SRC TR-14-009

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 FG007.4.009.006

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

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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 ............................................................................................... 17
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 5026-74-4

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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-oxiranemethanamine, 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 HPV3 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 Kow < 4.2 or 

chronic toxicity to Daphnia and toxicity to algae testing if log Kow ≥ 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 Kow of CASRN 5026-74-4 is estimated to be 0.871, which is < 4.2, the C2 aquatic 

toxicity testing requirement is considered 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 tests 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 preliminary 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-oxiranemethanamine, 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

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The aquatic toxicity studies identified above are evaluated for adequacy herein. This 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 to 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 satisfy the F1 

testing 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 testing

requirement for CASRN 5026-74-4. 

Evaluation of Data Adequacy for the F1 Testing Requirement:

Huntsman supplied preliminary results from 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 (component 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 testing requirement for CASRN 5026-74-4. EPA 

responds to Huntsman’s comments as follows:

 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 the 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 testing 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

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does not obviate the need for a reproduction/developmental toxicity screening test. As 

stated in the HPV3 test rule, the F1 testing 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 screening 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 F11. 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 toxicity of the proposed 

analog, CASRN 71604-74-5, indicate reproductive organ effects in females. Therefore, 

further testing is required to evaluate potential effects on reproductive and developmental 

endpoints. 

EPA currently considers the F1 testing 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 the 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/developmental 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 further 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/riskassessment/manualfortheassessmentofchemicals.htm as of April 10, 2014.
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 5026-74-4

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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:

Chronic toxicity to Daphnia and toxicity to algae data submitted by Huntsman are adequate to 

satisfy the C2 testing 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 required in the HPV3 test 

rule) are not considered adequate due to methodological deficiencies. Concentrations of the test 

substance were not verified by analytical monitoring. Given that a hydrolysis half-life of 4.1 

days was determined at a similar temperature and pH (Baltussen, 20121), 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 testing requirement. Adequate data have not yet been 

submitted to satisfy the F1 (repeated-dose, reproductive, and developmental toxicity) testing

requirement.

The repeated-dose, reproductive, and developmental toxicity endpoints (components of the 

F1 testing 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-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 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

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D. DATA FOR SIDS ENDPOINTS

D-1. Acute Toxicity to Fish

Summary of endpoint: Not adequate. However, acute 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: 96-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. Given that a 

hydrolysis half-life of 4.1 days was determined at a similar temperature and pH (Baltussen, 

20121), 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, 20112). 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 deficiencies 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 LC50 value. 

TEST SUBSTANCE 

Identity: 2-Oxiranemethanamine, 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-Hertogenbosch, The Netherlands. Project No. 496981. 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.]

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. 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. 

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

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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, proefacc, 

“De Haar Vissen”, L.U. Wageningen, the Netherlands. Mean length: 1.90 ± 0.27 cm (rangefinding test) and 1.96 ± 0.10 cm (definitive test); mean weight 0.30 ± 0.07 g (range-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 CaCO3/L and pH was 8.0 ± 0.2. 

Preparation of Test Concentrations: 

Range-Finding Test: No information was provided for preparation of test concentrations for the 

range-finding test. 

Definitive Test: “Stock solutions were prepared in acetone at 100, 56, 32, 18 and 10 mg/ml. A 

volume of 0.2 ml 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. Given that a

hydrolysis half-life of 4.1 days was determined at a similar temperature and pH (Baltussen, 

20121), 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-Hertogenbosch, The Netherlands. Project No. 496981. 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

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results from a study in algae demonstrate substantial loss (34-41%) of the test substance over a 

72-hour exposure period (Bouwman, 20111). 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 controls 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 substance-containing vessels (single replicates 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. 

Fish were observed for mortality 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 LC0 ≤ 3.2 mg/L 

96-hour LC50 ≤ 4.2 mg/L 

96-hour LC100 ≤ 5.6 mg/L 

**Endpoint values are expressed in terms of “less than or equal to” the reported LCx 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. 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

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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 dead in the 100 mg/L 

group; no other mortalities were observed at 24 hours. At 48 hours, 3/3 fish were dead 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, no 

deaths occurred within the first 24 hours of exposure. At 48 hours, 3/7 and 7/7 fish were dead in 

the 5.6 and 10 mg/L groups, respectively; surviving fish in the 5.6 mg/L group showed severe 

disturbances in swimming behavior 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 LC50 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. 

Given that a hydrolysis half-life of 4.1 days was determined at a similar temperature and pH 

(Baltussen, 20121), 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, 20112). 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 deficiencies 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 LC50 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 available 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 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. 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

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REFERENCE

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. Study report available 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

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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-epoxypropoxy)-N,N-bis(2,3-

epoxypropyl)aniline (semi-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 Straus (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 daphnids (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 100 mg/L; average

measured concentrations: 0.69 and 6.2 mg/L for the nominal concentrations of 1 and 10 mg/L, 

respectively (measured 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

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Definitive Test: Nominal concentrations: 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 test 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 CaCO3/L and a pH of 7.7 ± 0.3. 

Preparation of Test Solutions: 

The study 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, 20121). The study report does not provide any further information or comment on 

this discrepancy in test substance solubility.

Range-Finding Test:

Test solutions with nominal concentrations of 1, 10, and 100 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 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 (diluted with test medium). 

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 nominal 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 available online at http://www.regulations.gov/

#!documentDetail;D=EPA-HQ-OPPT-2009-0112-0233 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 found 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

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 follows: 

 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 

the 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 replicates/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 semistatic conditions with test solution renewal every 48 hours. The control group consisted of 20 

daphnids (1 daphnid/replicate). The exposure vessels were 60-mL glass chambers, covered with 

a Perspex plate, and contained ~50 mL of control or test medium; the loading rate was ~20

daphnids/L. The 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 body length of each surviving parent was measured at the end of the test. 

Appearance of first brood, number of newborn daphnids, 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 parental 

survival were evaluated using Fisher’s exact test. Effects of exposure on total reproduction were 

determined using an ANOVA test, followed by a mean comparison test (Bonferroni t-test). EC50
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 5026-74-4

13

values for reproduction were determined using log-linear 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 based on mean measured concentrations:

Parental mortality and body length:

21-day EC50 > 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 EC50 = 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 was 10/10, 9/10, 9/9, and 10/10 at concentrations of 0, 0.1, 1, and 10 mg/L, respectively.

Average measured 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

concentrations of 1 mg/L and 10 mg/L, respectively. “[T]est concentrations remained 

(relatively) stable during the 24 [days 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 study author did not provide an explanation for the decline in test 

substance concentrations between renewals. However, results are consistent with loss of test 

substance due to hydrolysis. The hydrolysis half-life of the test substance under similar 

experimental conditions was reported as 4.1 days (Baltussen, 20121). The geometric 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-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 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

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 renewal 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 of 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 this 

finding is consistent with a hydrolysis half-life of 4.1 days (Baltussen, 20122).

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. Times to first death were 14, 19, 11, 10, and 10 days in 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 calculated 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 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

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.” [Bouwman 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, 0.096, 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.” 

[Bouwman 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 daphnids was 

statistically significantly reduced (P < 0.05) at measured concentrations ≥ 1.7 mg/L, relative to 

the control.

Experimental 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 oxygen concentration ranged from 8.2 to 9.7 mg/L. Water 

hardness, measured in the control and highest test concentration, ranged from 179 to 214 mg

CaCO3/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.

The 21-day EC50 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 were 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,Nbis(2,3-epoxypropyl)aniline (semi-static). NOTOX B.V., ’s-Hertogenbosch, The Netherlands.

Project No. 496989. Sponsor: Huntsman Advanced Materials GmbH, Basel, Switzerland. 

Study report available online at http://www.regulations.gov/#!documentDetail;D=EPA-HQOPPT-2009-0112-0233 as of April 10, 2014.

 

Text in quotes is taken 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 algal growth inhibition test with p-(2,3-epoxypropoxy)-N,N-bis(2,3-

epoxypropyl)aniline

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 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 100 mg/L;

average 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 CaCO3/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 104cells/mL and maintained under the same conditions as used in the test. 

Preparation of Test Solutions: 

The study author stated that the test substance “was not completely soluble in test medium at 

loading rates of 1.0 mg/L and higher.” [Bouwman 2011, p. 10] However, the experimentally 

determined water solubility of the test substance has been reported as 3340 mg/L at 20 °C

(Baltussen, 20121). 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 Accommodated Fraction”2) was collected with a siphon and used 

for testing. The 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 medium. “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 vessels 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 104cells/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 light 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 report available online at http://www.regulations.gov/

#!documentDetail;D=EPA-HQ-OPPT-2009-0112-0233 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 found 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 104cells/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 test 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 concentration 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 the analytical method. 

Evaluation of Cell Densities and Appearance: 

Cell 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 

using 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 

2011, 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 EC50

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 EC50 and EC10 values was 

based on log-linear regression analysis of the percentages of growth rate reduction and the 
Final 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, pp. 12-13]

Acceptability Criteria: 

The test was considered acceptable if the following criteria were met:

1. “In the control, cell density increased 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 EC50 = 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 EC50 = 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 104cells/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 inhibition);

10 mg/L: 2.5% (growth rate inhibition), 13.0% (yield inhibition);

100 mg/L: 65.8% (growth rate inhibition), 97.6% (yield inhibition).

Initial (time = 0) measured test substance concentrations were 0.73 and 60 mg/L at nominal 

concentrations of 1 and 100 mg/L, respectively. At 24 hours, measured concentrations were 80-

81% 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 104 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 inhibition), 97.9% (yield inhibition);

100 mg/L: 76.1% (growth rate inhibition), 98.4% (yield inhibition).

Algal growth 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 rates 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 nominal 

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 

study 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, measured 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, respectively. 

Although the study author did 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, 

20121). 

The pH of the control and treated algal cultures ranged from 8.1 to 8.2 at 0 hours 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 incubator was maintained between 22.4 and 

23.5 °C. Temperature remained within the limits prescribed by the protocol (21-24 °C, constant 

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. 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 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

22

The definitive test met the criteria for acceptability prescribed by the protocol. 

CONCLUSIONS

Based on average measured concentrations, the 72-hour EC50 values for inhibition of growth rate 

and yield were 13 and 7.0 mg/L, respectively. The 72-hour NOEC values for inhibition of 

growth rate and yield were 4.2 and < 2.5 mg/L, respectively. The 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 in the test rule) must be tested at a purity of at 

least 99%. (2) Measured 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-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.

Text in quotes is taken directly from Bouwman (2011).