SRC TR

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HPV2 TEST RULE : RESPONSE TO AECOM COMMENTS DATED MARCH 5, 2014 ( FINAL )

1,3

- Propanediol, 2,2

- bis[(nitrooxy)methyl]

- , dinitrate (ester), CASRN 78

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- 5

Prepared by:

Amy Nguyen, Jennifer Rayner, Teresa Manyin, and William Richards

Defense and Environmental Solutions Division

SRC, Inc. 7502 Round Pond Road North Syracuse, NY 13212

Contract No. EP

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- 09

- 027 Task FG007.4.009.001

Submitted to:

OCSPP/OPPT/RAD (7403M) U.S. Environmental Protection Agency 1200 Pennsylvania Avenue Washington, D.C. 20460

Bryan Lobar, Project Officer David Brooks, Work Assignment Manager Amy Benson, Alternate Work Assignment Manager

April 22 , 201 4HPV

2 Test Rule: Response to AECOM (Final ) CAS RN 78

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1 On March 5 , 2014, AECOM submitted a memo to EPA 1 regarding difficulties in maxim izing the solubility of CASRN 78

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- 5 ( 1,3

- propanediol, 2,2

- bis[(nitrooxy)methyl]

- , dinitrate (ester) ; pentaerythritol tetranitrate [ PETN ] ) for the acute aquatic toxicity studies to fish and Daphnia required in the final test rule, “Testing of Certain High Production Volume Chemicals; Second Group of Chemicals” (76 FR 1067, January 7, 2011; referred to herein as HPV2 test rule ; available online at http://www.regulations.gov/#!documentDetail;D=EPA

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- 0579 as of April 22 , 2014 ). The memo was submitted in response to EPA’s data adequacy review for CASRN 78

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- 5 (available online at http://www.regulations.gov#!documentDetail; D=EPA

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- OPPT

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- 0211 as of April 22 , 2014)

, which determined that the acute toxicity studies o f fish and Daphnia that were previously submitted to EPA by the sponsor, Dyno Nobel Inc.

, on March 26, 2013 ( submission available online at http://www.regulations.gov/ #!documentDetail;D=EPA

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- OPPT

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- 0713 as of Ap ril 22 , 2014 ) were not adequate to satisfy the C4 aquatic toxicity testing requirement in the HPV2 test rule

. The memo addressed

some study deficiencies, reported the results of tests examining the effects of various factors on the solubility of CASRN 78

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, and presented possib le options for performing the toxicity
test s to fulfill test rule requirements

.

The comments and suggestions from AECOM are addressed below:

(1) Comment: AECOM had cited the use of EPA SW

- 846 Method 8330A (Nitroaromatics and Nitramines by High Performance Liquid Chrom a tography) for analytical determination of PETN concentrations in the previous ly submitted acute aquatic toxicity tests (Naddy, 2012, 2013)

. In its March 5, 2014 memo, AECOM clarified that a wavelength of 215 nm was used for the detection of PETN , rather than the wavelength of 254 nm specified in Method 8330A

. AECOM noted that the update of this method (8330B; Nitroaromatics, Nitramines, and Nitrate Esters b y High Performance Liquid Chroma tography) added PETN to the analyte list. In response to EPA’s comment that c hromatograms and limit of detection (LOD) values were not included in the study report s for the acute aquatic toxicity tests

, AECOM stated that this information is provided by the analytical laboratory only in higher level study report s

. EPA’s response: EPA considers the analytical method used in the acute toxicity to fish and Daphnia tests (i.e., EPA SW

- 846 Method 8330A /B with a wavelength of 215 nm 2 ) acceptable for measurement of PETN concentrations in these tests and any future aquatic toxicity studies. EPA continues to consider the absence of LOD values and chromatograms in the study reports to be deficiencies. EPA requests that the sponsor provi de sample chromatograms and the LOD value(s) of the analytical method in all subsequent study reports.

(2) Comment: AECOM noted that the purity of the test substance, which was not provided in the original study reports, was 98.43%.

1 Memo addressed to Mr

. John Schaeffer of EPA from Dr. Rami B. Naddy of AECOM , the performing laboratory for aquatic toxicity studies on fish and Daphnia that were previously submitted by Dyno Nobel Inc.

2 EPA SW

- 846 Method 8330B recommend s a similar wavelength ( 214 nm ) for detection of PETN

. EPA SW

- 846 Method s 8330A and 8330 B are a vailable online at http://www.epa.gov/osw/hazard/testmethods/sw846/pdfs/ 8330a.pdf and http://www.epa. gov/osw/hazard/testmethods/pdfs/8330b.pdf , respectively, as of April 22 , 2014

.HPV

2 Test Rule: Response to AECOM (Final ) CAS RN 78

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2 EPA considers the purity of 98.43% reported in the March 5, 2013 submission to be acceptable for the previous acute toxicity to fish and Daphnia tests and any future aquatic toxicity tests. I n all future study reports submitted to EPA , the sponsor should provide the test substance purity and the complete composition of the test material, including the percentages of all impurities.

(3) Comment: AECOM states “ Additional analytical samples were not measured in the 1.0 mg/L nominal PETN treatment for the fish beca use this was considered an extra treatment (i.e. the nominal 2.0 mg/L treatment was at the solubility limit for PETN and the idea was that if we could demonstrate no toxicity at that level, the lower treatments were not as important. ”

EPA’s response: It was unclear from the acute toxicity to fish study report (Naddy, 2013) that the definitive test was intended to be a limit test (i.e., a test conducted at the limit of solubility to demonstrate no toxicity at this concentration) ; if that was the case, then the use of an intermediary concentration of 1.0 mg/L was not necessary

. OPPTS 850.1075, p. 8, Section (f) “Performance of the test”, Sub

- section (2) “Test concentrations”, item (v) “Concentration analysis”, part (C) states: “In static

- renewal tests, the test substance should be measured in each replicate at the beginning and end of test and just before and after each renewal.” For future studies, analysis of t est substance concentrations should be performed on all replicates included in the study and should include samples taken just prior to (“old” test solutions) and after (“new” test solutions) each renewal for each replicate

.
(4) Comment: Regarding the fact that the acute toxicity to fish study report (Naddy, 2013) was missing experimental deta ils for the range

- finding (preliminary) test #2, AECOM states that the data sheets for the preliminary studies were included in Appendix D of the report.

EPA’s response: According to the study report, two preliminary tests were conducted, the first in No vember 2011 and the second in November 2012. Appendix D of the study report only includes the detailed results from the first (November 2011) preliminary test. The results of the second (November 2012) preliminary test are briefly summarized on page 11 o f the study report, but details are not provided.

(5) Comment: AECOM states that the concentration of PET N in the acetone stock solution (used in the definitive test) was “confirmed at 5,400 mg/L”.

EPA’s response: EPA appreciates this additional information , which demonstrates that the actual concentration of the stock solution was similar to th e nominal concentration of 5000 mg/L.

( 6 ) Comment: AECOM propose s the use of methanol or acetone in test solutions at concentrations in excess of the maximum solvent concentration recommended by standard aquatic toxicity testing guidelines to achieve target PETN test concentrations for the acute fish and Daphnia toxicity studies.

EPA’s response: EPA does not recommend the use of methanol , acetone, or other solvents at concentrations exceeding the maximum concentration recommended by aquatic toxicity testHPV

2 Test Rule: Response to AECOM (Final ) CAS RN 78

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3 guidelines. Specifically, solvent concentrations may not exceed 0.5 mL/L , as stated in ASTM E729

- 96 (test rule

- specified guideline for acute toxi city to fish and invertebrates).

R ecommendations for Further Testing to Satisfy Aquatic Toxicity Testing Requirements:

The sponsor should first perform a study to determine the solubility of CASRN 78

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. 1 The solubility study should be performed under similar conditions (e.g., temperature , test medi um , concentration of organic solvent

, etc. ) to those used in the aquatic toxicity tests. To conduct this solubility study, EPA recommends that the sponsor contract the same laboratory ( U.S. Army Center for Health Promotion and Preventative Medicine [ CHPPM ] ) that conducted the solubility test in deionized water (Hable et al., 2008). The sponsor should report the results of this solubility study to EPA. If t he measured solubility limit of CASRN 78

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- 5 in the fish and Daphnia test medium is less than or approximates the highest mean measured concentration in the fish and daphnid studies (i.e., 1218 μ g/L in the fish test and 943 μ g/L in the daphnid test), then EPA will accept the previously submitted toxicity studies in fish and daphnids (Naddy, 2012, 2013) as adequate and no further testing is needed. If the measured solubility limit of CASRN 78

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- 5 in the fish and Daphnia test medium exceeds the highest mean measured concentration in the fish and dap hnid studies, the sponsor will need to repeat the fish and daphnid studies.

If additional studies in fish and daphnids are needed , EPA provides the following

r ecommendations for conducting adequate studies :

( 1 ) Since the previously submitted toxicity to algae study (Pillard, 2008) was able to achieve test substance concentrations that approximated the water solubility limit of the chemical, EPA recommends that a similar strategy be used for preparation of test solutions for the additional fish and daphnid toxicity studies. 2 For the algae study , the testing laboratory received a n approximately saturated solution of PETN dissolved in water (nominal concentration of ~20 00 μ g/L; measured concentration of 2850 μ g/L) from CHPPM and added macro

- and mi cro

nutrients (salts) to this solution to prepare the highest test concentration. Mean measured test concentrations during the 96

- hour algae test were 3.2, 30, 310, and 2850 μg/L for nominal concentrations of 2, 20, 200, and 2000 μg/L , respectively, indicating that the laboratory was successful in preparing test concentrations that approximated the water solubility limit of PETN and that measured c oncentrations were similar to nominal concentrations.

To conduct the fish and daphnid toxicity studies, EPA suggests that CHPPM prepare a saturated solution of PETN in test medium and ship this solution to AECOM. The saturated solution would be used as the highest test concentration and could be diluted with test medium to attain lower test concentrations. Alternatively, CHPPM could send AECOM a saturated solution of PETN in distilled water , as was done for the algae test. In this case , AECOM would nee d to add

1 The test media used in the fish and daphnid studies (Naddy, 2012, 2013) appear to have been prepared using identical methods. Therefore, only one solubility test needs to be performed to determine solubility in the test medium; the results will be applicable to both the fish and daphnid tests.

2 AECOM stated “When we used this same technique [as used in the algae study] for the fish/invertebrate studies, it resulted in very low levels of PETN in solution, ~18 μg/L.” However, it is not clear exactly what technique was used.HPV

2 Test Rule: Response to AECOM (Final ) CAS RN 78

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4 salts to the stock solution to achieve a hardness level that falls within test guideline recommendations. For the algae study, the temperature of the saturated solution was i ncremental ly adjust ed to 30 oC before salt s were added and the solution was diluted ; a similar technique may be used for the fish and daphnid studies to aid in the dissolution of salts and solubilization of the test substance in solution

. If shipment volume is a concern, t he sponsor c ould consider performing the fish and daphnid toxicity tests with no renewal (i.e., static conditions ). In the previously submitted static toxicity to algae test (Pillard, 2008)

, concentrations were 82

- 90% of initial concentrations over the 96

- hour test period, suggesting that the te st material was stable for at least 96 hours in algae test medium

. Prior to conducting static fish and daphnid tests, EPA recommends that the sponsor conduct preliminary tests to determine whether test substance concentrations can be satisfactorily main tained over the test period (96 hours for fish and 48 hours for Daphnia ) in the test medi um

. If test concentrations are reasonably maintained over the test period, the required aquatic toxicity tests could be performed under static conditions. EPA adds that any future aquatic toxicity tests in fish and Daphnia should be conducted with analytic al monitoring every 24 hours to characterize test concentrations throughout the test period.

Test solutions in the exposure vessels should be thoroughly stirred to assure homogeneity before organisms are added to the vessels. The procedures for mixing th e concentrations should be adequately documented in the study reports . EPA notes that the study reports for the submitted acute toxicity to fish (Naddy

, 2013) and acute toxicity to Daphnia (Naddy

, 2012) tests did not adequately describe the procedures for mixing test solutions . Therefore, it is unknown if test solutions reached homogeneity prior to analytical sampling. If the test substance was not homogeneous ly distributed in the test vessels at study initiation , this may have contributed to the increas es observed in measured concentrations during the 24

- hour renewal periods.

( 2 ) If the techniques recommended above are not successful in producing consistent PETN concentrations up to the solubility limit in test medium, EPA recommends that the sponsor attempt to prepare a more concentrated (> 5000 mg/L) stock solution of CASRN 78

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acetone

. One of the possible options presented in AECOM’s memo involved the use of higher target concentrations (greater than 2000 or 2500 μg/L) in test solutions to a chieve higher PETN concentration s ; since the preparation of higher target test solution concentrations with a stock solution of 5000 mg/L in acetone would result in an acetone concentration greater than 0.5 mL/L in test solutions, AECOM requested permission to exceed the recommended solvent limit

. EPA agrees with AECOM that the use of target nominal test solution concentrations greater than (e.g., 2

- fold) the solubility limit of PETN in water may allow AECOM to attain a concentration that approxim ates the water solubility limit of PETN ( experimentally determined to be 2130 μg/L at 20 °C by Hable et al., 2008)

. As stated in OECD ’s Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD , 2002; p. 20, Section 3 “Media preparation and exposure systems for difficult test substances” ) : “...While the target should be to achieve exposure concentrations up to the aqueous solubility of the test substance, it may be necessary to utilize higher nominal concentrations to achieve it.” However, EPA does not recommend that AECOM exceed an acetone concentration of 0.5 mL/L in test solutions.HPV

2 Test Rule: Response to AECOM (Final ) CAS RN 78

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5 The nominal concentration of PETN in the stock solution used in the fish and Daphnia acute toxicity tests (Naddy

, 2013 and Naddy

, 2012, respectively) was

~ 5 0 00 mg/L in acetone; test solutions prepared from this stock solution wi th a nominal concentration of 2 5 00 μg /L contained a solvent concentration of 0.5 mL/L

. If a stock solution of 7000 or 8000 mg/L in acetone could be prepared , then test solutions w ith nominal concentrations of 3 5 00 or 4 0 00 μg /L, respectively, could be made from this stock solution without exceeding a solvent concentration of 0.5 mL/L. Prior to implementing this strategy for the fish and daphnid tests, EPA recommends that the sponsor conduct tests to determine if (1) a stock solution of 7000 or 8000 mg/L in acetone can be prepared successfully (including analytical verification of the concentration of PETN in the stock solution) and (2) if this stock solution can be used to prepare test concentrations that approximate the solubility limit of PETN in the test medium without exceeding an acetone concentration of 0.5 mL/L. Recommendations for achieving homogeneity of test solutions (described above) would also apply to this approach.

REFERENCES

ASTM. 2007. E729

- 96: Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians.

Hable, M.A., Oliver, C.G., and Johnson, M.S. 2008. Determination of the water solubility, octanol

- water partition coefficient and biodegradation potential of pentaerythritol

- tetranitrate (PETN) . Performing Laboratory: U.S. Army Center for Health Promoti on and Preventative Medicine, Aberdeen Proving Ground, MD. Project ID 82

- XE

- 07TY

- 08. Sponsor: Dyno Nobel, Inc., Salt Lake City, UT. Study report available online at http://www.regulations.gov/ #!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0062 as of April 22 , 2014.

Naddy, R. 2012. Acute toxicity of pentaerythritol tetranitrate (P ETN) to the cladoceran ( Daphnia magna ) under static

- renewal test conditions

. Performing Laboratory: AECOM Fort Collins Environmental Toxicology Laboratory, Fort Collins, CO

. Project ID 60225262

- 304

- 010. Sponsor: Dyno Nobel, Inc., West Jordan , UT. Study report available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0713 as of April 22 , 2014

. Naddy, R. 2013. Acute toxicity of pentaerythritol tetranitrate (PETN) to the fathead minnow ( Pimephales promelas ) under static

- renewal test conditions

. Performing Laboratory: AECOM Fort Collins Environmental Toxicology Laboratory, Fort Collins, CO

. Project ID 60279946

- 100

- 020. Sponsor: Dyno Nobel, Inc., West Jordan , UT. Study report available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0713 as of April 22 , 2014

.

OECD. 2002. “OECD Series on Testing and Assessment Number 23: Guidance document on aquatic toxicity testing of difficult substances and mixtures”. Available online at http://www.oecd

- ilibrary.org/environment/guidance

- document

- on

- aquatic

- toxicity

- testing

- of

difficult

- s ubstances

- and

- mixtures_9789264078406

- en as of April 22 , 2014.HPV

2 Test Rule: Response to AECOM (Final ) CAS RN 78

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6 Pillard, D.A. 2008. Toxicity of p entaerythri tol tetranitrate (PETN) to the u nicellular g reen a lga ( Selenastrum capricornutum ) under s tatic t est c onditions. Performing Laboratory: ENSR Fort Collins Environmental Toxicology Laboratory, Fort Collins, CO. Project ID 08503

- 131

- 073

- 002. Sponsor: Dyno Nobel, Inc., Salt Lake City, UT. Study report available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2005

- 0033

- 0294 as of April 22 , 2014.

US EPA. 1996. Ecological Effects Test Guidelines : OPPTS 850.1075

, Fish Acute Toxicity Test, Freshwater and Marine. Available online at http://www.epa.gov/ocspp/pubs/frs/home/ draftguidelines.htm as of April 22 , 2014.