Document ID: EPA-HQ-OPPT-2005-0033-0370
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2011-03-02T05:00Z

SRC TR-10-063

HPV1 TEST Rule STUDY – DATA Adequacy assessment

1,3-Propanediol, 2,2-bis[(nitrooxy)methyl]-, dinitrate (ester), CAS No.
78-11-5

Toxicity to Plants (Algae)

Prepared by:

Ashlee Aldridge, Teresa Manyin, and Joan D. Garey

Environmental Science Center

SRC, Inc.

7502 Round Pond Road

North Syracuse, NY 13212

Contract No. EP-W-09-027

Task FG007.0.009.004

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, 2010

HPV1 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

Prepared By:

SRC, Inc.                                                               
                                                                     
Environmental Science Center                                            
                                                            2451 Crystal
Drive, Suite 804                                                        
                                          Arlington, VA 22202

 

PREPARATION OF THE DATA ADEQUACY ASSESSMENT

This document contains the review of a toxicity study conducted in
response to the first HPV TSCA Section 4 Test Rule (71 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 always the test guideline required by 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 Report”.  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 controls and test substances.  Any deficiencies in the
descriptions of the study method, test system, animals studied, physical
conditions, dosage information, individual animal data, calculations,
and statistical analyses are described, and internal inconsistencies
within the study report are identified.  An overall assessment is
provided for each of the following: study conduct, data reliability, and
data utility.

TOXICITY TO PLANTS (ALGAE)

Title: Toxicity of Pentaerythritol tetranitrate (PETN) to the
Unicellular Green Alga (Selenastrum capricornutum) under Static Test
Conditions 

This study is:	  FORMCHECKBOX   Adequate		  FORMCHECKBOX   Not Adequate

TEST SUBSTANCE   

		

Identity:  Pentaerythritol tetranitrate (1,3-Propanediol,
2,2-bis[(nitrooxy)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); clear, 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

GLP compliant?: Yes

Year study was performed: 2008

Test species: Unicellular green algae (Pseudokirchneriella subcapitata,
formerly Selenastrum capricornutum); source: 4-day old in-house stock
cultures; starter culture obtained from Carolina Biological Supply
Company

Analytical monitoring: Yes

Exposure duration: 96 hours

Concentrations tested: Nominal: 0, 0.1, 1.0, 10, and 100% PETN (as
received), equivalent to 0, 2, 20, 200 and 2000 µg/L; Mean measured:
1.4, 3.2, 30, 310 and 2850 µg/L

Test method/conditions remarks: 

The dilution/control water used in this study (algal medium batch
#121807) 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
periodically analyzed for contaminants.  Additionally, the medium was
sterilized by filtration through a pre-washed 0.45 µm cellulose
membrane filter prior to mixing test solutions.

Macro- and micro-nutrients were added to the 100% test solution prior to
preparing test concentrations.  The test substance, with added
nutrients, was assumed to be 100% for purposes of test concentration
preparation.  Test concentrations are expressed as percent of PETN as
received; statistical endpoints are expressed as µg/L PETN, per the
analytical 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-mL (nominal volume) glass flasks containing
approximately 100 mL of test solution and capped with a plastic cup with
three small holes.  Test chambers were held in an environmental chamber
in a five by five pattern.  Test chambers were placed within the pattern
according to a computer-generated random design. 

The test chambers were housed in an environmental chamber under
continuous illumination at 400 ± 10% ft-c (4.3 K lux), which was
provided by a combination of 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 rpm on a mechanical shaker table to
keep the algal cells in suspension.  Test temperature was monitored
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 inoculum was 1.14 x 106 cells/mL. 
The inoculum was added to the test solution at a rate of 1 mL of
inoculum per 100 mL of test solution.  The initial cell density in the
test chambers was nominally 1.14 x 104 cells/mL.  This initial cell
density was verified in three control blanks that were preserved 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 was discarded at
test termination.

On the day of test initiation, temperature, pH, and conductivity were
measured and recorded for each test treatment.  Hardness (mg/L CaCO3),
alkalinity (mg/L CaCO3), total ammonia (mg/L as N), and total residual
chlorine (mg/L) were also measured in the control and 100% PETN
treatments at test initiation.  Temperature and pH were measured in each
concentration daily 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 Lugol's solution at a rate of 1% of
the final volume within 2 hours of test termination, and after
collection of analytical samples. Algal cell density in the test
solutions was later determined by direct microscopic counts (using a
hemocytometer and compound light microscope) and recorded.

Samples for PETN analysis were collected at test initiation, prior to
distributing the test solutions to the test chambers, from each freshly
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 pooled sample was
divided into two equal volumes.  One of these samples was filtered
through a pre-washed 0.45 µm cellulose membrane to remove algal cells. 
Both samples were collected for each treatment (one with cells and one
without) by pouring the composite 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)
guidelines to analyze for nitroaromatics and nitramines by HPLC. 
Analytical data from TestAmerica were reviewed by ENSR. All data were
determined to be valid and usable. 

Statistical Analysis 

Because cell density in any test concentration (including 100%) was not
reduced by more than 8% from the control, a 96-hour EC50 or IC50 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 derived.

RESULTS

Endpoint value(s): 96 hr EC50 > 2850 µg/L PETN 

Results remarks: 

At the time of test initiation, water quality measurements in dilution
water were: hardness = 18 mg/L CaCO3, alkalinity = 10 mg/L CaCO3, 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: hardness = 16 mg/L CaCO3, alkalinity = 11
mg/L CaCO3, pH = 7.7, conductivity = 82 µS/cm, total ammonia < 1.0 mg
N/L, and total residual chlorine < 0.03 mg/L.  The physical test
conditions monitored during the test were: pH = 7.5-9.4, conductivity
(measured only at test initiation) = 82-95 µS/cm, and temperature =
24-26 °C.  The conditions were within acceptable ranges for the
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 105 cells/mL for the control, 3.2, 30, 310,
and 2850 µg/L concentrations, respectively.  The EC50 value, based on
cell density and measured PETN concentrations, was > 2850 µg/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 PETN to S. capricornutum under static test conditions.  The
biological endpoint for this study was cell density (cells/ml).  A
96-hour EC50 was determined based on mean measured PETN concentration
(µg/L) and cell density. The EC50 was > 2850 µg/L PETN.  Because the
highest concentration tested (2850 µg/L of PETN) did not result in a
50% or greater reduction in growth, a definitive test is not required. 

STUDY RELIABILITY

[1] Reliable without restrictions.

REFERENCE

Pillard, D.A.  2008.  Toxicity of Pentaerythritol 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 tetranitrate (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 Environmental Toxicology Laboratory	,
Fort Collins, CO	

Test Standard: ASTM E 1218			

Date Study Received by EPA: N/A, posted to   HYPERLINK
"http://www.regulations.gov"  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 report 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 comments below.

 [D]    2. The final 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 standard or testing protocol
(CITB PM describe or attach) were not followed or are inadequately
documented.

 [D]    4. There is evidence that laboratory Standard Operating
Procedures (SOPs) were inadequately followed.

 [D]    5. The Statement of Quality Assurance (QA) inspections, their
dates, and findings were missing or not signed.  There is evidence that
QA procedures may be inadequate.

 [D]    6. There is evidence that Good Laboratory Practice Standards
(GLPS) were inadequately followed.

 [D]    7. The test, 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., deviations from temperature,
lighting, dosing) are apparent.

 [D]    9. The final study report inadequately describes the study
method. 

 [D]   10. The final study report inadequately describes the test
system.

 [D]   11. The final study report inadequately 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 data were not provided. 

 [NA] 13. The final study report 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 comments below as appropriate

Part A.  To be filled out by Reviewer

			

Study Conduct		1. The study was well-conducted.					[X]					2. The study
was generally well-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 significance.			

			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 Comments (continue on separate page, if needed):  

Although the test guideline states that each test chamber should be
inoculated at the rate of 2 x 104 cells/mL, the initial cell density in
this study was only 1.14 x 104 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 minimum acceptable growth rate.  Because cell densities in
test article-treated cultures were similar to controls, the EC50 value
exceeded the highest concentration tested; therefore, no statistics were
necessary.

Reviewer Name/Title/Branch/Division/Office: Ashlee Aldridge/SRC, Inc.

Reviewer Signature: David Brooks/HPVCB/RAD/OPPTS

Part B.  To be filled out by 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).   

Draft HPV1 Test Rule  – Data Adequacy – Toxicity to Plants (Algae)
CAS No. 78-11-5

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