SRC TR

- 1 4

- 009

HPV3 TEST RULE DATA ADEQUACY REVIEW (FINAL ) 2

- Oxiranemethanamine, N

- [4

- (2

- oxiranylmethoxy ) phenyl]

- N

- (2

- oxiranylmethyl )

- , CASRN 5026

- 74

- 4 Prepared by: Megan Nelson , Julie Klotzbach, Amy Nguyen, Teresa Manyin, and William L. Richards

Defense and Environmental Solutions Division

SRC, Inc. 7502 Round Pond Road North Syracuse, NY 13212
Contract No. EP

- W

- 09

- 027 Task FG007. 4 .009.00 6
Submitted to: OCSPP/OPPT/R AD (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 10 , 2014Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 5026 - 74 - 4

i TABLE OF CONTENTS A. SOURCE MATERIAL USED FOR ASSESSMENT
................................ .............................. 1 B. EVALUATING DATA ADEQUACY
................................ ................................ ..................... 2 C. SUMMARY OF DATA ADEQUACY FOR TESTING REQUIRED BY TEST RULE
........ 2 D. DATA FOR SIDS ENDPOINTS
................................ ................................ .............................. 5 D

- 1. Acute Toxicity to Fish
................................ ................................ ................................ ....... 5 D

- 2. Chronic Toxicity to Aquatic Invertebrates
................................ ................................ ....... 10 D

- 3. Toxicity to Aquatic Plants
................................ ................................ ............................... 17Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 5026 - 74 - 4

1 A. SOURCE MATERIAL USED FOR ASSESSMENT This document evaluates a submission from Huntsman Corporation (dated October 11, 2013) that contains data from toxicity testing of 2

- o xiranemethanamine, N

- [4

- (2

- oxiranylmethoxy)phenyl]

- N

- (2

- oxiranylmethyl)

- ( CASRN 5026

- 74

- 4 ) and was received by EPA in response to the final test rule, “Testing of Certain High Production Volume Chemicals; Third Group of Chemicals” ( 76 FR 65385 , October 21, 2011 ; referred to herein as HPV 3 test rule ; available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2009

- 0112

- 0086 as of April 10 , 2014 )
. The HPV3 test rule requires the following toxicological testing for CASRN 5026

- 74

- 4 : C2 (consisting of acute toxicity to Daphnia and toxicity to algae testing if log K ow < 4.2 or chronic toxicity to Daphnia and toxicity to algae testing if log K ow ≥ 4.2 ) and F1 (combined repeated dose toxicity study with the reproduction/developmental toxicity screening test OR repeated dose 28

- day oral toxicity study and reproduction/developmental toxicity screening test )
. Since the log K ow of CASRN 5026

- 74

- 4 is estimated to be 0.87 1 , which is < 4.2, the C2 aquatic toxicity testing requirement is cons idered to include acute toxicity to Daphnia and toxicity to algae testing. The October 11, 2013 submission from Huntsman Corporation (“Huntsman”), available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2009

- 0112

- 0233 as of April 10 , 2014, contained the following reports of toxicological test s conducted with CASRN 5026

- 74

- 4 :

 Bogers, L.M. 1996. 96

- Hour acute toxicity study in carp with TK 12759, Araldite MY 0510 . NOTOX B.V., ’s

- Hertogenbosch, The Netherlands. Project No. 169548. Sponsor: Ciba

- Geigy Ltd., Basel, Switzerland.

 Bouwman, L.M. 2012. Daphnia magna , reproduction test with p

- (2,3

- epoxypropoxy)

- N,N

- bis(2,3

- epoxypropyl)aniline (semi

- static). NOTOX B.V., ’s

- Hertogenbosch, The Netherlands. Project No. 496989. Sponsor: Huntsman Advanced Materials GmbH, Basel, Switzerland.

 Bouwman, L.M. 2011. Fresh water algal growth inhibition test with p - (2,3

- epoxypropoxy)

- N,N

- bis(2,3

- epoxypropyl)aniline . NOTOX B.V., ’s

- Hertogenbosch, The Netherlands. Project No. 496987. Sponsor: Huntsman Advanced Materials GmbH, Basel, Switzerland
. In its October 11, 2013 submission, Huntsman also supplied prelimi nary results from a 28

- day repeated

- dose oral toxicity study of a proposed analog, (m

- (2,3

- epoxypropoxy)

- N

- N

- bis(2,3

- epoxypropyl)aniline (CASRN 71604

- 74

- 5 ; synonym: 2

- o xiranemethanamine, N

- [3

- (2

- oxiranylmethoxy)phenyl]

- N

- (2

- oxiranylmethyl)

- ), in rodents.

1 U .S. EPA. 2012
. Estimation Programs Interface SuiteTM (EPI Suite) for Microsoft® Windows, v4.11. U.S. Environmental Protection Agency, Washington, DC, USA. Available online at http://www.epa.gov/opptintr/exposure/pubs/episuitedl.htm as of April 10 , 2014.Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 5026 - 74 - 4

2 The aquatic toxicity studies identified above are evaluated for adequacy herein. T his report also evaluates the use of the 28

- day repeated

- dose toxicity study of CASRN 71604

- 74

- 5 to satisfy components of the F1 testing requirement for CASRN 5026

- 74

- 4
. B. EVALUATING DATA ADEQUACY Data available for SIDS toxicity endpoints are evaluated for adequacy in Section D. Each study was summarized according to a standard 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 a study was determined t o be inadequate, a basis for that conclusion is supplied. C. SUMMARY OF DATA ADEQUACY FOR TESTING REQUIRED BY TEST RULE Evaluation of Adequacy of Proposed Analog for Read

- Across Strategy : Huntsman proposed the use of toxicological data for CASRN 71604

- 74

- 5 to s atisfy the F1 test ing requirement for CASRN 5026

- 74

- 4 . The proposed analog is a positional isomer of CASRN 5026

- 74

- 4 and the two chemicals have identical functional groups. On the basis of these similarities, the proposed analog is reasonably expected to have toxicity similar to that of the test rule chemical. Therefore, EPA would accept data from adequate repeated

- dose and reproductive/developmental toxicity studies of CASRN 71604

- 74

- 5 to satisfy the F1 test i ng requirement for CASRN 5026

- 74

- 4. Evaluation of Data Adequacy for the F1 Test ing Requirement: Huntsman supplied preliminary results f rom a 28

- day repeated

- dose oral toxicity study of the proposed analog, CASRN 71604

- 74

- 5 , in rodents (full study report to be submitted later)
. Preliminary results indicate a NOAEL of 50 mg/kg

- bw/day based on atrophy of the female reproductive organs at 150 mg/kg

- bw/day. Based on these data, Huntsman concluded that a reproduction/developmental toxicity screening test (co mponent of F1 testing requirement) for CASRN 5026

- 74

- 4 was not needed, implying that the 28

- day repeated

- dose oral toxicity study of CASRN 71604

- 74

- 5 would satisfy the F1 test ing requirement for CASRN 5026

- 74

- 4
. EPA responds to Huntsman’s comments as foll ows :

 Repeated

- dose Toxicity Component A full study report has not yet been submitted for the 28

- day repeated

- dose toxicity study of CASRN 71604

- 74

- 5. 1 EPA requires t he full study report in order to evaluate the study for adequacy
. If EPA determines the study to be adequate, then the study will satisfy the repeated

- dose toxicity component of the F1 testing requirement.

 Reproduction/Developmental Toxicity Component The 28

- day repeated

- dose oral toxicity study of CASRN 71604

- 74

- 5 is insufficient to satisfy the reproduction/developmental toxicity component of the F1 test ing requirement. The observation of effects on reproductive organs in the repeated

- dose toxicity study

1 In its October 11, 2013 submission, Huntsman stated that the final study report for the 28

- day repeated

- dose toxicity study of CASRN 71604

- 74

- 5 would be submitted to EPA once Huntsman had received it.Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 5026 - 74 - 4

3 does not obviate the need for a reproduction/developmental toxicity screening test. As stated in the HPV3 test rule, the F1 test ing requirement can be satisfied by conducting either (1) a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test or (2) a reproduction/developmental toxicity sc reening test and a repeated dose 28

- day oral toxicity study in rodents. Alternatively, EPA occasionally considers the combination of other specific tests acceptable to satisfy the reproduction/developmental toxicity component of F1 1 . For example, the F1 testing requirement can be fulfilled with a 90

- day repeated

- dose toxicity study that sufficiently documents examination of the reproductive organs and finds no histological effects in male or female reproductive organs, in combination with a developmental toxicity study. When repeated

- dose toxicity testing indicates histological effects on the reproductive organs, additional testing is required to evaluate potential effects on reproduction. Preliminary results supplied by the sponsor for repeated

- dose tox icity of the proposed analog, CASRN 71604

- 74

- 5, indicate reproductive organ effects in females. Therefore, further testing is required to evaluate potential effects on reproducti ve and developmental endpoints
. EPA currently considers the F1 test ing requirement for CASRN 5026

- 74

- 4 to be unfulfilled
. Once Huntsman submits the full study report for the 28

- day repeated

- dose toxicity study of CASRN 71604

- 74

- 5 , EPA will evaluate this study for adequacy to satisfy the repeated

- dose toxicity component of t he F1 testing requirement. There are two possible outcomes:  If EPA determines that the repeated

- dose toxicity study of CASRN 71604

- 74

- 5 is adequate, then Huntsman may satisfy the remaining component of the F1 testing requirement with a reproduction/develo pmental toxicity screening test (40 CFR 799.9355) of CASRN 5026

- 74

- 4.  If EPA determines that the repeated

- dose toxicity study of CASRN 71604

- 74

- 5 is not adequate, Huntsman may satisfy the F1 testing requirement by conducting (1) a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (40 CFR 799.9365) or (2) a reproduction/developmental toxicity screening test (40 CFR 799.9355) and a repeated dose 28

- day oral toxicity study in rodents (40 CFR 799.9305). Any fur ther testing conducted for the F1 testing requirement should be conducted with CASRN 5026

- 74

- 4, rather than CASRN 71604

- 74

- 5. EPA notes that the submitted chronic toxicity to Daphnia and toxicity to algae studies tested a substance with a purity of 94.33% , which does not satisfy the purity requirement in the test rule. According to “Testing of Certain High Production Volume Chemicals; Third Group of Chemicals” (76 FR 65385, October 21, 2011 ) , the test substance (a Class 1 chemical substance in the test rule) must be tested at a purity of at least 99%. The sponsor must request a modification from EPA before conducting any further tests of the test rule chemical with test substance purity less than 99%. As stated in the final test rule, “In instances in which the test sponsor(s) believes that a 99% level of purity is unattainable for a

1 See EPA’s “Guidance for Meeting SIDS Requirements” (Section 5.7 “Toxicity to Reproduction” ), available at http://www.epa.gov/chemrtk/pubs/general/sidsappb.htm as of April 10 , 2014 , which is based on the OECD Manual for the Assessment of Chemicals Chapter 4, Section 4.3 “Guidance for the Initial Assessment of Health Effects”, Sub

- section 4.3.7 “Reproduction/Developmental Toxicity”, available at http://www.oecd.org/chemicalsafety/risk

assessmen t/manualfortheassessmentofchemicals.htm as of April 10 , 2014
.Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 5026 - 74 - 4

4 given chemical substance, the sponsor may request a modification under the procedures described in 40 CFR 790.55.” Evaluation of Aquatic Toxicity Data Submitted on October 11, 2013: C hronic toxicity to Daphnia and toxicity to algae data submitted by Huntsman are adequate to satisfy the C2 test ing requirement for CASRN 5026

- 74

- 4. While acute toxicity to Daphnia (component of C2) data are not available, EPA considers the adequate chronic toxicity to Daphnia study supplied by Huntsman on October 11, 2013 acceptable to satisfy the acute toxicity to Daphnia component of the C2 testing requirement
. Data supplied by Huntsman for acute toxicity to fish ( endpoint not requi red in the HPV3 test rule) are not considered adequate due to methodological deficiencies. Concentrations of the test substance were not verified by analytical monitoring. G iven that a hydrolysis half

- life of 4.1 days was determined at a similar temperature and pH (Baltussen, 2012 1 ), it is anticipated that significant hydrolysis of the test substance would have occurred over the 96

- hour (static) exposure period of the acute toxicity to fish study. However, since acute toxicity to fish testing was not required for CASRN 5026

- 74

- 4 by the HPV3 test rule, the inadequacy of the submitted study does not affect the current testing requirements. Summary of Data Adequacy for Toxicological Testing Required by the Test Rule: Data supplied by Huntsman for chronic toxicity to Daphnia and toxicity to algae endpoints are considered adequate to satisfy the C2 test ing requirement. Adequate data have not yet been submitted to satisfy the F1 (repeated

- dose, reproductive, and developmental toxicity) test ing requirement
. The r epeated - dose, reproductive, and developmental toxicity endpoints (components of the F1 test ing requirement) are considered data gaps.

1 The half

- life of CASRN 5026

- 74

- 4 was determined to be 4.1 days at a temperature of 20 °C and a pH of 7.0. [Baltussen, E. 2012. Determination of physico

- chemical properties of p

- (2,3

- epoxypr opoxy)

- N,N

- bis(2,3

- epoxypropyl)aniline. NOTOX B.V., ’s

- Hertogenbosch, The Netherlands. Project No. 496981. Sponsor: Huntsman Advanced Materials GmbH, Basel, Switzerland. Study report a vailable online at http://www.regulations.gov/ #!documentDetail;D=EPA

- HQ

- OPPT

- 2009

- 0112

- 0233 as of April 10 , 2014.]Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 5026 - 74 - 4

5 D. DATA FOR SIDS ENDPOINTS D - 1. Acute Toxicity to Fish Summary of endpoint: Not a dequate
. However, a cute toxicity to fish testing is not required for CASRN 5026

- 74

- 4 by the HPV3 test rule. Study 1: Acute toxicity study in carp ( Bogers, 1996

) Title: 9 6

- Hour acute toxicity study in carp with TK 12759, Araldite MY 0510 This study is: Adequate Not Adequate This study is not considered adequate due to uncertainty regarding exposure concentrations. Concentrations of the test substance were not verified by analytical monitoring. G iven that a hydrolysis half

- life of 4.1 days was determined at a similar temperature and pH (Baltussen, 2012 1 ), it is anticipated that significant hydrolysis of the test substance would have occurred over the 96

- hour (static) exposure period of the acute toxicity to fish study. Analytical monitoring results from a study in algae demonstrate substantial loss ( 34

- 41 %) of t he test substance over a 72

- hour exposure period ( Bouwman, 2011 2 ). Based on these results, nominal concentrations of test substance used for the acute toxicity to fish study cannot be assumed to accurately reflect actual concentrations. Additional defici encies included the following: (1) Compliance with GLP standards was not specified; and (2) The study report did not indicate statistical methods used to determine the LC 50 value. TEST SUBSTANCE Identity: 2

- O xiranemethanamine, N

- [4

- (2

- oxiranylmethoxy ) phenyl]

- N

- (2

- oxiranylmethyl )

- , CASRN 5026

- 74

- 4 Remarks: Clear yellow liquid; Batch No. 50008M; Purity > 95%; Identification: TK 12759, Araldite MY 0510. 3 METHOD Method/guideline followed: EEC directive 92/69, Part C.1. (1992) “Acute toxicity for fish” and OECD TG 203 (1992) “Fish Acute Toxicity Test”

1 The half

- life of CASRN 5026

- 74

- 4 was determined to be 4.1 days at a temperature of 20 °C and a pH of 7.0. [Baltussen, E. 2012. Determination of physico

- chemical properties of p

- (2,3

- epoxypropoxy)

- N,N

- bis(2,3

- epoxypropyl)aniline. NOTOX B.V., ’s

- Hertoge nbosch, The Netherlands. Project No. 496981. Sponsor: Huntsman Advanced Materials GmbH, Basel, Switzerland. Study report a vailable online at http://www.regula tions.gov/ #!documentDetail;D=EPA

- HQ

- OPPT

- 2009

- 0112

- 0233 as of April 10 , 2014.] 2 Bouwman, L.M. 2011. Fresh water algal growth inhibition test with p

- (2,3

- epoxypropoxy)

- N,N

- bis(2,3

- epoxypropyl)aniline. NOTOX B.V., ’s

- Hertogenbosch, The Netherlands. Pro ject No. 496987. Sponsor: Huntsman Advanced Materials GmbH, Basel, Switzerland. Study report available online at http://www.regulations.gov/#!documentDetail;D= EPA

- HQ

- OPPT

- 2009

- 0112

- 0233 as of April 10 , 2014. 3 TK 12759 is a synonym for CASRN 5026

- 74

- 4 (see http://chem.sis.nlm.nih.gov/chemidplus/rn/5026

- 74

- 4#names ).Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 5026 - 74 - 4

6 Test type: Acute toxicity to fish (static) GLP compliant?: Not specified Year study was performed: 1996 Test species: Carp ( Cyprinus carpio ) from a single parent

- pair ; obtained from Zodiac, pro e facc, “De Haar Vissen”, L.U. Wageningen, the Netherlands. Mean length: 1.90 ± 0.27 cm ( r ange finding test) and 1.96 ± 0.10 cm ( definitive test); mean weight 0.30 ± 0.07 g ( r ange - finding test) and 0.21 ± 0.03 g ( definitive test). Analytical monitoring: No Exposure duration: 96 hours Concentrations tested: Range

- Finding Test : 0 ( negative control) , 0 (solvent control), 0.1, 1, 10 , and 100 mg/L (nominal) ; measured concentrations not reported. Definitive Test : 0 ( negative control), 0 (solvent control), 1.0, 1.8, 3.2, 5.6 , and 10 mg/ L (nominal) ; measured concentrations not reported. Test method/conditions remarks: Test Medium: The test medium was ISO medium , formulated using reverse osmosis

- purified tap water
. The hardness was 250 mg CaCO 3 /L and pH was 8.0 ± 0.2
. Prepa ration of Test Concentrations: Range

- Finding Test: No information was provided for preparation of test concentrations for the range - finding t est. Definitive Test: “Stock solutions were prepared in acetone at 100, 56, 32, 1 8 and 10 mg/ml. A volume of 0.2 m l was taken from each stock and mixed with 2 liters of test medium providing the test concentrations of 10, 5.6 , 3.2, 1.8 and 1.0 mg/l. The final test solutions were all clear without precipitation.” [Bogers 1996, p. 7 ] Concentrations of the test substance were not verified by analytical monitoring. G iven th at a hydrolysis half

- life of 4.1 days was determined at a similar temperature and pH (Baltussen, 2012 1 ) , it is anticipated that significant hydrolysis of the test substance would have occurred over the 96

- hour (static) exposure period of the acute toxicity to fish study
. Analytical monitoring

1 The half

- life of CASRN 5026

- 74

- 4 was determined to be 4.1 days at a temperature of 20 °C and a pH of 7.0. [Baltussen, E. 2012. Determination of physico

- chemical properties of p

- (2,3

- epoxypropoxy)

- N,N

- bis(2,3

- epoxypropyl)aniline. NOTOX B.V., ’s

- Hertoge nbosch, The Netherlands. Project No. 496981. Sponsor: Huntsman Advanced Materials GmbH, Basel, Switzerland. Study report a vailable online at http://www.regula tions.gov/ #!documentDetail;D=EPA

- HQ

- OPPT

- 2009

- 0112

- 0233 as of April 10 , 2014.]Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 5026 - 74 - 4

7 r esults from a study in algae demonstrate substantial loss ( 34

- 41 %) of the test substanc e over a 72

- hour exposure period ( Bouwman, 2011 1 ). Based on these results, nominal concentrations of test substance used for the acute toxicity to fish study cannot be assumed to accurately reflect actual concentrations. Exposure Conditions: Prior to transfer to the test vessels, the fish were acclimated for at least 12 days after delivery in a holding tank containing water with similar water quality parameters
. Test Procedures: Range

- Finding Test Procedure: Three fish per concentration were exposed to nominal concentrations of 0.1, 1 , and 10 mg /L
. Seven fish were exposed to the negative and solvent control s and to a test concentration of 100 mg/L . Fish were examined for mortality every 24 hours for a total of 96 hours. No further details regarding the experimental procedures for the range

- finding test were provided. Definitive Test Procedure: The exposure vessels were 5

- L glass vessels containing 2 L of test medium. Directly after preparation of the test media, the fish were introduced into the negative control, solvent control , or test s ubstance

- containing vessels (single replicate s of 7 fish each ). Fish loading rate was reported as 0.74 g fish/ L . Food was withheld for 24 hours prior to transferring the fish to the test vessels and thereafter throughout the test. Illumination was provided as a daily 16

- hour photoperiod and the test media were aerated continuously
. F ish were observed for mortal ity and other toxic effects at 2 , 24, 48, 72 , and 96 hours
. Fish were removed if no reaction was seen after touching the caudal peduncle and if visible breathing movements were absent
. Temperature, pH , and dissolved oxygen were monitored at the start of the test and daily throughout the test in all test vessels
. Statistical methods were not specified. RESULTS Endpoint value(s): Based on nominal concentrations: 96

- hour LC 0 ≤ 3.2 mg/L 96

- hour LC 50 ≤ 4.2 mg/L 96

- hour LC 100 ≤ 5.6 mg/L **Endpoint values are expressed in terms of “less than or equal to” the reported LC x values because test substance concentrations were not verified by analytical monitoring and are expected to have decreased during the test due to hydrolysis.**

1 Bouwman, L.M. 2011. Fresh water algal growth inhibition test with p

- (2,3

- epoxypropoxy)

- N,N

- bis(2,3

- epoxypropyl)aniline. NOTOX B.V., ’s

- Hertogenbosch, The Netherlands. Pro ject No. 496987. Sponsor: Huntsman Advanced Materials GmbH, Basel, Switzerland. Study report available online at http://www.regulations.gov/#!documentDetail;D= EPA

- HQ

- OPPT

- 2009

- 0112

- 0233 as of April 10 , 2014.Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 5026 - 74 - 4

8 Results remarks: Range

- Finding Test: No mortality occurred in the negative control, solvent control , or test solutions with concentrations ≤ 1.0 mg/L
. At 24 hours, 7/7 fish were de a d in the 100 mg/L group ; no other mortalities were observed at 24 hours . At 48 hours, 3/3 fish were de a d in the 10 mg/L group. No signs of toxicity were noted in surviving fish
. Definitive Test: No mortality or signs of toxicity were observed in the negative control, solvent control , or test solutions with concentrations ≤ 3.2 mg/L. In the 5.6 and 10 mg/L groups, n o deaths occurred within the first 24 hours of exposure. At 48 hours, 3/7 and 7/7 fish were de a d in the 5.6 and 10 mg/L groups, respectively; surviving fish in the 5.6 mg/L group showed severe disturbance s in swimming beha vior at 48 hours
. The remaining fish at 5.6 mg/L were dead by 72 hours. During the test, the temperature in the test vessels ranged from 20.4 to 20.5 °C, dissolved oxygen ranged from 8.6 to 9.2 mg/L , and pH ranged from 7.6 to 8.1
. CONCLUSIONS Based on nominal concentrations, the 96

- hour LC 50 for carp was ≤ 4.2 mg/L
. STUDY RELIABILITY [ 3 ] Not reliable
. This study is not considered reliable due to uncertainty regarding exposure concentrations. Concentrations of the test substance were not verified by analytical monitoring. G iven that a hydrolysis half

- life of 4.1 days was determined at a similar temperature and pH (Baltussen, 2012 1 ), it is anticipated that significant hydrolysis of the test substance would have occurred over the 96

- hour (static) exposure period of the acute toxicity to fish study
. Analytical monitoring results from a study in algae demonstrate substantial loss ( 34

- 41 %) of the test substance over a 72

- hour exposure period ( Bouwman, 2011 2 ). Based on these results, nominal concentrations of test substance used for the acute toxicity to fish study cannot be assumed to accurately reflect actual concentrations. Additional defic iencies included the following : (1) Compliance with GLP standards was not specified; and ( 2 ) The study report did not indicate statistical method s u sed to determine the LC 50 value.

1 The half

- life of CASRN 5026

- 74

- 4 was determined to be 4.1 days at a temperature of 20 °C and a pH of 7.0. [Baltussen, E. 2012. Determination of physico

- chemical properties of p

- (2,3

- epoxypropoxy)

- N,N

- bis(2,3

- epoxypropyl)aniline. NOTOX B.V., ’s

- Hertogenbosch, The Netherlands. Project No. 496981. Sponsor: Huntsman Advanced Materials GmbH, Basel, Switzerland. Study report a vailable online at http://www.regulations.gov/ #!documentDetail;D=EPA

- HQ

- OPPT

- 2009

- 0112

- 0233 as of April 10 , 2014.] 2 Bouwman, L.M. 2011. Fresh water algal growth inhibition test wi th p

- (2,3

- epoxypropoxy)

- N,N

- bis(2,3

- epoxypropyl)aniline. NOTOX B.V., ’s

- Hertogenbosch, The Netherlands. Project No. 496987. Sponsor: Huntsman Advanced Materials GmbH, Basel, Switzerland. Study report available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2009

- 0112

- 0233 as of April 10 , 2014.Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 5026 - 74 - 4

9 REFERENCE Bo gers , L.M. 1996
. 96

- Hour acute toxicity study in c arp with TK 12759, Araldite MY 0510
. NOTOX B.V., ’s

- Hertogenbosch, The Netherland s . Project No. 169548 . Sponsor: Ciba

- Geigy Ltd. , B asel , Switzerland
. Study report a vailable online at http://www.regulations.gov/ #!documentDetail;D=EPA

- HQ

- OPPT

- 2009

- 0112

- 0233 as of April 10 , 2014. Text in quotes is taken directly from Bogers (1996 ).Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 5026 - 74 - 4

10 D - 2. Chronic Toxicity to Aquatic Invertebrates Summary of endpoint: Adequate Study 1: Reproduction test in Daphnia magna (Bouwman, 2012 ) Title: Daphnia magna , reproduction test with p - (2,3

- e poxypropoxy)

- N,N

- b is(2,3

- e poxypropyl) a niline ( s emi

- static) This study is: Adequate Not Adequate TEST SUBSTANCE Identity: 2

- Oxiranemethanamine, N

- [4

- (2

- oxiranylmethoxy)phenyl]

- N

- (2

- oxiranylmethyl)

- , CASRN 5026

- 74

- 4 ; referred to as p

- (2,3

- epoxypropoxy)

- N , N

- bis(2,3

- epoxypropyl)aniline in the study report
. Remarks: Yellow liquid; Batch No. AA00373400 ; Purity : 94.33%
. METHOD Method/guideline followed: OECD TG 211 (2008) “ Daphnia magna Reproduction Test” ; ISO International Standard 10706 (2000) “Determination of long term toxicity of substances to Daphnia magna S traus ( Cladocera, Crustacea )” ; Commission Regulation ( EC ) No. 440/2008 Part C.20, “ Daphnia magna Reproduction Test” ; and OECD Series on Testing and Assessment No. 23 (2000) “Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures”
. Test type: Reproduction toxicity test in daphnid s ( semi

- static) GLP compliant?: Yes Year study was performed: 2011 Test species: Water flea ( Daphnia magna ); < 24 hours old; obtained from an in

- house laboratory culture Analytical monitoring: Yes Exposure duration: 21 days Concentrations tested: Range

- Finding Test : Nominal concentrations: 0 (control), 0.1 , 1, 10 , and 1 00 mg/L ; average measured concentrations : 0.6 9 and 6. 2 mg/L for the nominal concentrations of 1 and 10 mg/L, respectively (measure d concentrations not provided for nominal concentrations of 0.1 and 100 mg/L).Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 5026 - 74 - 4

11 Definitive Test : Nominal conc entrations: 0 (control), 0.1, 0.32, 1.0, 3.2 , and 10 mg/L ; corresponding average measured concentrations : 0, 0.04, 0.096, 0.42, 1.7 , and 4.8 mg/L , respectively
. Test method/conditions remarks: Test Medium: The t est medium was Elendt M7 medium , which was prepared by adding trace elements, macronutrients, and vitamins to adjusted ISO medium . (ISO medium was prepared by adding salts to tap water purified by reverse osmosis )
. The Elendt M7 medium had a hardness of 180 mg CaCO 3 /L and a pH of 7.7 ± 0.3
. Preparation of Test Solutions: The s tudy author stated that the test substance “ was not completely soluble in test medium at loading rates of 1.0 mg/ l and higher. ” [Bouwman 2012, p. 10] However, the experimentally determined water solubility of the test substance has been reported as 3340 mg/L at 20 ° C ( Baltussen, 2012 1 ). The study report does not provide any further information or comment on this discrepancy in test substance solubility. Range

- Finding Test: Test solution s with nominal concentrations of 1, 10 , and 100 mg/L were prepared separately. Solutions of test substance in mediu m were stirred for 2 days with a magnetic stirrer and then left to stabilize for 1 hour. A fraction (referred to by the study author as the “Water Accommodated Fraction” 2 ) was collected with a siphon and used for testing. The nominal concentration of 0.1 mg/L was prepared as a 10% dilution of the 1 mg/L test solution (dilut ed with test medi um )
. Control test solutions contained Elendt M7 medium only. “ The final test solutions were all clear and colourless.” [ Bouwman 2012, p. 10] Definitive Test: A test solution with a nominal concentration of 10 mg/L was prepared in medium and stirred with a magnetic stirrer for 2 days; the solution was then left to stabilize for 1

- 1.5 hours. A fraction (referred to by the study author as the “Water Accommodated Fraction”) was collected with a siphon and used as the highest test concentration; this solution was diluted with medium to achieve lower nomin al test concentrations of 0.1, 0.32, 1.0 , and 3.2 mg/L (dilutions of 1%, 3.2%, 10%, and 32% , respectively , of the 10 mg/L test solution) . Control test solutions contained Elendt M7 medium only.

1 Baltussen, E. 2012. Determination of physico

- chemical properties of p

- (2,3

- epoxypropoxy)

- N, N

- bis(2,3

- epoxypropyl)aniline. NOTOX B.V., ’s

- Hertogenbosch, The Netherlands. Project No. 496981. Sponsor: Huntsman Advanced Materials GmbH, Basel, Switzerland. Study report a vailable online at http://www.regulations.gov/ #!documentDetail;D=EPA

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- 0233 as of April 10 , 2014. 2 The term, “Water Accommodated Fraction”, refers to a solution prep ared from a complex multi

- component substance, which is only partially soluble in water (see OECD Series on Testing and Assessment No. 23). Since the main component, comprising ~94%, of the test substance used in this study was found to be soluble in wate r up to a concentration of 3340 mg/L, the use of this terminology appears to be questionable.Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 5026 - 74 - 4

12 Test Procedures: Range

- Finding Test Procedure: Procedures for the range

- finding test were similar to those for the definitive test (described below) , except as follow s :  Ten daphnids per concentration ( 2 replicates /concentration; 5 daphnids/replicate ) were exposed to nominal concentrations of 0 (control), 0.1, 1, 10 , and 100 mg/L;  “ Test duration was 7 days;  Test solutions were renewed on days 1, 3 and 6;  No body length was recorded at the end of the test;  Samples for analysis [of test substance concentration] were taken at days 0, 1 and 3 from th e freshly prepared solutions and at days 1, 3 and 6 from the old solutions;  Samples were taken from solutions containing the daphnia and food (algae), but also from an extra vessel incubated under the same conditions, but without daphnia and algae. ” [ Bouwman 2012, p . 11 ] Definitive Test Procedure: For the definitive test, neonate daphnids (10 replicate s/concentration; 1 daphnid /replicate) were exposed to nominal concentrations of 0.1, 0.32, 1.0, 3.2 , and 10 mg/L for 21 days under semi

static conditions with test solution renewal every 48 hour s . The control group consisted of 20 daphnids (1 daphnid/replicate). T he exposure vessels were 60

- mL glass chambers , covered with a Perspex plate , and contain ed ~ 5 0 mL of control or test medium ; the loadin g rate was ~ 20 daphnids/L
. T he photoperiod was 16 hours of light and 8 hours of darkness. The daphnids were fed an algal suspension (0.5 mL , corresponding to 0.2 mg C/daphnid ) daily
. Parents were examined daily for mortality , immobility , and the presence of eggs in the brood pouch. The b ody length of each surviving parent w as measured at the end of the test. Appearance of first brood, number of newborn daph n ids, presence of unhatched eggs , and offspring immobility were recorded when observed
. Temperature, dissolved oxygen , and pH were monitored at the start of the test, and just before and after renewal of the test solutions at each test concentration in one vessel with surviving daphnids. Analytical Monitoring: Samples (2 mL) for analysis of test substance concentrations were collected at the beginning and end of each renewal period in the range

- finding test and at the beginning and end of the day 0

- 2, 6

- 8, 12

- 14 , and 18

- 20 renewal periods for the definitive test. Samples were stored at ≤
- 15 ° C prior to analysis. Test concentrations were determined by HPLC (wavelength of 253 nm)
. Statistical Methods: Average measured concentrations for the definitive test were calculated by first taking the geometric mean of measurements obtained at the beginning and end of each renewal period for which samples were collected (days 0

- 2, 6

- 8, 12

- 14 , and 18

- 20 ) and then taking the arithmetic mean of these four values for each nominal concentration
. Effects of exposure on p arental survival were evaluated using Fisher’s exact test. Effects of exposure on t otal reproduction were determined using an ANOVA test , followed by a mean comparison test (Bonferroni t

- test)
. EC 50Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 5026 - 74 - 4

13 values for reproduction were determined using log

- linea r regression ( % reproductive reduction vs. log exposure concentration)
. Acceptability Criteria: The test was considered acceptable if the following criteria were met: 1. “The mortality of the parent animals (female Daphnia ) [in the controls] did not exceed 20% at the end of the test
... 2. The average cumulative number of young per female in the controls after 21 days was ≥ 60
... ” [ Bouwman 2012, p. 13] RESULTS Endpoint value(s): The following values were b ased on mean measured concentrations: Parental m ortality and body length : 21

- day EC 50 > 4.8 mg/L 21

- day NOEC = 4.8 mg/L (based on no effects on survival or body length at the highest concentration tested) Reproduction: 21

- day EC 50 = 1.4 mg/L (95% confidence interval : 0.91

- 2.2 mg/L ) 21

- day LOEC = 1.7 mg/L 21

- day NOEC = 0.42 mg/L Results remarks: Range

- Finding Test Results: Results of the range

- finding test are expressed in terms of nominal concentrations : At the highest concentration tested (100 mg/L) , 100% parental mortality was observed within the first 24 hours of exposure. At 1 mg/L, only 1 death was observed during the exposure ( on day 6 )
. No mortality was observed at 0.1 or 10 mg/L. On day 7, the number of daphnids with eggs in brood pouches w as 10/10, 9/10, 9/ 9 , and 10/10 at concentrations of 0, 0.1, 1 , and 10 mg/L, respectively. Average m easured concentrations of fresh test solutions ( arithmetic mean of concentrations at study initiation and at the start of each renewal period) were 81 and 70 % of the nominal concentration s of 1 mg/L and 10 mg/L , respectively
. “ [T] est concentrations remained (relatively) stable during the 24 [day s 0 to 1] and 48 hour [days 1 to 3] renewal periods (79

- 90% of initial), but decreased to 61

- 68% of initial during the 72 hour [days 3 to 6] renewal period.” [ Bouwman 2012, p. 14] The s tudy author did not provide an explanation for the decline in test substance concentrations between renewals . However, r esults are consistent with loss of test substance due to hydrolysis. The hydrolysis half

- life of the test substance under similar experimental conditions wa s reported as 4.1 days (Baltussen, 2012 1 )
. The geomet ric mean

1 The half

- life of CASRN 5026

- 74

- 4 was determined to be 4.1 days at a temperature of 20 °C and a pH of 7.0. [Baltussen, E. 2012. Determination of physico

- chemi cal properties of p

- (2,3

- epoxypropoxy)

- N,N

- bis(2,3

- epoxypropyl)aniline. NOTOX B.V., ’s

- Hertogenbosch, The Netherlands. Project No. 496981. Sponsor: Huntsman Advanced Materials GmbH, Basel, Switzerland. Study report a vailable online at http://www.regulations.gov/ #!documentDetail;D=EPA

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14 measured concentrations during the exposure period were 0.69 and 6.2 mg/L for nominal concentrations of 1 and 10 mg/L, respectively. 1 “Test conditions were maintained within the limits prescribed by the protocol.” [ Bouwman 2012, p. 14] Definitive Test Results: Based on analysis of samples obtained at the beginning and end of the day 0

- 2, 6

- 8, 12

- 14, and 18

- 20 renewal periods, average exposure concentrations were 0.040, 0.096, 0.42, 1.7, and 4.8 mg/L for the nominal concentrations of 0.1 , 0.32, 1.0, 3.2, and 10 mg/L, respectively. The study author stated that “in several samples test substance was detected at a concentration below the lowest calibration solution. These samples were reported as < 0.0800 mg/L.” [ Bouwman 2012, p.37]. In this case, the concentration was considered as 0.0400 mg/L (2

- fold less than the lowest calibration sample) when calculating the average . For all samples at the nominal concentration of 0.1 mg/L, measured concentrations at the beginning and end of each re newal period were reported as < 0.0800 mg/L. For the nominal concentration of 0.32 mg/L, samples were reported as < 0.0800 mg/L at beginning and end of the day 12

- 14 renewal period and at the end of the day 18

- 20 renewal period. Measured concentrations o f fresh test solutions ranged from 39 to 76% of nominal concentrations (excluding concentrations reported as < 0.0800 mg/L). Test substance concentrations at the end of the day 0

- 2, 6

- 8, 12

- 14, and 18

- 20 renewal periods were 78

- 84%, 72

- 78%, 63

- 72%, and 66

- 74%, respectively, of concentrations at the beginning of the renewal period (ranges exclude concentrations reported as < 0.0800 mg/L). Although the study author did not comment on loss of test substance during the 2

- day renewal periods, EPA notes that th is finding is consistent with a hydrolysis half

- life of 4.1 days (Baltussen, 2012 2 ). Results of the definitive test are expressed in terms of mean measured concentrations : No statistically significant (relative to control) treatment

- related effect was observed on parental mortality. Cumulative mortality on day 21 of exposure was 3/20 (15%), 1/10 (10%), 0/10 (0%), 1/10 (10%), 3/10 (30%) , and 1/10 (10%) at average measured concentrations of 0, 0.040, 0.096, 0.42, 1.7 , and 4.8 mg/L , respectively. Time s to first death w ere 1 4 , 19, 11, 1 0 , and 1 0 days i n the 0, 0.040, 0.42, 1.7 , and 4.8 mg/L groups, respectively. Average body length of parental daphnids on day 21 was 4.32, 4.29, 4.40, 4.39, 4.31, and 4.16 mm at average measured concentrations of 0, 0.040, 0.096, 0.42, 1.7, and 4.8 mg/L, respectively. Differences in body lengths of the surviving daphnids were “ not biologically significant (< 10%) [ compared to controls ] .” [ Bouwman 2012, p. 16 ]

“ In the control group, the presence of eggs in the brood pouch was recorded for the first time on day 4

- 6 and the first brood appeared between day 7 and day 9. The first recording of the

1 The geometric mean measured concentrations during the range

- finding test were c alculated by the EPA reviewer as the geometric mean of all measurements from fresh and old test solutions. 2 The half

- life of CASRN 5026

- 74

- 4 was determined to be 4.1 days at a temperature of 20 °C and a pH of 7.0. [Baltussen, E. 2012. Determination of physico

- chemical properties of p

- (2,3

- epoxypropoxy)

- N,N

- bis(2,3

- epoxypropyl)aniline. NOTOX B.V., ’s

- Hertogenbosch, The Netherlands. Project No. 496981. Sponsor: Huntsman Advanced Materials GmbH, Basel, Switzerland. Study report a vailable online at http://www.regulations.gov/ #!documentDetail;D=EPA

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15 presence of eggs in the brood pouch was similar to the control in treated solutions up to and including 4.8 mg/l. Hence, no delay in appearance of the first brood was observed in the treatments when compared to the control.” [ Bo uwman 2012, p. 15] “The average cumulative number of young per female in the control group after 21 days was 136 with a coefficient of variation of 11%. The reproduction curves [cumulative mean number of young per parent vs. time ] recorded at 0.040, 0.09 6 , and 0.42 mg/l closely followed the curve of the control. The curve recorded at 1.7 mg/l stayed behind from the start and daphnids exposed to 4.8 mg/l did not reproduce at all. There were some recordings of immobile young and/or aborted eggs in the control and the three lowest test concentrations, while significant amounts of aborted eggs were recorded at the two highest test concentrations throughout the 21

- day test period.” [ Bo uwman 2012, p. 15] At the end of exposure , the cumulative mean number of young per parental daphnid was 136, 132, 138, 135, 59, and 0 at average measured concentrations of 0, 0.040, 0.096, 0.42, 1.7, and 4.8 mg/L, respectively. The reproduction of daphni ds was statistically significantly reduced (P < 0.05) at measured concentrations ≥ 1.7 mg/L , relative to the control
. E xperimental conditions during the definitive test were maintained within the limits prescribed by the protocol. Throughout the study, the test temperature ranged from 19.4 to 20.5 °C , pH ranged from 7.5 to 8.4 , and dissolved oxyg en concentration ranged from 8.2 to 9.7 mg/L. W ater hardness , measured in the control and highest test concentration, ranged from 179 to 2 14 mg CaCO 3 /L. The total dissolved organic carbon content of the Elendt M7 medium was 0.42 mg/L. The definitive test met the criteria for acceptability prescribed by the protocol. CONCLUSIONS The 21

- day NOEC for parental mortality and body length was 4.8 mg/L (highest concentration tested) ; a LOEC value was not identified. T he 21

- day EC 50 for reproduction was calculated as 1.4 mg/L (95% CI : 0.91

- 2.2 mg/L ); the 21

- day NOEC and LOEC for reproduction w ere 0.42 and 1.7 mg/L , respectively
. STUDY RELIABILITY [ 2 ] Reliable with restrictions. The following deficiencies were noted: (1) The study report states that purity of the test substance was 94.33 %. However, according to “Testing of Certain High Production Volume Chemicals; Third Group of Chemicals” (76 FR 65385, October 21, 2011) , the test substance (a Class 1 chemical substance in the test rule ) must be tested at a purity of at least 99%. (2) Test substance concentrations were not measured for each renewal period; OECD TG 211 requires analytical measurement of all test concentrations, when freshly prepared and at renewal, if test substance concentrations are not expected to remain within ± 20% of the nominal concentrations. (3) Measured test substance concentrations varied greatly from nominal concentrations; average measured concentrations were 30

- 54 % of nominal concentrations.Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 5026 - 74 - 4

16 REFERENCE Bouwman, L.M. 2012
. Daphnia magna , reproduction test with p

- (2,3

- epoxypropoxy )

- N,N

bis(2,3

- epoxypropyl)aniline (semi

- static )
. NOTOX B.V., ’s

- Hertogenbosch, The Netherland s . Project No. 496989 . Sponsor: Huntsman Advanced Materials GmbH, B asel , Switzerland
. Study report a vailable online at http://www.regulations.gov/#!documentDetail;D=EPA

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- 0233 as of April 10 , 2014. Text in quotes is ta ken directly from Bouwman (2012 ).Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 5026 - 74 - 4

17 D - 3. Toxicity to Aquatic Plants Summary of endpoint: Adequate Study 1 : Toxicity test with Pseudokirchneriella subcapitata (Bouwman, 2011 ) Title: Fresh water alg al growth inhibition test with p - (2,3

- epoxypropoxy )

- N,N

- bis(2,3

- epoxypropyl)anilin e This study is: Adequate Not Adequate TEST SUBSTANCE Identity: 2

- Oxiranemethanamine, N

- [4

- (2

- oxiranylmethoxy)phenyl]

- N

- (2

- oxiranylmethyl)

- , CASRN 5026

- 74

- 4 ; referred to as p - (2,3

- e poxypropoxy)

- N , N

- b is(2,3

- e poxypropyl) a niline in the study report. Remarks: Yellow liquid; Batch No. AA00373400; Purity: 94.33 % METHOD Method/guideline followed: OECD TG 201 (2006) “Freshwater Alga and Cyanobacteria, Growth Inhibition Test” ; Commission Regulation (EC) No. 440/2008, Part C. 3 , Amended by EC No. 761/2009 , “Algal Inhibition Test” ; ISO International Standard 8692 (2004) “Water quality

– Freshwater algal growth inhibition test with unicellular green algae” ; and OECD Series on Testing and Assessment No. 23 (2000) “Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures”
. Test type: Toxicity to plants

– algae (static) GLP compliant?: Yes Year study was performed: 2011 Test species: Green algae ( Pseudokirchneriella subcapitata ); strain: NIVA CHL 1; obtained from an in

- house laboratory culture Analytical monitoring: Yes Exposure duration: 72 hours Concentrations tested: Limit/ Range

- Finding Test: Nominal concentrations: 0 (control), 0.1, 1.0 , 10 , and 10 0 mg/L ; a verage measured concentrations: 0.51 and 46 mg/L for the nominal concentrations of 1 and 100 mg/L, respectively (measured concentrations not provided for nominal concentrations of 0.1 and 10 mg/L )
.Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 5026 - 74 - 4

18 Definitive Test: Nominal concentrations: 0 (control), 4.6, 10, 22, 46 , and 100 mg/L ; corresponding average measured concentrations were 2.5, 2.6, 4.2, 26 , and 26 mg/L , respectively. Test method/conditions remarks: Culture medium for the study (M2 medium) was prepared according to OECD TG 201, using tap water purified by reverse osmosis (Milli

- RO water). The culture medium had a hardness of 24 mg CaCO 3 /L and a pH of 8.1 ± 0.2. Three days before the start of the study, cells from a stock culture (maintained in M1 medium that was prepared according to Nederlandse Praktijk Richtlijn no. 6505) were inoculated in M2 medium at a cell density of 1 x 10 4 cells/mL and maintained under the same conditions as used in the test. Preparation of Test Solutions: The s tudy author stated that the test substance “was not completely soluble in test medium at loading rates of 1.0 mg/L and higher.” [Bouwman 201 1 , p. 10] However, the experimentally determined water solubility of the test substance has been reported as 3340 mg/L at 20 ° C ( Baltussen, 2012 1 ). The study report does not provide any further information or comment on this discrepancy in test substance solubility. For both the limit/range

- finding and definitive tests, test solutions with nominal concentrations ≥ 1.0 mg/L were prepared separately. Solutions of test substance in medium were stirred for 2 days with a magnetic stirrer and then left to stabilize for 1

- 2 hours. A fraction (referred to by the study author as the “Water Accommoda ted Fraction” 2 ) was collected with a siphon and used for testing
. T he nominal concentration of 0.1 mg/L in the limit/range

- finding test was prepared by diluting the 1 .0 mg/L test solution with medi um. “The final test solutions were all clear and colourless.” [ Bouwman 2011, p. 10] Inoculation of Test Cultures and Environmental Conditions: After preparation of the test solutions, a 50

- mL volume of the control or test medium was added to each replicate vessel. Test vessels were glass, capped vesse ls with a volume capacity of 100 mL. A 1

- mL aliquot of algal suspension was added to each replicate vessel, providing an initial cell density of 1 x 10 4 cells/mL. The vessels were randomly distributed in the incubator and repositioned daily throughout the exposure period. Temperature was monitored continuously in a temperature control vessel and pH was measured in control and test vessels at beginning and end of the test . The algal cells were maintained in suspension throughout the exposure period by continuous shaking. The vessels were continuously illuminated with 30

- watt cool

- white TLD

- lamps with a li ght intensity in the range of 77 to 90 μE·m - 2 ·s - 1

.
1 Baltussen, E. 2012. Determination of physico

- chemical properties of p

- (2,3

- epoxypropoxy)

- N,N

- bis(2,3

- epoxypropyl)aniline. NOTOX B.V., ’s

- Hertogenbosch, The Netherlands. Project No. 496981. Sponsor: Huntsman Advanced Materials GmbH, Basel, Switzerland. Study repo rt a vailable online at http://www.regulations.gov/ #!documentDetail;D=EPA

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- 0233 as of April 10 , 2014. 2 The term, “Water Accommodated Fraction”, refers to a solution prepared from a complex multi

- component substance, which is only partially soluble in water (see OECD Series on Testing and Assessment No. 23). Since the main component, comprising ~94%, of the test substance used in this study was fo und to be soluble in water up to a concentration of 3340 mg/L, the use of this terminology appears to be questionable.Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 5026 - 74 - 4

19 Test Procedures: Limit/ Range

- Finding Test Procedure: Procedures for the combined limit/range

- finding test were similar to those for the definitive test (described below) , except as follows :  Three replicates of algae per concentration were exposed to nominal concentrations of 0 .1, 1, or 10 mg/L and six replicates per concentration were exposed to 0 (control) or 100 mg/L ;  “ One extra test vessel per concentration without algae was used as background for the determination of the algal cell density at each time interval ;  pH was only measured in the control and the highest test concentration.” [Bouwman 2011 , p. 10]  Test substance concentrations were measured at 0, 24 , and 72 hours in test solutions at nominal concentrations of 1 and 100 mg/L. Definitive Test Procedure: Freshwater algae ( initial cell density of 1 x 10 4 cells/mL) were exposed to nominal test substance concentrations of 4.6, 10, 22, 46 , and 100 mg/L (3 replicates/concentration) for 72 hours. A control (6 replicates) was tested concurrently. An additional replicate of each te s t concentration (with algae) , as well as two replicates of the 100 mg/L test solution without algae, were used for measurements of test substance concentrations
. Analytical Monitoring: Samples (2 mL) for analysis of test substance concentrations were taken during the definitive test at 0, 24 , and 72 hours and stored in a freezer (≤

- 15 °C) until analysis. “At the end of the exposure period, the replicates with algae were pooled at each concentrat ion before sampling.” [Bouwman 2011 , p. 11] Samples from replicates without algae were also taken for analysis. Test concentrations were determined by HPLC (wavelength of 253 nm). The study report did not state the limit of detection of t he analytical m ethod. Evaluation of Cell Densities and Appearance: C ell densities were recorded for each replicate at test initiation and every 24 hours thereafter in the limit/ range - finding and definitive tests. “At the beginning of the test, cells were counted usi ng a microscope and a counting chamber. Thereafter cell densities were determined by spectrophotometric measurement of samples at 720 nm using a spectrophotometer with immersion probe (pathlength = 20 mm). Algal medium was used as [a] blank.” [Bouwman 20 11, p. 11 ] Statistical Methods: Average measured concentrations were calculated as time

- weighted geometric means of concentrations measured at 0, 24 , and 72 hours. “For determination of the NOEC and the EC 50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant reduction of growth rate or inhibition of yield (ANOVA, Bonferroni t

- Test) ... Calculation of the EC 50 and EC 10 values was based on log

- linear regression analysis of the percentages of growth rate reduction and theFinal HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 5026 - 74 - 4

20 percentages of yield inhibition versus the logarithms of the corresponding average exposure concentrations of the test substance. ” [Bouwman 2011, p p . 1 2

- 13 ] Acceptability Criteria: The test was considered acceptable if the following criteria were met: 1. “In the control, cell density increas ed by an average factor of > 16 within two days. 2. The mean coefficient of variation for section

- by

- section specific growth rates in the control cultures did not exceed 35%
. 3. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7%. ” [Bouwman 2011 , p. 13] RESULTS Endpoint value(s): Based on mean measured concentrations : Growth Rate: 72

- hour EC 50 = 13 mg/L (95% confidence interval: 8.3

- 19 mg/L) 72

- hour NOEC = 4.2 mg/L 72

- hour LOEC = 26 mg/L Yield: 72

- hour EC 50 = 7.0 mg/L (95% confidence interval: 5.3

- 9.3 mg/L) 72

- hour NOEC < 2.5 mg/L 72

- hour LOEC = 2.5 mg/L Results remarks: Limit/ Range

- Finding Test Results: At 72 hours, mean cell densities at nominal concentrations of 0, 0.1, 1.0, 10 , and 100 mg/L were 227.2, 239.5, 219.2, 197.7 , and 6.4 x 10 4 cells/mL, respectively. Mean percentage values for inhibition of growth rate and inhibition of yield relative to the control at each nominal test substance concentration were as follows: 0. 1 mg/L:

- 1% (growth rate inhibition),

- 5.4 % (yield inhibition ) ; 1.0 mg/L: 0.7% (growth rate inhibition), 3.5 % (yield inhib ition) ; 10 mg/L: 2.5 % (growth rate inhibi tion), 13.0 % ( yield inhibition); 100 mg/L: 65.8% (growth rate inhibition), 97.6 % (yield inhibiti on)
. Initial ( time = 0 ) measured test substance concentrations were 0.7 3 and 60 mg/L at nominal concentrations of 1 and 100 mg/L, respectively. At 24 hour s , measured concentrations were 80

- 8 1% of initial measured concentrations
. At 72 hours, measured concentrations were 45

- 62% of initial measured concentrations. Time

- weighted average measured concentrations during the exposure period were 0.51 and 46 mg/L for nominal concentrations of 1 and 100 mg/L, respectively. “All test conditions were maintained within the limits prescribed by the protocol.” [Bouwman 2011, p. 13]Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 5026 - 74 - 4

21 Definitive Test Results: At 72 hours, mean cell densities at nominal concentrations of 0, 4.6 , 10, 22, 46 , and 100 mg/L were 158.5, 131.7, 145.6, 108.7, 4.2 , and 3.5 x 10 4 cells/mL, respectively. Mean percentage values for inhibition of growth rate and inhibition of yield relative to the control at each nominal test substance concentration were as follows: 4.6 mg/L: 3.6% (growth rate inhibition), 17.0% (yield inhibition); 10 mg/L: 1.6% (growth rate inhibition), 8.2% (yield inhibition); 22 mg/ L : 7.4% (growth rate inhibition), 31.6% (yield inhibition); 46 mg/L: 71.9% (growth rate inhibiti on), 97.9% (yield inhibition); 100 mg/L: 76.1% (growth rate inhibition), 98.4% (yield inhibition). Algal g rowth rates at nominal concentrations of 4.6 , 10 , and 22 mg/L were not statistically significantly different from the growth rate in control cultures. In contrast , the growth rate s of algae at nominal concentrations of 46 and 100 mg/L were significantly reduced (P < 0.05) compared to the control growth rate
. Decreases in yield, compared to the control, were statistically significant at nom inal concentrations of 4.6, 22, 46, and 100 mg/L ( P < 0.05 ) , but not at 10 mg/L. Inhibition of yield at a nominal concentration of 4.6 mg/L was approximately 2

- fold greater than at 10 mg/L. The s tudy author stated that there was no explanation for this disparity and that “ [s] tatistical analysis does not give a clear output on the NOEC for yield inhibition.” [Bouwman 2011, p. 15] However, since a statistically significant inhibition of yield was observed at 4.6 mg/L, EPA considers the NOEC to be less than the nominal concentration of 4.6 mg/L (average measured concentration of 2.5 mg/L )
. Initial (time = 0) measured test substance concentrations were 3.2, 3.3, 5.5, 33 , and 34 mg/L at nominal concentrations of 4. 6, 10, 22, 46 , and 100 mg/L, respectively. Over the 72

- hour exposure period, a duration

- dependent decrease in test substance concentration was observed. At 24 hours, measured concentrations were 82

- 87% of initial measured concentrations. At 72 hours, me asured concentrations were 59

- 66% of initial measured concentrations. For the nominal concentrations of 4.6, 10, 22, 46 , and 100 mg/L, time

- weighted average measured concentrations during the exposure period were 2.5, 2.6, 4.2, 26 , and 26 mg/L, respective ly. Although the study author d id not comment on loss of test substance over the course of the study, EPA notes that this finding is consistent with a hydrolysis half

- life of 4.1 days (Baltussen, 2012 1 )
. The pH of the control and tr eated algal cultures ranged from 8.1 to 8.2 at 0 hou rs and from 7.8 to 7.9 at 72 hours. “ The temperature of the test medium was 22.1 ° C at the start of the test. During the exposure period the temperature measured in the inc ubator was maintained between 22.4 and 23.5 °C. Temperature remained within the limit s prescribed by the protocol (21

- 24 °C, consta nt within 2 °C).” [Bouwman 2011, p. 16 ]

1 The half

- life of CASRN 5026

- 74

- 4 was determined to be 4.1 days at a temperature of 20 °C and a pH of 7.0. [Baltussen, E. 2012. Deter mination of physico

- chemical properties of p

- (2,3

- epoxypropoxy)

- N,N

- bis(2,3

- epoxypropyl)aniline. NOTOX B.V., ’s

- Hertogenbosch, The Netherlands. Project No. 496981. Sponsor: Huntsman Advanced Materials GmbH, Basel, Switzerland. Study report a vailable o nline at http://www.regulations.gov/ #!documentDetail;D=EPA

- HQ

- OPPT

- 2009

- 0112

- 0233 as of April 10 , 2014.]Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 5026 - 74 - 4

22 The definitive test met the criteria for acceptability prescribed by the protocol
. CONCLUSIONS Based o n average measured co ncentrations, the 72

- hour EC 50 v alues for inhibition of growth rate and yield were 13 and 7.0 mg /L, respectively
. T he 72

- hour NOEC values for inhibition of growth rate and yield were 4.2 and < 2.5 mg/L , respectively
. T he 72

- hour LOEC values for inhibition of growth rate and yield were 26 and 2.5 mg/L, respectively
. STUDY RELIABILITY [ 2 ] Reliable with restrictions. The following deficiencies were noted: (1) The study report states that purity of the test substance was 94.33%. However, according to “Testing of Certain High Production Volume Chemicals; Third Group of Chemicals” (76 FR 65385, October 21, 2011) , the test substance (a Class 1 chemical substance i n the test rule ) must be tested at a purity of at least 99%. (2) M easured test substance concentrations varied greatly from nominal concentrations; average measured concentrations were 19

- 56% of nominal concentrations. REFERENCE Bouwman, L.M. 2011 . Fresh water algal growth inhibition test with p - (2,3

- epoxypro poxy)

- N,N

- bis(2,3

- epoxypropyl)aniline
. NOTOX B.V ., ’s

- Hertogenbosch, The Netherland s . Project No. 496987 . Sponsor: Huntsman Advanced Materials GmbH , B asel , Switzerland
. Study report a vailable online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2009

- 0112

- 0233 as of April 10 , 2014. Text in quotes is ta ken directly from Bouwman ( 2011 ).