Attach_6_Sample_Work_Assignments_07-16-13.pdf

ATTACHMENT 6

ATTACHMENT 6

SAMPLE WORK ASSIGNMENTS

Sample Work Assignment A: Conduct of Studies to Evaluate the Toxicity and

Carcinogenicity of Amosite Asbestos in Laboratory Animals Exposed Via Nose Only

Inhalation

I. GENERAL WORK ASSIGNMENT OBJECTIVES

A. BACKGROUND INFORMATION

This WORK ASSIGNMENT involves the evaluation of the toxicologic and carcinogenic potential of amosite asbestos via nose-only inhalation in the Wistar Han rat. The WORK ASSIGNMENT includes prestart study activities, 14-day (plus a ninety-day post exposure phase) toxicity and tissue burden studies and 2-year toxicity/carcinogenicity and tissue burden studies.

B. PROJECT MANAGEMENT

This WORK ASSIGNMENT includes only one Task that is divided into PHASES. All activities associated with this test article are considered a TASK. All studies are to be conducted according to the "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, January 2011”.

C. PHASES WITHIN THE TASK

Phase 1: During Phase 1, the contractor shall perform those work activities associated with identity and purity determination of the bulk material, development of exposure generation and monitoring methods, development of methods for tissue amosite asbestos determinations, and health and safety concerns that must be addressed before study start, and submission of a prestart inhalation report.

Phase 2: Upon successful completion of Phase 1, the 14-day (plus a ninety-day post exposure phase) toxicity and tissue burden studies specified as Phase 2 shall be conducted.

Phase 3: Upon successful completion of Phase 2, the 2-year toxicity and tissue burden studies specified as Phase 3 shall be conducted

D. GOVERNMENT FURNISHED MATERIALS

- Test material.

- Equipment, software and user manuals for a government owned toxicology data collection system or access to an NTP specified data management system

II. PHASE 1 - PRESTART ACTIVITIES

A. TEST MATERIAL ANALYSIS

1. Receive the test material and schedule the generation and monitoring developmental work.

NOTE: The test material will be provided by NTP.

2. Develop identity and purity methods for the bulk material. NOTE: the Offeror must propose methods for determining identity and purity, to include both physical and chemical characteristics of the test agent.

3. Conduct identity and purity analysis of the test material using the methods developed.

Preliminary results shall be reported to the NTP for approval as soon as the results are available.

B. DEVELOPMENT OF AN EXPOSURE SYSTEM

1. Develop methods for generation and carousel monitoring at the protocol required concentrations in the study carousels. NOTE: Offerors are to propose appropriate methods for generation and monitoring of the test agent.

2. Validate the methods for generation and carousel monitoring at the protocol required concentrations.

3. Conduct detailed 3-day carousel characterization studies.

4. Determine measurement limits, specificity, precision and linearity of the carousel concentration monitoring system.

5. Determine T90, Tl0, carousel homogeneity, aerosol evaluation, and stability of the exposure concentration in all carousels. The stability of carousel concentration must be established for a period of 3 hours plus T90 for three days. All the data must be obtained in the same exposure room and under identical conditions that will be used during the animal exposures.

6. Perform particle evaluation.

7. Conduct stability studies of the test material in the generator reservoir and carousel.

(Identification of possible degradation products is the responsibility of the Contractor)

8. Develop and test method(s) of effluent exhaust treatment, during generation runs at the protocol required concentrations in all carousels under actual animal exposure conditions. Demonstrate effectiveness of the exhaust treatment unit immediately after the effluent treatment unit and at the point of exhaust from the building. Establish the percent efficiency of the exhaust treatment. The effluent exhaust treatment must be effective in removing the test material to an acceptable concentration (i.e. greater than 90% efficiency of removal by the treatment system and less than 50% of the TLV, if a TLV exists), or written documentation for a waiver from appropriate air regulatory agencies must be provided. Determine lifetime expectancy of any proposed filtration/treatment units and amount of treatment media required. Confirm that none of the exhausted test material is re-entrained.

9. Develop and test method(s) of room air monitoring during generation runs at protocol required concentrations in all carousels under actual animal exposure conditions. Define the lower limit of detection of the monitoring method(s) and document that this level provides an adequate safety margin for personnel.

10. Demonstrate containment of particulate test article during the generation runs at protocol required concentrations in all carousels under actual animal exposure conditions. Define the lower limit of detection of the monitoring method(s) and document that this level provides an adequate safety margin for personnel.

11. Prepare and submit a Prestart Inhalation Report of Phase I chemistry, exposure system, and health and safety activities and procedures to include:

(a) Description of bulk material identity and purity method development

(b) Confirmatory data on identity, purity and, if necessary, stability of the bulk material using methods developed, as well as copies of the SOPs for the bulk chemical reanalysis methods

(c) Description of nose only inhalation exposure carousels

(d) Generation system description and approach

(e) Carousel monitoring system description and calibration

(f) Homogeneity of test atmosphere in carousels

(g) Stability of carousel concentration over 3 days

(h) Physical and chemical evaluation of the fibers

(i) Carousel T90 and Tl0 times

(j) Test article stability studies in generator reservoir and carousel

(k) SOP for generation of test material

(l) SOP for generation maintenance/changeout

(m) SOP for carousel monitoring system including calibration approach

(n) SOP for bulk material identity and purity and reanalysis

(o) SOP for carousel homogeneity determinations

(p) SOP for evaluation of fibers

(q) Other SOPs as appropriate for the specific test material

(r) Description of effluent exhaust treatment and SOPs

(s) Effluent exhaust treatment monitoring data and percent efficiency of the effluent exhaust treatment

(t) Room air monitoring methodology, strategy, SOPs, frequency and results

12. Develop a preliminary, unvalidated method for the determination of amosite asbestos in lung, pleural tissue (diaphragm), bronchial and mediastinal lymph nodes, esophagus, forestomach, glandular stomach, small intestine, large intestine, kidney and ovary and testis.

Phase 1 shall include 1 month for NTP review of the Prestart Inhalation Report.

III. PHASE 2- 14-DAY (PLUS A NINETY-DAY POST EXPOSURE PHASE) TOXICITY AND TISSUE

BURDEN STUDY

A. TREATMENT

A sufficient number of Wistar Han dams and litters to place 270 weanling rats/sex on study (NOTE:

assume that three pups/sex from each litter will be placed to the study, with additional animals used for disease screening) with the same birth date shall be obtained. These animals shall arrive on ~PND10 to be quarantined. The rats shall be housed one dam/litter per cage and provided NIH-07 diet. On PND21, the rat pups shall be weaned, placed on NTP-2000 diet, and assigned randomly to exposure groups. Rats shall be acclimated to the carousels for one hour on PND21, two hours on PND22 and three hours on PND23; on PND24, exposures shall begin, At each of five exposure concentrations (0.1, 0.3, 1, 3, or 10 mg/m3) plus a control group, 35 animals per sex per group shall receive the test material via nose-only inhalation, 7 days/week for 14 days. The daily exposure time shall be three hours per day plus T90. All animals shall remain on the treatment regimen until the day prior to sacrifice. All rats shall be housed individually.

Animals Species Sexes Groups Total

Treatment 45 x 1 x 2 x 5 = 450 Controls 45 x 1 x 2 x 1 = 90 Sentinels 5 x 1 x 2 = 10

Total 550

*Sentinels are to be distributed amongst the control and treated carousels

B. OBSERVATIONS

All animals shall be observed two times daily, once in the early morning and once in the late afternoon, at least six hours apart (before 10:00 AM and after 2:00 PM), including holidays and weekends, for moribundity and mortality. All animals shall be weighed individually on day one on test, twice weekly during the exposure phase, weekly during the post exposure and at removal.

Formal clinical observations shall be performed and recorded daily during the exposure phase and weekly during the post-exposure phase. Signs of toxicity detected at times other than the formal observations shall also be noted and recorded.

C. NECROPSY AND HISTOPATHOLOGICAL EVALUATION

A complete necropsy shall be performed on all treated and control animals that either die or are sacrificed moribund during the exposure period. Five animals per sex per exposure group shall be randomly selected and a complete necropsy performed and lung weights determined the day after the final exposure and at the end of the post-exposure period. All tissues specified in the NTP Specifications shall be saved, trimmed, embedded, sectioned and stained with hematoxylin and eosin for possible histopathological evaluation. This shall be done for all scheduled and unscheduled animals in all groups.

Histopathological evaluation shall be done on the nasal cavity, trachea, larynx, pharynx, mediastinal and tracheobronchial lymph nodes, lungs and mainstem bronchi, pleural tissue and those organs and tissues showing gross evidence of treatment-related lesions to a no-effect concentration, plus corresponding controls.

D. TISSUE BURDEN STUDY

Immediately after the last exposure on day 14, the remaining animals per sex per species per exposure group shall be held without exposure for up to 90 days. At each of seven time points (0 days, 1 day, 2 days, 7 days, 14 days, 30 days and 90 days post termination of exposure) up to five rats per sex per exposure group shall be randomly selected and removed for assessment of lung, pleural, and bronchial and mediastinal lymph node amosite burden; the 90-day post exposure time point will coincide with the final histopathology time point (see section C above). These animals shall be weighed, sacrificed, and lung and pleural tissue collected, weighed, and subsequently frozen at -70°C for amosite asbestos determinations.

Preliminary analyses of samples from each tissue shall be performed from high and low exposure concentrations to determine an appropriate range for each tissue for which the method shall be validated. Inform NTP of this range. Depending upon whether amosite asbestos can be detected, NTP shall decide whether to proceed with method validation, or not. Once the method is validated a prestart toxicokinetic report shall be submitted for review and approval by NTP. Following approval of the prestart toxicokinetic report all samples shall be analyzed. Final tissue analysis shall include but not limited to tissue weight, concentration, burden, and normalized burden. For the lung, deposition and clearance rates and half-lives are to be calculated for amosite asbestos.

NOTE: offerors are to assume that all lung and pleural samples will be analyzed amosite asbestos.

Phase 2 shall include 1 month for NTP review of the 14-Day Toxicity and Tissue Burden

Study Report.

IV. PHASE 3- 2-YEAR STUDY

A sufficient number of Wistar Han dams and litters to place 450 weanling rats/sex on study (NOTE:

assume that three pups/sex from each litter will be placed to the study, with additional animals used for disease screening) with the same birth date shall be obtained. These animals shall arrive on ~PND10 to be quarantined. The rats shall be housed one dam/litter per cage and provided NIH-07 diet. On PND21, the rat pups shall be weaned, placed on NTP-2000 diet, and assigned randomly to exposure groups. Rats shall be acclimated to the carousels for one hour on PND21, two hours on PND22 and three hours on PND23; on PND24, exposures shall begin, At each of five exposure concentrations (TBD) plus a control group, 80 animals per sex per group shall receive the test material via nose-only inhalation, 7 days/week for 2 years. The daily exposure time shall be three hours per day plus T90. All animals shall remain on the treatment regimen until the day prior to sacrifice. All rats shall be housed individually.

Treatment 75 x 1 x 2 x 5 = 750 Controls 75 x 1 x 2 x 1 = 150 Sentinels 20 x 1 x 2 = 40

Total 940

*Sentinels are to be distributed amongst the control and treated carousels

Individual animal body weight for treated and control group animals shall be recorded on day one on test, once a week for the first 13 weeks, at 4-week intervals thereafter and at terminal sacrifice. If life threatening tumors develop or a significant number of deaths occur in the groups, the weighing frequency may be increased to every two weeks upon approval by the NTP Contract Officer’s Representative. It is estimated that animals shall be weighed every two weeks for the final thirteen weeks of the 2-year study.

Each animal shall be formally examined for clinical signs of toxicity at four-week intervals and these observations shall be recorded. Signs of toxicity detected at times other than the formal four-week observations shall be noted and recorded.

Animals shall be observed two times daily, once in the early morning and once in the late afternoon at least six hours apart (before 10:00 am and after 2:00 pm including holidays and weekends) for moribundity and mortality.

Complete necropsy and histopathological evaluation shall be performed on all animals in all treatment groups as described in the NTP Specifications.

D. TISSUE BURDEN STUDY

During the chronic study, lung, pleural tissue, bronchial and mediastinal lymph nodes, esophagus, forestomach, glandular stomach, small intestine, large intestine, kidney and ovary and testis shall be sampled and evaluated from up to 5 randomly selected male and female rats for amosite asbestos concentrations following 1 month, 3 months, 6 months, 12 months, and 18 months of treatment.

Preliminary results shall be submitted upon completion of analysis of each time point and the final results are to be included in the study report.

Preliminary analyses of samples from each tissue (except lung and pleural tissue) shall be performed from high and low exposure concentrations to determine an appropriate range for each tissue for which the method shall be validated. Inform NTP of this range. Depending upon whether amosite asbestos can be detected, NTP shall decide whether to proceed with method validation, or not.

Once the method is validated a prestart toxicokinetic report shall be submitted for review and approval by NTP. Following approval of the prestart toxicokinetic report all samples shall be analyzed. Final tissue analysis shall include but not limited to tissue weight, concentration, burden, and normalized burden. For the lung, deposition and clearance rates and half-lives are to be calculated for amosite asbestos. NOTE: Offerors are to assume that 5 tissues, including lung and pleural tissue, are to be analyzed for amosite asbestos in all animals removed for tissue burden analysis.

V. MILESTONE SCHEDULE AND DELIVERABLES

Narrative reports shall include charts, graphs, tabulations and pictures as necessary for explanation or support of the narrative. All final reports shall be dated and signed by the individual(s) responsible and the QAU.

Reporting requirements are listed in the table at the end of this section. Also listed in the tables are the due dates and number of copies required. All deliverables shall be sent directly to the appropriate recipient as indicated in the table and shall be delivered f.o.b. destination in accordance with the Specifications and by the dates specified herein.

A. PERIODIC REPORTS

1. Monthly Progress Report

A Monthly Progress Report shall be mailed by the fifteenth day of each month. Each Monthly Progress Report shall be submitted in the format specified in Specifications. No QAU audit of this progress report is required.

2. Water Analysis

Water samples shall be collected for analysis and a report of the results submitted to the NTP at least once per year. An analysis is required during the in-life portion of the 90-day study and once each year during the two-year study. The preliminary results of each analysis are to be submitted to the NTP at the time of their receipt.

B. PRESTART CHEMISTRY REPORT

This report shall be submitted in accordance with the prestart chemistry report format instructions in Specifications and the table in Section III.G. below.

C. 14-DAY (PLUS A 90-DAY POSTEXPOSURE HOLD) TOXICITY AND TISSUE BURDEN STUDY

REPORT

This report shall be submitted in accordance with the prechronic format instructions in Specifications and the table in Section III.G. below.

D. TWO-YEAR STUDY REPORT

This report shall be submitted in accordance with the instructions in Specifications and the table in Section III.G. below.

E. SUBMISSION OF ARCHIVAL MATERIALS

1. Archival Materials (Study Data) - See Specifications for details.

2. Histopathology Materials - See Specifications for details.

F. OTHER REPORTS OR SUBMISSIONS

Other reports/submissions of data are listed in Milestones and Deliverables (Section III.G. below). Any incident or unforseeable occurrences not listed in this document, or any physical modifications to the laboratory facilities, as well as any changes in personnel that might have an impact on the conduct and results of the studies shall be reported immediately to the NTP.

G. MILESTONES AND DELIVERABLES

PERIODIC SUBMISSIONS FREQUENCY DUE DATES DISTRIBUTION (Number of copies)

Monthly Progress Report 1/month Mail by 15th each month CC (2); CO-Cover page only

Water Analysis Minimum 1/year; 1/2-week study; 1/yr for 2-year study

CC(2)

PRESTART ACTIVITIES FREQUENCY DUE DATES DISTRIBUTION (Number of copies)

Submission of preliminary chemistry and generator and carousel characterization

1 TBD CC(1)

Submission of Prestart Inhalation Report

1 Within 3 months receipt of test material

CC (1 and DVD), CO-Cover page only

NTP Review and Approval to Proceed to Next Phase

NA Within 4 weeks of receipt of report

2-WEEK STUDY FREQUENCY DUE DATES DISTRIBUTION (Number of copies)

Animal Study Protocol (IACUC approved)

1/Species Prior to study start COR(1)

Start of 2-Week Study NA 4 Weeks from approval to proceed

Serum Samples for Disease Screening

See SPECS As collected SA-Send samples for evaluation

2-Week Study Report 1/Species Within 16 weeks from last day of necropsy CC (1 and DVD) CO-Cover page only

Electronic Submission of Non- NTP-Specified System Data

1 Within 1 week after approval of

CC-Transmittal letter and DVD

IANR Forms, Histology Processing Records, Slides, Blocks and Inventory;

Notification of Storage on TDMS

1/Species Within 4 weeks after approval of report

REP

CC, PC, QA-Copy of transmittal letter to include notification of pathology storage on TDMS

Wet Tissues and Inventory 1/Species Same as above REP

CC, PC, QA-Copy of transmittal letter

Archival Records for Study Raw Data and DVDs

1/Species Same as above REP CC-Copy of transmittal letter, inventory and DVDs

NA 4 weeks after approval of report

2-YEAR STUDY FREQUENCY DUE DATES DISTRIBUTION (Number of Copies)

1/Species Prior to study start COR(1)

2-Year Study Start NA 4 Weeks from approval

2-Year Study Report 1/Species 30 Weeks from last day of necropsy

CO (Cover page only)

1 Within 1 week of approval of

Blocks and Inventory;

Notification of Storage on TDMS

1/Species 4 Weeks after submission of 2-Year Study Report

REP

CC, PC, QA-Copy of transmittal letter to

Archival Records of Study Raw Data and DVDs

1/Species Same as above REP CC-Copy of transmittal letter, inventory

QA-Copy of transmittal letter

Shipment of 100 g Aliquot and Residual Test Material to Chemistry Contractor

1 At time of final bulk analysis CH CC-Copy of transmittal letter

IANR-Individual Animal Necropsy Record

TDMS-Toxicology Data Management System (also applicable to other NTP-specified data capture system

Abbreviations and General Addresses for Deliverables.

Exact names and addresses to be provided by the Contract Officer’s Representative.

CC Contracts Coordinator Research Triangle Park, NC 27713

CH Chemistry Contractor To Be Provided

CO Contracting Office NIEHS, Office of Acquisitions Research Triangle Park, NC 27713

PC Pathology Coordinator Research Triangle Park, NC 27709

COR Contract Officer’s Representative

NIEHS, DNTP, POB

Research Triangle Park, NC 27713

REP Repositories:

Archival Material - Raw Data

OR

Wet Tissues and Blocks Durham, NC 27713

SA Sentinel Animal Serum Samples To Be Provided

Sample Work Assignment B: Conduct of Studies to Evaluate the Toxicity and

Carcinogenicity of n-Nitrosodimethyl Amine in Laboratory Animals Administered Via

Dosed Feed of n-nitrosodimethyl amine via dosed feed in the Harlan Sprague Dawley rat and B6C3F1/N mouse.

Dosing in the rat study shall begin with in utero exposure and continue through lactation and after weaning for 2-years. The WORK ASSIGNMENT includes Prestart study activities and 2-year studies.

This WORK ASSIGNMENT includes only one TASK that is divided into PHASES. All activities associated with this test article are considered one TASK. All studies are to be conducted according to the "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," dated January 2011.

and purity determination of the bulk material, validation of dose formulation and analysis methods, and health and safety concerns that must be addressed before 2-year studies start. Phase 1 shall include 2 weeks for NTP review of the Prestart Chemistry Report.

Phase 2: Upon successful completion of Phase 1, the 2-year studies (Phase 2) shall be conducted.

- Procedures for bulk chemical reanalysis, dose formulation and formulation analysis.

- Equipment, software and user manuals for a government owned toxicology data collection system or access to an NTP specified data management system

- B6C3F1/N mice.

II. PHASE 1 - PRESTART ACTIVITIES

The contractor shall conduct Phase 1 activities and shall submit a Prestart Chemistry Report promptly for NTP review at the completion of Phase 1. Phase 1 activities shall include the following:

Receive the test material and perform bulk chemical analysis (identity and purity) according to the procedures provided. This shall be reported by telephone to the PO as soon as results are available.

Validate procedures provided for dose formulation and formulation analysis.

Prepare and submit a Prestart Chemistry Report to include chemistry results and chemical specific health and safety requirements/procedures.

Phase I shall include 2 weeks for NTP review of the Prestart Chemistry Report.

III. PHASE 2 – 2-YEAR DOSED FEED STUDY

A. PERINATAL EXPOSURE (RATS):

1. GESTATION PERIOD

Time-mated, female Harlan Sprague Dawley rats shall be 11-14 weeks of age and 200-240 g at receipt (gestation day 2, GD2), housed individually and provided NIH-07 diet. In addition, 10 age-matched non-pregnant females shall be received and used for disease screening prior to dosing the time-mated animals. On GD5 dams shall be randomly assigned to treatment and vehicle control groups (0, 1, 3, 10 or 30 ppb) and uniquely identified by tail tattoo. Starting on GD6 and continuing until postnatal day (PND) 28 the dams shall be provided the test agent in feed ad libitum. Dams shall be weighed on GD5 prior to dosing and then on GD6, 9, 12, 15, 18 and 21. Starting on GD9 food consumption shall be measured every three days during gestation. All dams shall be observed two times daily, once in the early morning and once in the late afternoon, at least six hours apart (before 10:00 AM and after 2:00 PM), including holidays and weekends, for signs of delivery, moribundity, and mortality. During the gestation and lactation exposure periods, any unexpected toxicity (such as mortality, body weight change, clinical signs in dams or pups) that may impact the continuation of the study shall be reported immediately. Dams that do not deliver within 3-4 days of the anticipated delivery date shall be euthanized, examined for uterine implantations/resorptions, and these findings recorded.

Pregnant dams Groups Total

TREATMEN

T

48 x 3 = 144

CONTROLS 48 x 1 = 48

TOTAL 192

2. LACTATION PERIOD

The lactation period includes the period from the day of delivery (PND0) until PND28, when the animals are weaned. PND28 is defined as 28 days post delivery of the last litter delivered.

Dams shall be weighed on PND1, 4, 7, 10, 14, 17, 21, 24 and 28 and individual pups shall be weighed on PND4, 7, 10, 14, 17, 21, 24 and 28. Food consumption measurements shall be made on PND4, 7, 10, 14 17, 21, 24 and 28 for the periods covered. All dams and pups shall be observed two times daily, once in the early morning and once in the late afternoon, at least six hours apart (before 10:00 AM and after 2:00 PM), including holidays and weekends, for morbidity, mortality, or abnormal maternal behavior.

a. PND1

The number, sex, and general appearance of the pups shall be recorded the day after delivery (PND1). Body weights shall be recorded for dams and litter weights for pups by sex and total.

b. PND4

Litters and pups shall be culled on PND4. An acceptable litter shall have eight pups per litter (randomly selected to 4 males and 4 females if possible with no litter having less than 2 male and 2 female pups). The minimum number of acceptable litters is 35 for each treatment and control group. In the event there are acceptable litters above the minimum requirement then additional litters with a maximum of 40 for each treatment and control group shall be utilized. Contact the COR for instruction if there are fewer than 35 acceptable litters in one or more groups. Each selected pup shall be tattooed (on a limb) for unique identification.

c. PND28

On PND28, 28 days after the last litter is delivered, two male and two female pups from each of 35 randomly selected acceptable litters (assumes acceptable litters in excess of this number will be included in the pool to select the 35 litters) shall be randomly selected and assigned to the 2-year study. Each pup shall be tail tattooed using unique consecutive numbers and records maintained to track the dam number for each selected pup. PND28 shall be designated Day 1 for the 2-year study. Sentinels shall be randomly chosen from control pups not selected for the 2-year study. Dams and pups not selected for continuation to the 2-year study are to be euthanized.

B. RAT AND MOUSE 2-YEAR STUDY

1. ANIMALS AND TREATMENT

Male and female Harlan Sprague Dawley rats chosen for the 2-year study shall be placed on NTP-2000 diet and continue to receive the test article in the diet at the same concentration as their dams (0, 1, 3, 10 or 30 ppb), for up to 104 weeks. All rats shall remain on the treatment regimen until the day of sacrifice. Male rats shall be housed 3 per cage and female rats shall be housed 5 per cage.

Male and female B6C3F1/N mice (5-6 weeks of age) shall be randomly assigned to treatment and control groups (0, 1, 3, 10 or 30 ppb) after a ten to fourteen day quarantine period. All mice shall remain on the treatment regimen until the day of sacrifice. Female mice shall be housed 5 per cage; male mice shall be housed individually.

Animals Sex Species Groups Total

Treatment 70 x 2 x 2 x 3 = 840 Controls 70 x 2 x 2 x 1 = 280 Sentinels 20 x 2 x 2 x = 80

Total 1200

2. OBSERVATIONS

Individual animal body weight for treated and control group animals shall be recorded on day

1, once a week for the first 13 weeks, at 4-week intervals thereafter, and at terminal sacrifice. If life threatening tumors develop or a significant number of deaths occur in the groups, the weighing frequency may be increased to every two weeks upon approval by the NTP COR. It is estimated that animals shall be weighed every two weeks for the final thirteen weeks of the 2-year study.

these observations shall be recorded. Signs of toxicity detected at times other than the formal four-week observations shall be noted and recorded.

Animals shall be observed two times daily, once in the early morning and once in the late afternoon at least six hours apart (before 10:00 am and after 2:00 pm including holidays and weekends) for moribundity and mortality.

Food consumption shall be measured and recorded once a week for the first 13 weeks and at 4-week intervals thereafter.

3. TISSUE DISTRIBUTION STUDY

During the 2-year study, after 3, 6, 12 and 18 months of exposure, up to 5 rats and mice from each group shall be randomly selected for collection of biological samples for tissue distribution. Rats and mice shall be bled into tubes containing anticoagulant and plasma (as much as possible, but a minimum of 0.25 mL) collected. Livers shall be collected following exsanguination. Liver and plasma samples shall be stored at -80C until shipment to an NTP-designated laboratory for analysis. Sample collection shall occur in the morning in a random order and be completed within as short a period of time as possible. The time of blood collection is to be recorded for each animal.

4. NECROPSY AND HISTOPATHOLOGICAL EVALUATION

A complete necropsy and histopathologic evaluation shall be performed on all animals in all treated and control groups in the 2-year studies as described in the NTP Specifications.

IV. MILESTONE SCHEDULE AND DELIVERABLES

Narrative reports shall include charts, graphs, tabulations and pictures as necessary for explanation or support of the narrative. All final reports shall be dated and signed by the individual(s) responsible and the QAU. Reporting requirements are listed in the table at the end of this section. Also listed in the tables are the due dates and number of copies required. All deliverables shall be sent directly to the appropriate recipient as indicated in the table and shall be delivered f.o.b. destination in accordance with the Specifications and by the dates specified here

A Monthly Progress Report shall be mailed by the fifteenth day of each month. Each Monthly

Water samples shall be collected for analysis and a report of the results submitted to the NTP at least once per year during the study. The preliminary results of each analysis are to be submitted to the NTP at the time of their receipt.

This report shall be submitted in accordance with the Prestart Chemistry Report format instructions in the Specifications.

C. TWO-YEAR STUDY REPORTS

These reports shall be submitted in accordance with the instructions in the Specifications and the table in Section IV.F. below. The report shall be dated and signed by the individual(s) responsible and the

QAU Auditor.

D. SUBMISSION OF ARCHIVAL MATERIALS

1. Archival Materials (Study Data) - See Specifications for details.

E. OTHER REPORTS OR SUBMISSIONS

Other reports/submissions of data are listed in Milestones and Deliverables (Section IV.F. below). Any incident or unforeseeable occurrences not listed in this document, or any physical modifications to the laboratory facilities, as well as any changes in personnel which might have an impact on the conduct and results of the studies shall be reported immediately to the NTP.

F. MILESTONES AND DELIVERABLES

PERIODIC SUBMISSIONS FREQUENCY DUE DATES DISTRIBUTION (Number of copies)

Monthly Progress Report 1/month Mail by 15th each month CC (2); CO-Cover page only

Water Analysis 1/yr for 2-year study CC(2)

Submission of Prestart Chemistry Report

1 Within 3 months receipt of test material

1/Species Prior to study start PO(1)

1 Within 1 week of approval of

Blocks and Inventory;

Notification of Storage on TDMS

1/Species Within 4 weeks after submission of report

REP

CC, PC, QA-Copy of transmittal letter to

Archival Records of Study Raw

Data and DVDs

1/Species Same as above REP

CC-Copy of transmittal letter, inventory

Chemistry Contractor

1 At time of final bulk analysis CH CC-Copy of transmittal letter

Exact names and addresses to be provided by the COR.

CH Chemistry Contractor To Be Provided

CO Contracting Office NIEHS, Office of Acquisitions Research Triangle Park, NC 27713

PC Pathology Coordinator Research Triangle Park, NC 27709

COR Contract Officer’s Representative

NIEHS, DNTP, POB

Research Triangle Park, NC 27713

REP Repositories:

Archival Material - Raw Data

OR

Wet Tissues and Blocks Durham, NC 27713

Sample Work Assignment C: Conduct of Studies to Evaluate the Toxicity and

Carcinogenicity of Hydrogen Sulfide in Laboratory Animals Exposed Via Whole Body

Inhalation of hydrogen sulfide (H2S) via whole body inhalation in the Harlan Sprague Dawley rat and B6C3F1/N mouse. The WORK ASSIGNMENT includes Prestart study activities and 13-week and

2-year studies.

to the "Specifications for the Conduct of Studies to Evaluate the Toxic and Carcinogenic Potential and purity determination of the bulk material, development of exposure generation and monitoring methods, health and safety concerns that must be addressed before study start, and submission of a prestart inhalation report.

Phase 2: Upon successful completion of Phase 1, the 13-week studies (Phase 2) shall be conducted.

Phase 3: Upon successful completion of Phase 2, the 2-year studies (Phase 3) shall be conducted.

- Equipment, software and user manuals for a government owned toxicology data collection system or access to an NTP specified data management system

- B6C3F1/N mice.

II. PHASE 1 - PRESTART ACTIVITIES

A. TEST MATERIAL ANALYSIS

1. Receive the test material and schedule the generation and monitoring developmental work.

NOTE: The test material shall be supplied by the Offeror.

2. Develop identity and purity methods for the bulk material. NOTE: the Offeror must propose methods for determining identity and purity of the test material.

2. Conduct identity and purity analysis of the test material using the methods developed.

Preliminary results shall be reported to the NTP for approval as soon as the results are available.

B. DEVELOPMENT OF AN EXPOSURE SYSTEM

1. Develop methods for generation and chamber monitoring at the protocol required concentrations in the study chambers. Preliminary results of generator and chamber characterization shall be reported to the NTP for approval as soon as the results are available.

2. Validate the methods for generation and chamber monitoring at the protocol required concentrations.

3. Conduct detailed 3-day chamber characterization studies.

4. Determine measurement limits, specificity, precision and linearity of the chamber concentration monitoring system.

5. Determine T90, Tl0, chamber homogeneity, aerosol evaluation, and stability of the chamber concentration in all chambers. The stability of chamber concentration must be established for a period of 6 hours plus T90 for three days. All the data must be obtained in the same exposure room and under identical conditions that will be used during the animal exposures.

6. Conduct stability studies of the test material in the generator reservoir and chamber.

Identification of possible degradation products is the responsibility of the Contractor.

7. Develop and test method(s) of effluent exhaust treatment, during generation runs at the protocol required concentrations in all chambers under actual animal exposure conditions. Demonstrate effectiveness of the exhaust treatment unit immediately after the effluent treatment unit and at the point of exhaust from the building. Establish the percent efficiency of the exhaust treatment.

The effluent exhaust treatment must be effective in removing the test material to an acceptable concentration (i.e. greater than 90% efficiency of removal by the treatment system and less than 50% of the TLV, if a TLV exists), or written documentation for a waiver from appropriate air regulatory agencies must be provided. Determine lifetime expectancy of any proposed filtration/treatment units and amount of treatment media required. Confirm that none of the exhausted test material is reentrained.

8. Develop and test method(s) of room air monitoring during generation runs at protocol required concentrations in all chambers under actual animal exposure conditions. Define lower limit of detection of the monitoring method(s) and document that this level provides an adequate safety margin for personnel.

9. Prepare and submit a Prestart Inhalation Report of Phase I chemistry, exposure system and health and safety activities and procedures to include:

(a) Confirmatory data on identity, purity and, if necessary, stability of the bulk material using methods developed, as well as copies of the SOPs for the bulk chemical reanalysis methods

(b) Description of inhalation exposure chamber

(c) Generation system description and approach

(d) Chamber monitoring system and calibration

(e) Homogeneity of test atmosphere in chambers

(f) Stability of chamber concentration over 3 days

(g) Evaluation for the presence of aerosols

(h) Chamber T90 and T10 times

(i) Test material stability studies in generator reservoir and chamber

(j) SOP for generation of test material

(k) SOP for generation maintenance/changeout

(l) SOP for chamber monitoring system including calibration approach

(m) SOP for bulk material identity and purity and reanalysis

(n) SOP for chamber homogeneity determinations

(o) SOP for evaluation for the presence of aerosols

(p) Other SOPs as appropriate for the specific test material

(q) Description of effluent exhaust treatment and SOPs

(r) Effluent exhaust treatment monitoring data and percent efficiency of the effluent exhaust treatment

(s) Room air monitoring methodology, strategy, SOPs, frequency and results

Phase 2 shall include 4 weeks for NTP review of the Prestart Chemistry Report.

III. PHASE 2- 13-WEEK STUDY

After a ten to fourteen day quarantine period, male and female Harlan Sprague Dawley rats and

B6C3F1/N mice shall be assigned at random to treatment and control groups. At each of five exposure concentrations plus a control group (0, 1.5, 5, 15, 50 or 150 ppm for rats; 0, 1, 3, 10, 30 or 100 ppm for mice), 10 animals per sex per species shall receive the test material via inhalation. The schedule will be five days per week, weekdays only and exclusive of holidays. The daily exposure time shall be six hours per day plus T90. All animals shall remain on the treatment regimen until the day (within 24 hours) of sacrifice. All rats and mice shall be housed individually.

Core Study Animals Treatment 10 x 2 x 2 x 5 = 200 Controls 10 x 2 x 2 x 1 = 40 Sentinels 5 x 2 x 2 = 20 Clinical Pathology rats Treatment 10 x 1 x 2 x 5 = 100 Controls 10 x 1 x 2 x 1 = 20

Total 380

*Sentinels are to be distributed amongst the control and treated chambers weekends, for moribundity and mortality. Core study animals shall be weighed individually on day one on test, after seven days and weekly periods thereafter, and at terminal sacrifice. Formal clinical observations shall be performed and recorded weekly. Signs of toxicity detected at times other than the formal weekly observations shall also be noted and recorded. Body weights and formal clinical observations are not required for the clinical pathology study animals.

C. SPECIAL STUDIES

1. CLINICAL LABORATORY STUDIES

Additional rats shall be exposed, both sexes, all treatment groups, for blood collection at days 4

1 and 21 2. The clinical pathology rats shall be sacrificed after the collection for day 21 and these animals discarded without further evaluation. The core study rats shall be bled at terminal sacrifice for clinical lab studies. Core study mice shall be bled at terminal sacrifice for hematology determinations.

All male and female animals shall be exposed the same number of days before collection of samples and all animals of a sex shall be bled on the same day. The period between the start of the study and the collection of the first sample at day 4 1 shall be continuous, not interrupted by a weekend.

HEMATOLOGY

Erythrocyte count Mean corpuscular volume Hemoglobin Hematocrit (Packed cell volume) Mean corpuscular hemoglobin Mean corpuscular hemoglobin concentration Erythrocyte morphologic assessment Leukocyte count Leukocyte differential Leucocyte morphological assessment Reticulocyte count Platelet count and morphologic assessment

CLINICAL CHEMISTRIES

Alanine aminotransferase Sorbitol dehydrogenase Alkaline phosphatase Total bile acids Creatinine Urea nitrogen Glucose Albumin Total protein Creatine kinase Cholesterol Triglycerides

2. BLOOD FOR MICRONUCLEI

Samples of blood (~200 µl) shall be collected in EDTA from rats and mice at termination of the 13-week study. Samples are to be refrigerated immediately after collection and remain refrigerated until shipped the day of collection. The samples shall be shipped refrigerated (not frozen) for overnight delivery to an NTP-designated laboratory for micronuclei determination.

3. SPERM COUNT AND VAGINAL CYTOLOGY EVALUATION (SCVC)

SCVC shall be conducted on rats and mice. Vaginal smears shall be taken for 16 consecutive days for females prior to terminal sacrifice. The Contract Officer’s Representative shall be notified at the end of the first 70 days of the study regarding mortality, body weight changes and clinical signs of toxicity for use in determining the treatment groups to be used for SCVC.

D. NECROPSY AND HISTOPATHOLOGICAL EVALUATION

sacrificed, and all tissues specified in the NTP Specifications shall be saved in formalin. All tissues required for complete histopathology as listed in the NTP Specifications shall then be trimmed, embedded, sectioned and stained with hematoxylin and eosin for possible histopathological evaluation. This shall be done for all animals in all groups.

Organ weights shall be determined from all animals surviving until the end of the study. Those organs to be weighed are: liver, thymus, left and right kidney, left and right testis, left and right epididymis, left and right ovary, heart, and lungs. Bilateral organs shall be weighed and recorded separately. These organs shall be weighed to the nearest 10 mg except for testis and thymus, which shall be weighed to the nearest 1.0 mg, and epididymis, which shall be weighed to the nearest 0.1 mg.

A complete histopathological evaluation inclusive of gross lesions shall be done on all control animals, all animals in the highest exposure group with at least 60% survivors at the time of sacrifice, plus all animals in higher exposure groups inclusive of early deaths and survivors.

Treatment-related lesions (target organs) shall be identified, and these organs plus gross lesions shall be examined to a no effect concentration. However, a complete histopathological evaluation shall be performed on all natural death/moribund sacrifice animals in lower exposure groups.

Phase 3 shall include 1 month for NTP review of the 13-Week Study Reports.

After a ten to fourteen day quarantine period, male and female Harlan Sprague Dawley rats and B6C3F1/N mice shall be assigned at random to treatment and control groups. At each of three exposure concentrations (TBA) plus a control group, 50 animals per sex per specie shall receive the test material via inhalation. The schedule will be five days per week, weekdays only and exclusive of holidays, for up to 104 weeks. The daily exposure time shall be six hours per day plus T90. All animals shall remain on the treatment regimen until the day (within 24 hours) of sacrifice. All rats and mice shall be housed individually.

Treatment 50 x 2 x 2 x 3 = 600 Controls 50 x 2 x 2 x 1 = 200 Sentinels 20 x 2 x 2 = 80

Total 880

*Sentinels are to be distributed amongst the control and treated chambers

Individual animal body weight for treated and control group animals shall be recorded on day one on test, once a week for the first 13 weeks, at 4-week intervals thereafter and at terminal sacrifice. If life threatening tumors develop or a significant number of deaths occur in the groups, the weighing frequency may be increased to every two weeks upon approval by the NTP Contract Officer’s Representative. It is estimated that animals shall be weighed every two weeks for the final thirteen weeks of the 2-year study.

these observations shall be recorded. Signs of toxicity detected at times other than the formal four-week observations shall be noted and recorded.

Animals shall be observed two times daily, once in the early morning and once in the late afternoon at least six hours apart (before 10:00 am and after 2:00 pm including holidays and weekends) for moribundity and mortality.

Complete necropsy and histopathological evaluation shall be performed on all animals in all treatment groups as described in the NTP Specifications.

Narrative reports shall include charts, graphs, tabulations and pictures as necessary for explanation or support of the narrative. All final reports shall be dated and signed by the individual(s) responsible and the QAU.

Reporting requirements are listed in the table at the end of this section. Also listed in the tables are the due dates and number of copies required. All deliverables shall be sent directly to the appropriate recipient as indicated in the table and shall be delivered f.o.b. destination in accordance with the Specifications and by the

Water samples shall be collected for analysis and a report of the results submitted to the NTP at least

This report shall be submitted in accordance with the prestart chemistry report format instructions in Specifications and the table in Section III.G. below.

C. PRECHRONIC STUDY REPORTS

These reports shall be submitted in accordance with the prechronic format instructions in Specifications and the table in Section III.G. below.

D. TWO-YEAR STUDY REPORTS

These reports shall be submitted in accordance with the instructions in Specifications and the table in Section III.G. below.

Other reports/submissions of data are listed in Milestones and Deliverables (Section III.G. below). Any incident or unforeseeable occurrences not listed in this document, or any physical modifications to the laboratory facilities, as well as any changes in personnel that might have an impact on the conduct and

PERIODIC SUBMISSIONS FREQUENCY DUE DATES DISTRIBUTION (Number of copies)

Monthly Progress Report 1/month Mail by 15th each month CC (2); CO-Cover page only

Water Analysis Minimum 1/year; 1/13-week chemistry and generator and chamber characterization

Report

1 Within 3 months receipt of test

13-WEEK STUDIES FREQUENCY DUE DATES DISTRIBUTION (Number of copies)

Start of 13-Week Study NA 4 Weeks from approval

Clinical Lab Study Data Per Design CC(1), PC(1)

Blood for Micronuclei (Rats and Mice)

1 Within 24 hours of collection MN-Blood and decoding information MI-Copy of transmittal letter and decoding

13-Week Study Report 1/Species Within 16 weeks from last day

1 Within 1 week after approval of

Blocks and Inventory;

Notification of Storage on TDMS

1/Species Within 4 weeks after approval of report

REP

CC, PC, QA-Copy of transmittal letter to

Archival Records for Study Raw Data and DVDs

1/Species Same as above REP

NTP Review And Approval to

2-Year Study Report 1/Species 30 Weeks from last day of CC (1 and DVD) necropsy CO (Cover page only)

TDMS Data

1 Within 1 week of approval of

Blocks and Inventory;

Notification of Storage on TDMS

1/Species 4 Weeks after submission of 2-Year Study Reports

REP

CC, PC, QA-Copy of transmittal letter to

Archival Records of Study Raw Data and DVDs

1/Species Same as above REP CC-Copy of transmittal letter, inventory

Chemistry Contractor

1 At time of final bulk analysis CH CC-Copy of transmittal letter

CH Chemistry Contractor To Be Provided

CO Contracting Office NIEHS, Office of Acquisitions Research Triangle Park, NC 27713

MI Micronuclei Decoding Information Research Triangle Park, NC 27709

MN Micronuclei Evaluation To Be Provided

PC Pathology Coordinator Research Triangle Park, NC 27709

COR Contract Officer’s Representative

NIEHS, DNTP, POB

Research Triangle Park, NC 27713

REP Repositories:

Archival Material - Raw Data

OR

Wet Tissues and Blocks Durham, NC 27713

Sample Work Assignment D: Conduct of Studies to Evaluate the Toxicity and

Carcinogenicity of Decabromodiphenylethane in Laboratory Animals Administered Via

Gavage of decabromodiphenylethane via gavage in the Harlan Sprague Dawley rat and B6C3F1/N mouse.

Dosing in the rat studies shall begin with in utero exposure and continue through lactation and after weaning for 13 weeks or 2 years. The WORK ASSIGNMENT includes Prestart study activities, thirteen-week studies and 2-year studies.

to the "Specifications for the Conduct of Studies to Evaluate the Toxic and Carcinogenic Potential and purity determination of the bulk material, validation of dose formulation and analysis methods, and health and safety concerns that must be addressed before 13-week studies start. Phase 1 shall include 2 weeks for NTP review of the Prestart Chemistry Report.

Phase 2: Upon successful completion of Phase 1, the 13-week studies (Phase 2) shall be conducted.

Phase 3: Upon successful completion of Phase 2, the 2-year studies (Phase 3) shall be conducted.

- Procedures for bulk chemical reanalysis, dose formulation and formulation analysis.

- Equipment, software and user manuals for a government owned toxicology data collection system or access to an NTP specified data management system

- B6C3F1/N mice.

II. PHASE 1 - PRESTART ACTIVITIES

The contractor shall conduct Phase 1 activities and shall submit a Prestart Chemistry Report promptly for NTP review at the completion of Phase 1. Phase 1 activities shall include the following:

Receive the test material and perform bulk chemical analysis (identity and purity) according to the procedures provided. This shall be reported by telephone to the PO as soon as results are available.

Validate procedures provided for dose formulation and formulation analysis.

Prepare and submit a Prestart Chemistry Report to include chemistry results and chemical…