SOW_NIHES2015063-082715.pdf

STATEMENT OF WORK FOR

SCREENING FOR CARDIOTOXICITY AND NEUROTOXICITY COMBINED WITH

HEPTATOTOXICITY IN ZEBRAFISH

NATIONAL INSTITUTES OF ENVIRONMENTAL HEALTH SCIENCES

1.0. BACKGROUND: The National Institute of Environmental Health Sciences (NIEHS), located in the Research

Triangle Park, North Carolina, is one (1) of the 27 Institutes and Centers composing the National Institutes of

Health. The NIEHS is home to the National Toxicology Program (NTP), a Health & Human Services (HHS) interagency program dedicated to testing and evaluating substances in our environment. The National Toxicology

Program Laboratory (NTPL) exists within the Division of the NTP to provide state-of-the-art laboratory research support that complements and extends the NTP’s scientific capabilities and contributes to the identification and understanding of potential health related issues related to environmental/chemical exposures.

The NTP has been interested in exploring effects of toxicants during early developmental life stages since they are often uniquely sensitive to environmental insults, due in part to the enormous changes in cellular differentiation, proliferation and migration required to form the required cell types, tissues and organs. As part of this, there has been recent interest across the NTP in exploring the utility of zebrafish as a potential model to be incorporated into screening efforts since it offers many practical advantages, including rapid external development, ease of maintenance, high fecundity, transparent embryos, and homology to mammals. While there are other ongoing efforts to assess teratogenicity and molecular end-points following early exposure, there are currently no studies in place to assess cardiotoxicity in conjunction with neurobehavior and hepatotoxicity by multiplexing assays in a Good

Laboratory Practice (GLP) environment. The Contractor shall have extensive experience conducting studies multiplexing various assays to provide composite information on systemic toxicity. Assays shall be conducted for screening cardiotoxicity, and a combination of behavioral alterations to assess neurotoxicity and hepatotoxicity.

These studies will provide valuable information in different target organs, and will be useful in identifying relative toxicity of compounds for further prioritization for in vivo hazard characterization.

Early developmental exposure can cause a large spectrum of health impairments affecting various biological systems.

Toxicant screening with high throughput in vitro assays can provide important information concerning the immediate effects on cellular function. Intermediate models like zebrafish, are important since they reduce the time and cost required for toxicology testing, thereby increasing the success rate of rapidly identifying potential target organs for environmental toxicants and help with prioritizing which chemicals should be then followed up with studies in mammalian models. This project will serve to demonstrate the usefulness of the zebrafish model for providing key information concerning cardiotoxicity, hepatotoxicity and neurotoxicity following developmental exposures.

2.0. OBJECTIVES: The Contractor shall reduce the time required to perform mammalian toxicity testing, and shall be able to screen a larger number of compounds more rapidly. The Contractor shall utilize an innovative method in which several assays shall be multiplexed using automation to conduct medium throughput high-content screening in zebrafish. The Contractor shall provide zebrafish based services in the preclinical area, mainly toxicology, as well as safety and efficacy.

3.0. SCOPE: The Contractor shall assess the impact of exposure during development on cardiotoxicity and neurotoxicity combined with heptatotoxicity of 30 compounds with unknown toxicological potential in zebrafish embryos.

4.0. TASKS: The Contractor shall conduct the assays as follows:

4.1. Maximal Tolerated Concentration (MTC): Embryos from the strain BBD-WT-006 (AB*) will be obtained and kept at 28.5ºC until they reach 2-4 hpf (hours post fertilization). At this time, fertilized embryos will be placed in 24 well plates (5 per well) and treated with the test items. 5 concentrations (0.1, 1, 10, 100, 1000 µM) will be assayed per compound and 10 embryos will be treated per experimental condition. Medium will be renewed at 48 hpf to ensure compound activity. A control group of vehicle treated embryos will be also included. Plates will be incubated at 28.5ºC and embryos will be analyzed at 2 and 4 dpf (days post fertilization) under the stereoscope. The incidence of lethality and the presence of obvious developmental defects will be recorded.

4.2. Cardiotoxicity Assay: Embryos expressing a green fluorescent protein in the heart, will be obtained and kept at

28.5ºC until they reach 48 hpf (hours post-fertilization). At this stage embryos will be treated with 5 different concentrations of the test items (1µM, 3µM, 10 µM, 30 µM, 100 µM ) although chosen concentrations could be different depending of the results obtained in the MTC carried out in the first assay). 10 embryos per condition will be used and compounds will be directly dispensed in the embryo media. Embryos will be incubated with the compound for

3 hours and analyzed for their potential to induce cardiotoxic effects. This analysis will be carried out by using Biobide´s automated image analysis system.

Terfenadine at 10 µM will be used as positive control and embryos untreated or treated with the vehicle will be used as negative control.

4.3. Neurotoxicity with Added Hepatotoxicity Signals: Embryos will be obtained and kept at 28.5ºC until they reach

4 dpf (days post fertilization). 16 embryos will be used per concentration and dispensed in individual 96 well plates. 5 concentrations will be tested per compound (0.1 µM, 1µM, 10 µM, 100 µM and 1000 µM), although chosen concentrations could be different depending of the results obtained in the MTC carried out in the previous assay) and they will be analyzed after 24 hours of incubation. A group of untreated or vehicle treated embryos will be also included.

Embryo movement will be recorded during 2 cycles of 10 minutes light-10 minutes dark each. Evaluation of different movement parameters will be combined with a gross morphology evaluation, including mortality, necrosis, trunk alterations, edemas, head alterations, swim bladder formation and equilibrium. Apart from behavour end points, the existence of opacity in the liver (indicative of hepatotoxicity) will be checked. Pictures to measure this effect will be taken at concentrations where this effect is detected.

4.4. Appendix: The Contractor shall utilize the following list of chemicals while performing the assays:

Chemical CAS RN

1. 4-Methylimidazole 822-36-6

2. Aspirin 50-78-2

3. Boric Acid 10043-35-3

4. Caffeine 58-08-2

5. Di-n-Pentyl phthalate 131-18-0

6. Dibutyl phthalate 84-74-2

7. Ethanol 64-17-5

8. Duloxetine (Cymbalta) 116539-59-4

9. Fluazifop-P-butyl 83066-88-0

10. HPTE 2971-36-0

11. Kaempferol 520-18-3

12. Linuron 330-55-2

13. Lisinopril 83915-83-7

14. Methoxychlor 72-43-5

15. Saccharin Sodium salt hydrate 82385-42-0

16. Vinpocetine 42971-09-5

17. Voriconazole 137234-62-9

18. Bisphenol A 80-05-7

19. Bisphenol AF 1478-61-1

20. Bisphenol S 80-09-1

21. Lead (II) acetate 6080-56-4

22. Methyl mercuric (II) chloride 115-09-3

23. Rotenone 83-79-4

24. Dieldrin 60-57-1

25. Triphenyl phosphate 115-86-6

26. Isopropylated phenyl phosphate 68937-41-7

27. L-ascorbic acid 50-81-7

28. Firemaster 550 860302-33-6

29. n-hexane 110-54-3

30. Firemaster 600

31. Dibenzo(a,l)pyrene 191-30-0

32. Phenanthrene 85-01-8

NOTE: Chemicals will be sent in batches as they become available.

5.0. DATA RIGHTS: The Government has unlimited rights to all documents/material produced under this contract to the extent permitted by the data rights clauses. The parties mutually acknowledge their understanding that this is the government’s intent. All documents and materials, to include the source codes of any software, produced under this task order shall be government owned and are the property of the government with all rights and privileges of ownership/copyright belonging exclusively to the government. These documents and materials may not be used or sold by the contractor without written permission from the contracting officer. All materials supplied to the

Government will be the sole property of the government and may not be used for any other purpose. This right does not abrogate any other government rights.

6.0. PLACE OF PERFORMANCE: The opportunities for sample degradation and compromising the quality of data are greater with the continued movement of samples to various sites for analysis, therefore; analysis shall take place in a single facility located within the contiguous United States (CONUS).

7.0. PERIOD OF PERFORMANCE: A base of 12 months with one (1) 1-year option period. Studies shall be completed within 75 days following receipt of compounds.

8.0. SAFETY AND HEALTH: The Contractor shall comply with all Federal, North Carolina State, Occupational

Safety and Health Administration (OSHA) standards and local laws and regulations concerning the environmental, occupational safety and health, and hazardous material. The contractor shall ensure employees have all required training and certifications related to OSHA requirements. The documentation shall be readily available for the

Contracting Officer to review at any time during the life of the contract.

9.0. DELIVERABLE SCHEDULE:

Deliverable Media Deliver To

A Final report will be provided containing all results observed, together with representative images and statistical analysis.

A CD will be provided containing raw data, and pictures

COR & CO