SRC TR - 10 - 063

HPV1 TEST RULE STU D Y

– DATA ADEQUACY ASSESS MENT 1,3 - Propanediol, 2,2 - bis [ (nitroox y)methyl] - , dinitrate (ester) , CAS No. 78 - 11 - 5 Toxicity to Plants (Algae ) Prepared b y: Ashlee Aldridge , Teresa Manyin , and Joan D. Garey

Environmental S cience Center

SRC, Inc. 7502 Round Pond Road North Sy racuse, NY 13212 Contract No. EP - W - 09 - 027 Task FG007.0.009.00 4 Submitted to: OPPT/RAD/HPVCB (7403M) U.S. Environmental Protection Agency 1200 Pennsylvania Avenue Washington, D.C. 20460 Elizabeth Margosches , Project Officer Meena Sonawane, Work Assignment Manager David Brooks, Alternate Work Assignment Manager

March 5 , 2010HPV 1 Test Rule – Data Adequacy Assessment

1,3 - Propanediol, 2,2 - bis [ (nitrooxy)methyl] - , dinitrate (ester), CAS No. 78 - 11 - 5 Toxicity to Plants (Algae ) March 2010

Submitted to: High Production Volume Chemicals Branch Risk Assessment Division Office of Pollution Prevention and Toxics Environmental Protection Agency WAM: Meena Sonawane Alternate WAM: David Brooks 1200 Pennsylvania Avenue, NW Washington, DC 20460 - 0001 Prepare d By : SRC , Inc. Environmental Science Center 2451 Cry stal Drive, Suite 804 Arlington, VA 22202Draft HPV 1 Test Rule

– Data Adequacy

– Toxicity to Plants (Algae) CAS No. 78 - 11 - 5

1 PREPARATION OF THE DATA ADEQUACY ASSESSMENT This document contains the review of a toxicity study conducted in respon se to the first HPV TSCA Section 4 Test Rule (7 1 FR 13708, March 16, 2006). Using the applicable test guideline and professional judgment as guides, the study was reviewed for adequacy and evaluated for compliance with the test guideline. (The applicable test guideline is not alway s the test guideline required b y the test rule, because some test sponsors requested and received approval from EPA to use a different guideline.) The study was summarized according to a standard format , assessed for adequacy , and assigned a Klimisch reliability code. If the study was determined to be inadequate, a basis for that conclusion is supplied. For studies already assigned Klimisch codes, an explanation is provided if the reviewer’s assessment of data adequacy differed from the pre - assigned Klimisch code. A detailed evaluation of the test report and study conduct is provided at the end of the summary document in a section entitled “Review of TSCA Section 4 Final Study Repo rt”. Any deviations from the applicable test guideline are described. The study is also evaluated for compliance with Standard Operating Procedures, Good Laboratory Practice Standards, and Quality Assurance procedures, as well as the use of appropriate c ontrols and test substances. Any deficiencies in the descriptions of the study method, test sy stem, animals studied, phy sical conditions, dosage information, individual animal data, calculations, and statistical analy ses are described, and internal incons istencies within the study report are identified. An overall assessment is provided for each of the following: study conduct, data reliability, and data utility .Draft HPV 1 Test Rule

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– Toxicity to Plants (Algae) CAS No. 78 - 11 - 5

2 TOXICITY TO PLANTS (ALGAE) Title: Toxicity of Pentaery thritol tetranitrate (PETN) to the Unicellular Green Alga (Selenastrum capricornutum ) under Static Test Conditions This study is: Adequate Not Adequate TEST SUBSTANCE Identity: Pentaery

thritol tetranitrate ( 1,3 - Propanediol, 2,2 - bis [ (nitroox y) methyl] - , dinitrate (ester) ; PETN ) , CAS No. 78 - 11 - 5 Remarks: Test substance was received as a solution of PETN (> 99% purity) dissolved in water (approximately

2100 μg/L); c lear, colorless liquid ; specific gravity: 1.77 g/cc; received from the United States Army

Center for Health Promotion and Preventative Medicine METHOD Method/guideline followed: ASTM E 1218 - 04 (ASTM 2006) and where applicable OPPTS 850.5400 (USEPA 1996) Test type: Toxicity

to Algae GL P compliant?: Yes Year study was performed: 2008 T est species: Unicellular green alga e ( Pseudokirchneriella subcapitata , formerly Selenastrum capricornutum ) ; source: 4 - day old in - house stock cultures; start er culture obtained from Carolina Biological Supply

Company Analytical monitoring : Yes Exposure du ration: 96 hours Concentrations tested: Nominal: 0, 0.1, 1.0, 10, and 100% PETN (as received), equivalent to 0, 2, 20, 200 and 2000 μ g/ L ; M ean m easured: 1.4, 3.2, 30, 310 and 2850 μ g/ L Test method/conditions remarks: The dilution/control water used in this study (algal medium batch #121 807) was ASTM algal growth medium with EDTA, prepared with sterile, deionized (Milli - Q®) water (ASTM type I) according to ASTM guidelines (ASTM 2006) . Milli - Q® water generated at the testing lab is periodi call y anal yz ed f

or contaminants. Additionally , the medium was sterilized by filtration through a pre - washed 0.45 μ m cellulose membrane filter prior to mixing test solutions .Draft HPV 1 Test Rule

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– Toxicity to Plants (Algae) CAS No. 78 - 11 - 5

3 Macro - and micro - nutrients were added to the 100% test solution prior t o preparing test concentr ations . The test substance, with added nutrients, was assumed to be 100% for purposes of t est concentration preparation. Test concentrations are expressed as percent of PETN as received; statistical endpoints are expressed as μg/L PETN, pe r the analy tical chemistry

data . S. capricornutum were exposed to PETN according to a 96 - hour static test design; test solutions were not renewed. S. capricornutum were exposed to four concentrations of PETN (using a dilution factor of 10), and a dilution water control . Nominal test concentrations were 100, 10, 1, 0.1 , and 0 % PETN. Test solutions were prepared on test Day 0 immediately

prior to test initiation. Test chambers were 250 - m L (nominal volume) glass flasks containing approximately 100 m L of test solution and capped with a plastic cup with three small holes . Test chambers were held in an environmental chamber in a five b y five pattern. Test chambers were placed within the pattern according to a computer - generated random design. The test chambers were hou sed in an environmental chamber under continuous illumination at 400 ± 10% ft - c (4 .3 K lux), which was provided by a combination o f incandescent and cool white fluorescent lamps to provide light output in the spectral range of 400 - 700 nm. Light intensity was monitored at the beginning of the test at the level of the test solutions and averaged 398 ft - c. Flasks were oscillated continuously at approximately

78 r pm on a mechanical shaker table to keep the algal cells in suspension . Test te mperature was moni tored using a minimum/maximum thermometer in the environmental chamber and was 25 °C throughout the test duration . The inoculum was prepared approximately 3 hours prior to test initiation . The cell density of the in oculum was 1.14 x 10 6 cells/mL . The in oculum was added to the test solution at a rate of 1 mL of inoculum per 100 mL of test solut ion . The initial cell d ensity in the test chambers was nominally

1.14 x 10 4 cells/mL . This initial cell density was verifi ed in three control blanks that were pre served with Lugol's solution at the rate of 1% of the blank volume within 2 hours of test initiation. Five replicates were tested per treatment; one replicate was used to monitor pH during the test and wa s discarded at test termination . O n the day

of tes t initiation, temperature, pH, and conductivity were measured and recorded for each test treatment. Hardness (mg/L CaCO 3 ) , alkalinity (mg/L CaCO 3 ), total ammonia (mg/L as N), and total residual chlorine (mg/L) were also measured in the control and 100% PE TN treatments at test initiation. Temperature and pH were measured in each concentration dail y and at test termination. Temperature within the environmental chamber was measured daily using a digital thermometer, and recorded. At test termination, test solutions destined for cell density determination were preserved with Lu gol's solution at a rate of 1% of the final volume within 2 hours of test termination, and after collection of anal yt ical samples. Algal cell density in the test solutions was later d etermined b y direct microscopic counts (using a hem o cy tometer and compound light microscope) and recorded.Draft HPV 1 Test Rule

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– Toxicity to Plants (Algae) CAS No. 78 - 11 - 5

4 Samples for PETN anal ysis were collected at test initiation, prior to distributing the test solutions to the test chambers, from each freshl y prepared test treatment and the dilution water . At test termination, samples of the old test solutions were collected after aliquots were removed for algal growth - response determination, and the replicates for each treatment were pooled into one sample. The po oled sample was divided into two equal volumes. One of these samples was filtered through a pre - washed 0.45 μ m cellulose membrane to remove algal cells. Both samples were collected for each treatment (one with cells and one without) by pouring the compos ite test solution into 250 - mL clear glass bottles, wrapped in aluminum foil . The samples were refrigerated until shipped (on ice) to TestAmerica, an independent laboratory, located in Knoxville, Tennessee. Method 8330 was used following USEPA (1986) guid elines to analyze for nitroaromatics and nitramines b y HP LC . Analy tical data from TestAmerica were reviewed by ENSR. All data were determined to be valid and usable. Statistical Analy sis Because cell density

in any test concentration (including 100%) w as not reduced b y more than 8% from the control, a 96 - hour EC 50 or I C 50 value (experimentally derived concentration of a test substance in dilution water that is calculated to affect 50% of a test population over 96 hours) could not be statistically

derive d. RESUL TS Endpoint value(s): 96 hr EC 50 > 2850 μ g/L PETN Results remarks: At the time of test initiation, water quality measurements in dilution water were: h ardness = 18 mg/L CaCO 3 , alkalinity = 10 mg/L CaCO 3 , pH = 7.5 , conductivity = 95 μS/cm , total ammonia < 1.0 mg N/L , and total residual chlorine < 0.02 mg/L . Water quality measurements of 100% PETN (with algal nutrients) were: h ardness = 16 mg/L CaCO 3 , alkalinity = 11 mg/L CaCO 3 , pH = 7.7 , conductivity = 82 μS/cm , total ammonia < 1.0 mg N/L , and total residual chlorine < 0.03 mg/L . The ph ys ical test conditions monitored during the test were : pH = 7.5 - 9.4, conductivity (measured onl y at test initiation) = 82 - 95 μS/cm , and temperature = 24 - 26 °C. The conditions were within acceptable ranges for th e survival of S. capricornutum and were in compliance with the protocol and ASTM requirements. The average cell densities after 96 hours of exposure were 25.63, 29.42, 31.71, 26.39, and 23.81 x 10 5 cells/mLfor the control, 3.2, 30, 310, and 2850 μg/L con centrations, respectivel y. The EC 50 value, based on cell density and measured PETN concentrations, was > 2850 μ g/ L ; cell density in any test concentration did not decrease more than 8% relative to the control . CONCLUSIONS The purpose of this study was to determine the short - term chronic toxicity of P ETN to S. capricornutum under static test conditions. The biological endpoint for this study was cell density (cells/ml). A 96 - hour EC 50 was determined based on mean measured PETN concentration ( μ g/ L) and cell density . The EC 50 was > 2850 μ g/L PETN. Because the highest concentration tested (2850 μ g/ L of PETN) did not result in a 50% or greater reduction in growth, a definitive test is not required.Draft HPV 1 Test Rule

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– Toxicity to Plants (Algae) CAS No. 78 - 11 - 5

5 STUDY RELIABILITY [1] Reliable without restrictions . REFERENCE Pillard, D.A . 2008. Toxicity of Pentaery thritol tetranitrate (PETN) to the Unicellular Green Alga (Selenastrum capricornutum ) under Static Test Conditions . ENSR Project ID 08503 - 131 - 073 - 002 , Study

#08503 - 131 - 073 - 002. Study completed 13 March 2008.Review of TSCA Section 4 Final Study Report

CITB Project Manager: Catherine Roman Chemical Name: 1,3 - Propanediol, 2,2 - bis [ (nitrooxy)methyl] - , dinitrate (ester) CAS Registry Number: 78 - 11 - 5 Report Title: Toxicity of Pentaerythritol tetrani trate (PETN) to the Unicellular Green Alga ( Selenastrum capricornutum ) under Static Test Conditions Test Sponsor: Dyno Nobel Inc., Salt Lake City, UT Testing Lab: ENSR Fort Collins Envir onmental Toxicology Laboratory , Fort Collins, CO Test Standard : ASTM E 1218 Date Study Received by EPA: N/A, posted to http://www.regulations.gov on 5/19/08 Date Study Due to EPA: 5/17/07 Special Instructions (e.g., regarding approved protocol modifications, etc.): Part I. Test Report and Study Conduct Enter one of the following codes into each of the checkboxes 1 through 13: Agree [ A ], Disagree [ D ], Not Applicable [ NA ], or Cannot Determine [ C ], and add comment, below , as needed [ D ] 1. The final study repor t indicates evidence of gross, unauthorized or undocumented deviation from the EPA test standard requirements, including “shall,” “must,” or “will” violations. List significant test standard deviations and “shall,” “must,” or “will” violations in comm ents below . [ D ] 2. The f

inal study report indicates evidence of deviations from the “should” statements in the study protocol that may have a bearing on the study conclusions. List these in comments below. [ D ] 3. Approved modifications of test s tandard or testing protocol (CITB PM describe or attach) we re not followed or are inadequately documented . [ D ] 4. There is e vidence that laboratory Standard Operating Procedures (SOPs) we re inadequately followed . [ D ] 5. The Statement of Quality A ssurance (QA) inspections, their dates, and findings were missing or not signed . There is evidence that QA procedures may be in adequate . [ D ] 6. There is e vidence that Good Laboratory Practice Standards (GLPS) were inadequately followed . [ D ] 7. T he t est, control , or reference substances are different from that specified in the section 4 test rule or Enforceable Consent Agreement (ECA). [ D ] 8. Animal identification is inadequate or unclear; use of unhealthy animals or other factors (e.g., dev iations from temperature, lighting, dosing) are apparent . [ D ] 9. The f

inal study r eport inadequately describes the study method . [ D ] 10. The f

inal study r eport inadequately describes the test system .[ D ] 11. The final study r eport inadequatel y describes the dosage information .

[ D ] 12. The final study report does not accurately reflect the individual animal data (or that of other discrete observations pertinent to the type of study conducted) , or individual d ata were not provided . [NA ] 1 3. The final study r eport inadequately describes the calculations, interpolations, data transformations, statistical analyses, or modeling parameters used . Part II. Conclusions Place a check mark beside the summary statement(s) that apply to this review and add comment s below as appropriate Part A. To be filled out by Reviewer Study Conduct 1. The study w as well - conducted. [X] 2. The study was generally we ll - conducted, with some minor [ ] deficiencies (list in notes below). 3. The study conduct was significantly deficient or questionable, (give [ ] reasons in notes below). Data Reliability 4. The study results are precise and accurate. [X] 5. Appropriate methods were used to determine [NA ] statistical si gnificance. 6. If repeated, the test results would be expected to be the [ X ] same. Data Utility 7. The data are useful for risk assessment for the endpoint tested. [ X ] 8. The data are useful for some other purpose (e.g., dose - setting). [ ] 9. The data are not useful for any purpose (describe in notes below). [ ] Reviewer C o mment s ( continue on separate page, if needed): Although the test guideline states that each test chamber should be inoculated at the rate of 2 x 10 4 cells/mL, the initial cell density in this study was only 1.14 x 10 4 cells/mL . It is unlikely that this violation has bearing on the study conclusion. Cell density in control cultures increased by 225x during the 96 - hour exposure period, which exceeds the minim

um acceptable grow

th rate. Because cell densities in test article - treated cultur es were similar to controls , the EC 50 value exceeded the highest concentration tested; therefore, no statistics were necessary. Reviewer Nam e/Title/Branch/Division/Office: As hlee Aldridg e /SRC , Inc. Reviewer Signature: David Brooks/HPVCB/RAD/OPPTS Part B . To be filled out b y CITB PM Tiered Testing Decision 1. The tiered testing trigger was met, additional testing is indicated. [ ] (when applicable) 2. The trigger for additional (tiered) testing was not met. [ ] Study Acceptability 3. The study is acceptable. [ ] 4. The study may not be acceptable [ ] (provide rationale in memo to CITB chief).