SRC TR

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HPV2 TEST RULE DATA ADEQUACY REVIEW ( REVISED FINAL ):

ACUTE TOXICITY TO FI SH AND DAPHNIA

1,3

- Propanediol, 2,2

- bis[(nitrooxy)methyl]

- , dinitrate (ester), CASRN 78

- 11

- 5

Prepared by:

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

Defense and Environmental Solutions Division

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

Contract No. EP

- W

- 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 4Final HPV 2 Test Rule Data Adequacy Review (April 2014) CAS RN 78

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1 A. SOURCE MATERIAL USED FOR ASSESSMENT

This document evaluates a submission from Dyno Nobel, Inc. (dated March 26, 2013 ) that contains data from toxicity testing of 1,3

- propanediol, 2,2

- bis[(nitrooxy)methyl]

- , dinitrate (ester) (CASRN 78

- 11

- 5; synonym: pentaerythritol tetranitrate or PETN ) and was received by EPA in response to 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;
available online at http://www.regulations.gov/#!documentDetail;D=EPA

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- 2007

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- 0579 as of A pril 16 , 201 4 ).

The HPV2 test rule requires the following toxicological testing for CASRN 78

- 11

- 5: C4 (consisting of acute toxicity to fish and acute toxicity to Daphnia testing if log K ow < 4.2 or chronic toxicity to Daphnia testing if log K ow ≥ 4.2). Since the log K ow of CASRN 78

- 11

- 5 has been experimentally determined to be 2.04 1 , which is < 4.2, the C4 aquatic toxicity testing requirement is considered to include acute toxicity to fish and acute toxicity to Daphnia testing.

The March 26, 2013 submission from Dyno Nobel, Inc. (available online at http://www.regulations.gov/#!documentDetail;D=EPA

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- 0713 as of A pril
16 , 201 4 ) contained the following study reports, which were submitted in response to the C4 testing requirement:

 Acute Toxicity of Pentaerythritol Tetranitrate (PETN) to the Fathead Minnow ( Pimephales promelas ) under Static

- Renewal Test Conditions

 Acute Toxicity of Pentaerythritol Tetranitrate (PETN) to the Cladoceran ( Daphnia

magna ) under Static

- Renewal Test Conditions

These newly submitted data are evaluated for adequacy herein. No other toxicity data are evaluated in this report.

B. EVALUATING DATA ADEQUACY

The submitted data are evaluated for adequacy in Section D. Each study was summarized according to a stan dard format and assigned a Klimisch reliability code based on its scientific merit and conduct, using professional judgment. To determine whether data were adequate to satisfy SIDS endpoints, study details were compared to current standard test guidelines to establish whether the study met current testing requirements. If the data available for the endpoint were determined to be inadequate, a basis for that conclusion is supplied.

C. SUMMARY OF DATA ADEQUACY FOR TESTING REQUIRED BY TEST RULE

Data submitted by Dyno Nobel, Inc. on March 26, 2013 are not considered adequate to satisfy the C4 testing requirement for CASRN 78

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- 5 promulgated in the final HPV2 test rule. Both the acute toxicity to fish study and the acute toxicity to Daphnia study are considered unreliable
because the concentrations tested were less than the solubility limit of the test item and the tests

1 The log K ow of PETN in water was reported in a letter of October 15, 2008 and its attached partition coefficient study report (without appendices) from Mr. Neal Olsen of Dyno Nobel, Inc. to the EPA (available online at http://www.regulations.gov/#!documentDetail;D=EPA

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- 2007

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- 0062 as of April 16, 2014 ).Final HPV 2 Test Rule Data Adequacy Review (April 2014) CAS RN 78

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2 failed to identify an LC 50 or EC 50 for fish and daphnids, respectively. The mean measured test
substance concentration at the h ighest concentration tested was 1218 and 943 μg/L , respectively,
for the fish and Daphnia studies, compared to the reported water solubility limit of 2130 μg/L at 20 ° C 1 . Furthermore, a previously submitted study of the toxicity of PETN to algae (Pillard
2 008) 2 achieved a mean measured test substance concentration of 2850 μg/L, suggesting that the highest concentrations tested in the fish and daphnid studies may have been as low as one half to one third of the potential solubility limit. In a cover letter attached to the acute toxicity to fish study, the study director speculates that the maximum measured PETN concentration in the fish and daphnid tests was less than the reported water solubility limit of 2130 μg/L due to “increased polarity of the water us ed in the toxicity studies (i.e., the required laboratory test water has a target water hardness and alkalinity of 170 and 115 mg/L as CaCO 3 , respectively).” However, the study reports do not provide experimental data examining the relationship between solubility of PETN and water hardness and/or alkalinity to support this statement.

Additional deficiencies of the acute toxicity to fish and acute toxicity to Daphnia tests include the following: (1) The acute toxicity to fish and daphnid test reports did not explain why measured concentrations of PETN showed an apparent increase from the beginning to the end of
the 24

- hour renewal intervals. (2) OPPTS 850.1075 (test guideline cited in the acute toxicity to fish study report) requires analysis of test item concen trations for replicate samples at the start and end of the test, as well as just before (“old” test solutions) and after (“new” test solutions) each renewal in
static

- renewal tests. However, measurements in the acute toxicity to fish test were not perform ed on “old” samples collected on days 2 or 4 for the nominal concentration of
1000 μ g/L. In addition, the “old” test solution from only a single replicate (of 3 replicates) was analyzed on days 1 and 3 for the nominal concentration of 1000 μg/L

. (3) OPPTS 850 .1010 (test guideline cited in the acute toxicity to Daphnia study report) requires that measured concentrations not vary by more than 20% among replicate test chambers. However, variation in test substance concentration among the replicates in the

acute toxicity to daphnid test for “old” samples (taken just before each renewal with fresh medium) collected on day 2 was greater than 20%. (4) Details regarding the analytical measurements of test substance concentrations were missing from the fish and daphnid st udy reports, including the limit of detection ( LOD ) for the analytical method and chromatogram s for the analytical measurements. Also, the fish study report was missing experimental details for range

- finding test 2.

Conclusion: The fish and daphnid studies should be repeated to assure that PETN is tested up to its solubility limit in a test medium appropriate for fish and Daphnia . The water solubility study report for this compound (available online at http://www.regulations.gov/#!documentDetail;

1 The solubility of PETN in water was reported in a letter of October 15, 2008 and its attached water solubility s tudy report (without appendices) from Mr. Neal Olsen of Dyno Nobel, Inc. to the EPA (available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0062 as of April 16, 2014 ).

2 Pillard, D.A. 2008. Toxicity of pentaerythritol tetranitrate (PETN) to the unicellular green alga ( Selenastrum capricornutum ) under static test conditions. Performing Laboratory: ENSR Fort Collins Environmental Tox icology 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 16, 2014.Final HPV 2 Test Rule Data Adequacy Review (April 2014) CAS RN 78

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3 D=EPA

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- 0062 as of A pril 16 , 201 4 ) stated that “the process for PETN equilibration in aqueous solutions at room temperature i s slow” based on the achievement of maximum solubility after 48 hours or longer; therefore, the laboratory should ensure that sufficient time is allowed for mixing of test solutions. It is recommended that the study design for further acute toxicity testi ng in fish and Daphnia includes the conduct of preliminary testing to determine what procedures are necessary to assure that PETN reaches maximum possible solubility in the intended test medium (i.e., dilution water) at each concentration that is to be

tes ted in the main toxicity study. In addition, the study report should include the chromatogram s and LOD for the analytical measurements to allow an independent assessment of the validity of
the method used for quantitation of this test substance.Final HPV 2 Test Rule Data Adequacy Review (April 2014) CAS RN 78

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4 D. DATA FOR SIDS ENDPOINTS

D

- 1 . Acute Toxicity to Fish

Summary of endpoint: Not A dequate

Study 1: 96

- h our a cute toxicity study in fathead minnow ( Naddy , 2013 )

Title: Acute toxicity of pentaerythritol tetranitrate (PETN) to the fathead minnow ( Pimephales promelas ) under static

- renewal test conditions

This study is: Adequate Not Adequate

This study is considered unreliable because the test was not conducted up to the solubility limit
of the test item and the test failed to identify an LC 50 for fish . The mean measured concentration
of test item of 1218 μg/L at the highest concentration tested is approximately 2

- fold lower than the reported water solubility of 2130 μg/L at 2 0 ° C 1

.

TEST SUBSTANCE

Identity: 1,3

- Propanediol, 2,2

- bis[(nitrooxy)methyl]

- , dinitrate (ester); referred to as pentaerythritol tetranitrate (PETN) in study report; CASRN 78

- 11

- 5 (synonyms: penthrit; pentrite; nitropenta; nitropentaerytritol)

Remarks: Received as a s tock solution in acetone with a nominal concentration of 5000 mg PETN/L and a measured concentration of 5 4 00 mg PETN/L 2 ; c lear, colorless liquid ; p urity : 98.43% 3

.

METHOD

Method/guideline followed: OPPTS 850.1075 (“Fish Acute Toxicity Test, Freshwater and Marine”) and ASTM E729

- 96 (“ Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinver t ebrates, and Amphibians”)

Test type: Acute fish toxicity t est ( s tatic

- renewal )

GLP compliant?: Yes , with the exception that “ analytical data were generated following NELAP ( National Environmental Laboratory Accreditation Conference ) guidelines .” [Naddy 2013, p. iv]

1 The solubility of PETN in water was reported in a letter of October 15, 2008 and its attached water solubility study report (without appendices) from Mr. Neal Olsen of Dyno Nobel, Inc. to the EPA (available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2007

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- 0062 as of A pril 16 , 201 4 ).

2 The measured concentration of the stock solution was reported in a m emo , dated March 5, 2014, from Dr. Rami B. Naddy of AECOM to Mr

. John Schaeffer of EPA.

3 The purity of the test substance was reported in a m emo , dated March 5, 2014, from Dr. Rami B. Naddy of
AECOM to Mr

. John Schaeffer of EPA.Final HPV 2 Test Rule Data Adequacy Review (April 2014) CAS RN 78

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5 Year study was performed: 201 2

Test species: Fathead minnow ( Pimephales promelas ) obtained from in

- house laboratory stocks

Analytical monitoring: Yes (definitive test only)

Exposure duration: 96 hours

Concentrations tested: Preliminary test 1 (nominal) : 0, 100, 500, and 2 000 μg/L Preliminary test 2 (nominal) : 0, 1000, and 2000 μg/L Definitive test: Nominal concentrations: 0, 1000 , and 2 0 00 μ g/L; m ean measured concentrations : 0, 723 , and 1218 μ g /L

. M ean measured concentrations we re based on the average of n ewly prepared test solutions (“new” samples) and test solutions collected at the time of renewal (“old” samples)

. New samples were collected for analysis on days 0, 1, 2, and 3 and old samples were collected for analysis on day s 1, 2, 3, and 4.

Test method/conditions remarks: Test Species: Fathead minnow ( Pimephales promelas ); 14

- days old at initiation; obtained from in

- house laboratory stocks; mean length of 8.5 mm and mean weight of 3.4 mg at initiation. Mean length and weight were based on 10 fish from the same batch as those used for the test. Fish were acclimatized to test conditions for at least 7 days prior to exposure and were not fed in the 48 hours prior to initiation of exposure or during the study.

Test Item and Test Item Preparation:
The test item was received as a stock solution in acetone with a nominal concentrat ion of
5000 mg PETN/L (measured concentration of 5 4 00 mg PETN/L 1 ) from the United States Army Center for Health Promotion and Preventative Medicine (CHPPM ). The stock solution was received and stored in an amber jar at 4°C and was rewarmed to room temperature prior to use

. “The test substance was assumed to be 100% active ingredient for purposes of stock solution and exposure concentrations calculation and preparation.” [Naddy 2013, p. 2 ]

“Each day, the highest test concentration, 2000 μg PETN/L (nominal), was prepared by a ddition of 0.8 mL of the PETN stock solution in acetone ... [ nominal concentration of 5 0 00 mg PETN/L ] to 2 L of dilution water. A 650 mL aliquot of the highest concentration was then diluted to 1.3 L to prepare the 1000 μg PETN/L (nominal) treatment daily. This was sufficient volume for biological and analytical sampling.” [Naddy 2013 , p. 4 ]

The dilution water, which was periodically analyzed for contaminants, was 100% Milli

- Q ® water (ASTM type I; Millipore, Billerica MA) that was adjusted to a target hardn ess of 170 ± 10 mg/L
as CaCO 3 and alkalinity of 115 ± 5 mg/L as CaCO 3

. At test initiation, t he dilution water had a pH of 8.3, with hardness and alkalinity (as CaCO 3 ) of 174 and 115 mg/L, respectively. Total

1 The measured concentration of the stock solution was reported in a m emo , dated March 5, 2014, from Dr. Rami B. Naddy of AECOM to Mr

. John Schaeffer of EPA.Final HPV 2 Test Rule Data Adequacy Review (April 2014) CAS RN 78

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6 residual chlorine was < 0.02 mg/L and total ammonia (as N) was < 1.0 mg/L. Conductivity was
593 μ S/cm and resistance was 18 MΩ·cm

.

Test Procedures: Preliminary Test Procedure: Two preliminary , 96

- hour test s were conducted using a stock
solution of 1000 mg PETN/L in methanol. Nominal concentrations in test 1 were 0, 100, 500,
and 2 000 μg/L. Nominal concentrations in test 2 were 0, 1000, and 2000 μg/L. Analytical monitoring was not conducted in the preliminary studies, although analytical monitoring was conducted on test substance solutions that were similarly prepared

. For preliminary test 1, t wo replicates, with 5 fish per replicate, were tested. Test conditions were similar to those in the definitive test, except that test organisms were 4 days old at the start of exposure and media w ere

renew ed only once (at 48 hours) during the 96

- hour exposure. No experimental details were reported for preliminary test 2.

Definitive Test Procedure : Juvenile fish (10/replicate ; 3 replicates/concentration) were exposed

to the test item under static

- renewal conditions for 96 hours; test solutions were renewed once daily at the beginning of each 24

- hour period . Fish loading rate was ≤ 0.5 g / L of test medium

. Solvent (0.4 mL acetone/L dilution water) and dilution water controls were exposed to the same
conditions in the absence of the test item

.

“ Test chambers were new, 500

- m L (nominal volume) plastic cups containing ~250 m L of test
solution

... Test chambers were covered during the test to minimize evaporation and possible air

borne contamination. ” [Naddy 2013, p. 4 ]

“ The exposure chambers were housed in a temperature

- controlled environmental chamber that maintained the test solution temperature at 25 ± 2 °C. Lighting wa s controlled to provide 16 hours of light per 24

- hour period; light intensity was ambient (approximately 50

- 100 footcandles). ” [Naddy 2013, p. 5 ]

F ish were observed daily for survival. A fish was considered dead if it was non

- responsive to gentle prodd ing. Following euthanasia, length measurements were recorded for all surviving fish in the control and treatment groups

. Fish weight was recorded for control groups only.

Hardness and alkalinity (both as mg CaCO 3 /L), total ammonia ( as mg N /L), and total residual chlorine were recorded at initiation in the controls and the highest test item concentration. Dissolved oxygen, pH, and conductivity were recorded for one replicate at initiation and daily thereafter. Temperature was recorded for each replicate and concentration throughout the study.

For a nalytical m onitoring

, “ s amples for PETN analysis were collected from bulk solutions at test initiation, prior to distributing the test solutions to the test chambers, from each freshly prepared
test treatment, the solvent control, and the dilution water. On days 1, 2, and 3, samples were
collected from bulk solutions of all freshly prepared test treatments after distributing to test chambers, as well as from all replicates of old test solutions for treatments after organisms had
been transferred to new solutions. Only one replicate of old test solutions wa s analyzed on days 1 and 3

... [ for the nominal concentration of 1000 μg/L

. None of the day 2 or day 4 old test solution samples were analyzed for the 1000 μg/ L concentration ]... Each replicate of the old testFinal HPV 2 Test Rule Data Adequacy Review (April 2014) CAS RN 78

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7 solutions was sampled from all treatments at test termination after removing the test organisms

... All samples were poured directly into certified clean 500

- m L glass amber bottles. The samples

were refrigerated until shipped (on ice) [to an independent laboratory for analysis].” [ Naddy 2013, p. 6]

“ EPA Method 8330A was used following USEPA (1986) guidelines to analyze for
nitroaromatics and nitramines by HPLC [ using a wavelength of 215 nm 1

, 2 ]

. ” [ Naddy 2013, p. 6]

The study report did not include the limit of detection (LOD) for the analytical method

.

Regarding s tatistical analyses, “ g iven that there was not ≥ 50% mortality observed in any treatment of this study, statistical analyses were not required. The concentrations estimated to cause death (or immobility) of 50% of the test organisms after 24, 48, 72, and 96 hours of exposure (the 24

- , 48

- , 72

- , and 96

- hour LC 50 , respectively) were determined by inspection. The
LC 50 values are expressed as average measured PETN concentrations measured in new and old test solutions (μg PETN/L). ” [ Naddy 2013, p. 6]

RESULTS

Endpoint value(s): The following endpoint values are based on mean measured concentrations:
96

- hour LC 50 > 1218 μ g/L
96

- hour LOEC (mortality) = 1218 μ g/L
96

- hour NOEC (mortality) = 723 μ g/L

Results remarks: Preliminary Test Results: Mortality was observed at the highest (nominal) concentration of 2000 μg/L in both preliminary

test s. In test 1 , mortality percentages at 96 hours were 0%, 0%, 0%, and 60% for the nominal concentrations of 0 (dilution water control), 100, 500, and 2 000 μg/L, respectively. In test 2

, mortality percentages at 96 hours were 0%, 0%, 0%, and 70% for the nominal concent rations of 0 (dilution water control), 0 (solvent control), 1000, and 2000 μg/L, respectively.

The concentrations of test item in media were not measured in the preliminary tests

. Analys i s of
solutions prepared similarly to those used in test 1 showed measured concentrations of approximately 85% and 86% of nominal values for the nominal concentrations of 500 and 1000 μg/L

, respectively. A sample corresponding to the 2000 μg/L nominal concentration was not analyzed.

Definitive Test Results:

1 The wavelength of 215 nm was reported in a m emo , dated March 5, 2014, from Dr. Rami B. Naddy of AECOM to Mr

. John Schaeffer of EPA

. 2 EPA SW

- 846 Method 8330B (Nitroaromatics, Nitramines, and Nitrate Esters by High Performance Liquid
Chrom a tography), which is the updated version of EPA SW

- 846 Method 8330A, recommends a similar wavelength (214 nm) for detection of PETN. EPA SW

- 846 Methods 8330A and 8330B are available online at http://www.epa.gov/osw/hazard/testmethods/sw846/pdfs/8330a.pdf and http://www.ep a.gov/osw/hazard/ testmethods/pdfs/8330b.pdf , respectively, as of April 16, 2014.Final HPV 2 Test Rule Data Adequacy Review (April 2014) CAS RN 78

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8 Mortality data are summarized in Table 1. After 96

- hours of exposure, 26.7% m ortality was observed in the 2000 μg/L group , compared to 0% in the dilution water control and 3.3% in the solvent control and 1000 μg/L groups (statistical significance was not reported)

. No deaths occurred in the 1000 or 2000 μg/L groups during the first 72 hour s of the exposure period

.

Table 1

. Mortality (% of total number of fish exposed per group ) in fish exposed to PETN
Nominal concentration ( μ g/L) Observation Time (h r )

0 24 48 72 96 0 (solvent) 0% 0% 3% (1/30) a 3% (1/30) 3 .3 % (1/30) 0 (dilution water) 0% 0% 0% 0% 0% (0/30)

1000 0% 0% 0% 0% 3 .3 % (1/30)

2000 0% 0% 0% 0% 26.7 % (8/30)

a The n umber in parentheses is the cumulative number of dead fish per total number exposed

.

At the 96

- hour observation, concentration

- related “weakness” was observed in surviving fish as follows: solvent control, 1/29 (3.4%); dilution water control , 0/30 (0%); 1000 μg/L, 7 / 29 (24.1%); and 2000 μg/L, 18/22 (81.8%). No w eakness was observed from 0 to 72 hours in control or test item treatment groups

.

Exposure to the test material did not appear to affect fish growth as assessed by length , although no summary statistics were reported.

Test chamber temperatures, measured daily, ranged from 23.0 t o 25.1 o C. The pH range was 8. 2

- 8. 4 and the d issolved oxygen range was 6. 2

- 6.9 mg O 2 /L. Conductivity was 5 72

- 631 μ S/cm. These values “ were in compliance with the protocol and ASTM requirements .” [Naddy 201 3 , p. 7]

Mean measure d concentrations (based on combined “old” and “new” samples) for the 0 (solvent control), 0 (dilution control), 1000 μ g/L

, and 2000 μ g/L groups were < LOD

, < LOD , 723 μ g/L

, and 1218 μ g/L , respectively. As shown in T able 2 , measured concentrations (combined new and old) for the 1000 and 2000 μ g/L groups ranged from 5 0 to 100% and 49 to 78% of nomi n al
concentrations , respectively.Final HPV 2 Test Rule Data Adequacy Review (April 2014) CAS RN 78

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9 Table 2

. Measured Concentrations of PETN in Test Water a

Nominal C oncentration ( μ g/L) New Samples Old Samples Mean
Measured Conc entration ( μ g/L) b Study Day

0 1 2 3 1 2 3 4 0 (solvent) < LOD < LOD < LOD < LOD < LOD < LOD < LOD < LOD < LOD 0 (dilution water) < LOD < LOD < LOD < LOD < LOD < LOD < LOD < LOD < LOD

1000 530

( 53% ) 500

( 50% ) 790

( 79% ) 820

( 82% ) 570

( 57% ) NA c 1000

( 100% ) NA 723 ( 72% )

2000 1100

( 55% ) 1000

( 50% ) 1000

( 50% ) 1400

( 70% ) 1167

( 58% ) 973

( 49% ) 1533

( 77% ) 156 7

( 78% ) 1218

( 61% )

a Expressed as μ g/L; values in () are % of nominal concentration

. b Mean of combined new and old samples

.

c NA = Not a vailable ; o ld samples were not measured on study days 2 or 4 for the 1000 μ g/L nominal
concentration

.

CONCLUSIONS

The 96

- hour LC 50 and NOEC ( for mortality ) for Pimephales promelas were > 1218 μ g/L and
723 μ g/L, respectively, based on mean measured concentrations. The LOEC was 1218 μ g/L based on increased mortality , relative to controls, at the highest concentration tested

.

STUDY RELIABILITY

[ 3 ] Not reliable

. This study is considered un reliable because the test was not conducted up to the solubility limit of the test item and the test failed to identify an LC 50 for fish

. The study author did not report any pre cipitation in the test media, indicating that the solubility limit was not
exceeded. The mean measured concentration of test item of 1218 μg/L at the highest
concentration tested is approximately 2

- fold lower than the reported water solubility of 2130 μg/L at 20 ° C 1

. Furthermore, a previously submitted study of the toxicity of PETN to aquatic plant s (Pillard 2008) achieved a mean measured test substance concentration of 2850 μg/L. Given that increased fish mortality was observed at a mean measured concentration of 1 218 μg/L , testing at concentrations up to the solubility limit of 2 130 μg/L (or 2 85 0 μg/L based on the solubility limit reported in the algae study) may provide sufficient data to determine an LC 50 value for PETN in freshwater fish

.

Additional deficiencies include the following: (1) The test report did not explain why measured concentrations of PETN showed an apparent increase from the beginning to the end of the 24

- hour renewal intervals. (2) OPPTS 850.1075 (test guideline cited in the study report) requires analysis of test item

concen trations for replicate samples at the start and end of the test, as well as just before (“old” test solutions) and after (“new” test solutions) each renewal in static

- renewal tests. However, measurements were not performed on “ old ” samples collected on da ys 2 or 4

1 The solubility of PETN in water was reported in a letter of October 15, 2008 and its attached water solubility study report (without appendices) from Mr. Neal Olsen of Dyno Nobel, Inc. to the EPA (available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0062 as of A pril 16 , 201 4 ).Final HPV 2 Test Rule Data Adequacy Review (April 2014) CAS RN 78

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10 for the nominal concentration of 1000 μ g/L. In addition, the “old” test solution from only a single replicate (of 3 replicates) was analyzed on days 1 and 3 for the nominal concentration of 10 00 μg/L

. (3) Several experimental details are missing from the study report, including the LOD for the analytical method, chromatogra m s for the analytical measurements, and experimental details for range

- finding test 2.

REFERENCES

Naddy

, R

. 201 3

. Acute toxicity of p entaerythritol t etranitrate (PETN) to the fathead minnow ( Pimephales promelas ) u nder static

- renewal t est c onditions

. 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 A pril
16 , 201 4

.

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, For t 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

- 200 5

- 0033

- 0294 as of A pril
16 , 201 4

.

Text in quotes is taken directly from Naddy (201 3 ).Final HPV 2 Test Rule Data Adequacy Review (April 2014) CAS RN 78

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11 D

- 2. Acute Toxicity to Aquatic Invertebrates

Summary of endpoint: Not Adequate

Study 1 : Acute toxicity study in Daphnia magna ( Naddy
, 2012 )

Title: Acute toxicity of pentaerythritol tetranitrate (PETN) to the cladoceran ( Daphnia magna ) under static

- renewal test conditions

This study is: Adequate Not Adequate

This study is considered unreliable because the test was not conducted up to the solubility limit
of the test item and the test failed to identify an EC 50 for daphnids . The mean measured
concentration of test item of 943 μg/L at the highest concentration tested is approximately 2.2

- fold lower than the reported water solubility of 2130 μg/L at 2 0 ° C 1

.

TEST SUBSTANCE

Identity: 1,3

- Propanediol, 2,2

- bis[(nitrooxy)methyl]

- , dinitrate (ester); referred to as pentaerythritol tetranitrate (PETN) in study report; CASRN 78

- 11

- 5 (synonyms: penthrit; pentrite; nitropenta; nitropentaerytritol)

Remarks: Received as a stock solution in acetone with a nominal concentration of 5000 mg PETN/L and a measured concentration of 5 4 00 mg PETN/L 2 ; c lear, colorless liquid ; p urity : 98.43% 3

.

METHOD

Method/guideline followed: OPPTS 850

. 1010 (“Aquatic Invertebrate Acute Toxicity Test,
Freshwater Daphnids” ) and ASTM E729

- 96 (“ Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinver t ebrates, and Amphibians”)

Test type: Acute toxicity t est in daphnids ( s tatic

- renewal )

GLP compliant?: Yes , with the exception that “ analytical data were generated following NELAP ( National Environmental Laboratory Accreditation Conference ) guidelines .” [Naddy 2012, p. iv]

Year study was performed: 2012

1 The solubility of P ETN in water was reported in a letter of October 15, 2008 and its attached water solubility study report (without appendices) from Mr. Neal Olsen of Dyno Nobel, Inc. to the EPA (available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0062 as of A pril 16 , 201 4 ).

2 The measured concentration of the stock solution was reported in a m emo , dated March 5, 2014, from Dr. Rami B. Naddy of AECOM to Mr

. John Schaeffer of EPA.

3 The purity of the test substance was reported in a m emo , dated March 5, 2014, from Dr. Rami B. Naddy of
AECOM to Mr

. John Schaeffer of EPA.Final HPV 2 Test Rule Data Adequacy Review (April 2014) CAS RN 78

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12 Test species: Water flea ( Daphnia magna ); < 24 hours old; obtained from in

- house stocks ; Batch Number 052912

Analytical monitoring: Yes (definitive test only)

Exposure duration: 48 hours

Concentrations tested: Preliminary test (nominal): 0, 268, 419, 547, 809, and 943 μ g/L Definitive test: Nominal concentrations: 0 (solvent control), 0 (dilution water control)

, 500

, 800

, 1100

, 1700, and 2500 μ g/L; m ean m easured concentrations : 0 (solvent control)

, 0 (dilution water control), 268, 419, 547, 809, and 943 μ g/L

. Mean measured concentrations were based on the average of newly prepared test solutions (“new” samples) and test solutions collected at the time of renewal (“old” samples). New samples were collected for analysis on days 0, 1, and 2 and old samples were c ollected for analysis on days 1, 2, and 3.

Test method/conditions remarks: Test Species: Daphnia magna neonates (< 24 hours old) were obtained from AECOM FCETL in

- house
cultures (Batch number 052912)

. “ Organisms were cultured in an environmental chamber at 25°C, and were moved to a 20oC environmental chamber at least 48 hours prior to test initiation.
Brood D. magna were also maintained in 100% dilution water for at least 48 hours prior to test
initiation ... Organisms were not fed during testing. ” [ Naddy 2012 , p. 3 ]

Test Item and Test Item Preparation:
The test item was received as a stock solution in acetone with a nominal concentration of
5000 mg PETN/L (measured concentration of 5 4 00 mg PETN/L 1 ) from the United States Army Center for Health Promotion and Preventative Medicine (CHPPM). The stock solution was received and stored in an amber jar at 4°C and was rewarmed to room temperature prior to use

. “The test substance was assumed to be 100% active ingredient for purposes of stock solution and exposure concentrations calculati on and preparation.” [Naddy 2012 , p. 2]

“ The highest test concentration, 2.5 mg PETN/L (nominal), was prepared by addition of the appropriate volume of the PETN stock solution in acetone to 10 L of dilution water. This was sufficient volume for the entire duration of the study. An appropriate volume of the highest concentration was then diluted to 1.4 L to prepare all other test solutions daily. This was sufficient volume for biological and analytical sampling. ” [ Naddy 2012, pp. 3

- 4]

The dilution water, which was periodically analyzed for contaminants, was 100% Milli

- Q ® water (ASTM type I; Millipore, Billerica MA) that was adjusted to a target hardness of 170 ± 10 mg/L
as CaCO 3 and alkalinity of 115 ± 5 mg/L as CaCO 3

. At test initiation, t he dilution water had a pH of 8.5, with hardness and alkalinity (as CaCO 3 ) of 172 and 116 mg/L, respectively. Total residual chlorine was < 0.02 mg/L and total ammonia (as N) was < 1.0 mg/L. Conductivity was 579 μ S/cm and resistance was 18 M Ω ·cm.

1 The measured concentration of the stock solution was r eported in a m emo , dated March 5, 2014, from Dr. Rami B. Naddy of AECOM to Mr

. John Schaeffer of EPA.Final HPV 2 Test Rule Data Adequacy Review (April 2014) CAS RN 78

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13 Test Procedures: Preliminary Test Procedure: A 48

- hour test was conducted using a stock solution concentration
of 1000 mg PETN /L in methanol . Nominal concentrations were 0 (dilution water control)

, 100, 500, and 2000 μg/L. Analytical monitoring was not conducted in the preliminary study, although analytical monitoring was conducted on test substance solutions that were similarly prepared

. Two replicates per group , with 5 organisms per replicate, were tested under similar conditions as the definitive te s t, except that media was not renewed during exposure and a solvent control group was not included

.

Definitive Test Procedure: Daphnids (5/replicate; 4 replicates per concentration) were exposed to
the test item at nominal concentrations of 0 (solvent control)

, 0 (dilution water control), 500, 800, 1100, 1700, and 2500 μ g/L under static

- renewal conditions for 48 hours

. T est solutions were renewed once daily

. Daphnid loading was equi valent to 2 5 daphnids per liter of test
medium. Solvent (0.5 mL acetone/L dilution water) and dilution water controls were exposed to
the same conditions in the absence of the test item

.

“ Test chambers were 500

- m L (nominal volume) plastic cups containing ~200 m L of test
solution

... Test chambers were covered during the test to minimize evaporation and possible air

borne contamination. ” [Naddy 2012, p. 4] “ The exposure chambers were housed in a temperature

- controlle d environmental chamber that maintained the water temperature at 20 ±
2°C. Lighting was controlled to provide 16 hours of light per 24

- hour period; light intensity was ambient (approximately 50

- 100 foot

- candles). ” [Naddy 2012, p. 5]

Daphnids were observed daily for survival. A daphnid was considered dead (immobilized) if it

was non

- responsive to gentle prodding.

Hardness and alkalinity (both as mg CaCO 3 /L), total ammonia ( as mg N /L), and total residual chlorine were measured at initiation in the controls and the highest test item concentration. Dissolved oxygen, pH, and conductivity were recorded for one replicate at initiation and daily
thereafter. Temperature was recorded for each replicate and concentration throughout the study.
For a nalytical m onitoring

, “ s amples for PETN analysis were collected from bulk solutions at test initiation, after distributing the test solutions to the test chambers

... , from each freshly prepared

te st treatment, the solvent control, and the dilution water. On day 1, samples were collected from bulk solutions of all freshly prepared test treatments after distributing to test chambers, as well as from all replicates of old test solutions after organis ms had been transferred to new solutions. At test termination, each replicate of the old test solutions was sampled. All samples were poured directly into certified clean 500

- m L glass amber bottles. The samples were refrigerated until shipped (on ice) [to an independent laboratory for analysis].” [ Naddy 2012, pp. 5

- 6]Final HPV 2 Test Rule Data Adequacy Review (April 2014) CAS RN 78

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14 “EPA Method 8330A was used following USEPA (1986) guidelines to analyze for
nitroaromatics and nitramines by HPLC [ using a wavelength of 215 nm 1

, 2 ] .” [ Naddy 2012, p. 6]

The study report did not include the limit of detection (LOD) for the analytical method.

Regarding s tatistical m ethods

, “ g iven that there was no mortality observed in any treatment of this study, statistical analyses were not required. The concentrations estimated to cause death (or immobility) of 50% of the test organisms after 24 and 48 hours of exposure (the 24

- and 48

- hour
EC 50 , respectively) were determined by inspection. The EC 50 values are expressed as average
measured PETN concentrations measured in new and ol d test solutions (μg PETN/L). ” [ Naddy 2012, p. 6]

RESULTS

Endpoint value(s): The following endpoint values are based on mean measured concentrations:
48

- hour EC 50 > 943 μ g/L
48

- hour LOEC (immobility) > 943 μ g/L
48

- hour NOEC (immobility) = 943 μ g/L

Results remarks: Preliminary Test Results: No mortalities or signs of immobility were observed in the preliminary test

. The concentrations
of test item in media were not measured. Analyses of solutions prepared similarly to those used in the test showed measured concentrations of approximately 86% of nominal values f or the
nominal concentrations of 500 and 1000 μ g/L

.

Definitive Test Results:
No mortalities or signs of toxicity were observed in control or test item groups

. Test chamber temperatures, measured daily, ranged from 19 to 22 o C. The pH was 8.5

- 8.6. Dissolved oxygen
was 6.9

- 7.3 mg O 2 /L. Conductivity was 563

- 585 μ S/cm. These values “were in compliance with the protocol and ASTM requirements.” [Naddy 2012, p. 7]

M easured concentrations are summarized in Table 3. B ased on combined “old” and “new ” samples for the 0 (solvent control), 0 (dilution control), 500, 800, 1100, 1700, and 2500 μ g/L

groups , mean measured concentrations were < LOD, < LOD, 268 (54% of nominal), 419 (52% of nominal), 547 (50% of nominal), 809 (48% of nominal), and 943 (38% of nominal) μ g/L

, respectively.

1 The wavelength of 215 nm was reported in a m emo , dated March 5, 2014, from Dr. Rami B. Naddy of AECOM to Mr

. John Schaeffer of EPA

. 2 EPA SW

- 846 Method 8330B (Nitroaromatics, Nitramines, and Nitrate Esters by High Performance Liquid
Chrom a tography), which is the updated version of EPA SW

- 846 Method 8330A, recommends a similar wavelength (214 nm) for detection of PETN. EPA SW

- 846 Methods 8330A and 8330B a re available 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 16, 2014.Final HPV 2 Test Rule Data Adequacy Review (April 2014) CAS RN 78

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15 Table 3. Measured Concentrations of PETN Test Water a

Nominal concentration ( μ g/L) New Samples Old Samples Mean
measured concentration ( μ g/L) b Study Day

0 1 1 2 0 (solvent) < LOD < LOD < LOD < LOD < LOD
0 (dilution water) < LOD < LOD < LOD < LOD < LOD

500 240 ( 48% ) 240

( 48% ) 288

( 58% ) 305

( 61% ) 268

( 54% )

800 360 ( 45% ) 380

( 48% ) 463

( 58% ) 475

( 59% ) 419

( 52% )

1100 480 ( 44% ) 500

( 45% ) 638

( 58% ) 570

( 52% ) 547

( 50% )

1700 670 ( 39% ) 620

( 36% ) 978

( 58% ) 968

( 57% ) 809

( 48% )

2500 390 ( 16% ) 895

( 36% ) 1350

( 54% ) 1138

( 46% ) 943

( 38% )

a Expressed as μ g/L; values in () are % of nominal concentration as reported in the study report

. b Mean of combined new and old samples

.

CONCLUSIONS

The 48

- hour EC 50 and NOEC for Daphnia magna were > 943 μ g/L and 943 μ g/L, respectively,
based on mean measured concentrations. The LOEC was > 943 μ g/L , based on no effects at the highest concentration tested

.

STUDY RELIABILITY

[ 3 ] Not r eliable. This study is considered unreliable because the test was not conducted up to the solubility limit of the test item and the test failed to identify an EC 50 for daphnids

. The study author did not report any prec ipitation in the test media , indicating that the solubility limit was
not exceeded. The mean measured concentration of te st item of 943 μ g/L at the highest
concentration tested is approximately 2.2

- fold lower than the reported water solubility of 2130

μ g/L at 2 0 ° C 1

. Furthermore, a previously submitted study of the toxicity of PETN to aquatic plants (Pillard 2008) achieved a mean measured test substance concentration of 2850 μ g/L

.

Additional deficiencies included the following: (1) The test report did not explain why measured concen trations of PETN showed an apparent increase from the beginning to the end of the 24

- hour renewal intervals. (2) OPPTS 850.1010 (test guideline cited in the study report) requires that measured concentrations not vary by more than 20% among replicate test chambers. However, variation in test substance concentration among the replicates for “ old ” samples (taken just before each renewal with fresh medium) collected on day 2 was greater than 20%.

1 The solubility of PETN in water was reported in a letter of October 15, 2008 and its attached water solubility study report (without appendices) from Mr. Neal Olsen of Dyno Nobel, Inc. to the EPA (available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0062 as of A pril 16 , 201 4 ).Final HPV 2 Test Rule Data Adequacy Review (April 2014) CAS RN 78

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16 (3) Details regarding the analytical measurements of test substance concentrations were missing from the study report, including t he LOD for the analytical method and chromatogram s for the analytical measurements,

.

REFERENCES

Naddy

, R . 201 2

. Acute toxicity of p entaerythritol t etranitrate (PETN) to the c ladoceran ( Daphnia magna ) u nder static

- renewal t est c onditions

. 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 A pril
16 , 201 4

.

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, For t 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 A pril
16 , 201 4

.

Text in quotes is taken directly from Naddy (201 2 ).Draft Addendum to HPV2 Test Rule Data Adequacy Review ( April 2014

) CASRN 78

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A

- 1 Addendum: Test Guideline Source Material to Support Reliability Ratings Assigned to Submitted Studies That Are Considered Inadequate to Satisfy Test Rule Requirements

This addendum provides additional information regarding deficiencies of each of the stud ies that is considered inadequate to satisfy a test rule requirement. For each deviation from relevant test guidelines (i.e., standard test guidelines purportedly followed by each study according to the study report), quotes of relevant passages from the gui delines are supplied, followed by a brief scientific interpretation of the quoted material. Emphasis has been added to quotes (in the form of italics) by the reviewer.

In the ASTM E729

- 96 guideline, p. 3, Section 3 “Terminology”, Sub

- section 3.6 , the wor ds “must ” and “should” hav e very specific meanings: “‘Must’ is used to express an absolute requirement, that is, to state that the test ought to be designed to satisfy the specified condition,
unless the purpose of the tes t requires a different design. ‘ Must’ is only used in connection with factors that directly relate to the acceptability of the test... ‘Should’ is used to state that the specified condition is recommended and ought to be met if possi ble. Although violation of one ‘should’ is rarely a ser ious matter, violation of several will often render the results questionable. ”

Study 1: Acute Toxicity to Fish (Naddy 2013) Relevant Guidelines: Study was conducted according to OPPTS 850.1075 and ASTM E729

- 96

[A] Deviation 1: This study failed to identify an LC 50 because mortality at the highest test concentration was less than 50% in the definitive test. The study only determined that the LC 50 was greater than 1218 μg/L (i.e., 1.218 mg/L) , which is considerably less than the reported water solubility limit of the test substance (2130 μg/L at 20 ° C ). OPPTS 850.1075, p. 1, Section (c) “Principle of the test”, Sub

- section (1) “Definitive test” states: “ The goal of the definitive test is to determine concentration

- response curves for fish mortality, the LC50’s , and the 95 percent confidence intervals for each species tested at 24, 48, 72, and 96 h in a static, static

- renewal, or flow

- through system. ”; and Sub

- section (2) “Range

- finding or limit testin g” states: “ D efinitive testing may be waived if limit testing with at least 30 organisms shows LC50 levels to be greater than 1,000 mg/L based on 100 percent active ingredients (AI), or the limits of water solubility or dispersibility. ” OPPTS 850.1075, p. 10, Section (g) “Data and reporting”, Sub

- section (2) (i) “Test report” states: “The test report must include the following:”; and item (viii) states: “The
LC 50 concentration

- response curves, LC 50 values, and associated 95 percent CI should be
determined fo r 24, 48, 72, and 96 h, whenever sufficient data exist.”; and item (xi) states: “If
no LC 50 value is determined, but it can be demonstrated that the concentrations tested were the highest possible due to the test chemical’s aqueous solubility limit

, self

- dispersibility limit, or other physicochemical limitations, then the data will be considered for acceptance.”
ASTM E729

- 96, p. 9, Section 9 “Test Material”, Sub

- section 9.3 “Test Concentration(s)”, item 9.3.2 states: “In some (usually regulatory) situ ations, it is necessary only to determine (1) whether a specific concentration of test material is acutely toxic to the test species, or (2) whether the LC 50 , EC 50 , or IC 50 is above or below a specific concentration. For example, the specific concentratio n might be the concentration occurring in a surface water, the concentration resulting from the direct application of the material to a body of water, or the solubility limit of the material in water . When there is interest only in a specificDraft Addendum to HPV2 Test Rule Data Adequacy Review ( April 2014

) CASRN 78

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- 2 concentratio n, it is often necessary only to test that concentration (see 11.1.1.2) and it is not necessary to actually determine the LC 50 , EC 50 , or IC 50 .”

Interpretation: A primary objective of an acute toxicity to fish test is to determine a discrete
LC 50 value. I f a discrete LC 50 value i s not determined because insufficient (< 50%) mortality occurs at the highest concentration tested and this concentration is below the aqueous solubility or dispersibility limit, the test is not considered acceptable.

[ B ] Deviation 2 : Measured c oncentration s increase d from the beginning to the end of the 24

- hour renewal intervals , but the study report did not provide a valid explanation

. OPPTS 850.1075, p. 2, Section (c) “Principle of the test”, Sub

- section (3) “Informat ion on the test substance” states: “... A reliable analytical method for quantification of test
substance concentrations must be available.” OPPTS 850.1075, p. 2, Section (d) “Validity of the test”, Sub

- section (4) states: “... [T]here must be evidence that test concentrations remained at least 80 percent of the nominal concentrations throughout the test or that mean measured concentrations are an accurate representation of exposure levels maintained throughout the test period. OPPTS 850.107 5, p. 10, Section (g) “Data and reporting”, Sub

- section (2) (i) “Test report” states: “ The test report must include the following:” and item ( x iii) states: “Any protocol deviations or occurrences which may have influenced the final results of the test.” ASTM E729

- 96, p. 1 8 , Section 15 “Report”, Sub

- section 15.1 states: “The record of the results of an acceptable acute toxicity test should include the following information either directly or by reference to available documents:” and item 15.1.3 states: “ An ything unusual about the test, any deviation from these procedures, and any other relevant information. ” ASTM E729

- 96, p. 16, Section 11 “Procedure”, Sub

- section 11.9.3 “Test Material”, item 11.9.3.1 states: “... If the test material is not uniformly dispersed in the test chamber in static and renewal tests, the whole volume of solution in the test chamber should be (1) used as the sample or (2) treated appropriately (for example, by adding acid, base, or surfactan t and mixing thoroughly) to uniformly distribute the test material before a sample is taken.”

Interpretation: Test concentrations must be determined by a reliable analytical method that provides an accurate representation of exposure levels. The report must address any deviations from the protocol or occurrences that could impact the results. If the test substance is not homogeneously distributed in the fluid in the test chambers, additional measures should be taken to ensure that samples collected for analysis are representative of actual mean concentrations of
test substance in the test chambers.

[ C ] Deviation 3 : Test concentrations were not measured for each test substance renewal and
some test concentration measurement s were performed on single replicate samples. Specifically, test concentrations were not measured on “old” samples (taken just before each renewal with fresh medium) collected on days 2 or 4 for the lowest test concentration ( i.e., nominal
concentration o f 1000 μg/L). In addition, for the same concentration, only a single replicate was measured for “old” samples collected on day s 1 and 3. OPPTS 850.1075, p. 8, Section (f) “Performance of the test”, Sub

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

- renewalDraft Addendum to HPV2 Test Rule Data Adequacy Review ( April 2014

) CASRN 78

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- 3 tests, the test substance should be measured in each replicate at the beginning and end of test and just before and after each renewal.”

Interpretation: Test concentration analyses should be performed on all replicates. Test fluid samples should be collected from each replicate at the beginning of the test, just before and after each test substance renewal , and at the end of the test

. All samples should be analyzed to
determine tes t substance concentrations.

[ D ] Deviation 4 : Details regarding the analytic al measurements of test substance concentrations

w ere missing from the study report, including the limit of detection of the analytical method and chromatogram s for the analytical measurements. OPPTS 850.1075, p p . 10

- 11 , Section (g) “Data and reporting”, Sub

- section (2) (i) “Test report” states: “ The test report must include the following:” and item ( xv ii) states: “Methods used in analysis of test concentrations of the test chemical should be described.
The accuracy of the method (i.e., detection limit and quantification limit) should be given.” OPPTS 850.1075, p. 2, Section (c) “Principle of the test”, Sub

- section (3) “Information on the test substance” states: “... A reliable analytical method for quantification of test
substance concentrations must be available.” OPPTS 850.1075, p. 2, Section (d) “Validity of the test”, Sub

- section (4) states: “... [T]here must be ev idence that test concentrations remained at least 80 percent of the nominal concentrations throughout the test or that mean measured concentrations are an accurate representation of exposure levels maintained throughout the test period.” ASTM E729

- 96, p. 18, Section 15 “Report”, Sub

- section 15.1 states: “The record of the results of an acceptable acute toxicity test should include the following information either directly or by reference to available documents:” and item 15.1.9 states: “ Me thods used for, and results (with standard deviations or confidence limits) of, chemical analyses of water quality and concentrations(s) of test material, impurities, and reaction and degradation products, including validation studies and reagent blanks.”
ASTM E729

- 96, p. 16, Section 12 “Analytical Methodology”, Sub

- section 12.2 states: “Chemical and physical data should be obtained using appropriate ASTM standards whenever possible. For those measurements for which ASTM standards do not exist or are not sensitive enough, methods should be obtained from other reliable sources.”

Interpretation: The analytical method use d for measurement of test substance concentrations should be fully described. The results of validation studies of the analytical method should be provided. The limit of detection should be provided. The analytical results, including all data that are g enerated (e.g., chromatogram s) and used to determine test substance concentrations in samples taken from test chambers over the course of the study, should be fully described and
documented

. The test report must provide evidence that the analytical method is reliable and

accurate

. The lack of complete details precludes an evaluation of whether or not the analytical
method adhere s to acceptable standards, such as ASTM.

REFERENCE

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

- renewal test conditions

. Performing Laboratory: AECOMDraft Addendum to HPV2 Test Rule Data Adequacy Review ( April 2014

) CASRN 78

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A

- 4 Fort Collins Environmental Toxicology Laboratory, Fort Collins, CO

. Project ID 60279946

- 100

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

- HQ

- OPPT

- 2007

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- 0713 as of April
1 6 , 201 4

.Draft Addendum to HPV2 Test Rule Data Adequacy Review ( April 2014

) CASRN 78

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- 5 Study 2: Acute Toxicity to Daphnia (Naddy 2012) Relevant Guidelines : Study was conducted according to OPPTS 850.1010 and ASTM E729

- 96

[A] Deviation 1: This study failed to identify an EC 50 because mortality and immobility at the highest test concentration were less than 50% in the definitive test. The study only determined
that the E C 50 was greater than 943 μ g/L (i.e., 0.943 mg/L) , which is considerably less than the reported water solubility limit of the test substance (2130 μg/L at 20 ° C ). OPPTS 850.1010, p. 2, Section (d) “Test procedures”, Sub

- section (3) “Definitive test”, item (i ) states: “ The purpose of the definitive test is to determine the concentration

- response curves and the 24

- and 48

- h EC 50 values .” OPPTS 850.1010, p. 3, Section (d) “Test procedures”, Sub

- section (3) “Definitive test”, item (ii) states: “ The concentration ranges should be selected to determine the concentration

resp onse curves and EC50 values at 24 and 48 h.” OPPTS 850.1010, p. 3, Section (d) “Test procedures”, Sub

- section (3) “Definitive test”, item (v) states: “... Concentration

- response curves and 24

- h and 48

- h EC 50 values for
immobilization should be determined along with their 95 percent confidence limits.” OPPTS 850.10 10 , p. 8 , Section ( f ) “ Reporting” states: “ ... In addition to the reporting requirements prescribed in 40 CFR Part 792

– Good Laboratory Practice Standards, the reporting of test data should include the following: ”; and item ( 10 ) states: “ (10) The 24

- and
48

- h EC50 values and their respective 95 percent confidence limits using the mean measured test chemical concentration, and the methods u sed to calculate both the EC50 values and their
confidence limits. ” ASTM E729

- 96, p. 9, Section 9 “Test Material”, Sub

- section 9.3 “Test Concentration(s)”, item 9.3.2 states: “In some (usually regulatory) situations, it is necessary only to determine (1) whether a specific concentration of test material is acutely toxic to the test species, or (2) whether the LC 50 , EC 50 , or IC 50 is above or below a specific concentration. For example, the specific concentration might be the concentration occurring in a su rface water, the concentration resulting from the direct application of the material to a body of
water, or the solubility limit of the material in water . When there is interest only in a specific concentration, it is often necessary only to test that con centration (see 11.1.1.2) and it is not necessary to actually determine the LC 50 , EC 50 , or IC 50 .”

Interpretation: A primary objective of an acute toxicity to Daphnia test is to determine a discrete EC 50 value. I f a discrete E C 50 value i s not determined because insufficient (< 50%) immobilization occurs at the highest concentration tested and this concentration is below the aqueous solubility or dispersibility limit, the test is not considered acceptable.

[ B ] Deviation 2 : Measured c oncentration s increase d from the beginning to the end of the 24

- hour renewal intervals , but the study report did not provide a valid explanation

. OPPTS 850.1010, p. 4, Section (d) “Test procedures”, S ub

- s ection (4) “Analytical measurements” , item (i) “Test chemical” s tates: “... The analytical method used to measure the amount of test chemical in a sample should be validated before beginning the test by appropriate laboratory practices.” OPPTS 850.1010, p. 8 , Section ( f ) “ Reporting” states: “

... In addition to the reporting requirements prescribed in 40 CFR Part 792

– Good Laboratory Practice Standards, the reporting of test data should include the following:” ; and item (11) states: “ All chemicalDraft Addendum to HPV2 Test Rule Data Adequacy Review ( April 2014

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- 6 analyses of water quality and test chemical concentrations, including methods, method validations, and reagent blanks.” ; and item (13) states: “ Any deviation from this test guideline and anything unusual about the test, e.g. diluter failure, temperature fluctuations,

etc. ” ASTM E729

- 96, p. 1 8 , Section 15 “Report”, Sub

- section 15.1 states: “The record of the results of an acceptable acute toxicity test should include the following information either directly or by reference to available documents:” and item 15.1.3 states: “ Anything unusual
about the test, any deviation from these procedures, and any other relevant information. ” ASTM E729

- 96, p. 16, Section 11 “Procedure”, Sub

- section 11.9.3 “Test Material”, item 11.9.3.1 states: “... If the test material is not uniformly dispersed in the test chamber in static and renewal tests, the whole volume of solution in the test chamber should be (1) used as the sample or (2) treated appropriately (for example, by adding acid, base, or surfac tant and mixing thoroughly) to uniformly distribute the test material before a sample is taken.”

Interpretation: Test concentrations should be determined with a validated analytical method. The report should address any deviations from the protocol or u nusual occurrences that could impact the results. If the test substance is not homogeneously distributed in the fluid in the test chambers, additional measures should be taken to ensure that samples collected for analysis are representative of actual mean concentrations of test substance in the test chambers.

[ C ] Deviation 3 : Variation in test item concentrations among the replicates for “old” samples (taken just before each renewal with fresh medium) collected on day 2 was greater than 20 percent. OPPTS 850.1010, p. 3, Section (d) “Test procedures”, Sub

- section (3) “Definitive test”, item (viii) states: “... Among replicate test chambers of a treatment concentration, the
measured concentration of the test chemical should not vary more than ± 20 percen t.”

Interpretation: Measured c oncentrations of samples taken from replicate test vessels of a given

test item concentration should not vary by more than 20 percent from each other

.

[ D ] Deviation 4 : Details regarding the analytical measurements of test substance concentrations

were missing from the study report, including the limit of detection of the analytical method and chromatogram s for the analytical measurements. OPPTS 850.1010, p. 4, Section (d) “Test procedures”, Sub

- section (4) “Analytical measurements”, item (i) “Test chemical” states: “... The analytical method used to measure the amount of test chemical in a sample should be validated before beginning the test by appropriate laboratory practices.” OPPTS 850.1010, p. 8 , Section ( f ) “ Reporting” states: “ ... In addition to the reporting requirements prescribed in 40 CFR Part 792

– Good Laboratory Practice Standards, the reporting of test data should include the following:” and item (11) states: “All chemical analyses of water quality and test chemical concentrations, including methods, method validations, and reagent blanks.” ASTM E729

- 96, p. 18, Section 15 “Report”, Sub

- section 15.1 states: “The rec ord of the results of an acceptable acute toxicity test should include the following information either directly or by reference to available documents:” and item 15.1.9 states: “ Methods used for, and results (with standard deviations or confidence limits) of, chemical analyses of waterDraft Addendum to HPV2 Test Rule Data Adequacy Review ( April 2014

) CASRN 78

- 11

- 5

A

- 7 quality and concentrations(s) of test material, impurities, and reaction and degradation products

, including validation studies and reagent blanks.”
ASTM E729

- 96, p. 16, Section 12 “Analytical Methodology”, Sub

- section 12.2 states: “Chemical and physical data should be obtained using appropriate ASTM standards whenever possible. For those measurements for which ASTM standards do not exist or are not sensitive enough, methods should be obtained from other reliable sources.”

Interpretation: The analytical method used for measurement of test substance concentrations should be fully described. The results of validation studies of the analytical method, including the limit of detection, should be provided. The analytical resul ts, including all data that are generated (e.g., chromatogram s) and used to determine test substance concentrations in samples taken from test chambers over the course of the study, should be fully described and
documented . The measurement of t est substance concentrations should be conducted using a validated analytical method. The lack of complete details regarding the analytical method precludes a full evaluation of the validity of the analytical method, including whether or not it
adhered to acceptable standards, such as ASTM.

REFERENCE

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

- renewal test conditions

. Performing Laboratory: AECOM Fort Collins Environmental Toxico logy Laboratory, Fort Collins, CO

. Project ID 60225262

- 304

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

- HQ

- OPPT

- 2007

- 0531

- 0713 as of April
1 6 , 201 4

.