SRC TR-14-005

HPV3 TEST RULE DATA ADEQUACY REVIEW (REVISED FINAL):

1-Propanesulfonic acid, 2-hydroxy-3-(2-propen-1-yloxy)-, sodium salt (1:1), 

CASRN 52556-42-0

Prepared by:

Amy Nguyen, Ashlee Aldridge, Megan Nelson, Jennifer Rayner, William L. Richards, and 

Teresa Manyin

Defense and Environmental Solutions Division

SRC, Inc.

7502 Round Pond Road

North Syracuse, NY 13212

Contract No. EP-W-09-027

Task FG007.4.009.006

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 18, 2014
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 52556-42-0

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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.............................................................................................. 4

D-1. Acute Toxicity to Fish ....................................................................................................... 4

D-2. Acute Toxicity to Aquatic Invertebrates............................................................................ 8

D-3. Toxicity to Aquatic Plants ............................................................................................... 12

D-4. Acute Toxicity.................................................................................................................. 18

D-5. Repeated-Dose, Reproductive, and Developmental Toxicity.......................................... 21

D-6. Genetic Toxicity – Gene Mutation................................................................................... 32

D-7. Genetic Toxicity – Chromosomal Aberrations................................................................ 38

E. DATA ADEQUACY SUMMARY TABLE........................................................................... 45

APPENDIX: Test Guideline Source Material to Support Reliability Ratings Assigned to 

Submitted Studies That Are Considered Inadequate to Satisfy Test Rule Requirements ............ 46
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 52556-42-0

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A. SOURCE MATERIAL USED FOR ASSESSMENT

This document evaluates a submission (dated August 28, 2013) from the COPS-1 Consortium, a 

cost-sharing consortium formed and managed under the Society of Chemical Manufacturers and 

Affiliates (SOCMA), that contains data from toxicity testing of 1-propanesulfonic acid, 2-

hydroxy-3-(2-propen-1-yloxy)-, sodium salt (1:1) (CASRN 52556-42-0) 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; 

available online at http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT2009-0112-0086 as of April 18, 2014). 

The HPV3 test rule requires the following toxicological testing with CASRN 52556-42-0: 

C1 (consisting of Test Group 1 [acute toxicity to fish, acute toxicity to Daphnia, and toxicity to 

algae] if log Kow < 4.2 or Test Group 2 [chronic toxicity to Daphnia and toxicity to algae testing] 

if log Kow ≥ 4.2), D (acute mammalian toxicity test), E1 (bacterial reverse mutation test), E2 

(chromosome aberration or micronucleus test), and F1 (combined repeated dose toxicity study 

with the reproduction/developmental toxicity screening test). Based on an estimated log Kow

of -5.001, Test Group 1 testing is required to determine the toxicity of CASRN 52556-42-0 to 

aquatic organisms.

The August 28, 2013 submission from the COPS-1 Consortium (available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0112-0146 as of April 

18, 2014) contained the following study reports:

 96-Hour Acute Toxicity Study in Fathead Minnow with 1-Propanesulfonic acid, 2-

hydroxy-3-(2-propen-1-yloxy)-, sodium salt (1:1) (Static), submitted for the C1 testing 

requirement

 Acute Toxicity Study in Daphnia magna with 1-Propanesulfonic acid, 2-hydroxy-3-(2-

propen-1-yloxy)-, sodium salt (1:1) (Static), submitted for the C1 testing requirement

 Fresh Water Algal Growth Inhibition Test with 1-Propanesulfonic acid, 2-hydroxy-3-(2-

propen-1-yloxy)-, sodium salt (1:1), submitted for the C1 testing requirement

 Assessment of Acute Oral Toxicity with 1-Propanesulfonic acid, 2-hydroxy-3-(2-propen1-yloxy)-, sodium salt (1:1) in the Rat (Up and Down Procedure), submitted for the D 

testing requirement

 Evaluation of the Mutagenic Activity of 1-Propanesulfonic acid, 2-hydroxy-3-(2-propen1-yloxy)-, sodium salt (1:1) in the Salmonella typhimurium Reverse Mutation Assay and 

the Escherichia coli Reverse Mutation Assay (with Independent Repeat), submitted for 

the E1 testing requirement

 Evaluation of the Ability of 1-Propanesulfonic acid, 2-hydroxy-3-(2-propen-1-yloxy)-, 

sodium salt (1:1) to Induce Chromosome Aberrations in Cultured Peripheral Human 

Lymphocytes (with Repeat Experiment), submitted for the E2 testing requirement

 Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental 

Toxicity Screening Test of 1-Propanesulfonic acid, 2-hydroxy-3-(2-propen-1-yloxy)-, 

sodium salt (1:1) in Rats by Oral Gavage, submitted for the F1 testing requirement

 

1 The estimated log Kow value of -5.00 for CASRN 52556-42-0 was presented as a printout from KOWWIN in a 

letter from the COPS-1 Consortium to EPA dated August 29, 2012 available at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0112-0118 as of April 18, 2014. 
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 52556-42-0

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

evaluated in this report.

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. A table summarizing data 

availability/adequacy for each SIDS endpoint is included in Section E of this document. An 

appendix provides additional information regarding deficiencies of each submitted study that is 

considered inadequate to satisfy a requirement in the test rule. 

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

Data submitted by the COPS-1 Consortium on August 28, 2013 are adequate to satisfy the acute 

toxicity to fish (component of C1), acute toxicity to Daphnia (component of C1), toxicity to 

algae (component of C1), and combined repeated-dose/reproductive/developmental toxicity (F1) 

testing requirements for CASRN 52556-42-0 promulgated in the final HPV3 test rule. 

However, data submitted for mammalian acute toxicity (D), bacterial reverse mutation (E1), and 

mammalian chromosome aberration (E2) endpoints are not considered adequate due to the 

following deviations from relevant test guidelines and specific guidance provided to the 

submitter by EPA: 

 The mammalian acute toxicity study was not adequate because the administered dose of 

active ingredient (a.i., CASRN 52556-42-0) was less than the dose required for an acute 

toxicity limit test. This study failed to identify a discrete LD50 because no mortality was 

observed at the only dose tested (nominal dose of 2000 mg test substance/kg-bw; 

equivalent to 772.8 mg a.i./kg-bw based on a concentration of 38.64% CASRN 52556-

42-0 (the a.i.) in the test substance). The study only determined that the LD50 was greater 

than 772.8 mg a.i./kg-bw, which is considerably less than the limit dose of 2000 mg 

a.i./kg-bw or 5000 mg a.i./kg-bw specified in the acute toxicity test guidelines (i.e., 

OECD TG 425 and OPPTS 870.1100). Also, the selection of the limit dose was not 

based on the concentration of a.i. in the test substance (i.e., 38.64% CASRN 52556-42-0) 

as was specified in EPA’s response to the sponsor’s request for modification regarding 

the test substance composition (Richards et al., 2012). Although no adequate data are 

available for the acute mammalian toxicity endpoint, EPA does not currently consider 

acute mammalian toxicity testing to be necessary given that adequate data are available 

for the repeated-dose toxicity endpoint. Therefore, EPA is not requiring further testing 

for the D test rule requirement.

 The bacterial reverse mutation study was not adequate because the highest test 

concentration (1932 μg a.i./plate, calculated based on a concentration of 38.64% CASRN 

52556-42-0 (the a.i.) in the test substance) was substantially lower than the maximum 
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 52556-42-0

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concentration of 5 mg a.i./plate (5000 μg a.i./plate) that is recommended by the cited test 

guidelines (OECD TG 471 and TSCA 799.9510) and did not produce an increased 

number of revertant colonies, cytotoxicity, or precipitation during the test. Also, test 

concentrations were not selected based on the concentration of a.i. in the test substance 

(i.e., 38.64% CASRN 52556-42-0) as was specified in EPA’s response to the sponsor’s 

request for modification regarding the test substance composition (Richards et al., 2012). 

Therefore, testing is still required to satisfy the E1 test rule requirement.

 The mammalian chromosomal aberration test was not adequate because the highest 

concentration (~843 μg a.i./mL or 0.0039 M a.i., calculated based on a concentration of 

38.64% CASRN 52556-42-0 (the a.i.) in the test substance) was substantially lower than 

the maximum concentration (defined as 5 μL a.i./mL, 5 mg a.i./mL (5000 μg a.i./mL), or 

0.01 M a.i., whichever value is lowest) recommended by the cited guidelines (OECD TG 

473 and TSCA 799.9537) and did not produce an increased number of chromosome 

aberrations, cytotoxicity, or precipitation during the definitive chromosomal aberrations 

test. Also, test concentrations were not selected based on the concentration of a.i. in the 

test substance (i.e., 38.64% CASRN 52556-42-0) as was specified in EPA’s response to 

the sponsor’s request for modification regarding the test substance composition (Richards 

et al., 2012). Therefore, testing is still required to satisfy the E2 test rule requirement.

The bacterial reverse mutation and mammalian chromosome aberration endpoints are 

considered data gaps and testing is still required for these endpoints.

The study designs of the submitted bacterial reverse mutation and chromosome aberration tests 

are acceptable with the exception that the selection of doses/concentrations, reporting of 

dose/concentration vs. response, and values of toxicity endpoints were based on mg or g of the 

test substance (Bimax Product CS-AHPS) rather than on mg or g of the a.i., CASRN 52556-42-

0, present in the test substance. Given that the results for genotoxicity were negative in the 

mutagenicity and chromosomal aberrations tests, these studies must be repeated using 

maximum/limit doses or concentrations that are selected based on the concentration of a.i. in the 

test substance (i.e., 38.64% CASRN 52556-42-0) as was specified in EPA’s review of the 

sponsor’s study plan (Richards et al., 2012). Specifically, in accordance with test guidelines: If 

not precluded by cytotoxicity or precipitation, the maximum concentrations of the a.i. (CASRN 

52556-42-0) shall be 5 mg a.i./plate (5000 μg a.i./plate) in the mutagenicity study and 5 μL 

a.i./mL, 5 mg a.i./mL (5000 μg a.i./mL), or 0.01 M a.i. (whichever value is lowest) in the 

chromosome aberrations study.

REFERENCE

Richards, W.L., Manyin, T., and Morlacci, L. 2012. HPV3 test rule: Response to submitter’s 

request for modification regarding test substance composition, 1-Propanesulfonic acid, 2-

hydroxy-3-(2-propen-1-yloxy)-, sodium salt (1:1), CASRN 52556-42-0. SRC Technical Report 

No. TR-12-092. 
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D. DATA FOR SIDS ENDPOINTS

D-1. Acute Toxicity to Fish

Summary of endpoint: Adequate

Study 1: Acute toxicity study in fathead minnow (Bouwman, 2013a) 

Title: 96-Hour acute toxicity study in fathead minnow with 1-propanesulfonic acid, 2-hydroxy3-(2-propen-1-yloxy)-, sodium salt (1:1) (static)

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Bimax Product CS-AHPS1; commercial product that contains 38.64% 

1-propanesulfonic acid, 2-hydroxy-3-(2-propen-1-yloxy)-, sodium salt (1:1) (CASRN 52556-42-

0), also known as COPS-1.

Remarks: Clear colorless liquid; Batch No. 12E1101; Composition: 38.64% 1-propanesulfonic 

acid, 2-hydroxy-3-(2-propen-1-yloxy)-, sodium salt (1:1) (CASRN 52556-42-0), 2.26% 3-allyl1,2-propanediol (CASRN 123-34-2), and 59.1% water (CASRN 7732-18-5). 

METHOD

Method/guideline followed: OPPTS 850.1075 (1996) “Fish Acute Toxicity Test, Freshwater 

and Marine” and OECD TG 203 (1992) “Fish Acute Toxicity Test”

Test type: Acute toxicity to fish (static)

GLP compliant?: Yes

Year study was performed: 2013

Test species: Fathead minnow (Pimephales promelas); obtained from an in-house culture; mean 

length: 1.9 ± 0.1 cm; mean weight: 0.14 ± 0.04 g

Analytical monitoring: Yes

Exposure duration: 96 hours

 

1 EPA accepted the submitter’s request to test Bimax Product CS-AHPS in the toxicity tests required for CASRN 

52556-42-0 given that the only component in this commercial product other than water and CASRN 52556-42-0 is 

CASRN 123-34-2, a presumed byproduct of the synthesis of CASRN 52556-42-0. 
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 52556-42-0

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Concentrations tested: Nominal concentrations: 0 (control) and 1000 mg test substance/L

(equivalent to ~ 386 mg a.i.1/L based on a concentration of 38.64% CASRN 52556-42-0 in the 

test substance).

“No correction was made for the purity/composition of the test substance.” [Bouwman 2013a, p. 

8]

Test method/conditions remarks: 

Test Medium: The test medium was prepared with ISO medium formulated with reverse 

osmosis-purified tap water and adjusted to a hardness of 180 mg CaCO3/L and pH of 7.7 ± 0.3. 

Preparation of Test Concentrations: The nominal test concentration of 1000 mg test substance/L 

(corresponding to 386 mg a.i./L) was prepared by adding the test substance to test medium and 

stirring the mixture for 15 minutes with a magnetic stirrer. “The final test concentration was 

clear and colorless.” [Bouwman 2013a, p. 8]

Exposure Conditions: Prior to transfer to the test vessels, the fish were acclimated to the test 

conditions in a holding tank containing water with similar water quality parameters. During 

acclimation, pH and temperature variations were less than 0.5 unit and 0.5 °C, respectively. The 

study report did not specify the duration of the acclimation period.

The exposure vessels were 6.5-L glass vessels containing 5 L of test medium. Within 40 minutes 

of test media preparation, the fish were placed in the control vessels (one replicate of 7 fish) or 

test substance-containing vessels (two replicates of 7 fish per replicate). Fish loading was 0.20 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 not aerated.

The fish were observed for mortality and other toxic effects at 6, 24, 48, 72, and 96 hours. “In 

addition [test vessels were checked], every afternoon and morning from day 1, except for the 

afternoon at day 2, to observe for any dead or severely distressed fish.” [Bouwman 2013a, p. 10] 

Temperature, pH, and dissolved oxygen were monitored at the start of the test and daily 

throughout the test in all test vessels. 

Analytical Monitoring: Samples (1.5 mL) for analysis of test substance concentrations were 

collected from each replicate vessel at test initiation (0 hours) and at 48 and 96 hours. Samples 

were stored in the freezer (≤ -15 °C) until analysis. Concentrations were measured according to 

a validated analytical method2using an HPLC instrument equipped with a refractive index 

detector.

 

1 Active ingredient (a.i.) = CASRN 52556-42-0

2 The study report for the development and validation of the analytical method was submitted as a separate report, 

“Development and validation of an analytical method for the analysis of 1-propanesulfonic acid, 2-hydroxy-3-(2-

propen-1-yloxy)-, sodium salt (1:1) in water”, available at http://www.regulations.gov/#!documentDetail;D=EPAHQ-OPPT-2009-0112-0146 as of April 18, 2014.
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 52556-42-0

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

 No mortality occurred in the control vessel.

 Test conditions were maintained throughout the test.

 Dissolved oxygen concentrations were at least 60% of the air saturation value (i.e., > 5 

mg/L) throughout the test.

 Measured test substance concentrations were at least 80% of nominal concentrations 

throughout the test.

RESULTS

Endpoint value(s): Based on nominal concentrations:

96-hour LC50 > 1000 mg test substance/L (> 386 mg a.i./L)

96-hour NOEC = 1000 mg test substance/L (386 mg a.i./L)

Results remarks: No mortality occurred and no clinical signs were observed during the test. 

During the test, the temperature in the test vessels ranged from 22.5 to 23.0 °C, dissolved oxygen 

ranged from 6.8 to 9.2 mg/L, and pH ranged from 7.5 to 7.9. 

Measured test substance concentrations in samples of the 1000 mg test substance/L solution 

collected at 0, 48, and 96 hours ranged from 101 to 104% of nominal concentrations. “Analysis 

of the samples taken at the start of the test showed that the actual test concentration was in 

agreement with nominal (102–103%). This concentration remained stable during the test period 

(99–101% of initial at the end of the test). Given these results, the effect parameters were based 

on the nominal exposure concentration.” [Bouwman 2013a, p. 10]

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

CONCLUSIONS

Based on nominal concentrations, the 96-hour LC50 for fathead minnow was > 1000 mg test 

substance/L (> 386 mg a.i./L); the 96-hour NOEC was 1000 mg test substance/L (= 386 mg 

a.i./L). 

STUDY RELIABILITY

[2] Reliable with restrictions. The duration of acclimation of the test organisms was not 

provided in the study report and both OPPTS 850.1075 and OECD TG 203 require a minimum 

12-day acclimation period with a minimum of 7 days in test dilution water (OPPTS 850.1075) or 

water of the quality to be used in the test (OECD TG 203). Although an LC50 was not 

determined and the highest concentration was less than the limit test concentration of 1000 mg 

a.i./L stipulated by OPPTS 850.1075, this is not a clear deficiency since the highest 

concentration tested (386 mg a.i./L) was greater than the limit test concentration of 100 mg a.i./L 

stipulated by OECD TG 203. 
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 52556-42-0

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REFERENCE

Bouwman, L.M. 2013a. 96-Hour acute toxicity study in fathead minnow with 1-

propanesulfonic acid, 2-hydroxy-3-(2-propen-1-yloxy)-, sodium salt (1:1) (static). WIL 

Research Europe B.V., The Netherlands; Laboratory Project No. 500677. Sponsor: Society of 

Chemical Manufacturers and Affiliates (SOCMA), Washington, DC. Study report available 

online at http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0112-0146 as 

of April 18, 2014.

Text in quotes is taken directly from Bouwman (2013a).
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D-2. Acute Toxicity to Aquatic Invertebrates

Summary of endpoint: Adequate

Study 1: Acute toxicity study in Daphnia magna (Bouwman, 2013b)

Title: Acute toxicity study in Daphnia magna with 1-propanesulfonic acid, 2-hydroxy-3-(2-

propen-1-yloxy)-, sodium salt (1:1) (static)

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Bimax Product CS-AHPS1; commercial product that contains 38.64% 

1-propanesulfonic acid, 2-hydroxy-3-(2-propen-1-yloxy)-, sodium salt (1:1) (CASRN 52556-42-

0), also known as COPS-1.

Remarks: Clear colorless liquid; Batch No. 12E1101; Composition: 38.64% 1-propanesulfonic 

acid, 2-hydroxy-3-(2-propen-1-yloxy)-, sodium salt (1:1) (CASRN 52556-42-0), 2.26% 3-allyl1,2-propanediol (CASRN 123-34-2), and 59.1% water (CASRN 7732-18-5). 

METHOD

Method/guideline followed: OPPTS 850.1010 (1996) “Aquatic Invertebrate Acute Toxicity 

Test, Freshwater Daphnids” (EPA 712-C-96-114) and OECD TG 202 (2004) “Daphnia sp., 

Acute Immobilisation Test” 

Test type: Acute toxicity to Daphnia (static)

GLP compliant?: Yes

Year study was performed: 2013

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

laboratory culture 

Analytical monitoring: Yes

Exposure duration: 48 hours

 

1 EPA accepted the submitter’s request to test Bimax Product CS-AHPS in the toxicity tests required for CASRN 

52556-42-0 given that the only component in this commercial product other than water and CASRN 52556-42-0 is 

CASRN 123-34-2, a presumed byproduct of the synthesis of CASRN 52556-42-0. 
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 52556-42-0

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Concentrations tested: 

Range-Finding Test: Nominal concentrations: 0 (control), 0.10, 1.0, 10, 100, and 1000 mg test 

substance/L; Nominal concentrations adjusted for percentage active ingredient (a.i.)1based on a 

concentration of 38.64% CASRN 52556-42-0 in the test substance: ~ 0 (control), 0.039, 0.39, 

3.9, 39, and 386 mg a.i./L.

Definitive Test: Nominal concentrations: 0 (control) and 1000 mg test substance/L; Nominal 

concentrations adjusted for percentage a.i. based on a concentration of 38.64% CASRN 52556-

42-0 in the test substance: ~ 0 (control) and 386 mg a.i./L.

“No correction was made for the purity/composition of the test substance.” [Bouwman 2013b, p. 

9]

Test method/conditions remarks: Test medium (adjusted ISO medium) was prepared with tap 

water purified by reverse osmosis. 

Preparation of Test Solutions: 

To prepare the test solutions for the definitive test, an amount of test substance corresponding to 

a nominal concentration of 1000 mg test substance/L (386 mg a.i./L) was added to the test 

medium. The solution was stirred for fifteen minutes using a magnetic stirrer “... to accelerate 

the dissolving of the test substance in the test medium. The lower test concentrations for the 

range-finding test were prepared by subsequent dilutions of the 1000 mg/L concentration in test 

medium. The final test solutions were all clear and colorless.” [Bouwman 2013b, p. 9] Control 

test medium contained adjusted ISO medium only.

Range-Finding Test Procedure: 

For the range-finding test, the “[t]est procedure and conditions were similar to those applied in 

the limit [definitive] test [described below] with the following exceptions: 

 Ten daphnids per concentration (in duplicate, 5 per vessel) were exposed to nominal 

concentrations of 0.10, 1.0, 10, 100 and 1000 mg [test substance]/L, containing 0.039, 

0.39, 3.9, 39, and 386 mg a.i. per litre, respectively;

 Dissolved oxygen concentrations and pH were only measured in the control and the 

highest test concentration;

 No sampling for determination of actual test concentrations was performed.” [Bouwman 

2013b, p. 9]

Definitive Test Procedure:

For the definitive test, daphnids (4 replicate vessels; 5 daphnids/replicate) were exposed to a 

nominal concentration of 0 or 1000 mg test substance/L for 48 hours under static conditions. 

The exposure vessels consisted of 100 mL glass test vessels containing 80 mL of control or test 

medium. The loading rate was calculated to be 62.5 daphnids/L. Daphnids were introduced 

within 20 minutes after preparation of test solutions. During the test, the photoperiod was 16 

hours of light, the test vessels were not aerated, and the daphnids were not fed. 

 

1 Active ingredient (a.i.) = CASRN 52556-42-0
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The daphnids were observed for immobility/mortality at 24 and 48 hours. The temperature in a 

temperature control vessel was monitored continuously throughout the exposure period. 

Dissolved oxygen and pH were measured at 0 and 48 hours.

Analytical Monitoring: 

Samples (1.5 mL) for analysis of test substance concentrations were collected during the 

definitive test at 0 and 48 hours and stored in the freezer (≤ -15 °C) until analysis. Analyses 

were performed with an HPLC instrument equipped with a refractive index detector. “At the end 

of the exposure period, the replicates were pooled at each concentration before sampling” 

[Bouwman 2013b, p. 10]

Acceptability Criteria: 

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

 No daphnids became immobilized in the control.

 The oxygen concentration was ≥ 3 mg/L in the control and test vessels at the end of the 

test.

RESULTS

Endpoint value(s): Based on nominal concentrations:

48-hour EC50 > 1000 mg test substance/L (> 386 mg a.i./L)

48-hour NOEC = 1000 mg test substance/L (386 mg a.i./L)

Results remarks: 

Range-Finding Test Results: 

No immobilization was noted at any concentration during the range-finding test. “All test 

conditions were maintained within the limits prescribed by the protocol.” [Bouwman 2013b, p. 

11]

Definitive Test Results:

No immobilization was noted in any of the control or test replicates throughout the study. 

Throughout the study, the test temperature was maintained between 19.4 and 20.5 °C. The pH 

ranged from 7.6 to 7.9 and the dissolved oxygen concentration ranged from 8.8 to 9.2 mg/L. 

Measured test substance concentrations in samples of the 1000 mg test substance/L solution 

collected at 0 and 48 hours were 100% and 102% of the nominal concentrations, respectively. 

“Analysis of the sample taken at the start of the test showed that the actual test concentration was 

in agreement with nominal (100%). This concentration remained stable during the test period 

(102% of initial at the end of the test). Given these results, the effect parameters were based on 

the nominal exposure concentration.” [Bouwman 2013b, p. 11]

The definitive test met the criteria for acceptability prescribed by the protocol.
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 52556-42-0

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CONCLUSIONS

Based on nominal concentrations, the 48-hour EC50 for Daphnia magna was greater than 1000 

mg test substance/L (> 386 mg a.i./L); the 48-hour NOEC was 1000 mg test substance/L (= 386 

mg a.i./L).

STUDY RELIABILITY

[1] Reliable without restrictions. 

REFERENCE

Bouwman, L.M. 2013b. Acute toxicity study in Daphnia magna with 1-propanesulfonic acid, 

2-hydroxy-3-(2-propen-1-yloxy)-, sodium salt (1:1) (static). WIL Research Europe B.V., The 

Netherlands; Laboratory Project No. 500676. Sponsor: Society of Chemical Manufacturers and 

Affiliates (SOCMA), Washington, DC. Study report available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0112-0146 as of April 

18, 2014. 

Text in quotes is taken directly from Bouwman (2013b).
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D-3. Toxicity to Aquatic Plants

Summary of endpoint: Adequate

Study 1: Toxicity to Pseudokirchneriella subcapitata (Bouwman, 2013c)

Title: Fresh water algal growth inhibition test with 1-propanesulfonic acid, 2-hydroxy-3-(2-

propen-1-yloxy)-, sodium salt (1:1)

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Bimax Product CS-AHPS1; commercial product that contains 38.64% 

1-propanesulfonic acid, 2-hydroxy-3-(2-propen-1-yloxy)-, sodium salt (1:1) (CASRN 52556-42-

0), also known as COPS-1.

Remarks: Clear colorless liquid; Batch No. 12E1101; Composition: 38.64% 1-propanesulfonic 

acid, 2-hydroxy-3-(2-propen-1-yloxy)-, sodium salt (1:1) (CASRN 52556-42-0), 2.26% 3-allyl1,2-propanediol (CASRN 123-34-2), and 59.1% water (CASRN 7732-18-5). 

METHOD

Method/guideline followed: OCSPP 850.4500 (2012) “Algal Toxicity” and OECD TG 201 

(2006) “Freshwater Alga and Cyanobacteria, Growth Inhibition Test”

Test type: Toxicity to plants – algae (static)

GLP compliant?: Yes

Year study was performed: 2013

Test species: Green algae (Pseudokirchneriella subcapitata); strain: NIVA CHL 1; obtained 

from an in-house laboratory culture 

Analytical monitoring: Yes

Exposure duration: 96 hours

 

1 EPA accepted the submitter’s request to test Bimax Product CS-AHPS in the toxicity tests required for CASRN 

52556-42-0 given that the only component in this commercial product other than water and CASRN 52556-42-0 is 

CASRN 123-34-2, a presumed byproduct of the synthesis of CASRN 52556-42-0. 
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 52556-42-0

13

Concentrations tested: 

Range-Finding Tests: Nominal concentrations: 0 (control), 0.10, 1.0, 10, 100, and 1000 mg test 

substance/L; Nominal concentrations adjusted for percentage active ingredient (a.i.)1based on a 

concentration of 38.64% CASRN 52556-42-0 in the test substance: ~ 0 (control), 0.039, 0.39, 

3.9, 39, and 386 mg a.i./L

Definitive Test: Nominal concentrations: 0 (control) and 1000 mg test substance/L; Nominal 

concentrations adjusted for percentage a.i. based on a concentration of 38.64% CASRN 52556-

42-0 in the test substance: ~ 0 (control) and 386 mg a.i./L.

“No correction was made for the purity/composition of the test substance.” [Bouwman 2013c, p. 

10]

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 pH of 8.1 ± 0.2. Three to four 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: 

To prepare the test solutions, an amount of test material corresponding to a nominal 

concentration of 1000 mg test substance/L (386 mg a.i./L) was added to the test medium. 

“Approximately fifteen minutes of magnetic stirring was applied to accelerate the dissolving of 

the test substance in the test medium. The lower test concentrations for the range-finding tests 

were prepared by subsequent dilutions of the 1000 mg [test substance]/L concentration in test 

medium. The final test solutions were all clear and colorless.” [Bouwman 2013c, p. 10] Control 

cultures contained M2 medium without test material added.

Inoculation of Test Cultures and Environmental Conditions: 

After preparation of the test solutions, 50 mL of the control or test medium was added to each 

replicate vessel. Test vessels were glass, capped vessels with a volume capacity of 125 mL. An 

aliquot (1 mL) of algal suspension was added to each replicate vessel, providing an initial cell 

density of 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 at 0 and 96 hours. 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 59 to 66 μE·m-2·s-1.

Range-Finding Test Procedure: 

“Two range-finding tests were performed to provide information about the range of 

concentrations to be used in the final test. Test procedure and conditions were similar to those

applied in the limit [definitive] test [described below] with the following exceptions: 

 Exponentially growing algal cultures were exposed to nominal concentrations of 0.10, 

 

1 Active ingredient (a.i.) = CASRN 52556-42-0
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 52556-42-0

14

1.0, 10, 100, and 1000 mg [test substance]/L, containing 0.039, 0.39, 3.9, 39, and 386 mg 

a.i. per liter, respectively;

 Three replicates were tested per concentration and three replicates in the control group;

 During the second range-finding test pH was only measured in the control and the highest 

test concentration; 

 At the end of the second range-finding test algae were not observed to verify a normal 

and healthy appearance;

 No sampling for determination of actual test concentrations was performed.” [Bouwman 

2013c, p. 10]

The M2 medium used for the first range-finding test contained slightly different concentrations 

than specified by OECD TG 2011, with 2.6 μg ZnCl2/L, 2 μg CoCl2·6H2O/L, 0.017 μg 

CuCl2·2H2O/L, and 12.6 μg Na2MoO4·2H2O/L. 

Definitive Test Procedure: 

Freshwater algae (cell density of 1 x 104cells/mL) were exposed (6 replicates/concentration) to a 

nominal concentration of 0 or 1000 mg test substance /L for 96 hours. An additional replicate of 

the control and 1000 mg test substance/L test concentrations, as well as two replicates of the 

1000 mg test substance/L solution without algae, were employed for measurements of test 

substance concentration and algal cell density. 

Analytical Monitoring: 

Samples (1.5 mL) for analysis of test substance concentrations were taken during the definitive 

test at 0, 24, and 96 hours and stored in a freezer (≤ -15 °C) until analysis. Samples from 

replicates without algae were also taken for analysis. Analyses were performed with an HPLC 

instrument equipped with a refractive index detector. “At the end of the exposure period, the 

replicates with algae were pooled at each concentration before sampling.” [Bouwman 2013c, p. 

11]

Evaluation of Cell Densities and Appearance: 

Mean cell densities were recorded at test initiation and every 24 hours thereafter in the rangefinding 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. One extra test vessel per concentration without 

algae was used as [a] background [control] for the determination of the algal cell density at each 

time interval.” [Bouwman 2013c, p. 12] The cells were also observed by microscopy for any 

abnormalities in appearance at the end of the first range-finding test at the highest test 

concentration (1000 mg test substance/L) and in the definitive test. 

 

1 The concentrations specified by OECD TG 201 are 3 μg ZnCl2/L, 1.5 μg CoCl2·6H2O/L, 0.01 μg CuCl2·2H2O/L, 

and 7 μg Na2MoO4·2H2O/L. 
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 52556-42-0

15

Acceptability Criteria: 

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

1. “In the controls, cell density increased by an average factor of > 100 within 3 days. 

2. The coefficient of variation of average specific growth rates during the whole test period 

in replicate control cultures did not exceed 15%. 

3. The coefficient of variation of average yield during the whole test period in replicate 

control cultures did not exceed 15%.

4. Test conditions and test set-up were [performed] according to the guideline... ” 

[Bouwman 2013c, p. 13]

RESULTS

Endpoint value(s): Based on nominal concentrations:

96-hour EC50 (growth rate, yield, and biomass) > 1000 mg test substance/L (> 386 mg a.i./L) 

96-hour NOEC = 1000 mg test substance/L (386 mg a.i./L)

Results remarks: 

Range-Finding Test Results: 

First Range-Finding Test: At 96 hours, the mean cell densities at 0, 0.10, 1.0, 10, 100, and 1000 

mg test substance/L were 270.9, 269.0, 186.6, 236.3, 188.5, and 187.8 x 104 cells/mL, 

respectively. 

Mean percentage values for inhibition of growth rate, yield, and biomass relative to the control at 

each nominal test substance concentration were as follows:

0.1 mg/L: 0.1% (growth rate inhibition), 0.7% (yield inhibition), -0.2% (biomass inhibition); 

1.0 mg/L: 7.5% (growth rate inhibition), 31.2% (yield inhibition), 33.2% (biomass inhibition);

10 mg/L: 2.6% (growth rate inhibition), 12.8% (yield inhibition), 15.5% (biomass inhibition);

100 mg/L: 7.0% (growth rate inhibition), 30.5% (yield inhibition), 31.7% (biomass inhibition);

1000 mg/L: 7.4% (growth rate inhibition), 30.8% (yield inhibition), 34.8% (biomass inhibition).

The data suggested inhibition of algal growth, yield, and biomass relative to the control at 1.0 mg 

test substance /L and above, although statistical significance was not indicated in the study 

report. At 1.0 mg test substance /L and above, growth was inhibited by 2.6 to 7.5%, yield was

inhibited by 12.8 to 31.2%, and biomass was inhibited by 15.5 to 34.8%, relative to the control. 

Given that a concentration-response relationship was not apparent, it is unclear whether the 

inhibition of growth, yield, and biomass observed at concentrations ≥ 1.0 mg test substance/L 

could be attributable to exposure to the test substance. 

According to the study author, cell densities were highly variable among the three replicates at 

each concentration in the first range-finding test; however, replicate data were not provided, so 

these data could not be analyzed for statistical significance. “Although the [first range-finding] 

test proved to be valid a second range-finding test was performed because of the large variation 

in cell density between replicate vessels of the test concentrations.” [Bouwman 2013c, p. 14]

Second Range-Finding Test: No effects of the test substance on growth rate, yield, or biomass 

were observed at any test concentration relative to the control. At 96 hours, the mean cell 
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 52556-42-0

16

densities at 0, 0.10, 1.0, 10, 100, and 1000 mg test substance/L were 457.3, 453.8, 458.9, 456.9, 

450.3 and 453.1 x 104cells/mL, respectively. 

Mean percentage values for inhibition of growth rate, yield, and biomass relative to the control at 

each nominal test substance concentration were as follows:

0.1 mg/L: 0.1% (growth rate inhibition), 0.8% (yield inhibition), 0.1% (biomass inhibition); 

1.0 mg/L: -0.1% (growth rate inhibition), -0.4% (yield inhibition), 2.1% (biomass inhibition);

10 mg/L: 0.0% (growth rate inhibition), 0.1% (yield inhibition), 2.3% (biomass inhibition);

100 mg/L: 0.2% (growth rate inhibition), 1.5% (yield inhibition), 1.9% (biomass inhibition);

1000 mg/L: 0.1% (growth rate inhibition), 0.9% (yield inhibition), 9.5% (biomass inhibition).

Measurements of pH and temperature during the range-finding tests were not provided in the 

study report. According to the study author, “pH [during the second range-finding test] was 

maintained within the limits of the protocol. Temperature [during the second range-finding test] 

varied by more than 2 °C during the test period... [but remained within the limits prescribed by 

the protocol (22 to 26 °C)].” [Bouwman 2013c, p. 15]

Definitive Test Results:

No effects of the test substance on growth rate, yield, or biomass were observed in the definitive 

test. At 96 hours, the mean cell densities at 0 and 1000 mg test substance/L were similar, with 

values of 220.5 x 104cells/mL and 207.4 x104cells/mL, respectively. Mean percentage values 

for inhibition of growth rate, yield, and biomass at 1000 mg test substance/L were 1.1%, 6.0%, 

and 7.0%, relative to the control. “Microscopic observations at the end of the test revealed a 

normal and healthy appearance of the exposed cells when compared to the control.” [Bouwman 

2013c, p. 17]

The pH for the control and treated algal cultures was 8.0 at 0 hours; the pH values recorded at 96 

hours were 9.6 and 9.5 for the control and limit test concentrations, respectively. “The pH was 

within the limits prescribed by the protocol (6.0-9.0) at the start of the test. However, the pH of 

both the control and the test concentration slightly exceeded the upper limit at the end of the test 

and... varied by 1.5-1.6 unit [from the initial pH]... The temperature of the test medium was 

23.5 °C at the start of the test. During the exposure period the temperature measured in the 

incubator was maintained between 24.2 and 24.9 °C. Temperature remained within the limits 

prescribed by the protocol (22-26 °C, constant within 2 °C).” [Bouwman 2013c, p. 17]

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

Analytical Monitoring: Measured test substance concentrations in samples of the 1000 mg test 

substance/L solution collected at 0, 24, and 96 hours were 101%, 101%, and 103%, respectively, 

of the nominal concentrations. “Analysis of the sample taken at the start of the test showed that 

the actual test concentration was in agreement with nominal (101%). This concentration 

remained stable during the test period (102% of initial at the end of the test). Given these results, 

the effect parameters were based on the nominal exposure concentration.” [Bouwman 2013c, p. 

16] Measured test substance concentrations in replicates without algae were 102%, 107%, and 

92% of nominal concentrations at 0, 24, and 96 hours, respectively.
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 52556-42-0

17

CONCLUSIONS

Based on nominal concentrations, the 96-hour EC50 values for inhibition of growth, yield, and 

cell growth were all greater than 1000 mg test substance/L (> 386 mg a.i./L); the 96-hour NOEC 

was 1000 mg test substance/L (= 386 mg a.i./L).

STUDY RELIABILITY

[2] Reliable with restrictions. Replicate data were not provided for the range-finding tests and it 

is unclear whether statistical analyses were performed. The test methods deviated from OECD 

TG 201 because the pH of the control culture increased by more than 1.5 units during the 

definitive test. Actual increases at 0 and 1000 mg test substance/L were 1.6 and 1.5 units, 

respectively. Because the difference from the value stipulated by the guideline is relatively small 

(i.e., 0.1 unit), this deviation is considered minor and is not likely to have impacted the results of 

the study. 

The study author did not provide an explanation for the discrepancy between the results of the 

first range-finding test and those of the second range-finding and definitive tests. In the first 

range-finding test, inhibition of growth (2.6 to 7.5%), yield (12.8 to 31.2%), and biomass (15.5 

to 34.8%) were observed at 1 mg test substance/L and above. By contrast, inhibition of growth, 

yield, and biomass were not observed in either the second range-finding test, which was 

performed under similar conditions as the first range-finding test, or the definitive test. The 

study author indicated that cell density measurements were highly variable among replicate 

vessels in the first range-finding test. However, the study author did not provide an explanation 

for this variability and did not provide the replicate data for either of the range-finding tests to 

demonstrate variability among replicates. The discrepancies between the results of the first 

range-finding test and those of the second range-finding test and definitive test raise uncertainties 

about the consistency of the test procedures. Nevertheless, the agreement of the results from the 

second range-finding test and the definitive test suggests that these data are reliable.

Although an EC50 was not determined and the highest concentration was less than the limit test 

concentration of 1000 mg a.i./L recommended by OCSPP 850.4500, this is not a clear deficiency 

since the highest concentration tested (386 mg a.i./L) was greater than the limit test concentration 

of 100 mg a.i./L stipulated by OECD TG 201. 

REFERENCE

Bouwman, L.M. 2013c. Fresh water algal growth inhibition test with 1-propanesulfonic acid, 2-

hydroxy-3-(2-propen-1-yloxy)-, sodium salt (1:1). WIL Research Europe B.V., The 

Netherlands; Laboratory Project No. 500678. Sponsor: Society of Chemical Manufacturers and 

Affiliates (SOCMA), Washington, DC. Study report available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0112-0146 as of April 

18, 2014. 

Text in quotes is taken directly from Bouwman (2013c).
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 52556-42-0

18

D-4. Acute Toxicity

Summary of endpoint: Not adequate. Although no adequate data are available for the acute 

mammalian toxicity endpoint, EPA does not currently consider acute mammalian toxicity testing 

to be necessary given that adequate data are available for the repeated-dose toxicity endpoint. 

Study 1: Acute oral toxicity study in rats (van Huygevoort, 2013)

Title: Assessment of acute oral toxicity with 1-propanesulfonic acid, 2-hydroxy-3-(2-propen-1-

yloxy)-, sodium salt (1:1) in the rat (up and down procedure)

This study is: Adequate Not Adequate

The study is not adequate due to the following deficiencies: The administered dose of active 

ingredient (a.i.) was less than the dose required for an acute toxicity limit test. This study failed 

to identify a discrete LD50 because no mortality was observed at the only dose tested (nominal 

dose of 2000 mg test substance/kg-bw; equivalent to 772.8 mg a.i./kg-bw based on a 

concentration of 38.64% CASRN 52556-42-0 (the a.i.) in the test substance). The study only 

determined that the LD50 was greater than 772.8 mg a.i./kg-bw, which is considerably less than 

the limit dose of 2000 mg a.i./kg-bw or 5000 mg a.i./kg-bw specified in the acute toxicity test 

guidelines (i.e., OECD TG 425 and OPPTS 870.1100). Also, the selection of the limit dose was 

not based on the concentration of a.i. in the test substance (i.e., 38.64% CASRN 52556-42-0) as 

was specified in EPA’s response to the sponsor’s request for modification regarding the test 

substance composition.

TEST SUBSTANCE 

Identity: Bimax Product CS-AHPS1; commercial product that contains 38.64% 

1-propanesulfonic acid, 2-hydroxy-3-(2-propen-1-yloxy)-, sodium salt (1:1) (CASRN 52556-42-

0), also known as COPS-1.

Remarks: Clear colorless liquid; Batch No. 12E1101; Composition: 38.64% 1-propanesulfonic 

acid, 2-hydroxy-3-(2-propen-1-yloxy)-, sodium salt (1:1) (CASRN 52556-42-0), 2.26% 3-allyl1,2-propanediol (CASRN 123-34-2), and 59.1% water (CASRN 7732-18-5). 

METHOD

Method/guideline followed: OECD TG 425 (2008) “Acute Oral Toxicity – Up-and-Down 

Procedure (UDP)”2and OPPTS 870.1100 (2002) “Acute Oral Toxicity”

Test type: Acute Oral Toxicity (Up-and-Down Procedure)

 

1 EPA accepted the submitter’s request to test Bimax Product CS-AHPS in the toxicity tests required for CASRN 

52556-42-0 given that the only component in this commercial product other than water and CASRN 52556-42-0 is 

CASRN 123-34-2, a presumed byproduct of the synthesis of CASRN 52556-42-0.

2 OECD TG 425 is equivalent to the method described in 40 CFR 799.9110(d)(1)(i)(A), which is specified for the 

acute mammalian toxicity testing requirement in the HPV3 Test Rule.
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 52556-42-0

19

GLP compliant?: Yes

Year study performed: 2013

Test species: Female Wistar rats (strain Crl:WI); supplied by Charles River Deutschland, 

Sulzfeld, Germany; age at initiation: 9–10 weeks; weight at initiation: 153–190 g 

Number of animals/sex/dose: 5 females/dose

Route of administration: Oral (gavage)

Vehicle: None

Doses/concentrations tested: 2000 mg test substance/kg-bw; equivalent to 772.8 mg a.i./kgbw, based on a concentration of 38.64% CASRN 52556-42-0 (the a.i.) in the test substance.

“The test substance was dosed undiluted as delivered by the sponsor. No correction was made 

for the purity of the test substance.” [van Huygevoort 2013, p. 8]

Statistical methods used: Not applicable

Test method/conditions remarks: A single dose (2000 mg test substance/kg-bw) of the

undiluted test material was administered to each of five rats at a dosing volume of 1.71 mL/kg. 

The first rat was treated and observed for mortality before the remaining rats were treated. Each 

rat was fasted overnight prior to test substance administration and until 3–4 hours after dosing;

water was available ad libitum during the fasting period. Following dosing, the animals were 

monitored for mortality twice daily for 14 days. Clinical signs were recorded at 2 and 4 hours 

post-dosing, and once daily thereafter for 14 days. Body weights were recorded before treatment 

on study day 1, and on study days 8 and 15. All rats were sacrificed on day 15 and examined by 

gross necropsy.

RESULTS

Endpoint value(s): LD50 > 2000 mg test substance/kg-bw; LD50 > 772.8 mg a.i./kg-bw (based 

on a concentration of 38.64% CASRN 52556-42-0 (the a.i.) in the test substance)

Results remarks: No mortalities occurred. Hunched posture (two rats) and piloerection (one 

rat) were seen on the day of dosing. No other clinical signs were observed on the day of dosing 

or during the 14-day post-treatment observation period. All rats gained weight during the 

observation period and no adverse effects on body weight gain were apparent. No abnormalities 

were found during necropsy. 
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 52556-42-0

20

CONCLUSIONS

The oral LD50 for female rats was > 2000 mg test substance/kg-bw, corresponding to > 772.8 mg 

a.i./kg-bw based on a concentration of 38.64% CASRN 52556-42-0 (the a.i.) in the test 

substance.

STUDY RELIABILITY

[3] Not reliable. The administered dose of a.i. was less than the dose required for an acute 

toxicity limit test. This study failed to identify a discrete LD50 because no mortality was 

observed at the only dose tested (nominal dose of 2000 mg test substance/kg-bw; equivalent to 

772.8 mg a.i./kg-bw based on a concentration of 38.64% CASRN 52556-42-0 (the a.i.) in the test 

substance). The study only determined that the LD50 was greater than 772.8 mg a.i./kg-bw, 

which is considerably less than the limit dose of 2000 mg a.i./kg-bw or 5000 mg a.i./kg-bw 

specified in the acute toxicity test guidelines (i.e., OECD TG 425 and OPPTS 870.1100). Also, 

the selection of the limit dose was not based on the concentration of a.i. in the test substance (i.e., 

38.64% CASRN 52556-42-0) as specified in EPA’s response to the sponsor’s request for 

modification regarding the test substance composition.

REFERENCE

van Huygevoort, A.H.B.M. 2013. Assessment of acute oral toxicity with 1-propanesulfonic 

acid, 2-hydroxy-3-(2-propen-1-yloxy)-, sodium salt (1:1) in the rat (up and down procedure). 

WIL Research Europe B.V., The Netherlands; Laboratory Project No. 500679. Sponsor: 

Society of Chemical Manufacturers and Affiliates (SOCMA), Washington, DC. Study report 

available online at http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-

0112-0146 as of April 18, 2014.

Text in quotes is taken directly from van Huygevoort (2013).
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 52556-42-0

21

D-5. Repeated-Dose, Reproductive, and Developmental Toxicity

Summary of Repeated-Dose Toxicity Endpoint: Adequate

Summary of Reproductive Toxicity Endpoint: Adequate

Summary of Developmental Toxicity Endpoint: Adequate

Study 1: Repeated-dose oral toxicity study in rats (van Otterdijk, 2013)

Title: Combined 28-day repeated dose toxicity study with the reproduction/developmental 

toxicity screening test of 1-propanesulfonic acid, 2-hydroxy-3-(2-propen-1-yloxy)-, sodium salt 

(1:1) in rats by oral gavage

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Bimax Product CS-AHPS1; commercial product that contains 38.64% 

1-propanesulfonic acid, 2-hydroxy-3-(2-propen-1-yloxy)-, sodium salt (1:1) (CASRN 52556-42-

0), also known as COPS-1.

Remarks: Clear colorless liquid; Batch No. 12E1101; Composition: 38.64% 1-propanesulfonic 

acid, 2-hydroxy-3-(2-propen-1-yloxy)-, sodium salt (1:1) (CASRN 52556-42-0), 2.26% 3-allyl1,2-propanediol (CASRN 123-34-2), and 59.1% water (CASRN 7732-18-5). 

METHOD

Method/guideline followed: OECD TG 422 (1996) “Combined Repeated Dose Toxicity Study 

with the Reproduction/Developmental Toxicity Screening Test" and OPPTS 870.3650 (2000) 

“Combined Repeated Dose Toxicity with the Reproduction/Developmental Toxicity Screening 

Test”

Test type: Combined Repeated-Dose Toxicity Study with Reproductive/Developmental 

Toxicity Screening

GLP compliant?: Yes

Year study performed: 2013

Test species: Crl:WI(Han) strain rats; obtained from Charles River Deutschland, Sulzfeld, 

Germany; at initiation, rats were approximately 11 weeks old and weighed 302-339 g (males) 

and 191-215 g (females). Individual body weights were within 20% of the sex mean body 

weight.

 

1 EPA accepted the submitter’s request to test Bimax Product CS-AHPS in the toxicity tests required for CASRN 

52556-42-0 given that the only component in this commercial product other than water and CASRN 52556-42-0 is 

CASRN 123-34-2, a presumed byproduct of the synthesis of CASRN 52556-42-0. 
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 52556-42-0

22

Number of animals/sex/dose: 10

Route of administration: Oral (gavage)

Vehicle: Purified water (Millipore)

Exposure duration: Males: 29 days; Females: 42-48 days (throughout 14 days of pre-mating, 

mating, and post-coitum, and during at least 4 days of lactation until one day prior to scheduled 

euthanasia and necropsy)

Doses/concentrations tested: Nominal doses: 0 (control), 100, 300, and 1000 mg test 

substance/kg-bw/day; equivalent to approximately 0, 38.6, 116, and 386 mg a.i.1/kg-bw/day, 

respectively, based on a concentration of 38.64% CASRN 52556-42-0 in the test substance.

“No correction was made for the purity/composition of the test substance.” [van Otterdijk 2013, 

p. 11]

Statistical methods used: “Group means were calculated for continuous data and medians were 

calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the 

basis of exact values for means and pooled variances. Individual values, means, and standard 

deviations may have been rounded off before printing. Therefore, two groups may display the 

same printed means for a given parameter, yet display different test statistics values.” [van 

Otterdijk 2013, p. 21]

The Dunnett test based on a pooled variance estimate was used to compare treated and control 

groups for each sex if the variables could be assumed to follow a normal distribution. The Steel

test (many-to-one rank test) was used to compare treated and control groups for each sex if the 

data could not be assumed to follow a normal distribution. Frequency data were evaluated with 

the Fisher Exact test. Motor activity data were evaluated for intergroup differences with the 

Kruskal-Wallis nonparametric ANOVA test. When intergroup differences were found, the 

Wilcoxon test was applied to motor activity data to compare control and treated groups. All tests 

were two-sided and a p-value of less than 0.05 indicated statistical significance.

The standard unit of assessment for analyses of pup data was the litter.

Test method/conditions remarks: 

Preparation and Analyses of Test Dose Formulations:

Dosing formulations (w/w) were prepared fresh each day by diluting the test substance in the 

vehicle, purified water, and were used within 6 hours of preparation. Prepared formulations 

were assessed visually for homogeneity. After the in-life phase of the study was completed, 

similarly prepared test substance formulations were evaluated for accuracy (all concentrations) 

 

1 Active ingredient (a.i.) = CASRN 52556-42-0
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 52556-42-0

23

and homogeneity (highest and lowest concentrations) according to a validated analytical method1

using an HPLC instrument equipped with a refractive index detector. Test formulations were 

evaluated after the in-life phase of the study on similarly prepared formulations because a

validated analytical method was not available during the in-life phase of the study. “The 

accuracy of preparation was considered acceptable if the mean measured concentrations were 

90–110% of the target concentration. Homogeneity was demonstrated if the coefficient of 

variation was ≤ 10%.” [van Otterdijk 2013, p. 12]

Dosing and Environmental Conditions:

Groups of rats (10/sex/group) were orally administered the test substance (7 days/week) via 

gavage at dose levels of 100, 300, and 1000 mg test substance/kg-bw/day (approximately 38.6, 

116, and 386 mg a.i./kg-bw/day, respectively) at a constant dosing volume of 5 mL/kg-bw. A 

concurrent control group was treated with the vehicle only. Treatment continued throughout the 

pre-mating (14 days), mating, and post-mating periods. Exposure continued in males and 

females until the day prior to scheduled euthanasia, for total exposure periods of 29 consecutive 

days (males) or 42-48 consecutive days (females). 

Males and females were housed in groups of five until mating, with food and water available ad 

libitum throughout the study. During the mating period, males and females were paired one-toone until there was evidence of mating (i.e., evidence of sperm in the vaginal lavage or the 

appearance of an intravaginal copulatory plug). Following evidence of mating (designated as 

day 0 post-coitum), males were returned to their original cages. Mated females were housed 

individually throughout gestation and with their litters after delivery and during lactation. “The 

females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter 

was found completed (i.e., membranes and placentas cleaned up, nest build up and/or feeding of 

pups started). Females that were littering were left undisturbed.” [van Otterdijk 2013, p. 12]

Observations:

Parental animals were observed at least twice daily for mortality and viability, and detailed 

clinical observations were performed cage-side on a daily basis. Detailed clinical observations 

were also performed outside of the assigned cage in a standard arena prior to the start of 

treatment and at weekly intervals during the treatment period. “Clinical observations ([cageside] clinical signs and arena) were conducted after dosing... within a similar time period after 

dosing for the respective animals. The time of onset, grade, and duration of any observed sign 

was recorded. Signs were graded for severity and the maximum grade was predefined a[s] 3 or 

4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3), and very severe 

(grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored.” [van 

Otterdijk 2013, p. 14]

Parental animals were weighed on the first day of exposure and then weekly throughout the 

study. Females were weighed on post-coitum days 0, 4, 7, 11, 14, 17, and 20 and on lactation 

days 1 and 4. Food consumption was determined weekly in all animals except during mating

 

1 The study report for the development and validation of the analytical method was submitted as a separate report,

“Development and validation of an analytical method for the analysis of 1-propanesulfonic acid, 2-hydroxy-3-(2-

propen-1-yloxy)-, sodium salt (1:1) in water”, available at http://www.regulations.gov/#!documentDetail;D=EPAHQ-OPPT-2009-0112-0146 as of April 18, 2014.
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 52556-42-0

24

and for females with no evidence of mating. Food consumption of mated females was 

determined on post-coitum days 0, 4, 7, 11, 14, 17, and 20, and on lactation days 1 and 4. 

“Subjective appraisal [of water consumption] was maintained during the study, but no 

quantitative investigation was introduced as no treatment related effect was suspected.” [van 

Otterdijk 2013, p. 15]

Selected animals (5/sex/group) were observed for signs of neurobehavioral toxicity during 

treatment. Functional observations were performed after animals were observed for clinical 

signs and included hearing ability, pupillary reflex, static righting reflex, grip strength, and 

locomotor activity. Males were tested during week 4 of treatment. Selected females in the 

control and low-dose group were evaluated toward the end of the lactation period (from lactation 

day 4 onwards), and the females from the mid- and high-dose groups were evaluated at the end 

of the post-coitum period as no females in those groups delivered offspring. 

Hematology and Clinical Chemistry Measurements:

Blood samples were collected on the day of scheduled necropsy from 5 parental 

animals/sex/group. All animals selected for hematology and clinical chemistry determinations 

were deprived of food overnight (maximum of 24 hours) on the night prior to scheduled 

necropsy. The following hematological parameters were evaluated in blood prepared with K3-

EDTA as an anti-coagulant: white blood cell (WBC) count, differential leukocyte counts

(neutrophils, lymphocytes, monocytes, eosinophils, and basophils, as a % of WBC), red blood 

cell (RBC) count, reticulocytes (as a % of RBC), RBC distribution width, hemoglobin, 

hematocrit, mean corpuscular volume, mean corpuscular hemoglobin (MCH), MCH 

concentration, and platelet count. Clotting parameters evaluated in plasma (prepared with citrate 

as an anti-coagulant) included prothrombin time and activated partial thromboblastin time.

Clinical chemistry parameters evaluated in blood treated with lithium heparin as an anticoagulant included alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, 

total protein, albumin, total globulin, albumin/globulin ratio, total bilirubin, bile acids, urea, 

creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, and inorganic phosphate. 

“All parameters were determined in plasma, except for bile acids which were determined in 

serum.” [van Otterdijk 2013, p. 17]

Gross Necropsy and Histopathology:

Animals were euthanized on the day following the last exposure (day 30 in parental males, and 

postpartum/lactation day 5–7 in parental females). Females that failed to deliver were 

necropsied on post-coitum days 25–27. Females with total litter loss were necropsied within 24 

hours of total litter loss. Terminal body weights were recorded for all males and 5 females/group 

at the time of necropsy. All parental adults were examined by necropsy and macroscopic 

abnormalities were recorded. Organ weights were recorded for 5 parental rats/sex/group for the 

adrenal glands, brain, prostate, heart, kidneys, liver, ovaries, spleen, thymus, uterus with cervix, 

seminal vesicles with coagulating glands, and thyroid including parathyroid. Organ weights of 

the testes and epididymides were recorded for parental males of all dose groups.

The following tissues were examined by histopathology for 5 parental animals/sex from the 

control and high-dose group: adrenal glands, brain (cerebellum, mid-brain, and cortex), cecum, 
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 52556-42-0

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colon, duodenum, eyes (with optic nerve if detectable and Harderian gland), femur including 

joint, heart, ileum, jejunum, kidneys, liver, lung, lymph nodes (mandibular and mesenteric), 

female mammary gland area, Peyer’s patches (jejunum and ileum) if detectable, pituitary gland, 

rectum, sciatic nerve, skeletal muscle, spinal cord (cervical, midthoracic, and lumbar), spleen, 

sternum with bone marrow, stomach, thymus, thyroid including parathyroid if detectable, 

trachea, and urinary bladder. Spleen tissue samples collected from 5 parental females per group 

in the low- and mid-dose groups were also examined microscopically. 

Reproductive/Developmental Data:

Pregnant females were observed daily and the following reproductive and developmental 

parameters were recorded: male number paired with, mating date, confirmation of pregnancy, 

and day of delivery, the number of females with evidence of mating, number of pregnant 

females, number of females bearing live pups, numbers of live male and female pups at first litter 

check, and numbers of live pups and dead pups before scheduled necropsy. Calculated 

reproductive and developmental parameters included mating index, fertility index, conception 

index, gestation index, duration of gestation, percentage of live male pups at first litter check, 

percentage of live female pups at first litter check, percentage of postnatal loss, and pup viability 

index. 

“Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage 

debris of pregnant females was examined to detect signs of abortion or premature birth. Any 

deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left 

scattered and cold, physical abuse of pups, or apparently inadequate lactation or feeding) were 

examined.” [van Otterdijk 2013, p. 15]

At scheduled necropsy, the numbers of implantation sites and corpora lutea were recorded for 

each paired female. Non-gravid uteri were stained to detect any former implantation sites when 

no macroscopically visible implantation sites were present. Spermatogenesis staging was 

performed on 5 selected males from both the control and high-dose groups, as well as all males 

that were suspected to be infertile. Reproductive organs were weighed, as described above. 

Histological examinations were performed on the following reproductive tissues from all 

parental animals: clitoral gland, epididymides, ovaries, preputial glands, prostate gland, seminal 

vesicles, coagulating glands, testes, uterus, cervix, and vagina. Reproductive tissues were also 

examined microscopically for all males that failed to sire a litter and any females that failed to 

deliver. 

Detailed clinical observations of offspring were made for all pups at least once daily. The 

number of live and dead pups was determined daily beginning on Day 1 of lactation. The cause 

of death or defects in dead pups was evaluated when possible. Pups were weighed and sexed on 

Days 1 and 4 of lactation.

Pups surviving until planned necropsy were euthanized on lactation days 5–7. “All pups were 

sexed and descriptions of all external abnormalities were recorded. The stomach of pups not 

surviving to the scheduled necropsy date were examined for the presence of milk. If possible, 

defects or cause of death were evaluated.” [van Otterdijk 2013, p. 19]
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 52556-42-0

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RESULTS

Endpoint value(s): 

Systemic Toxicity:

LOAEL (female, systemic toxicity) = 300 mg test substance/kg-bw/day (= 116 mg a.i./kgbw/day), based on decreased body weight, body weight gain, food consumption, and relative 

food consumption; NOAEL (female, systemic toxicity) = 100 mg test substance/kg-bw/day (= 

38.6 mg a.i./kg-bw/day).

NOAEL (male, systemic toxicity) = 1000 mg test substance/kg-bw/day (= 386 mg a.i./kgbw/day).

Reproductive Toxicity:

LOAEL (reproductive toxicity) = 100 mg test substance/kg-bw/day (= 38.6 mg a.i./kg-bw/day), 

based on reductions in the mean numbers of corpora lutea and implantation sites per dam.

NOAEL (reproductive toxicity) = Not established.

Developmental Toxicity:

LOAEL (developmental toxicity) = 100 mg test substance/kg-bw/day (= 38.6 mg a.i./kgbw/day), based on reductions in the mean numbers of corpora lutea and implantation sites per 

dam.

NOAEL (developmental toxicity) = Not established.

Results remarks: 

Analyses of Test substance Formulations:

Measurement of test substance concentrations in similarly prepared dosing formulations showed 

that the formulations (nominal doses of 100, 300, and 1000 mg test substance/kg-bw/day) were 

within 99–102% of the nominal doses. The formulations for the nominal doses of 100 and 1000 

mg test substance/kg-bw/day were homogenous, with a coefficient of variation ≤ 10%.

Clinical Observations:

No mortality occurred during the treatment period. One female in the 100 mg test substance/kgbw/day group was euthanized before scheduled necropsy due to total litter loss on lactation 

day 5. Alopecia (control) and scabs (100 mg test substance/kg-bw/day) were seen in 5 to 15% of 

males during pre-mating and/or mating. Similar findings were not observed in other male dose 

groups or in females, and the incidence was within the range of background findings for the 

strain and age of rats; therefore, these findings were not considered to be toxicologically 

relevant.

Body Weight and Food/Water Consumption:

No changes in body weight, body weight gain, or food consumption were observed in males

during the test or in females during pre-mating or mating. Females in the high-dose group (1000 

mg test substance/kg-bw/day) were excluded from the body weight and food consumption

analyses because there were no pregnancies in this group. Body weight and food consumption 

analyses of females in the mid-dose group (300 mg test substance/kg-bw/day) during the postcoital period and lactation were based on the mean value for the two pregnant females in this 
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 52556-42-0

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group. 

At 300 mg test substance/kg-bw/day, mean female body weights were significantly (p < 0.01) 

reduced on post-coitum days 17 and 20 by 16% and 24%, respectively, relative to controls. 

Mean female body weight gain (as a % of initial body weight) was significantly (p < 0.01) 

reduced at 300 mg test substance/kg-bw/day on post-coitum days 14, 17, and 20 by 41%, 61%, 

and 70%, respectively, relative to controls. The decreased body weight and body weight gain 

may have been due, at least in part, to decreased food consumption as food consumption during 

this time period (post-coitum day intervals 14–17 and 17–20) was significantly reduced at 300 

mg test substance/kg-bw/day by 30% (p < 0.01) and 28% (p < 0.05), respectively, relative to 

controls. Relative food consumption (measured as g/kg body weight/day) during post-coitum 

day interval 14-17 was significantly (p < 0.01) reduced in females at 300 mg test substance/kgbw/day by 21%, relative to controls.

Neurological Assessments:

No toxicologically relevant effects were seen during functional tests that assessed hearing ability, 

pupillary reflex, static righting reflex, and grip strength. “For motor activity, all groups showed 

a similar habituation profile with high activity in the first interval that decreased over the 

duration of the test period. The higher motor activity of females at 300 and 1000 mg [test 

substance]/kg (statistically significant [p < 0.05] at 1000 mg/kg) was considered due to these 

females not having delivered offspring. This variation was therefore considered to be of no 

toxicological relevance.” [van Otterdijk 2013, p. 22]

Hematology and Clinical Chemistry Measurements:

There was no effect of treatment on hematology parameters in male animals.

“All statistically significant changes in clinical biochemistry parameters in males at 100 and 300 

mg [test substance]/kg were considered to be of no toxicological significance as they occurred in 

the absence of a dose-related trend and remained within the range considered normal for rats of 

this age and strain. These changes consisted of higher total bilirubin, creatinine, bile acid and 

sodium levels and lower cholesterol levels.” [van Otterdijk 2013, p. 23]

Reticulocytes (% RBC) were significantly (p < 0.05) reduced (approximately 67% lower) in 

females at 1000 mg test substance/kg-bw/day relative to the control group. According to the 

study author “[this finding was] considered to be due to the pregnancy status (small litter size or 

absence of litters), and to be within the physiological range of non-pregnant females.” [van 

Otterdijk 2013, p. 23]

Alanine aminotransferase (ALAT) activity was significantly reduced in females in the 300 and 

1000 mg test substance/kg-bw/day groups by 53% (p < 0.01) and 36% (p < 0.05), respectively, 

relative to controls. Urea was significantly reduced in females in the 300 and 1000 mg test 

substance/kg-bw/day groups by 29% (p < 0.01) and 22% (p < 0.05), respectively, relative to 

controls. Sodium was significantly (p < 0.05) increased in females in the 1000 mg test 

substance/kg-bw/day group, by 1%, relative to the control group and significantly (p < 0.05) 

reduced in females in the 100 mg test substance/kg-bw/day group, by 1%, relative to the control 

group. Chloride was significantly increased in females in the 300 and 1000 mg test 
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 52556-42-0

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substance/kg-bw/day groups by 4.0% (p < 0.05) and 5.1% (p < 0.01), respectively, relative to the 

control group. A significant (p < 0.01) increase in albumin level (by 13%) was seen in females 

at 300 mg test substance/kg-bw/day, relative to controls. The study report noted that the clinical 

chemistry values observed at 300 and 1000 mg test substance/kg-bw/day were within the range 

of values expected for non-pregnant females of the same strain and age. Therefore, the changes 

in clinical chemistry values at 300 and 1000 mg test substance/kg-bw/day had no clear 

toxicological relevance.

Gross Necropsy and Histopathology:

No macroscopic abnormalities were observed in males or females that were considered related to 

the treatment. 

There were no treatment-related effects on male organ weight or organ to body weight ratios. 

Absolute liver weights of females were significantly (p < 0.01) reduced at 300 and 1000 mg test 

substance/kg-bw/day by 16% and 26%, respectively, relative to the control group. Relative liver 

weights of females were significantly reduced at 300 and 1000 mg test substance/kg-bw/day (p < 

0.05 and p < 0.01, respectively) by 15% and 21%, respectively, relative to controls. Absolute 

thymus weights were significantly (p < 0.01) increased in females at 300 and 1000 mg test 

substance/kg-bw/day by 91% and 65%, respectively, relative to controls. Relative thymus 

weights were significantly (p < 0.01) increased in females at 300 and 1000 mg test substance/kgbw/day by 95% and 77%, respectively, relative to controls. Absolute ovary weights were 

significantly (p < 0.05) increased at 300 and 1000 mg test substance/kg-bw/day by 35% and 

29%, respectively, relative to controls. Relative ovary weights were significantly (p < 0.05) 

increased at 300 and 1000 mg test substance/kg-bw/day by 36% and 38%, respectively, relative 

to controls. Based on the absence of corroborating histopathological effects in the liver, thymus, 

and ovaries, the changes in organ weights were not clearly related to treatment. Relative thyroid 

weights were significantly increased (p < 0.05) in females at 1000 mg test substance/kg-bw/day, 

but the increase was within the normal range for rats of the same strain and age; therefore, this 

change was not considered to be toxicologically relevant. 

No treatment-related histopathological changes were observed. “The microscopic findings for 

one female treated at 1000 mg [test substance]/kg were regarded to be related to a gavage 

accident, because of the single incidence and the recorded disruption of the normal glandular 

stomach architecture.” [van Otterdijk 2013, p. 24]

Reproductive/Developmental Data: 

The mating index and pre-coital time were similar between treated and control animals. The 

fertility and conception indices at 0, 100, 300, and 1000 mg test substance/kg-bw/day were 90%, 

90%, 20%, and 0%, respectively (both indices had equivalent values at each dose level). The 

gestation index in the 0, 100, and 300 mg test substance/kg-bw/day groups was 100%, 88.9%, 

and 0%, respectively. The gestation index could not be calculated for the 1000 mg test 

substance/kg-bw/day group due to the absence of implantations or pregnancies.

There were 9, 9, 2, and 0 pregnant females in the 0, 100, 300, and 1000 mg test substance/kgbw/day treatment groups, respectively, which delivered 9, 8, 0, and 0 litters, respectively. The 
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 52556-42-0

29

mean number of corpora lutea per dam in the 100 and 300 mg test substance/kg-bw/day groups 

was decreased by 22% and 25%, respectively, relative to the control group. The mean number of 

implantation sites per dam in the 100 and 300 mg test substance/kg-bw/day groups was

decreased by 19% and 88%, respectively, relative to the control group. The mean number of 

implantation sites per dam at 300 mg test substance/kg-bw/day was based only on two 

pregnancies. Although the study author did not find the reduced numbers of corpora lutea and 

implantation sites in the low- and mid-dose animals to be statistically significantly different from 

the controls, the reductions are considered to be of sufficient magnitude to warrant concern. 

Therefore, the EPA reviewer considers the reduced numbers of corpora lutea and implantation 

sites in the low- and mid-dose groups to be biologically significant; these effects form the basis 

of the LOAEL for reproductive and developmental toxicity at 100 mg test substance/kg-bw/day.

One female in the 100 mg test substance/kg-bw/day group, having 2 corpora lutea and 1 

implantation site, delivered a single pup which died on lactation day 5. Another female in the 

same group did not deliver a litter and had one corpora lutea and one implantation site. Only two 

females in the 300 mg test substance/kg-bw/day group were determined to be pregnant based on 

the presence of implantation sites, but did not deliver any litters; one female had 20 corpora lutea 

and 1 implantation site, while the other had 2 corpora lutea and 2 implantation sites. No 

pregnancies and no implantation sites were observed in the 1000 mg test substance/kg-bw/day 

group. One female in the 1000 mg test substance/kg-bw/day group had 19 corpora lutea, but 

none were observed in the other females.

Examination of Reproductive Organs: Blood from the vagina was recorded on the day of 

scheduled necropsy in one female in the 300 mg test substance/kg-bw/day group. An early 

implantation site and hemorrhagic contents in one uterine horn were seen in this female. A large 

fetus in one uterine horn was found at necropsy. “For the remaining females, no histological 

correlation could be made for the lack of offspring. These females showed a normal 

reproductive cycle.” [van Otterdijk 2013, p. 159]

“For the males, no abnormalities were seen in the reproductive organs which could account for 

their infertility. Furthermore, the spermatogenic staging profiles were normal for all examined 

males.” [van Otterdijk 2013, p. 159]

Clinical Examinations of Offspring: One pup from the control group was missing on lactation 

day 2 and was thought to have been cannibalized. Three pups in the 100 mg test substance/kgbw/day group were found dead at the first litter check and showed severe autolysis. A pup in the 

100 mg test substance/kg-bw/day group found dead on lactation day 5 had shown a lean 

appearance from first litter check onwards and absence of milk in the stomach from lactation day 

2. “No toxicological relevance was attributed to these dead/missing pups since the mortality 

incidences remained within the range considered normal for pups of this age.” [van Otterdijk 

2013, p. 25]
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 52556-42-0

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Offspring Viability and Growth: Offspring viabilities in the control and 100 mg test 

substance/kg-bw/day test groups were similar, with indices of 99.1 and 98.8%, respectively, 

relative to controls. “No toxicologically relevant effects on gestation index and duration, 

parturition, maternal care, and early postnatal pup development (mortality, clinical signs, body 

weight, and macroscopy) were observed at 100 mg [test substance]/kg. At 300 and 1000 mg

[test substance]/kg no pups were delivered, and hence developmental effects [as evidenced in 

pup development] could not be assessed for these dose levels.” [van Otterdijk 2013, p. 25]

F1 Offspring Necropsy: Findings in pups in the control and 100 mg test substance/kg-bw/day 

groups at necropsy consisted of the presence of a wound, scabbing, absence of milk in the 

stomach, a blue spot on the hind foot, and an abnormal posture of the hind leg. These findings 

occurred at similar incidences in control and treated pups and did not appear to be related to the 

treatment. 

CONCLUSIONS

Systemic Toxicity:

The LOAEL for systemic toxicity in females is 300 mg test substance/kg-bw/day (116 mg 

a.i./kg-bw/day), based on decreased body weight, body weight gain, food consumption, and 

relative food consumption. The NOAEL for systemic toxicity in females is 100 mg test 

substance/kg-bw/day (38.6 mg a.i./kg-bw/day). 

The NOAEL for systemic toxicity in males is 1000 mg test substance/kg-bw/day (386 mg a.i./kgbw/day).

Reproductive Toxicity:

The LOAEL for reproductive toxicity is 100 mg test substance/kg-bw/day (38.6 mg a.i./kgbw/day), based on reductions in the mean numbers of corpora lutea and implantation sites per 

dam. The NOAEL for reproductive toxicity is not established.

Developmental Toxicity:

The LOAEL for developmental toxicity is 100 mg test substance/kg-bw/day (38.6 mg a.i./kgbw/day), based on reductions in the mean numbers of corpora lutea and implantation sites per 

dam. The NOAEL for developmental toxicity is not established.

STUDY RELIABILITY

[2] Reliable with restrictions. Calculations for pre-implantation loss were not conducted. The 

study did not analyze concentration, stability, or homogeneity of the prepared formulations used 

during the study. Instead, concentration, stability, and homogeneity of similarly prepared test 

formulations were analyzed after the in-life phase of the study because a validated analytical 

method was not available during the in-life phase of the study.
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 52556-42-0

31

REFERENCE

van Otterdijk, F.M. 2013. Combined 28-day repeated dose toxicity study with the 

reproduction/developmental toxicity screening test of 1-propanesulfonic acid, 2-hydroxy-3-(2-

propen-1-yloxy)-, sodium salt (1:1) in rats by oral gavage. WIL Research Europe B.V., The 

Netherlands; Laboratory Project No. 500682. Sponsor: Society of Chemical Manufacturers and 

Affiliates (SOCMA), Washington, DC. Study report available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0112-0146 as of April 

18, 2014.

Text in quotes is taken directly from van Otterdijk (2013).
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 52556-42-0

32

D-6. Genetic Toxicity – Gene Mutation

Summary of endpoint: Not adequate 

Study 1: Mutagenicity study in Salmonella typhimurium and Escherichia coli (VerspeekRip, 2013)

Title: Evaluation of the mutagenic activity of 1-propanesulfonic acid, 2-hydroxy-3-(2-propen-1-

yloxy)-, sodium salt (1:1) in the Salmonella typhimurium reverse mutation assay and the 

Escherichia coli reverse mutation assay (with independent repeat)

This study is: Adequate Not Adequate

The study is not adequate due to the following deficiency: The highest test concentration (1932 

μg a.i./plate, calculated based on a concentration of 38.64% CASRN 52556-42-0 (the a.i.) in the 

test substance) was substantially lower than the maximum concentration of 5 mg a.i./plate (5000 

μg a.i./plate) that is recommended by the cited test guidelines (OECD TG 471 and TSCA 

799.9510) and did not produce an increased number of revertant colonies, cytotoxicity, or 

precipitation during the test. Also, test concentrations were not selected based on the 

concentration of a.i. in the test substance (i.e., 38.64% CASRN 52556-42-0) as was specified in 

EPA’s response to the sponsor’s request for modification regarding the test substance 

composition.

TEST SUBSTANCE 

Identity: Bimax Product CS-AHPS1; commercial product that contains 38.64% 

1-propanesulfonic acid, 2-hydroxy-3-(2-propen-1-yloxy)-, sodium salt (1:1) (CASRN 52556-42-

0), also known as COPS-1.

Remarks: Clear colorless liquid; Batch No. 12E1101; Composition: 38.64% 1-propanesulfonic 

acid, 2-hydroxy-3-(2-propen-1-yloxy)-, sodium salt (1:1) (CASRN 52556-42-0), 2.26% 3-allyl1,2-propanediol (CASRN 123-34-2), and 59.1% water (CASRN 7732-18-5).

METHOD

Method/guideline followed: TSCA 799.9510 (equivalent to OECD TG 471 (1997) and Method 

B.13/14 of Commission Regulation (EC) No. 440/2008)

Test type: Bacterial Reverse Mutation Test

GLP compliant?: Yes

 

1 EPA accepted the submitter’s request to test Bimax Product CS-AHPS in the toxicity tests required for CASRN 

52556-42-0 given that the only component in this commercial product other than water and CASRN 52556-42-0 is 

CASRN 123-34-2, a presumed byproduct of the synthesis of CASRN 52556-42-0. 
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 52556-42-0

33

Year study performed: 2013

Test species: Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 (obtained 

from Trinova Biochem GmbH, Germany) and Escherichia coli strain WP2 uvrA (obtained from 

The National Collections of Industrial and Marine Bacteria, Aberdeen, UK)

Metabolic activation: With (phenobarbital and β-naphthoflavone-induced rat liver S9 

microsomal fraction) and without

Concentrations tested:

Range-Finding Toxicity Test Concentrations:

TA100 and WP2 uvrA (without and with 5% S9): 3, 10, 33, 100, 333, 1000, 3330, and 5000 μg

test substance/plate; equivalent to approximately 1.2, 3.9, 12.8, 38.6, 129, 386, 1287, and 1932 

μg a.i./plate, respectively, based on a concentration of 38.64% CASRN 52556-42-0 (the a.i.) in 

the test substance.

Definitive Mutagenicity Assay Concentrations:

Experiment 1:

TA98, TA1535, and TA1537 (without and with 5% S9): 100, 333, 1000, 3330, and 5000 μg test 

substance/plate; equivalent to approximately 38.6, 129, 386, 1287, and 1932 μg a.i./plate, 

respectively, based on a concentration of 38.64% CASRN 52556-42-0 (the a.i.) in the test 

substance.

Experiment 2:

TA98, TA100, TA1535, TA1537, and WP2 uvrA (without and with 10% S9): 100, 333, 1000, 

3330, and 5000 μg test substance/plate; equivalent to approximately 38.6, 129, 386, 1287, and 

1932 μg a.i./plate, respectively, based on a concentration of 38.64% CASRN 52556-42-0 (the 

a.i.) in the test substance.

“No correction was made for the purity/composition of the test compound.” [Verspeek-Rip 2013, 

p. 8]

Solvent: Milli-Q water

Statistical methods used: No formal testing was conducted.

Test method/conditions remarks: A stock solution of the test substance was prepared by 

dissolving the test substance in Milli-Q water and the solution was filter sterilized using a 0.22 

μm filter. Test concentrations were prepared by diluting the filtered stock solution in Milli-Q 

water and were used within 2.5 hours of preparation. The test concentrations selected for the 

mutagenicity assay were based on the results of a range-finding toxicity test in Salmonella 

typhimurium strain TA100 and Escherichia coli strain WP2 uvrA.

Bacterial Cultures and Plating:

“Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth... and 

incubated in a shaking incubator (37°C, 150 spm) until the cultures reached an optical density of 
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 52556-42-0

34

1.0 ± 0.1 at 700 nm (109cells/ml). Freshly grown cultures of each strain were used for [all 

tests].” [Verspeek-Rip 2013, p. 9]

“Agar plates (9 cm) contained 25 ml glucose agar medium. Glucose agar medium contained [the 

following] per liter: 18 g purified agar... in Vogel-Bonner Medium E [and] 20 g glucose... The 

agar plates for the test with the Salmonella typhimurium strains also contained 12.5 μg/plate 

biotin... and 15 μg/plate histidine... and the agar plates for the test with the Escherichia coli

strain contained 15 μg/plate tryptophan...” [Verspeek-Rip 2013, p. 9] 

Top agar was prepared by heating Milli-Q water containing 0.6% (w/v) bacteriological agar and 

0.5% (w/v) sodium chloride until the agar was dissolved. The top agar was then transferred to 

glass tubes and autoclaved for 20 minutes. For each test, top agar was melted by heating to 45°C 

and the following solutions were successively added to 3 mL of molten top agar: 0.1 mL fresh 

bacterial culture (109cells/mL), 0.1 mL of diluted test substance preparation in Milli-Q water, 

and either 0.5 mL S9-mix (for assays with S9 metabolic activation) or 0.5 mL 0.1 M phosphate 

buffer (for assays without S9 metabolic activation). The ingredients were mixed by vortexing 

and melted top agar was poured onto each plate. After solidification, the plates were inverted 

and the bacteria were incubated at 37.0 ± 1.0°C in the dark for 44 to 52 hours. 

Preparation of Rat Liver S9 Fractions: 

Adult male Wistar rats (obtained from Charles River, Sulzfeld, Germany) were orally 

administered a suspension of phenobarbital (80 mg/kg-bw/day) and β-naphthoflavone (100 

mg/kg-bw/day) in corn oil for three consecutive days. The rats were denied access to food for 3 

to 4 hours before each dosing. The rats were sacrificed on the day following the third dose and 

liver homogenates were prepared from liver tissue isolated from each rat. The homogenates

were centrifuged and the supernatant (S9) was collected and stored in liquid nitrogen. The S9-

mix for the study was freshly prepared for each test from frozen S9, sterilized with a 0.22 μm 

filter, and stored on ice until use.

“Before use, all S9 batches were characterized with the mutagens Benzo-(a)-pyrene... and 2-

aminoanthracene..., which require metabolic activation, in tester strain TA98 at concentrations 

of 5 μg/plate and 1 μg/plate, respectively.” [Verspeek-Rip 2013, p. 10]

Positive Controls: 

The following positive controls were evaluated without S9-mix:

Chemical Amount/plate and Strain

Sodium azide 5 μg/plate for TA1535

ICR-191 2.5 μg/plate for TA1537

2-Nitrofluorene 10 μg/plate for TA98

Methylmethanesulfonate 650 μg/plate for TA100

4-Nitroquinoline N-oxide 10 μg/plate for WP2 uvrA
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 52556-42-0

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The following positive controls were evaluated with S9-mix:

Chemical Amount/plate and Strain Concentration of S9-mix

2-Aminoanthracene 2.5 μg/plate for TA1535 5%; 10%

2-Aminoanthracene 2.5 μg/plate for TA1537 5%

2-Aminoanthracene 5 μg/plate for TA1537 10%

2-Aminoanthracene 1 μg/plate for TA98 5%; 10%

2-Aminoanthracene 1 μg/plate for TA100 5%

2-Aminoanthracene 2.5 μg/plate for TA100 10%

2-Aminoanthracene 10 μg/plate for WP2 uvrA 5%; 10%

Solvents for the positive controls were saline for sodium azide and dimethylsulfoxide for all 

other chemicals. 

Range-Finding Toxicity Test Procedure:

Salmonella typhimurium strain TA100 and Escherichia coli strain WP2 uvrA were evaluated in 

triplicate at concentrations of 0 (solvent control), 3, 10, 33, 100, 333, 1000, 3330, and 5000 μg 

test substance/plate, with and without 5% (v/v) S9-mix. At the end of the incubation period, 

each plate was examined for test substance precipitation, and toxicity and the mean number of 

revertant colonies per plate were determined. Results at were included as part of Experiment 1 

of the definitive mutagenicity assay.

Definitive Mutagenicity Assay Procedure:

Salmonella typhimurium strains TA98, TA1537, and TA1538 were evaluated in triplicate in two 

independent experiments (Experiments 1 [with and without 5% (v/v) S9-mix] and 2 [with and 

without 10% (v/v) S-9 mix]) at concentrations of 0 (solvent control), 100, 333, 1000, 3330, and 

5000 μg test substance/plate. Salmonella typhimurium strain TA100 was evaluated in triplicate 

in Experiment 2 at concentrations of 0 (solvent control), 333, 1000, 3330, and 5000 μg test 

substance/plate, with and without 10% (v/v) S9-mix, and at 100 μg test substance/plate with 10% 

S9-mix1. Escherichia coli strain WP2 uvrA was evaluated in triplicate in Experiment 2 at 

concentrations of 0 (solvent control), 100, 333, 1000, 3330, and 5000 μg test substance/plate, 

with and without 10% (v/v) S9-mix. At the end of the incubation period, each plate was 

examined for test substance precipitation, and toxicity and the mean number of revertant colonies 

per plate were determined.

Mutagenicity and Toxicity Assessment:

“The revertant colonies (histidine independent or tryptophan independent) were counted 

manually if less than 40 colonies per plate were present. If more than 40 colonies were present, 

these were counted automatically with a Biocount 4000 Pro-S-colony counter. Plates with 

abundant test substance precipitate which interfered with automated colony counting were 

counted manually and the evidence of test substance precipitate on the plates was recorded. The 

condition of the bacterial background lawn was evaluated, both macroscopically and 

microscopically by using a dissecting microscope.” [Verspeek-Rip 2013, p. 11]

 

1 TA100 was not tested at 100 μg test substance/plate in the absence of 10% S9 in Experiment 2 due to a procedural 

error.
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The test substance was considered to be toxic if there was a reduction in the bacterial background 

lawn and/or a biologically relevant decrease in the mean number of revertant colonies per plate. 

Criteria for Positive Results: 

“A test substance is considered positive (mutagenic) in the test if: 

a) The total number of revertants in tester strain TA100 is greater than two (2) times the 

concurrent control, or the total number of revertants in tester strains TA1535, TA1537, 

TA98, or WP2uvrA is greater than three (3) times the concurrent vehicle control.

b) In case a repeat experiment is performed when a positive response is observed in one of

the tester strains, the positive response should be reproducible in at least one 

independently repeated experiment.” [Verspeek-Rip 2013, p. 11]

Acceptability Criteria: 

“A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation 

assay is considered acceptable if it meets the following criteria:

a) The negative control data (number of spontaneous revertants per plate) should be within 

the laboratory historical range for each tester strain.

b) The positive control chemicals should produce responses in all tester strains, which are 

within the laboratory historical range documented for each positive control substance. 

Furthermore, the mean plate count should be at least three times the concurrent vehicle 

control group mean.

c) The selected dose range should include a clearly toxic concentration or should exhibit 

limited solubility as demonstrated by the preliminary toxicity range-finding test or should 

extend to 5 mg/plate.” [Verspeek-Rip 2013, p. 11]

RESULTS 

Genotoxicity result: Negative with and without metabolic activation

Cytotoxic concentration (if applicable): No cytotoxicity was observed at the concentrations 

tested with or without activation.

Results remarks: The test substance showed no indication of induction of mutations with or 

without metabolic activation in any of the five strains tested in the range-finding or definitive 

tests. Test substance precipitation was not seen on any of the plates. The test substance was not 

cytotoxic at any test concentration in either the absence or presence of metabolic activation. 

“The negative and strain-specific positive control values were within the laboratory historical 

control data ranges indicating that the test conditions were adequate and that the metabolic 

activation system functioned properly.” [Verspeek-Rip 2013, p. 13]

Only two of three acceptability criteria were satisfied during the assay: 

 The negative control data were within the laboratory historical control range for each 

tester strain.

 The positive control chemicals produced responses in all tester strains, which were within 

the laboratory historical range documented for each positive control substance. In 
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 52556-42-0

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addition, each positive control mean plate count was at least three times the concurrent 

vehicle control group mean.

The third acceptability criterion was not satisfied because none of the concentrations were 

cytotoxic, the solubility of the test solutions was not limited as evidenced by the lack of 

precipitation, and the active ingredient portion of the test substance was not tested up to

5 mg/plate (the highest test concentration was only 1932 μg a.i./plate).

CONCLUSIONS

The test substance did not induce gene mutations in bacteria either with or without metabolic 

activation under the conditions of this study.

STUDY RELIABILITY

[3] Not reliable. The highest test concentration (1932 μg a.i./plate, calculated based on a 

concentration of 38.64% CASRN 52556-42-0 (the a.i.) in the test substance) was substantially 

lower than the maximum concentration of 5 mg a.i./plate (5000 μg a.i./plate) that is 

recommended by the cited test guidelines (OECD TG 471 and TSCA 799.9510) and did not 

produce an increased number of revertant colonies, cytotoxicity, or precipitation during the test. 

Also, test concentrations were not selected based on the concentration of a.i. in the test substance 

(i.e., 38.64% CASRN 52556-42-0) as was specified in EPA’s response to the sponsor’s request 

for modification regarding the test substance composition.

Due to a procedural error, Salmonella typhimurium strain TA100 was not tested at 100 μg test 

substance/plate in the absence of S9-mix in Experiment 2. Therefore, only four concentrations in 

the definitive test were tested rather than five concentrations as recommended by OECD TG 471 

and TSCA 799.9510. This deviation does not affect the integrity of the study results as the strain 

was tested at four higher concentrations and no toxicity, precipitation, or increase in revertant 

colonies was observed. Additionally, assays at 100 μg test substance/plate in the absence of S9-

mix were conducted in triplicate in the range-finding study and showed no toxicity, precipitation, 

or increase in revertant colonies.

REFERENCE

Verspeek-Rip, C.M. 2013. Evaluation of the mutagenic activity of 1-propanesulfonic acid, 2-

hydroxy-3-(2-propen-1-yloxy)-, sodium salt (1:1) in the Salmonella typhimurium reverse 

mutation assay and the Escherichia coli reverse mutation assay (with independent repeat). WIL 

Research Europe B.V., The Netherlands; Laboratory Project No. 500680. Sponsor: Society of 

Chemical Manufacturers and Affiliates (SOCMA), Washington, DC. Study report available 

online at http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0112-0146 as 

of April 18, 2014. 

Text in quotes is taken directly from Verspeek-Rip (2013).
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 52556-42-0

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D-7. Genetic Toxicity – Chromosomal Aberrations

Summary of endpoint: Not adequate 

Study 1: Chromosome aberration assay in human lymphocytes (Verbaan, 2013)

Title: Evaluation of the ability of 1-propanesulfonic acid, 2-hydroxy-3-(2-propen-1-yloxy)-, 

sodium salt (1:1) to induce chromosome aberrations in cultured peripheral human lymphocytes 

(with repeat experiment)

This study is: Adequate Not Adequate

The study is not adequate due to the following deficiency: The highest concentration (~843 μg

a.i./mL or 0.0039 M a.i., calculated based on a concentration of 38.64% CASRN 52556-42-0

(the a.i.) in the test substance) was substantially lower than the maximum concentration (defined 

as 5 μL a.i./mL, 5 mg a.i./mL (5000 μg a.i./mL), or 0.01 M a.i., whichever value is lowest)

recommended by the cited guidelines (OECD TG 473 and TSCA 799.9537) and did not produce 

an increased number of chromosome aberrations, cytotoxicity, or precipitation during the 

definitive chromosomal aberrations test. Also, test concentrations were not selected based on the 

concentration of a.i. in the test substance (i.e., 38.64% CASRN 52556-42-0) as was specified in 

EPA’s response to the sponsor’s request for modification regarding the test substance 

composition.

TEST SUBSTANCE 

Identity: Bimax Product CS-AHPS1; commercial product that contains 38.64% 

1-propanesulfonic acid, 2-hydroxy-3-(2-propen-1-yloxy)-, sodium salt (1:1) (CASRN 52556-42-

0), also known as COPS-1.

Remarks: Clear colorless liquid; Batch No. 12E1101; Composition: 38.64% 1-propanesulfonic 

acid, 2-hydroxy-3-(2-propen-1-yloxy)-, sodium salt (1:1) (CASRN 52556-42-0), 2.26% 3-allyl1,2-propanediol (CASRN 123-34-2), and 59.1% water (CASRN 7732-18-5). 

METHOD

Method/guideline followed: TSCA 799.9537 and OECD TG 473 (1997; equivalent to Method 

B.10 of Commission Regulation (EC) No. 440/2008). The chromosomal aberration test was 

carried out as described by Evans (1984) with minor modifications. 

Test type: Mammalian Chromosome Aberration Test (in vitro)

GLP compliant?: Yes

 

1 EPA accepted the submitter’s request to test Bimax Product CS-AHPS in the toxicity tests required for CASRN 

52556-42-0 given that the only component in this commercial product other than water and CASRN 52556-42-0 is 

CASRN 123-34-2, a presumed byproduct of the synthesis of CASRN 52556-42-0. 
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 52556-42-0

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Year study performed: 2013

Test species: Human peripheral blood lymphocytes; obtained from healthy, non-smoking adult 

males; average generation time: 12.9–13.5 hours.

Metabolic activation: With (phenobarbital and β-naphthoflavone-induced rat S9 microsomal 

fraction) and without 

Concentrations tested: Nominal concentrations (range-finding and/or definitive tests

combined): 1, 10, 33, 100, 333, 1000, 1500, and 2182 μg test substance/mL culture medium; 

equivalent to approximately 0.39, 3.9, 12.8, 38.6, 129, 386, 580, and 843 μg a.i./mL, 

respectively, based on a concentration of 38.64% CASRN 52556-42-0 (the a.i.) in the test 

substance.

Range-Finding Test Concentrations:

Without S9: 3- or 24-h exposure, cell harvest at 24 h: 33, 100, 333, 1000, and 2182 μg test 

substance/mL

Without S9: 48-h exposure, cell harvest at 48 h: 33, 100, 333, 1000, and 2182 μg test 

substance/mL

With S9: 3-h exposure, cell harvest at 24 h: 33, 100, 333, 1000, and 2182 μg test substance/mL

Definitive Chromosomal Aberrations Assay Concentrations:

Experiment 1:

Without S9: 3-h exposure, cell harvest at 24 h: 10, 33, 100, 1000, and 2182 μg test substance/mL

With S9: 3-h exposure, cell harvest at 24 h: 10, 33, 100, 1000, and 2182 μg test substance/mL

Experiment 2:

Without S9: 24-h exposure, cell harvest at 24 h: 33, 100, 1000, 1500, and 2182 μg test 

substance/mL

Without S9: 48-h exposure, cell harvest at 48 h: 33, 100, 1000, 1500, and 2182 μg test 

substance/mL

With S9: 3-h exposure, cell harvest at 48 h: 1, 33, 100, 1000, and 2182 μg test substance/mL

“No correction was made for the purity/composition of the test compound.” [Verbaan 2013, p. 9]

Statistical methods used: “The incidence of aberrant cells (cells with one or more 

chromosome aberrations, gaps included or excluded) for each exposure group outside the 

laboratory historical control data range was compared to that of the solvent control using Chisquare statistics.” [Verbaan 2013, p. 13]

Vehicle: Culture medium

Test method/conditions remarks: Test concentrations were prepared by dissolving the test 

substance in culture medium. Prepared concentrations were filter sterilized with a 0.22 μm filter 

and used within 2.5 hours of preparation. The pH and osmolarity of the highest test 

concentration (nominal concentration of 2182 μg test substance/mL culture medium) were 

recorded. 
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Cell Isolation and Culturing Conditions:

Human lymphocytes were isolated from a blood sample collected from the peripheral circulation 

of three healthy, non-smoking, adult male volunteers, ages 30, 33, and 36. Lymphocyte cultures 

were initiated immediately after blood collection in RPMI 1640 medium containing heparin and 

phytohemagglutinin. After approximately 48 hours of mitogenic stimulation with 

phytohemagglutinin, duplicate cultures were treated with the test substance (or appropriate 

control) in the presence (3 h) or absence (3 h, 24 h, or 48 h) of S9 metabolic activation at 37°C. 

“All incubations were carried out in a controlled environment in the dark, in which optimal 

conditions were a humid atmosphere of 80–100% (actual range 49–95%), containing 5.0 ± 0.5% 

CO2 in air, at a temperature of 37.0 ± 1.0°C (actual range 35.9–37.4°C). Temperature and 

humidity were continuously monitored throughout the experiment. The CO2 percentage was 

monitored once on each working day.” [Verbaan 2013, p. 10]

Preparation of Rat Liver S9 Fractions:

Adult male Wistar rats (obtained from Charles River, Sulzfeld, Germany) were orally 

administered a suspension of phenobarbital (80 mg/kg-bw/day) and β-naphthoflavone (100 

mg/kg-bw/day) in corn oil for three consecutive days. The rats were denied access to food for 3 

to 4 hours before each dosing. The rats were sacrificed on the day following the third dose and 

liver homogenates were prepared from liver tissue isolated from each rat. The homogenates 

were centrifuged and the supernatant (S9) was collected and stored in liquid nitrogen. The S9-

mix for the study was freshly prepared for each test from frozen S9, sterilized with a 0.22 μm 

filter, and stored on ice until use. For assays conducted with metabolic activation, a 

concentration of 1.8% (v/v) S9-mix in the exposure medium was used.

“The S9 batch was characterized in a bacterial reverse mutation assay in Salmonella 

typhimurium tester strain TA98 with the mutagens Benzo-(a)-pyrene... and 2-

aminoanthracene... at concentrations of 5 μg/plate and 1 μg/plate, respectively. These mutagens 

require metabolic activation for exerting their mutagenic effects.” [Verbaan 2013, pp. 10-11]

Positive and Negative Controls:

The following positive controls were included in the chromosomal aberrations assay:

Without S9-mix: Mitomycin C

3-h exposure, 0.5 and 0.75 μg/mL

24-h exposure, 0.2 and 0.3 μg/mL

48-h exposure, 0.1 and 0.15 μg/mL

With S9-mix: Cyclophosphamide

3-h exposure, 10 μg/mL

The solvent for all positive controls was Hanks’ Balanced Salt Solution (HBSS) without calcium 

and magnesium. 

Negative controls for the range-finding test and definitive chromosomal aberrations assay 

consisted of culture medium without the test substance.
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Range-Finding Test Procedure: 

A range-finding test was performed in lymphocytes exposed for 3, 24, or 48 hours in the absence 

of S9-mix or for 3 hours in the presence of S9-mix to concentrations of 0 (solvent control), 33, 

100, 333, 1000, or 2182 μg test substance/mL. After 3 hours of exposure in the absence or 

presence of S9-mix, the cells were collected, rinsed with HBSS, and incubated in culture 

medium for 20 to 22 hours (post-exposure incubation period). Cells were harvested after a total 

of 24 hours (exposure + post-exposure incubation period) had elapsed. The cells that were 

exposed for 24 or 48 hours in the absence of S9-mix were not rinsed after exposure and were 

immediately fixed at the end of the exposure period. Cytotoxicity of the test substance was 

determined from the mitotic index by counting the number of metaphases from at least 1000 

cells.

Definitive Chromosomal Aberrations Assay Procedure:

In the chromosomal aberrations assay, lymphocytes were exposed to the test substance in two 

independent experiments (Experiments 1 and 2), with and without S9 metabolic activation, at 

concentrations of 0 (solvent control), 1, 10, 33, 100, 1000, 1500, or 2182 μg test substance/mL. 

Selected concentrations in Experiments 1 and 2 were based on the results of the range-finding 

test.

Experiment 1: “Lymphocytes were cultured for 48 ± 2 h and thereafter exposed in duplicate to 

selected [concentrations of the test substance] for 3 h in the absence and presence of S9-mix. 

After 3 h exposure, the cells were separated from the exposure medium by centrifugation... The 

supernatant was removed and the cells were rinsed once with 5 ml HBSS. After a second 

centrifugation step, HBSS was removed and cells were resuspended in 5 ml culture medium and 

incubated for another 20–22 h [post-exposure incubation period].” [Verbaan 2013, pp. 11-12]

Experiment 2: “Lymphocytes were cultured for 48 ± 2 h and thereafter exposed in duplicate to 

selected [concentrations of the test substance] for 24 h and 48 h in the absence of S9-mix and for 

3 h in the presence of S9-mix.” [Verbaan 2013, p. 12]

“After 3 h exposure, the cells exposed to [the test substance] in the presence of S9-mix were 

separated from the exposure medium by centrifugation... The supernatant was removed and the 

cells were rinsed once with 5 mL of HBSS and incubated in 5 mL culture medium for another 

44–46 h [post-exposure incubation period]. The cells that were treated for 24 h and 48 h in the 

absence of S9-mix were not rinsed after exposure but were fixed immediately after 24 h and 48 

h...” [Verbaan 2013, p. 12]

Chromosome Preparation and Slide Preparation:

Lymphocytes were arrested in mitosis with colchicine (0.5 μg/mL culture medium) 2.5 to 3 

hours before scheduled harvest. Cells were treated with hypotonic 0.56% (w/v) potassium 

chloride solution at 37°C, fixed with methanol/acetic acid (3:1 v/v mixture), and mounted on 

slides. Slides were stained with 5% (v/v) Giemsa solution in water and embedded in a 1:10 

mixture of xylene/pertex. All slides were randomly coded for evaluation. 
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 52556-42-0

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Mitotic Index Determination and Concentration Selection for Scoring of Metaphases:

“The mitotic index of each culture was determined by counting the number of metaphases from 

at least 1000 cells (with a maximum deviation of 5%). At least three analysable concentrations 

were used for scoring of the cytogenetic assay.” [Verbaan 2013, p. 12]

Analysis of Slides for Chromosomal Aberrations:

“One hundred metaphase chromosome spreads per culture were examined by light microscopy 

for chromosome aberrations. In case the number of aberrant cells, gaps excluded, was ≥ 25 in 50 

metaphases, no more metaphases were examined. Only metaphases containing 46 ± 2 

centromeres (chromosomes) were analyzed. The number of cells with aberrations and the 

number of aberrations were calculated.” [Verbaan 2013, p. 12]

Criteria for Positive Results: 

“A test substance is considered positive (clastogenic) in the chromosome aberration test if: 

a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) 

increase in the number of cells with chromosome aberrations.

b) A statistically significant and biologically relevant increase in the frequencies of the 

number of cells with chromosome aberrations was observed in the absence of a clear 

dose-response relationship.” [Verbaan 2013, p. 13]

Acceptability Criteria: 

“A chromosome aberration test is considered acceptable if it meets the following criteria: 

a) The number of chromosome aberrations found in the solvent control cultures should 

reasonably be within the laboratory historical control data range...

b) The positive control substances should produce a statistically significant (Chi-square test, 

one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.

c) A homogenous response between the replicate cultures is observed.

d) A possible precipitate present on the slides should not interfere with the scoring of 

chromosome aberrations.” [Verbaan 2013, p. 13]

RESULTS

Genotoxicity result: Negative

Cytotoxic dose: No cytotoxicity was observed at the concentrations tested during the definitive 

chromosomal aberrations assay.

Results remarks: The test substance did not induce an increase in the number of cells with 

chromosomal aberrations in the presence or absence of metabolic activation. The test substance

did not induce an increase in the number of polyploid cells or cells with endoreduplicated 

chromosomes. Although some evidence of cytotoxicity was observed in the range-finding test 

(up to 61% inhibition of the mitotic index at the highest concentration tested), a biologically 

significant (> 50% reduction) in mitotic index was not observed at any test concentration during 

the definitive chromosomal aberrations assay. No precipitation of the test substance in the 

culture medium was observed.
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 52556-42-0

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The following concentrations were scored for chromosomal aberrations: 

Experiment 1: Without and with S9-mix: 100, 1000, and 2182 μg test substance/mL (3-h 

exposure time, cell harvest at 24 h)

Experiment 2: Without S9-mix: 33, 1000, and 2182 μg test substance/mL (24-h and 48-h 

exposure time, cell harvest at 24 h and 48 h, respectively)

With S9-mix: 100, 1000, and 2182 μg test substance/mL (3-h exposure time, cell harvest at 48 

h)

“The number of cells with chromosome aberrations found in the solvent control cultures was 

within the laboratory historical control data range... The number of polyploid cells and cells 

with endoreduplicated chromosomes in the solvent control cultures was within the laboratory 

historical control data range... The positive control chemicals [mitomycin C and 

cyclophosphamide] both produced statistically significant increases in the frequency of aberrant 

cells. It was therefore concluded that the test conditions were adequate and that the metabolic 

activation system (S9-mix) functioned properly.” [Verbaan 2013, p. 16]

“The pH and osmolarity of a concentration of 2182 μg [test substance]/ml were 7.95 and 285 

mOsm/kg, respectively (compared to 7.93 and 284 mOsm/kg in the solvent control).” [Verbaan 

2013, p. 14]

CONCLUSIONS

Under the conditions of this test, the test substance did not induce chromosomal aberrations in 

human lymphocytes in vitro.

STUDY RELIABILITY

[3] Not reliable. The highest concentration (~843 μg a.i./mL or 0.0039 M a.i., calculated based 

on a concentration of 38.64% CASRN 52556-42-0 (the a.i.) in the test substance) was 

substantially lower than the maximum concentration (defined as 5 μL a.i./mL, 5 mg a.i./mL 

(5000 μg a.i./mL), or 0.01 M a.i., whichever value is lowest) recommended by the cited 

guidelines (OECD TG 473 and TSCA 799.9537) and did not produce an increased number of 

chromosome aberrations, cytotoxicity, or precipitation during the definitive chromosomal 

aberrations test. Also, test concentrations were not selected based on the concentration of a.i. in 

the test substance (i.e., 38.64% CASRN 52556-42-0) as was specified in EPA’s response to the 

sponsor’s request for modification regarding the test substance composition.

REFERENCE

Verbaan, I.A.J. 2013. Evaluation of the ability of 1-propanesulfonic acid, 2-hydroxy-3-(2-

propen-1-yloxy)-, sodium salt (1:1) to induce chromosome aberrations in cultured peripheral 

human lymphocytes (with repeat experiment). WIL Research Europe B.V., The Netherlands; 

Laboratory Project No. 500681. Sponsor: Society of Chemical Manufacturers and Affiliates 

(SOCMA), Washington, DC. Study report available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0112-0146 as of April 

18, 2014. 
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 52556-42-0

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Text in quotes is taken directly from Verbaan (2013).
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 52556-42-0

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E. DATA ADEQUACY SUMMARY TABLE

Endpoint Study Information 

Available

Acceptable Data Gap

Aquatic Effects

Acute Toxicity to Fish Yes Yes No

Acute Toxicity to Aquatic Invertebrates Yes Yes No

Toxicity to Aquatic Plants Yes Yes No

Health Effects

Acute Toxicity Yes No No1

Repeated-Dose Toxicity Yes Yes No

Gene Mutation Yes No Yes

Chromosomal Aberrations Yes No Yes

Reproductive Toxicity Yes Yes No

Developmental Toxicity Yes Yes No

1 Although no adequate data are available for the acute mammalian toxicity endpoint, EPA does 

not currently consider acute mammalian toxicity testing to be necessary given that adequate data 

are available for the repeated-dose toxicity endpoint.
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 52556-42-0

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APPENDIX: Test Guideline Source Material to Support Reliability Ratings Assigned to 

Submitted Studies That Are Considered Inadequate to Satisfy Test Rule Requirements

This appendix provides additional information regarding deficiencies of each submitted study 

that is considered inadequate to satisfy a requirement in the test rule. For each deviation from 

relevant test guidelines (i.e., standard test guidelines purportedly followed by each study 

according to the study report), quotes of relevant passages from the guidelines and EPA 

correspondence with the submitter are supplied, followed by a brief summary of the quoted 

material and, where appropriate, an interpretation by a qualified scientist. Emphasis has been 

added to quotes (in the form of italics) by the reviewer.

Study 1: Acute Oral Toxicity Study in Rats (van Huygevoort 2013)

Relevant Guidelines: OECD TG 425 (2008) “Acute Oral Toxicity – Up-and-Down Procedure 

(UDP)”1and OPPTS 870.1100 (2002) “Acute Oral Toxicity”

Deviation 1: The administered dose of active ingredient (a.i.) was less than the dose required for 

an acute toxicity limit test. This study failed to identify a discrete LD50 because no mortality was 

observed at the only dose tested (nominal dose of 2000 mg test substance/kg-bw; equivalent to 

772.8 mg a.i./kg-bw based on a concentration of 38.64% CASRN 52556-42-0 (the a.i.) in the test 

substance). The study only determined that the LD50 was greater than 772.8 mg a.i./kg-bw, 

which is considerably less than the limit dose of 2000 mg a.i./kg-bw or 5000 mg a.i./kg-bw 

specified in the acute toxicity test guidelines (i.e., OECD TG 425 and OPPTS 870.1100). Also, 

the selection of the limit dose was not based on the concentration of a.i. in the test substance (i.e., 

38.64% CASRN 52556-42-0) as was specified in EPA’s response to the sponsor’s request for 

modification regarding the test substance composition (Richards et al., 2012).

Richards et al. (2012) states: “EPA notes that for all toxicity studies, the selection of 

doses/concentrations,... reporting of dose/concentration vs. response, and values of toxicity 

endpoints shall be based on mg or g of the active ingredient, CASRN 52556-42-0.” 

OECD TG 425, p. 1, Section “Initial Considerations”, item 6 states: “The method permits 

estimation of an LD50 with a confidence interval and the results allow a substance to be 

ranked and classified according to the Globally Harmonized System for the classification of 

chemicals which cause acute toxicity...”

OECD TG 425, p. 2, Section “Principle of the Limit Test”, item 12 states: “The Limit 

Test is a sequential test that uses a maximum of 5 animals. A test dose of 2000, or 

exceptionally 5000 mg/kg, may be used... (see paragraphs 23-25 for limit test at 2000 mg/kg 

and paragraphs 26-30 for limit test at 5000 mg/kg).” 

OECD TG 425, p. 10, Section “Data and Reporting”, Sub-section “Test report”, item 48 

states: “The test report must include the following information:”; and part entitled 

“Results” states: “LD50 data”.

OPPTS 870.1100, pp. 1-2, Section (c) “Definitions” states: “... The following definitions 

also apply to this test guideline.” and “LD50 (median lethal dose), oral, is a statistically 

derived single dose of a substance that can be expected to cause death in 50 per cent of 

animals when administered by the oral route. The LD50 value is expressed in terms of weight 

 

1 OECD TG 425 is equivalent to the method described in 40 CFR 799.9110(d)(1)(i)(A), which is the specified 

guideline for the acute mammalian toxicity testing requirement in the HPV3 Test Rule.
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 52556-42-0

47

of test substance per unit weight of test animal (mg/kg).” and “Limit dose refers to a dose at 

an upper limitation on testing (2000–5000 mg/kg).”

OPPTS 870.1100, p. 5, Section (f) “Principle of the limit test” states: “The limit test is a 

sequential test that uses a maximum of 5 animals (see paragraphs (i)(2)(i) through (i)(2)(iv) of 

this guideline). A test dose of 5000 mg/kg is used.” and p. 9, Section (i) “The up-and-down 

testing procedure”, Sub-section (2) “Implementation of the limit test”, item (v) states: 

“If a limit test is performed at 2000 mg/kg, animals should be dosed sequentially and testing 

should be performed on all five animals.” 

OPPTS 870.1100, pp. 15-16, Section (l) “Test reporting” states: “The test report must

include the following information:” and Sub-section (5) “Results:”, item (v) states: “LD50

and CI”.

Summary of Quoted Material: A primary objective of an acute toxicity study is to determine a 

discrete LD50 value. In lieu of a full study (with multiple test doses), a limit test may be 

conducted in which only one dose (the limit dose) is tested to determine if the LD50 is greater 

than the limit dose; if the LD50 is not greater than the limit dose, additional (lower) doses need to 

be tested. In acute toxicity tests, the test substance dose in the limit test is either 2000 mg/kg-bw 

or 5000 mg/kg-bw. Interpretation: To adequately conduct an acute oral toxicity limit test, an 

appropriate limit dose needs to be at least 2000 mg a.i./kg-bw. 

REFERENCES

van Huygevoort, A.H.B.M. 2013. Assessment of acute oral toxicity with 1-propanesulfonic 

acid, 2-hydroxy-3-(2-propen-1-yloxy)-, sodium salt (1:1) in the rat (up and down procedure). 

WIL Research Europe B.V., The Netherlands; Laboratory Project No. 500679. Sponsor: 

Society of Chemical Manufacturers and Affiliates (SOCMA), Washington, DC. Study report 

available online at http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-

0112-0146 as of April 18, 2014.

Richards, W.L., Manyin, T., and Morlacci, L. 2012. HPV3 test rule: Response to submitter’s 

request for modification regarding test substance composition, 1-Propanesulfonic acid, 2-

hydroxy-3-(2-propen-1-yloxy)-, sodium salt (1:1), CASRN 52556-42-0. SRC Technical Report 

No. TR-12-092. 
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 52556-42-0

48

Study 2: Mutagenicity Study in Salmonella typhimurium and Escherichia coli (Verspeek-Rip 

2013)

Relevant Guidelines: TSCA 799.9510 (equivalent to OECD TG 471 (1997) and Method 

B.13/14 of Commission Regulation (EC) No. 440/2008)

Deviation 1: The highest test concentration (1932 μg a.i./plate, calculated based on a 

concentration of 38.64% CASRN 52556-42-0 (the a.i.) in the test substance) was substantially 

lower than the maximum concentration of 5 mg a.i./plate (5000 μg a.i./plate) that is 

recommended by the cited test guidelines (OECD TG 471 and TSCA 799.9510) and did not 

produce an increased number of revertant colonies, cytotoxicity, or precipitation during the test. 

Also, test concentrations were not selected based on the concentration of a.i. in the test substance 

(i.e., 38.64% CASRN 52556-42-0) as was specified in EPA’s response to the sponsor’s request 

for modification regarding the test substance composition (Richards et al., 2012).

Richards et al. (2012) states: “EPA notes that for all toxicity studies, the selection of 

doses/concentrations,... reporting of dose/concentration vs. response, and values of toxicity 

endpoints shall be based on mg or g of the active ingredient, CASRN 52556-42-0.” 

TSCA 799.9510, p. 419, Section (e) “Test method”, Sub-section (2) “Description”, item 

(ii) “Test conditions”, sub-item (B) “Exposure concentrations”, part (1) states: 

“Amongst the criteria to be taken into consideration when determining the highest amount of 

test substance to be used are cytotoxicity and solubility in the final treatment mixture... The 

recommended maximum test concentration for soluble non-cytotoxic substances is 5 mg/plate 

or 5 μl/plate. For non-cytotoxic substances that are not soluble at 5 mg/plate or 5 μl/plate, 

one or more concentrations tested should be insoluble in the final treatment mixture. Test 

substances that are cytotoxic already below 5 mg/plate or 5 μl/plate should be tested up to a 

cytotoxic concentration.”

Summary of Quoted Material: The maximum concentration tested should induce cytotoxicity, 

be insoluble (i.e., induce precipitation), or be equivalent to 5 mg/plate (5000 μg/plate). 

Interpretation: To adequately conduct a bacterial reverse mutation assay, the selected 

concentration range needs to include a clearly toxic concentration, exhibit limited solubility by 

producing visible precipitation, or extend to 5 mg a.i./plate (5000 μg a.i./plate).

REFERENCES

Verspeek-Rip, C.M. 2013. Evaluation of the mutagenic activity of 1-propanesulfonic acid, 2-

hydroxy-3-(2-propen-1-yloxy)-, sodium salt (1:1) in the Salmonella typhimurium reverse 

mutation assay and the Escherichia coli reverse mutation assay (with independent repeat). WIL 

Research Europe B.V., The Netherlands; Laboratory Project No. 500680. Sponsor: Society of 

Chemical Manufacturers and Affiliates (SOCMA), Washington, DC. Study report available 

online at http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0112-0146 as 

of April 18, 2014.

Richards, W.L., Manyin, T., and Morlacci, L. 2012. HPV3 test rule: Response to submitter’s 

request for modification regarding test substance composition, 1-Propanesulfonic acid, 2-
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 52556-42-0

49

hydroxy-3-(2-propen-1-yloxy)-, sodium salt (1:1), CASRN 52556-42-0. SRC Technical Report 

No. TR-12-092. 
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 52556-42-0

50

Study 3: Chromosome aberration assay in human lymphocytes (Verbaan, 2013)

Relevant Guidelines: TSCA 799.9537 and OECD TG 473 (1997; equivalent to Method B.10 of 

Commission Regulation (EC) No. 440/2008).

Deviation 1: The highest concentration (~843 μg a.i./mL or 0.0039 M a.i., calculated based on a 

concentration of 38.64% CASRN 52556-42-0 (the a.i.) in the test substance) was substantially 

lower than the maximum concentration (defined as 5 μL a.i./mL, 5 mg a.i./mL (5000 μg a.i./mL), 

or 0.01 M a.i., whichever value is lowest) recommended by the cited guidelines (OECD TG 473 

and TSCA 799.9537) and did not produce an increased number of chromosome aberrations, 

cytotoxicity, or precipitation during the definitive chromosomal aberrations test. Also, test 

concentrations were not selected based on the concentration of a.i. in the test substance (i.e., 

38.64% CASRN 52556-42-0) as was specified in EPA’s response to the sponsor’s request for 

modification regarding the test substance composition (Richards et al., 2012).

Richards et al. (2012) states: “EPA notes that for all toxicity studies, the selection of 

doses/concentrations,... reporting of dose/concentration vs. response, and values of toxicity 

endpoints shall be based on mg or g of the active ingredient, CASRN 52556-42-0.” 

OECD TG 473, p. 3, Section “Description of the Method”, Sub-section entitled “Test 

conditions”, item entitled “Exposure concentrations”, sub-item 14 states: “Among the 

criteria to be considered when determining the highest concentration are cytotoxicity, 

solubility in the test system, and changes in pH or osmolality.”

OECD TG 473, p. 3, Section “Description of the Method”, Sub-section entitled “Test 

conditions”, item entitled “Exposure concentrations”, item 16 states: “At least three

analysable concentrations should be used. Where cytotoxicity occurs, these concentrations 

should cover a range from the maximum to little or no toxicity... At the time of harvesting, 

the highest concentration should show a significant reduction in degree of confluency, cell 

count or mitotic index, (all greater than 50%)... For relatively non-cytotoxic compounds the 

maximum concentration should be 5 μl/ml, 5 mg/ml, or 0.01M, whichever is the lowest.”

OECD TG 473, p. 3, Section “Description of the Method”, Sub-section entitled “Test 

conditions”, item entitled “Exposure concentrations”, item 17 states: “For relatively 

insoluble substances that are not toxic at concentrations lower than the insoluble 

concentration, the highest dose used should be a concentration above the limit of solubility in 

the final culture medium at the end of the treatment period.”

TSCA 799.9537, pp. 431-432, Section (f) “Description of the method”, Sub-section (2) 

“Test conditions”, item (ii) “Exposure concentrations”, sub-item (A) states: “Among the 

criteria to be considered when determining the highest concentration are cytotoxicity, 

solubility in the test system, and changes in pH or osmolality.” and sub-item (C) states: “At 

least three analyzable concentrations should be used. Where cytotoxicity occurs, these 

concentrations should cover a range from the maximum to little or no toxicity... At the time 

of harvesting, the highest concentration should show a significant reduction in degree of 

confluency, cell count or mitotic index, (all greater than 50%)... For relatively non-cytotoxic 

compounds the maximum concentration should be 5 μ[l]/ml1, 5 mg/ml, or 0.01M, whichever 

is the lowest.” and sub-item (D) states: “For relatively insoluble substances that are not 

 

1 This value is shown as 5 μg/ml as the first criterion of maximum concentration in TSCA 799.9537, but has been 

assumed herein (and edited herein) to be 5 μl/ml as presented in similar guidelines for this mammalian chromosomal 

aberrations test such as OECD TG 473.
Final HPV3 Test Rule Data Adequacy Review (April 2014) CASRN 52556-42-0

51

toxic at concentrations lower than the insoluble concentration, the highest dose used should be 

a concentration above the limit of solubility in the final culture medium at the end of the 

treatment period.”

Summary of Quoted Material: The highest concentration tested should induce cytotoxicity, be 

insoluble (i.e., induce precipitation), or be equivalent to 5 μL/mL, 5 mg/mL (5000 μg/mL), or 

0.01 M, whichever is the lowest. Interpretation: To adequately conduct an in vitro mammalian 

chromosome aberration test, the selected concentration range needs to include a clearly toxic 

concentration or exhibit limited solubility as demonstrated by precipitation during the test, or the 

maximum concentration needs to be the lowest of 5 μL a.i./mL, 5 mg a.i./mL (5000 μg a.i./mL),

or 0.01 M a.i.

REFERENCES

Verbaan, I.A.J. 2013. Evaluation of the ability of 1-propanesulfonic acid, 2-hydroxy-3-(2-

propen-1-yloxy)-, sodium salt (1:1) to induce chromosome aberrations in cultured peripheral 

human lymphocytes (with repeat experiment). WIL Research Europe B.V., The Netherlands; 

Laboratory Project No. 500681. Sponsor: Society of Chemical Manufacturers and Affiliates 

(SOCMA), Washington, DC. Study report available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0112-0146 as of April 

18, 2014.

Richards, W.L., Manyin, T., and Morlacci, L. 2012. HPV3 test rule: Response to submitter’s 

request for modification regarding test substance composition, 1-Propanesulfonic acid, 2-

hydroxy-3-(2-propen-1-yloxy)-, sodium salt (1:1), CASRN 52556-42-0. SRC Technical Report 

No. TR-12-092.