AND NEED

II. REFERENCES

III. STATEMENT OF WORK

A. Summary of Studies B. GLP Compliance & Other Regulations C. Deliverables & Reporting Requirements D. Section 508 Compliance

E. Electronic Data Submission & Database

F. Other Considerations

1. Data Rights

2. Replication,Dissemination & Use of the Results

3. Government Furnished Materials

4. Site Visits

5. Project Management

6. Travel Requirements

APPENDICES

NTP SPECIFICATIONS FOR THE CONDUCT OF STUDIES TO EVALUATE THE

TOXIC & CARCINOGENIC POTENTIAL OF CHEMICAL, BIOLOGICAL AND PHYSICAL

AGENTS IN LABORATORY ANIMALS .............................................................................. APPENDIX A

NTP IMMUNOTOXICITY STUDY PATHOLOGY SPECIFICATIONS ................................ APPENDIX B

Studies to Evaluate the Potential for Test Agents Selected by the National Toxicology Program to Induce Immunotoxicity

I. BACKGROUND, HISTORY AND NEED

The National Toxicology Program (NTP) organizes and conducts a comprehensive testing and research program focused on determining potential human health hazards due to exposure to chemicals, drugs, food additives, dietary supplements or environmental agents. Contracts within the Toxicology Branch (TB) support these efforts through: (1) the development and establishment of methods with predictive value for potentially immunotoxic chemicals and drugs; (2) examination of selected agents employing a defined panel of immune function tests and observational measures and (3) mechanistic studies to define the cellular and molecular events associated with immunotoxicity.

Immunotoxicity is used to describe the potential of an agent to alter the normal structure and/or function of the immune system as determined by established immunological techniques.

Immunotoxicity can be divided into three major research areas: (1) studies of altered hematopoietic or immunological events associated with exposure of humans and animals to chemical or therapeutic agents, including altered host resistance; (2) studies of respiratory or dermal hypersensitivity responses resulting from xenobiotic exposures; and (3) studies on the ability of chemical or therapeutic agents to induce or exacerbate autoimmune disease (van der Laan and van Loveren 2005; Germolec 2004). The tasks in the Statement of Work (SOW) described below are designed to address these research areas. In the first case the immune system acts as a passive target (non-specific) of the xenobiotic and may result in an increased incidence of infectious disease or neoplasia due to the inability to adequately respond to a disease-causing agent via immunodeficiency. For example, the use of immunosuppressive drugs such as those administered to organ transplant or cancer patients has been associated with an increased incidence of infections and neoplastic disease (Penn, 1985; Ehrke and Mihich, 1987).

Less commonly, xenobiotics may also stimulate specific immune effectors or pathways, potentially leading to hypersensitivity or autoimmunity. In hypersensitivity, the immune system responds to chemically-modified ("non-self") compounds (haptens) as part of a "specific" immune response. The most likely health consequences include respiratory tract allergies (e.g., asthma, rhinitis) or allergic contact dermatitis. Autoimmune disease occurs when the immune system recognizes host tissue as foreign and mounts an immunologic response against it resulting in structural or functional damage.

Immunotoxicity induced by xenobiotics has been well characterized in animals. However, there is considerably less information on xenobiotic-induced immunotoxicity in humans (Luebke et al 2006). Evaluation of immunotoxicity in humans is notably more complex than in animals considering the limited number of non-invasive tests and biological responses in the general population, which are relatively heterogeneous. In addition, with the exception of controlled clinical studies, exposure levels of the agent (i.e., dose) are often difficult to verify. However, a number of xenobiotics have been demonstrated to alter immune function in humans after accidental environmental or occupational exposure. Exposure to contaminated rice oil containing polychlorinated biphenyls and polychlorinated dibenzofurans in Japanese and

Taiwanese populations resulted in long-term suppression of T-cell numbers and function, decreased serum IgA and IgM and increased rates of respiratory infections (Lee and Chang, 1985). Chronic occupational exposure to benzene has been associated with bone marrow hypoplasia, decreased serum complement and decreased circulating immunoglobulins in exposed workers (rev. in ATSDR, 2007). Reductions in T-cell numbers and increased incidence of upper respiratory tract infections have been reported following occupational exposure to tricothecene mycotoxins from Stachybotrys chartarum (Johanning et al., 1996). In addition, persistent decreases in specific lymphocyte subpopulations and depressed antigen-specific T-Helper cell function have been demonstrated in workers exposed to high concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin at a chemical plant (Tonn et al, 1996; Jung et al., 1998).

Acute hypersensitivity disease or allergy represents one of the most common diseases with an immunologic pathogenesis affecting millions of Americans. Approximately 2-15 percent of these cases are thought to be of industrial origin (Isola et al., 2008). The characteristic that sets this response apart from normal immune mechanisms involved in host defense is that the reaction is excessive, and often leads to tissue damage. A large number of industrial materials have known or presumed allergic etiologies in humans and include, among others, trimellitic anhydride, formaldehyde, various diisocyanates, beryllium and possibly organophosphates. In addition, many metals can produce allergic contact hypersensitivity. Chemical-induced hypersensitivity can be both antibody (IgE)-mediated as well as T cell-mediated.

A number of examples of drug-induced autoimmunity (e.g., gold salts) and an increasing number of examples where environmental chemicals have been found to initiate or exacerbate preexisting autoimmune disease in humans and experimental animals. The best example of the latter is mercury-induced glomerulonephritis in rodents (Bagenstose et al., 1999). In humans, exposure to a number of chemicals, including aromatic amines, chlordane, formaldehyde and trichloroethylene has been shown to produce syndromes similar to systemic lupus erythematosus (Bigazzi, 1997). Clinical studies suggest that the therapeutic use of recombinant cytokines has led to increased incidence of organ-specific autoimmune disorders.

These observations have spurred numerous experimental reports in the recent literature which has increased our awareness of the immunotoxicity of environmental agents and therapeutic drugs and extended the list of chemicals that are known to produce immunosuppressive effects or act as sensitizers. Previous contracts supported by the NTP have developed and validated a comprehensive testing panel that can reproducibly characterize immune alterations that occur following in vivo chemical exposure in experimental animals (Luster et al., 1988). These efforts have continued in studies designed to evaluate selected xenobiotics for their ability to alter immune function as well as assess chemical-induced hypersensitivity. Results of these studies have been collected and are publicly available as a developing database for predicting no effect levels and potential human health risk. These data have also been analyzed in an attempt to develop an improved testing configuration for detecting immunosuppressive agents and provide insight into the qualitative and quantitative relationships between the immune tests and various host resistance assays (Luster et al., 1992, 1993). Recent efforts have evaluated additional endpoints for use as screening tools to predict chemical-induced effects on the immune system (Germolec et al. 2004). The results of these and similar studies have led to specific regulatory requirements within the Environmental Protection Agency and the Food and Drug

Administration for assessing potential immunosuppressive effects of chemicals. The EPA's guidelines for immunotoxicology testing fall under both its Toxic Substances Control Act and its Federal Insecticide, Fungicide and Rodenticide Act Series 870 Health Effects Test guidelines, which are used by the Office of Pesticide Programs and the Office of Pollution Prevention and Toxics.

Routine parameters measured in toxicology studies, such as blood or tissue cellularity, organ weights and histopathology are often considerably less sensitive indicators of immunotoxicity than immune function tests. The lymphocyte and macrophage, in particular, possess a number of characteristics that make them an appropriate model for examining the effects of various agents on proliferation, differentiation and function. Among these are their capacity to undergo modulation or division in vitro in response to antigens or nonspecific stimuli; expression of gene products that can be utilized as markers of differentiation, and their potential to undergo terminal differentiation resulting in production of soluble mediators (e.g., cytokines, proteolytic enzymes or antibodies) or providing effector function (e.g., tumor target cell killing).

A major concern in risk assessment has involved extrapolation of dose response data from effect to no effect level or from rodent model systems to humans. So that the most accurate estimate possible of chemical safety can be obtained, there is a need to increase the existing data base regarding the ability of chemicals to affect the immune system as well as to understand the basic cellular and subcellular mechanisms responsible for altered immune function. Furthermore, there exist a large number of environmental agents and therapeutics that have not been examined for immunotoxicity in animal models and represent a potential adverse health effect in humans.

The studies to be conducted under this contract are the only available mechanism within the federal or private sector for assessing these potential human health effects. Thus, the Core Tasks (I-V) within this request for proposal (RFP) specify the utilization of a comprehensive immune testing panel that evaluates the major components of the immune system for potential suppression or enhancement, as well as hypersensitivity and autoimmunity. Option III shall involve the development, evaluation, and validation of novel assays to determine the potential of xenobiotics to induce alterations in immune parameters and the investigation of the mechanistic basis for chemical-induced perturbations of immune function.

II. REFERENCE MATERIALS

ATSDR. Toxicological Profile for Benzene. US Department of Health and Human Services.

2007.

Bagenstose LM, Salgame P, Monestier M. Murine mercury-induced autoimmunity: a model of chemically related autoimmunity in humans. Immunol Res. 20: 67-78, 1999

Bigazzi, P.E.: Autoimmunity caused by xenobiotics. Toxicology 119: 1-21, 1997.

Ehrke, MJ and Mihich, E. Effects of anticancer agents on immune responses. Trends Pharmacol Sci 6: 412-418, 1987.

Germolec, DR. Sensitivity and predictivity in immunotoxicity testing: immune endpoints and disease resistance. Toxicol Lett. 149: 109-14, 2004 .

Germolec, DR, Nyska, A, Kashon, M, Kuper, CF, Portier, C, Kommineni, C, Johnson, KA and Luster, MI. Extended histopathology in immunotoxicity testing: interlaboratory validation studies. Toxicol. Sci. 78: 107-15, 2004.

Isola D, Kimber I, Sarlo K, Lalko J, Sipes IG. Chemical respiratory allergy and occupational asthma: what are the key areas of uncertainty? J Appl Toxicol. 28: 249-53, 2008

Johanning, E, Biagini, RE, Hull, D, Morey, P, Jarvis, B, and Landisbergis, P. Health and immunology study following exposure to toxigenic fungi (Stachybotrus chartarum) in a water-damaged office environment. Int Archiv Occup Environ Health 68: 207-218, 1996.

Jung, D, Berg, PA, Edler, L, Ehrenthal, W, Fenner, D, Flesch-Janys,D, Huber, C, Klein, R, Koitka, C, Lucier, G, Manz, A, Muttray, A, Needham, L, Papke, O, Pietsch, M, Portier, C, Patterson, D, Prellwitz, W, Rose, DM, Thews, A, and Konietzko, J.

Immunologic findings in workers formerly exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin and its Congeners. Environ Health Perspect 106S: 689-695, 1998.

Lee, TP and Chang, KJ. Health effects of polychlorinated biphenyls. In:

Immunotoxicology and Immunopharmacology, Dean, JH, Luster, MI, Munson, AE and Amos, H, eds, pp.415-422, Raven Press, New York, NY, 1985.

Luster, M.I., Munson, A.E., Thomas, P., Holsapple, M.P., Fenters, J., White, K., Lauer, L.D., and Dean, J.H.: Development of a testing battery to assess chemical-induced immunotoxicity.

Fund. Appl. Toxicol. 10:2-19, 1988.

Luster, M.I., Portier, C., Pait, D.G., White, K.L., Gennings, C., Munson, A.E., and Rosenthal, G.J.: Risk assessment in immunotoxicology. I. Sensitivity and predictability of immune tests.

Fund. Appl. Toxicol. 18:200-210, 1992.

Luebke RW, Chen DH, Dietert R, Yang Y, King M, Luster MI; Immunotoxicology Workgroup.

The comparative immunotoxicity of five selected compounds following developmental or adult exposure. J Toxicol Environ Health B Crit Rev. 9: 1-26, 2006.

Luster, M.I., Portier, C., Pait, D., Rosenthal, G.J., Germolec, D.R., Corsini, E., Blaylock, B.L., White, K.L., Munson, A.E., and Comment, C.E.: Risk assessment in immunotoxicology.II.

Relationship between immune and host resistance tests. Fund. Appl. Toxicol. 21:71-82, 1993.

Penn, I. Neoplastic consequences of immunosuppression. In: Immunotoxicology and Immunopharmacology, Dean, JH, Luster, MI, Munson, AE and Amos, H, eds, pp.79-89, Raven Press, New York, NY, 1985.

Tonn, R, Esser, C, Schneider, ES, Steinmann-Steiner-Haldenstatt, W, and Gleichmann, E.

Persistence of Decreased T-Helper Cell Function in Industrial Workers 20 Years after Exposure to 2,3,7,8-Tetrachlorodibenzo-p-Dioxin. Environ Health Perspect. 104: 422- 426, 1996.

van der Laan JW, van Loveren H. Current status and burning issues in immunotoxicity testing of drugs. Toxicol Appl Pharmacol. 207(2 Suppl): 435-40, 2005

III. STATEMENT OF WORK

October 9, 2012

Independently, and not as an agent of the Government, the Contractor shall provide all the necessary services, qualified personnel, materials (including animals not specified in the Statement of Work), equipment, and facilities not otherwise provided by the Government as needed to examine the immune system in laboratory animals following exposure to xenobiotics.

Tasks I – V are defined as the Core Studies of this contract and shall be conducted as specified in Part 58 “Good Laboratory Practices for Non-Clinical Studies (Federal Register, Friday, December 22, 1978, Part II and any late interpretations published by the FDA). The Government will provide the test articles to be evaluated as described in Tasks I - V on an "as needed basis" to be determined by the Contracting Officer’s Representative (COR). In general, there will be a desire to maintain consistency with other NTP testing efforts and thus, the Government will furnish the test material. Compounds used as positive controls and test articles that can be purchased through commercial sources where there is no requirement for consistency with other NTP studies shall be purchased by the Contractor.

A. STUDIES TO BE CONDUCTED

Task I – Evaluation of the Potential to Induce Immunotoxicity

I-A: Screening (formerly designated Range-finding) studies on Immunomodulation

The Contractor shall test approximately four (4) xenobiotics per year using tests described in Task I-A-1 through 5. Exposure regimen will typically consist of daily dosing for 28 days employing oral gavage, drinking water, intratracheal, diet or intraperitoneal injection of the test article (route to be determined by the COR) at five (5) dose levels in addition to a vehicle control. Eight (8) animals per dose level shall be employed for the tests described in items I-A-1 through I-A-5. Previous studies at the NIEHS have shown that certain of the required assays can be performed from tissue derived from the same animal. Appropriate positive and vehicle controls shall be used for each immune assay, with the understanding that the positive controls may be different for specific immune function tests. When necessary, as determined by the COR, the results from the studies shall be confirmed by a repeat study in the Contractor's laboratory. In most circumstances, these studies shall be performed in female B6C3F1/N mice.

Females are suggested since they have been shown to be more responsive than males in many immune tests and they can be group housed. However, in certain instances the COR may determine that the use of male B6C3F1/N mice, female Harlan Sprague-Dawley (HSD) rat or another mouse or rat strain or stock is necessary. This determination may be based on pharmacokinetic considerations or demonstrated species- or strain-specific effects in other toxicology studies. In rare instances (such as when chemical exposure via inhalation is required) the test article exposure may be conducted at other NTP testing facilities and the Immunotoxicology Contractor shall receive appropriately treated animals, immune cells and/or tissues to assay for immune function.

The test articles and procedures for dose formulation will be provided by the NTP as specified above. Dose analysis of the test article and/or analysis of biological samples (i.e. serum, plasma or urine) will be conducted by the NTP on an as needed basis to be determined by the COR. The NTP will provide procedures for collection of samples and submission to an NTP- designated Chemistry Services laboratory for dose or biological sample analysis when the COR determines that such analyses are required. The COR will supply the address and contact information for shipment of samples to be analyzed. If background information on a test article procured by the Contractor is not available, as determined by the COR, the purity of the chemical will be determined by the NTP, through a separate NTP Chemistry Services Contractor. Results of these analyses shall be disseminated to the Contractor as soon as they are available.

I-A-1. Immunopathology - The Contractor shall measure standard toxicity parameters including body weight, selected organ weights (spleen, thymus, liver, lung, and kidney with adrenals unless otherwise specified) and hematological profile (complete blood count and differential).

Liver, lung, kidney, adrenal glands, spleen, bone marrow (femur), mesenteric lymph node chain, submandibular lymph nodes and popliteal lymph nodes, gastrointestinal tract (stomach to rectum with Peyer’s patches) and thymus shall be fixed in accordance with NTP protocols (Appendix B) for later histological evaluation by the NTP. Wet tissues with animal identification as specified in Appendix B (species, sex, strain/stock, age) and study data (copies of scientific protocol, study design, dose group identification, list of tissues collected, body and organ weights and individual animal necropsy reports [including descriptions of any gross abnormalities]) shall be shipped within 72 hours to an NTP designated contract lab for processing and histological evaluation. It is strongly recommended that a board certified veterinary pathologist be present at all necropsies where tissues are collected for Immunopathology. For selected studies the COR may request that approximately 100 mg of tissue from the spleen, thymus, liver or other organs be collected, flash frozen in liquid nitrogen and stored at –80° C until transfer to the NTP frozen tissue bank or an NTP designated contract laboratory for molecular biology studies. The Contractor shall collect and archive serum or other biological samples on a case-by-case basis as determined by the COR for assessment of test article levels under a separate NTP Chemistry Services Contract as described above.

Animals found moribund during the course of the study should be humanely euthanized, and a complete necropsy as described above should be performed to ascertain if the morbidity is treatment or handling related.

I-A-2. Humoral Mediated Immunity - The Contractor shall assess humoral mediated immunity by quantitating antigen-specific antibody response following intravenous administration of sheep red blood cells (SRBC) by both ELISA and the antibody forming cell (AFC) plaque assay. In addition, the Contractor shall assess the response to an alternative T-dependent antigen (i.e.

Keyhole limpet hemocyanin) via ELISA or ELISPOT.

I-A-3. Cell Mediated Immunity - The Contractor shall quantitate cell-mediated immunity by assessment of the lytic ability of splenic cytotoxic T lymphocytes.

I-A-4. Cell Quantification – The Contractor shall quantitate splenic cellularity, splenic differentials, and B cell, T cell, T-cell subsets, NK cells and monocyte enumeration by cytometric analysis.

I-A-5. Non-specific Immunity - The Contractor shall assess nonspecific immunity by quantitation of natural killer cell activity. In some instances the COR may require studies to evaluate macrophage function, (i.e. phagocytosis or tumor cell killing assays) as a measure of non-specific immunity.

Task I-B. Definitive Studies in Immunomodulation and Evaluation of Host Resistance Endpoints

The Contractor shall test approximately two (2) xenobiotics per year (selected from those tested in Task I-A) using more comprehensive testing methods to evaluate the specific immune cell targeted by the chemical of interest. This may involve repeating some portion of the tests described in Task I-A, as well as evaluating effects in alternate tests that measure the target immune cell population (for example evaluating IgG rather than IgM responses in the AFC assay). The COR will select the test article(s) and appropriate test method(s) to be used. The exposure regimen shall be similar to that described under Task I-A with the exception that three

(3) rather than five (5) dose levels shall be employed in addition to vehicle control groups. Eight

(8) animals per treatment group shall be employed for these tests and the bone marrow studies described in Task I-B-1. For some Task I-B studies, quantification of the expression of relevant activation markers as specified by the COR and determined on a case-by-case basis shall be required. In most circumstances these studies shall be performed in female B6C3F1/N mice.

However, in certain instances, the COR may select rats or another mouse strain/stock for Task I- B studies.

I-B-1. Changes in Hematopoiesis – The Contractor shall quantitate bone marrow cell numbers and differentials, DNA synthesis and colony formation from bone marrow stem cells. The progenitor cell populations (i.e. erythroid, myeloid, lymphoid) to be evaluated shall be determined by the COR on a case-by-case basis.

I-B-2. Host Resistance Assays - The Contractor shall assess the ability of the selected test article to alter disease resistance following challenge with at least two established host resistance models. For all host resistance studies a minimum of ten (10) animals per dose group and challenge level shall be employed. The appropriate tumor cell and/or infectious agent models shall be determined by the COR in consultation with the Contractor and may include, but are not limited to the following:

(a) The growth of transplantable syngeneic tumor cells (PYB6) using a cell concentration that induces a TD20 in control mice.

(b) The quantitation of melanoma cells (B16F10) in the lung using a concentration of cells that induces a TD20 in control mice.

(c) Liver and spleen bacterial counts following challenge with Listeria monocytogenes

(ED20).

(d) Moribundity or bacterial cell counts following challenge with Streptococcus (ED20).

(e) Moribundity or tissue viral load following challenge with Influenza (ED20).

(f) Quantitation of parasitized erythrocytes following infection with plasmodium.

(ED20).

Moribund animals shall be humanely euthanized and death shall not be used as an endpoint for any host resistance studies. Animals found moribund during the course of the study should be humanely euthanized, and a complete necropsy as described above should be performed to ascertain if the morbidity is treatment or handling related.

Task II - Evaluation of the Potential to Induce Hypersensitivity

The Contractor shall evaluate approximately four (4) xenobiotics per year for their potential to produce dermal hypersensitivity in the mouse using tests described below. The COR will select the test articles to be evaluated. The dose levels to be tested for dermal sensitizing capability shall be based on solubility. Female BALB/c mice shall be purchased by the Contractor and used for these studies. A minimum of four (4) animals per dose group shall be used.

II-A-1. Mouse ear swelling test (MEST) - The Contractor shall test allergenic or immunogenic potential using the mouse ear-swelling test. For sensitization in the MEST assay, the compound shall be administered topically by applying the appropriate concentration directly to the skin on the shaven interscapular area of the upper back for three (3) days. The animals shall be rested for approximately four (4) days. For challenge, the test article shall be applied to the ears. Ear swelling shall be quantitated at 24 and 48 hours following challenge. An appropriate positive control shall be used for each study.

II-A-2. Local lymph node assay (LLNA) - The Contractor shall examine the sensitization potential of the test articles of interest in BALB/c mice and matching controls using the LLNA.

The LLNA shall be performed in accordance with the current ICCVAM guidelines (http://iccvam.niehs.nih.gov/methods/immunotox/llna.htm). The test article shall be applied to the dorsum of the ear for three (3) consecutive days. The animals shall be rested for two (2) days and proliferation in the draining lymph nodes evaluated on day six (6).

Animals found moribund during the course of the study should be humanely euthanized, and a complete necropsy as described above should be performed to ascertain if the morbidity is treatment or handling related.

Task III – Evaluation of the Potential to Influence the Development and/or Progression of Autoimmune Disease

The Contractor shall test approximately one (1) xenobiotic per year for the potential to influence autoimmune disease. The COR will select the chemical(s) and the appropriate animal model.

Using a rodent model for autoimmune disease (e.g. NZB mouse for human SLE, NOD mouse for insulin-dependent diabetes or Brown-Norway rat for mercury-induced glomerulonephritis) the xenobiotic shall be administered for up to 20 weeks and the animals monitored for the development or exacerbation of autoimmune disease using appropriate pathological (e.g., histopathology of target organ) and biochemical (e.g., changes in circulating autoantibodies or other serum proteins, proinflammatory or other cytokines or cytokine receptors, glucose levels) tests. The COR will determine the appropriate endpoints to be evaluated for each specific model based on the disease endpoints they are meant to represent. In some instances, the xenobiotic shall be administered prenatally to assess developmental effects.

To examine gender-specific differences in the development or exacerbation of autoimmunity, chemical effects shall be evaluated in both sexes. Approximately 25 animals per treatment group and three (3) test article-dose levels and a vehicle control shall be employed.

Animals found moribund during the course of the study should be humanely euthanized, and a complete necropsy as described above should be performed to ascertain if the morbidity is treatment or handling related.

Task IV – Evaluation of Immunomodulation in the Developing Immune System

The Contractor shall test approximately four (4) xenobiotics per year using tests described in Task I A-1-5 for the potential to modulate the immune system in the developing organism. The exposure regimen shall normally (but not always) consist of daily dosing of time-mated female HSD rats beginning on day 6 of gestation (GD 6) and continuing through Post Natal Day (PND)

4. Pups shall be dosed daily when dosing of the dams is discontinued on PND 4 and continue generally through PND 42. Dosing of the pups may be delayed until weaning at PND 28 depending on the bioavailability of the chemical via lactation in which case maternal dosing shall continue through weaning on PND 28. The COR shall specify the dosing regimen and route of exposure. Routes of administration may include oral gavage, drinking water, or dosed feed at three (3) dose levels in addition to a vehicle control. A minimum of eight (8) pups per gender per treatment group shall be employed for the tests described in items I A-1-5. As a litter is considered a single unit, all treatment groups shall be composed of pups from different litters. In most circumstances, these studies shall be performed in HSD rat pups. However, in rare instances another rat or outbred mouse strain/stock may be employed (to be determined by the COR). In rare instances test article exposure may be conducted by the NTP and the Contractor shall receive appropriately treated animals and/or immune cells and/or tissues shipped on wet ice to assay for immune function. The COR will provide study protocols, animal health status and relevant study data (body and organ weights, etc.) prior to shipment of any animals or materials.

Shipment of animals, cells or tissues will be coordinated between the COR and the appropriate representatives at the NTP designated contractors at least 60 days in advance of a study start.

Animals found moribund during the course of the study should be humanely euthanized, and a complete necropsy as described above should be performed to ascertain if the morbidity is treatment or handling related.

Summary of Studies to be conducted under the Base Contract:

Study Type Task Estimated number of tasks per year Immunomodulation -Screening I-A 4 Immunomodulation – Definitive I-B 2 Hypersensitivity II 4 Autoimmunity III 1 Developmental IV 4

The Base Contract shall consist of the performance of the tasks described above for a base period of one (1) year with nine (9) optional one-year periods of performance (Option I) dependent on programmatic needs, Contractor performance and availability of funds.

Option II - Evaluation of Additional Xenobiotics on an as Needed Basis

The Contractor shall have the capability of performing additional test article evaluations on an as needed basis that will not exceed 50 (fifty) tasks for the life of the contract or a maximum number of 5 additional tasks per year. The option to conduct additional Tasks as described in I- IV above may be exercised any time during the life of the contract based on programmatic needs and availability of funds.

Option III - Development and/or Evaluation of New and Improved Technology

Immunology is a developing science and, as such, it is expected that new and improved methodology will be required to maintain "state of the art" evaluation. In consultation with the COR, the Contractor shall identify research gaps and conduct methods development and validation on a continuing basis beginning in year 2 of the contract. A defined protocol and cost proposal for the specific task as identified by the Contractor, shall be prepared by the Contractor and submitted to the COR for approval prior to conducting the evaluation. All work conducted under this task shall have prior approval by the COR. The direction of research shall be dictated by both previous research and needs in the area. For example, it may be necessary to evaluate methods to assess the relevance of changes in cytokine levels, monitor additional cell surface markers or quantitate certain biomedical changes in chemical models of autoimmune disease.

Assessment of the utility of traditional screening methods or development of new strategies to evaluate immune perturbations in neonatal or aging animals shall be given a high priority. In addition, the validation of novel methods to determine the potential for xenobiotics to induce or potentiate autoimmune disease should be considered. The Contractor is expected to develop strategies for assessing the chemical-induced development of autoimmune disease in rodents, such as the use of activation markers in immune cell phenotyping or use of immunoprecipitation to assay for autoantibodies. Alternative endpoints for disease resistance assays, in particular those that reduce the numbers of test animals needed may be a targeted research area. For xenobiotics shown to be sensitizers, it may be useful to further characterize specific antibody production including classes (e.g., IgE) and IgG subtypes, or cytokine profiles using molecular techniques such as the RNAse protection assay. Alternatively, follow-up studies to help establish cellular and/or molecular mechanisms of action for immune perturbations observed during Tasks I – V performance may be appropriate. Development of novel screening strategies including the use of molecular techniques such as analysis of gene expression by microarray to assess xenobiotic-induced immunologic alterations would be desirable. Activities performed under Option III shall be no more than 10% of the base contract level of effort per year. The Option III portion of the contract will not be available in year one of the contract, but may be exercised each year for years 2 – 10, and shall be optional based on the COR's discretion and the availability of funds.

B. GLP COMPLIANCE & OTHER REGULATIONS

The contractor shall have the facilities, demonstrated ability and suitably trained personnel to function under the FDA Good Laboratory Practice regulations. Tasks I – IV are defined as the Core Studies of this contract and shall be conducted as specified in Part 58 “Good Laboratory Practices for Non-Clinical Studies (Federal Register, Friday, December 22, 1978, Part II and any late interpretations published by the FDA). The Government reserves the right to ensure compliance with FDA Good Laboratory Practice regulations as specified in Part 58 “Good Laboratory Practices for Non-Clinical Studies and to conduct Quality Assurance audits. The NTP Specifications for the Conduct of Studies to Evaluate the Toxic and Carcinogenic Potential of Chemical, Biological and Physical Agents in Laboratory Animals (NTP Specifications) and any subsequent revisions thereof apply to work performed under this contract (January 2011, Appendix A), including the use of serology testing to establish the health status of the animals, use of NTP approved diet, and a minimum four (4) day quarantine period for all animals prior to use in any study. Animal care facilities shall be of a design and operation (as specified in section V of Appendix A) such that an appropriate barrier minimizes the risk of infection influencing the outcome of experiments and shall comply with the NTP Specifications for the Conduct of Studies to Evaluate the Toxic and Carcinogenic Potential of Chemical, Biological and Physical Agents in Laboratory Animals (January 2011, Appendix A). The Contractor shall be responsible for ensuring the appropriate institutional review and safety assessment for each test article.

C. DELIVERABLES/REPORTING REQUIREMENTS:

Archival Materials (Study Data) – Shall be submitted to the NTP archives within 180 calendar days of completion of each individual task. Completion of a task is defined as the conclusion of all of the in-life phases of the study (i.e. euthanasia of the last animal).

Archival study data materials should be submitted to the NTP archives as specified in Appendix A.

Histopathology Materials – Shall be submitted to an NTP designated contractor. See the SOW and Appendix B for details.

Technical Reports

All reports shall be submitted in an electronic format that meets the NIEHS/NTP hardware and software standards. Any electronic documents (data, word processing, page layout, interchange formats, www content) must conform to the latest version of Microsoft Office.

In addition to those documents required by the other terms of this contract, the Contractor shall prepare and submit the following reports in the manner stated below:

The following reports are required deliverables:

1. Status Reports: On the 20th day of each month, the COR shall receive a brief (1-3 page) summary of the work currently in progress. This report shall include the status of all agents on test, reports and manuscripts and shall be submitted to the COR via electronic mail. It shall include any difficulties encountered in ongoing tasks, assay results outside of historical control ranges and remedial actions taken, including any deviations from the laboratory study protocols. No QAU audit of this status report is required.

2. Test Article Study Report: A draft test article study report shall be submitted to the COR 180 calendar days after completion of each study. Completion of a study is defined as the conclusion of all of the in-life phases of the study (i.e. euthanasia of the last animal. This report shall include a complete laboratory protocol, individual animal data, a summary of individual assays conducted in the testing of agents performed as part of Tasks I-IV and a summary of experimental findings. Individual animal data for each test conducted in the study shall be submitted as an excel spreadsheet or other COR-specified format and as a separate file, along with the draft study report for entry into the NTP database. It shall also include difficulties encountered and remedial action taken, including any deviations from the laboratory study protocol. The COR will review the draft and return comments to the Principal Investigator to be incorporated in the final report. Draft reports need not be 508 compliant. Within 60 calendar days of receipt of the COR’s requested revisions the Contractor shall supply a full copy of a revised report including covers and signature pages that is Section 508 compliant in pdf format and with all graphics attached on a DVD. A separate DVD containing MS Word files of the report and included tables that are Section 508 compliant shall also be submitted.

3. Annual Progress Report: A brief (1-2 page) electronic summary of the progress made during the previous 12-month period for studies conducted under Option III. The due date shall be 30 calendar days after the anniversary date of the contract, beginning in year

3. Annual Progress reports must be section 508 compliant. No annual progress report will be required during the final year of the contract.

4. Animal use protocols approved by the appropriate Institutional Animal Care and use Committee and Health and Safety Summaries and Chemical Hygiene Plan. Animal use and health and safety materials approved by the Contractor’s internal review committees shall be provided to the NTP no later than 7 calendar days prior to the commencement of the specific studies that they relate to.

Reports as specified below shall be sent to the COR electronically or at the address specified at time of award.

Summary of Reports

Description Due Date

(1) Status Report Monthly via email on the 20th day of the month

(2) Test Article Study Report First draft to be received180 calendar days after completion of Task. Final draft 60 calendar days after comments received from NTP

(3) Annual Progress Report (Option III) 30 calendar days after the contract anniversary beginning in Year 3.

This report would only be required if the Option III is exercised.

An electronic copy of the Title Page of each report shall be sent to the Contracting Officer:

D. SECTION 508 COMPLIANCE

Final, electronic test article reports shall be prepared, formatted, and checked for conformance with Section 508 standards, prior to delivery to NIEHS. Based on the US Access Board’s standards, HHS has written procedures and checklists for preparing documents to meet all requirements of Section 508 standards, using either Microsoft Word or Adobe Professional, version 8.0 or higher.

The checklists and standards are found on HHS’s Section 508 website at:

http://www.hhs.gov/web/508/checklists/index.html. The Contractor shall refer to the website for further guidance. Submission of electronic documents which satisfy the appropriate HHS Checklists and Standards shall be considered acceptable. Should an accessibility problem be found in the documents, the Government will return them for remediation, with a timeframe to be negotiated at that time.

The following Section 508 accessibility standards apply to the content or communications material identified in this SOW:

“36 CFR part 1194.41 Information, Documentation, and Support”

Section 508 compliance for PDF files: The NIH Section 508 Coordinator approved a Section 508 accommodation for certain NTP reports. As the accommodation must be renewed annually, the

COR will inform the Contractor about any changes. The accommodation applies only to the deliverables identified in the table below : Contract Deliverables covered in Accommodation.

Table. Contract Deliverables covered in Accommodation.

Deliverables identified in the SOW Accommodation Document Types Test Article Study Reports Study Reports

If the contractor cannot make these reports fully Section 508 compliant, PDF Documents containing these reports are acceptable for Section 508 compliance provided the following minimum standards are met:

1. The language in PDF document properties must be set to the appropriate language.

2. The text in the PDF document must be searchable to the extent possible (this may require Optical Character Recognition processing). Handwritten notes, images, drawings and some text not recognized by OCR are not required to be searchable.

3. Tags must be added to the PDF document.

4. Reading order must be set in the PDF document.

The Contractor shall contact the COR for additional information.

E. ELECTRONIC DATA SUBMISSION AND DATABASE

It is suggested that the Contractor work closely with the COR and database staff as the NTP is currently transitioning to a new Provantis data management system. Databases prepared and/or maintained for the NIEHS shall be accessible for query and modification by SQL commands (i.e.

Microsoft SQL, MySQL, Oracle, etc.), and the electronic data and management systems used for the Immunotoxicity studies to be conducted under this contract shall be approved by the COR.

The Contractor shall use the NTP version of the Provantis system for collection of study data.

NTP will provide licensed access to this system. NTP’s version of this leased system is currently under development and the NTP will provide additional information and system support when the system becomes available. It is anticipated that the system will be available by the time of the contract award. In cases where data for a portion of a study is not collected in Provantis, or until the Provantis system is available, all study data shall be delivered to NTP database personnel as either excel spreadsheet or database/text files in a tab-delimited format with an agreed upon character as a repeating field character for fields in the database(s) that are defined with repeating entities.

The COR may request unaudited interim or preliminary data in the form of data tables or individual animal data on a case-by-case basis (generally for comparison with results from other NTP toxicity studies). Should data need to be extracted from a Contractor’s database on a regular basis, a specified time interval shall be named and maintained as well as the method of receivership of this data (electronic, CD-ROM, DVD, etc.).

F. OTHER CONSIDERATIONS

(1) DATA RIGHTS, PATENTS, COPYRIGHTS:

All original source documentation is the property of the NTP. Data summaries and other relevant information generated during the performance of this contract shall be submitted to the COR in the form of status reports and agent specific reports as specified in the Deliverables section above. In addition, annual reports shall be submitted for Option III activities as specified in the Deliverables section. To the extent possible, the Government will retain unrestricted access and rights to all tissues, data and technical information generated under the contract. The Contractor shall arrange for delivery of all raw data to the NTP Archives within 180 calendar days of the completion of each individual Task.

(2) REPLICATION, DISSEMINATION, OR USE OF THE RESULTS:

It is anticipated that the results of the research conducted under the Contract shall be published in the open, peer-reviewed scientific literature and in an NTP technical report series dedicated to Immunotoxicology studies. Specific details concerning authorship and technical issues surrounding the publication of the research shall be discussed and agreed upon by the Principal Investigator and the NTP COR on a case-by-case basis.

Because premature release of information developed under the contract could be harmful to the public, the Contractor and the Government agree that it is important to consider issues related to the timing of the release of research results when submitting information for publication. With regard to public disclosures of the research to be carried out pursuant to this contract, the Contractor agrees to submit all proposed articles to the COR at least thirty (30) calendar days in advance of submission for publication. Within this thirty-day period the COR shall inform the Contractor if there are serious concerns raised by the timing of said publication. Upon receipt of a notification that the Government objects to the publication of said information the Contractor agrees to withhold submission of said publication for an additional thirty (30) calendar days while the author and the COR discuss the issues of concern. The Contractor agrees to give good faith consideration to all such issues and further agrees that it will not knowingly submit for publication any article, abstract or other item which would be harmful to the public welfare; however, the final decision on whether to submit an article for publication shall rest with the author of said publication after the procedures set forth above are followed.

Similar procedures shall apply for scientific presentations and meeting abstracts.

(3) GOVERNMENT FURNISHED MATERIALS, EQUIPMENT, AND FACILITIES

The NTP will provide the test articles to be evaluated as described in Tasks I-IV (unless otherwise specified). The NTP will provide the information on test article identity and purity and methods for dose formulation. Dose analysis will be performed by the NTP on an “as needed basis” to be specified by the COR. The government will furnish B6C3F1/N mice. The Government will provide licensed access to the Provantis system for collection of study data. The Contractor shall provide all other chemicals, materials, animals and equipment. Information on NTP-designated suppliers such as those for test animals and disease screening will be provided after award.

(4) SITE VISITS

The COR will conduct periodic site visits, meetings with the Principal Investigator, review of laboratory study protocols and chemical reports, electronic mail and telephone discussion, as required. In addition, NTP Quality Assurance site visits may be conducted to inspect and audit study data and records. NTP Laboratory Animal Medicine site visits may be conducted to ensure that all facilities, practices and procedures of husbandry, care and maintenance of animals used in the performance of this contract are in accordance with the latest issue in effect of the Guide for the Care and Use of Laboratory Animals.

NTP may also perform health and safety inspections.

(5) PROJECT MANAGEMENT

This contract shall be managed on a “Task” basis with individual laboratory protocols prepared for each Task and test article. Each Task will be initiated by the COR, will include a detailed description of the work to be performed, and will be sent directly to the Contractor. The Contractor shall submit a laboratory protocol and cost proposal to include a description of how the work shall be conducted and shall include those details specific for the proposed Task. Laboratory study protocols shall be submitted to the COR for review within 3-5 weeks of receipt of the assignment of the Task. A protocol approved by the Contractor’s IACUC must be received at least one week prior to the proposed commencement of the study. The Contractor may not proceed with any work until they have received an approved, signed laboratory protocol from the NTP and must sign and return a copy of the laboratory protocol to the COR. Any incident or unforeseeable occurrences not listed in this document, or any physical modification to the laboratory facilities, as well as any changes in key personnel that would have a significant impact on the conduct or results of study activities (Principal Investigator, Deputy Principal Investigator, QA officer, Veterinarian, Health and Safety Officer), shall be reported immediately to the COR.

(6) TRAVEL REQUIREMENTS:

The Principal Investigator shall be expected to travel to NIEHS at least once a year for a period of one (1) day for a conference with the COR to discuss progress.

SPECIFICATIONS

FOR THE CONDUCT OF STUDIES

TO EVALUATE THE TOXIC AND CARCINOGENIC POTENTIAL

OF CHEMICAL, BIOLOGICAL AND PHYSICAL AGENTS

IN LABORATORY ANIMALS

FOR THE NATIONAL TOXICOLOGY PROGRAM (NTP)

January 2011

APPENDIX A 1

BLANK PAGE

(For Double-side Printing)

APPENDIX A 2

TABLE OF CONTENTS

I. PERSONNEL

A. KEY PERSONNEL

B. DISCIPLINE LEADERS

C. OTHER CRITICAL STAFF

D. SUPPORT STAFF

II. FACILITY

A. FLOOR PLAN

B. EMERGENCY FACILITY SUPPORT

C. ANIMAL FACILITIES

D. SUPPORTING FACILITIES

III. HEALTH AND…