SOW _NIHES2015062.pdf

STATEMENT OF WORK FOR

ASSESSMENT OF INTERNAL DOSE/ BIOAVAILABILITY IN CONJUNCTION WITH MEDIUM

THROUGHPUT HIGH CONTENT IN VIVO ASSAYS 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 zebrafish is an important complementary tool to mammalian toxicity since it can be used to screen a larger number of compounds more rapidly and provide information on prioritizing compounds for subsequent mammalian toxicity studies. There are several ongoing efforts at the NTP exploring the utility of zebrafish as a potential model to screen compounds to prioritize for further mammalian testing. One critical consideration in determining relative toxicity of compounds is their ability to enter the fish. To date, there are no projects that evaluate the bioavailability or internal concentration in zebrafish. Another important requirement is the assessment of ototoxicity following developmental exposure, an important target organ that is currently not being evaluated in other studies. To address this, the NIEHS has requires a Contractor that can multiplex assays to assess ototoxicity and developmental defects and evaluate bioavailability using the HPLC-MS-MS method in a GLP-environment. These studies will provide important complementary information to other ongoing zebrafish efforts to enable prioritization of compounds for further mammalian testing that are currently not being covered.

2.0. OBJECTIVES: The Contractor shall perform the assessment of bioavailability following exposure to 30 compounds with potential for developmental toxicity in zebrafish embryos. The Contractor shall provide high content screening information on multiple target organs under the same roof using a GLP-environment. Ongoing projects the

NTP focus on assessing teratogenicity and molecular end-points in zebrafish following early in life exposure. However, one (1) critical piece of information key to this effort is determining how much of the chemical enters inside the zebrafish.

To date the NIEHS does not have a mechanism to assess bioavailability/ internal dosing. The Contractor shall multiplex ototoxicity and developmental defects to be able to design more efficient experiments using fewer fish, to reduce time.

The Contractor shall be capable of providing zebrafish based services in the preclinical area, mainly toxicology through early detection of adverse effects in a GLP-setting. The Contractor shall assesse bioavailability /internal dosing following chemical exposure.

3.0. SCOPE: The Contractor shall perform the assessment of internal dose/ bioavailability in conjunction with medium throughput high content in vivo assays in zebrafish to evaluate developmental effects, ototoxicity and internal concentrations in 30 compounds over five (5) concentrations. Two (2) independent toxicity assays to screen for developmental toxicity and ototoxicity will be carried out at the concentrations selected after the Maximum Tolerated

Concentration (MTC) is developed. Once assays are complete, the embryos shall be frozen and will be used to test internal exposure in the concentrations tested by HPLC-Ms-Ms. The widely used AB* zebrafish strain is proposed for use in these studies.

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

4.1. Evaluation of Developmental Defects: Embryos from the same strain shall be obtained and treated as described in the previous section. Eight concentrations per compound, chosen based in the results obtained in the MTC phase, will be assayed and 15 embryos will be treated per experimental condition. Apart from the control group of vehicle treated embryos, a negative control of embryos treated with 1000µM sucrose will be included. As a positive control of the assay, embryos treated with retinoic acid at 100 nM will be also used. Plates will be incubated at 28.5ºC and embryos will be analyzed at 2 and 4 dpf under the stereoscope. A detailed analysis of developmental defects found and the incidence of lethality will be recorded.

EC50 and LC50 will be calculated and a teratogenic index (TI) established (ratio between LC50 and EC50) at 2 and 4 dpf to conclude about the teratogenic potential of each compound.

4.2. Ototoxicity Test: Embryos from line BBD-WT-006 will be obtained and grown at 28.5ºC until they reach approximately 5dpf. At this stage embryos will be treated with 5 different concentrations of the test items (3 µM, 10µM, 30µM, 100 µM and 300 µM) although chosen concentrations could be different depending of the results obtained in the MTC carried out in the first study). 10 embryos per condition will be used and compounds will be directly dispensed in the embryo media. Embryos will be incubated with compounds for 24 hours and analyzed for their potential to induce ototoxicity at 6 dpf. For this purpose, embryos will be stained with DASPEI anesthetized with Tricaine at 0.003% and immobilized in methyl-cellulose (3%). Images of all the embryos will be captured in order to quantify the number of the neuromasts.

Kanamycin at 50 M shall be used as positive control and embryos untreated or treated with the vehicle will be used as negative control.

4.3. Bioavailability Test: The Contractor shall ensure that at the end of the selected experiment(s), embryos are collected, washed and frozen for the evaluation of the bioavailability. The real exposure to the compounds at each concentration shall be carried out by HPLC-Ms-Ms of smashed embryos (10 embryos per concentration) that will allow elucidating real exposure to the test items, even discharging false negative results.

4.4. Appendix: The Contractor shall use the following list of chemicals in performance of 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

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.

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

Provide a Complete Protocol (in consultation with the NTP team).

Electronically via FTP site. Large files use NIEHS hard drive.

COR & CO

Provide a Detailed Final Report no later than _____ after task completion.

Includes raw data, DVD containing embryo´s representative images etc.

COR & CO

GLP environment

Continued follow up