SRC TR

- 1 4

- 005

HPV 3 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 .009.00 6
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 , 201 4Final HPV 3 Test Rule Data Adequacy Review ( April 2014) CASRN 52556 - 42 - 0

i TABLE OF CONTENTS A. SOURCE MATERIAL USED FOR ASSESSMENT
................................ .............................. 1 B. EVALUATING DATA ADEQUACY
................................ ................................ ..................... 2 C. SUMMARY OF DATA ADEQUACY FOR TESTING REQUIRED BY TEST RULE
........ 2 D. DATA FOR SIDS ENDPOINTS
................................ ................................ .............................. 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
............ 46Final HPV 3 Test Rule Data Adequacy Review ( April 2014) CASRN 52556 - 42 - 0

1 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

- OPPT

2009

- 0112

- 0086 as of April 18 , 2014). The HPV3 test rule requires the following toxicolog ical 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 K ow < 4.2 or Test Group 2 [chronic toxicity to Daphnia and toxicity to algae testing] if log K ow ≥ 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 K ow of

- 5.00 1 , 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 i n 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 s alt (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 Toxicit y with 1

- Propanesulfonic acid, 2

- hydroxy

- 3

- (2

- propen 1

- 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

- propen 1

- yloxy)

- , s odium 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

- pr open - 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 K ow 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 HPV 3 Test Rule Data Adequacy Review (April 2014) CASRN 52556 - 42 - 0

2 These newly submitted data are evaluated for adequacy herein. No other toxicity data ar e 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 tha t 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 inade quate 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 toxic ity to Daphnia (component of C1), toxicity to algae (component of C1), and combined repeated

- dose / reproducti ve /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 pr ovided 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 identif y a discrete LD 50 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 o nly determined that the LD 50 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 maximumFinal HPV 3 Test Rule Data Adequacy Review (April 2014) CASRN 52556 - 42 - 0

3 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 o f 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 concen tration (~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 compositio n (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 endpoin ts. 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 (Richa rds et al. , 2012). Specifically, in accordance with test guidelines: I f 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.Final HPV 3 Test Rule Data Adequacy Review (April 2014) CASRN 52556 - 42 - 0

4 D . DATA FOR SIDS ENDPOINTS D - 1. Acute Toxicity to Fish Summary of endpoint: A dequate Study 1: Acute toxicity study in fathead minnow ( Bouwman , 2013 a ) Title: 96

- Hour acute toxicity study in fathead minnow 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

- AHPS 1 ; 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 s alt (1:1) (CASRN 52556

- 42

- 0), 2.26% 3

- allyl

1,2

- 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 comm ercial 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 HPV 3 Test Rule Data Adequacy Review (April 2014) CASRN 52556 - 42 - 0

5 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 2013 a , p. 8] Test method/conditions remarks: Test Medi um : The t est medium was prepared with ISO medium formulated with reverse osmosis

- purified tap water and adjusted to a hardness of 180 mg CaCO 3 /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 2013 a , p. 8] Exposure Conditions: Prior to transfer to the test vessels, t he fish were acclimat ed 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 no t 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. I llumination wa s provided as a daily 16

- hour photoperiod and the t est media were not aerated. The f ish 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 2013 a , p. 10] Temperature, pH, and d issolved 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
. C oncentrations were measured according to a validated analytical method 2 using 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 metho d 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=EPA

HQ

- OPPT

- 2009

- 0112

- 0146 as of April 18 , 2014.Final HPV 3 Test Rule Data Adequacy Review (April 2014) CASRN 52556 - 42 - 0

6 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 concentration s were at least 60% of the air saturation value (i.e., > 5 mg/L) throughout the test.  Measured t est substance concentrations we re at least 80% of nominal concentrations throughout the test. RESULTS Endpoint value(s): Based on nominal concentrations: 96

- hour LC 50 > 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 (9 9

– 101% of initial at the end of the test). Given these results, the effect parameters were based on the nominal exposure concentration.” [ Bouwman 2013 a , p. 10] The test met the criteria for acceptability prescribed by the protocol. CONCLUSIONS Based on nominal concentrations, t he 96

- hour LC 50 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 r estrictions
. T he duration of acclim at i on of the test organisms was not provided in the study report and both OPPTS 8 5 0.1075 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 LC 50 was not determined and t he 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 HPV 3 Test Rule Data Adequacy Review (April 2014) CASRN 52556 - 42 - 0

7 REF ERENCE Bouwman, L.M. 2013 a . 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 , 201 4
. Text in quotes is taken directly from Bouwman (2013 a ).Final HPV 3 Test Rule Data Adequacy Review (April 2014) CASRN 52556 - 42 - 0

8 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

- AHPS 1 ; 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 s alt (1:1) (CASRN 52556

- 42

- 0), 2.26% 3

- allyl

1,2

- 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 HPV 3 Test Rule Data Adequacy Review (April 2014) CASRN 52556 - 42 - 0

9 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.) 1 based 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 (contro l) 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 (ad j usted 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 s ubstance 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] Cont rol 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 nomi nal 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

- 0Final HPV 3 Test Rule Data Adequacy Review (April 2014) CASRN 52556 - 42 - 0

10 The daphnids were observed for immobility/mortality at 24 and 48 hours. The temperat ure 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 a t 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 contr ol and test vessels at the end of the test. RESULTS Endpoint value(s): Based on nominal concentrations: 48

- hour EC 50 > 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 immobiliz ation 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 con centration.” [ Bouwman 2013b, p. 11] The definitive test met the criteria for acceptability prescribed by the protocol.Final HPV 3 Test Rule Data Adequacy Review (April 2014) CASRN 52556 - 42 - 0

11 CONCLUSIONS Based on nominal concentrations, the 48

- hour EC 50 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).Final HPV 3 Test Rule Data Adequacy Review (April 2014) CASRN 52556 - 42 - 0

12 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

- AHPS 1 ; 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; Composit ion: 38.64% 1

- propanesulfonic acid, 2

- hydroxy

- 3

- (2

- propen - 1

- yloxy)

- , sodium salt (1:1) (CASRN 52556

- 42

- 0), 2.26% 3

- allyl

1,2

- 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 pr oduct 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 HPV 3 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.) 1 based 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 concentr ations: 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/compos ition 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 h ardness of 24 mg CaCO 3 /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 c ell density of 1 x 10 4 cells/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 b y 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 C ultures 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 s uspension was added to each replicate vessel, providing an initial cell density of 10 4 cells/mL. The vessels were randomly distributed in the incubator and repositioned daily throughout the exposure period. Temperature was monitored continuously in a tem perature 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 in tensity 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

- 0Final HPV 3 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, an d 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 201 1 , with 2.6 μg ZnCl 2 /L, 2 μg CoCl 2 ·6H 2 O/L, 0.017 μg CuCl 2 ·2H 2 O/L, and 12.6 μg Na 2 MoO 4 ·2H 2 O/L. Definitive Test Procedure: Freshwater algae (cell density of 1 x 10 4 cells/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 t est 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 t he 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 range

finding and definitive tests. “At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter cell densities were dete rmined 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 [contr ol] 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 ZnCl 2 /L, 1.5 μg CoCl 2 ·6H 2 O/L , 0.01 μg CuCl 2 ·2H 2 O/L , and 7 μg Na 2 MoO 4 ·2H 2 O/L
.Final HPV 3 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 EC 50 (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 10 4 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 concentrati ons ≥ 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, replicat e 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 replic ate 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 cellFinal HPV 3 Test Rule Data Adequacy Review (April 2014) CASRN 52556 - 42 - 0

16 den sit ies 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 10 4 cells/m L , 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% (gr owth 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 inhibi tion). 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 [duri ng 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 10 4 cells/mL and 207.4 x10 4 cells/mL, respectively. Mean percentage values for inhibitio n 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 contro l.” [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).” [Bou wman 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 HPV 3 Test Rule Data Adequacy Review (April 2014) CASRN 52556 - 42 - 0

17 CONCLUSIONS Based on nominal concentrations, the 96

- hour EC 50 values for inhibition of growth, yield, and cell growth were all greater than 1000 mg test substance/L (> 386 mg a.i./L); t he 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 te sts. 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 eith er 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 ran ge - 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 definit ive test suggests that these data are reliable. Although an EC 50 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 HPV 3 Test Rule Data Adequacy Review (April 2014) CASRN 52556 - 42 - 0

18 D - 4. Acute Toxicity Summary of endpoint: Not a dequate
. 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 toxic ity 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) Th is study is: Adequate Not Adequate The study is not adequate due to the following deficiencies: T he administered dose of active ingredient (a.i.) was less than the dose required for a n acute toxicity limit test . T his study failed to identify a discrete LD 50 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 LD 50 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

- 4 2

- 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

- AHPS 1 ; commercial product that contains 38.64% 1

- p ropanesulfonic 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

- 4 2

- 0), 2.26% 3

- allyl

1,2

- propanediol (CASRN 123

- 34

- 2), and 59.1% water (CASRN 7732

- 18

- 5). METHOD Method/guideline followed: OECD TG 425 (200 8 ) “Acute Oral Toxicity

– Up

- and

- Down Procedure (UDP)” 2 and 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 52 556

- 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 T est Rule.Final HPV 3 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 ini tiation: 9

– 10 w eeks; 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./kg

bw , 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 t he undiluted test material was administered to each of five rat s 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, t he a nimals were monitored for mortality twice daily for 14 days. C linical signs were recorded at 2 and 4 hours post
- dosing, and o nce 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 e xamined by g ross necropsy . RESULTS Endpoint value(s): LD 50 > 2000 mg test substance /kg

- bw; LD 50 > 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 du ring the observation period and no adverse effects on body weight gain were apparent. No abnormalities were found during necropsy.Final HPV 3 Test Rule Data Adequacy Review (April 2014) CASRN 52556 - 42 - 0

20 CONCLUSIONS The oral LD 50 for female rats was > 2000 mg test substance /kg

- bw, corresponding to > 772.8 mg a.i./kg

- bw base d 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. T his study failed to identify a discrete LD 50 because no mortality was observed at the only dose tested (n ominal 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 LD 50 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. S ponsor: 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 , 201 4
. Text in quotes is taken directly from van Huygevoort (2013).Final HPV 3 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 E ndpoint: A dequate Summary of Reproductive Toxicity Endpoint: A dequate Summary of Developmental Toxicity Endpoint: A dequate 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

- AHPS 1 ; 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 s alt (1:1) (CASRN 52556

- 42

- 0), 2.26% 3

- allyl

1,2

- propanediol (CASRN 123

- 34

- 2), and 59.1% water (CASRN 7732

- 18

- 5). METHOD Method/guideline followed: O ECD 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 Reproducti ve /Develo pmental 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 1 1 weeks old and weighed 302

- 3 3 9 g (males) and 191

- 21 5 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 HPV 3 Test Rule Data Adequacy Review (April 2014) CASRN 52556 - 42 - 0

22 Number of animals/sex/dose: 1 0 Route of administration: Oral (gavage) Vehicle: Purified water (Millipore) Exposure duration: Males: 29 days; Females: 4 2

- 4 8 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 1 000 mg test substance /kg

- bw/day ; equivalent to approximately 0, 38 .6 , 11 6 , 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 Otterdi jk 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 contro l groups for each sex if the data could not be assumed to follow a normal distribution. Frequency data were evaluat ed with the Fisher Exact test. Motor activity data were evaluated for intergroup differences with the Kruskal

- Wallis non parametric 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 t est substance formulations were evaluated for accuracy (all concentrations)

1 Active ingredient (a.i.) = CASRN 52556

- 42

- 0Final HPV 3 Test Rule Data Adequacy Review (April 2014) CASRN 52556 - 42 - 0

23 and homogeneity (highest and lowest concentrations) according to a validated analytical method 1 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 acceptab le 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 , 11 6 , 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 expos ure periods of 29 consecutive days (males) or 42

- 4 8 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

- to
one 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 the ir or iginal cage s . 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 complet ed (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 d etailed 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 ( [cage

side] clinical signs and arena) were conducted after dosing... within a similar time period after dosing for the respective animals. The time of o nset, 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 cer tain 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 l actation 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=EPA

HQ

- OPPT

- 2009

- 0112

- 0146 as of April 18 , 201 4
.Final HPV 3 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 a nimals (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 str ength, and locomotor activity. M ales were tested during week 4 of treatment. S elected 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 ni ght prior to scheduled necropsy . The following hematological parameters were evaluated in blood prepared with K 3 - 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), R BC 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 tim e and activated partial thromboblastin time
. Clinical chemistry parameters evaluated in blood treated with lithium heparin as an anti

coagulant 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 post partum

/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 we ights 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 gland s, br ain, prostate, h eart, kidneys, liver, ovaries, spleen, thymus, uterus with cervix , seminal vesicles with coagulating glands, and thyroid including parathyroid
. O rgan 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: a drenal glands, brain (cerebellum, mid

- bra in, and cortex), cecum,Final HPV 3 Test Rule Data Adequacy Review (April 2014) CASRN 52556 - 42 - 0

25 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 detec table, 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 sa mples 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 deliver y , 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 la ctation or feeding) were examined.” [ van Otterdijk 2013, p. 15] At scheduled necropsy, t he numbers of implan t ation 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 mal es that were suspected to be infertile. Reproductive organs were weighed, as described above. Histological examinations were performed on t he following reproductive tissues from all parental animals: clitoral gland, epididymides, ovaries, preputial gland s, 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 ob servations of offspring were made for all pups at least once daily. The number of live and dead pups was determined d aily beginning on Day 1 of lact ation. The cause of death or defects in dead pups was evaluated when possible. Pups were weighed and sexe d 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 d ate were examined for the presence of milk. If possible, defects or cause of death were evaluated.” [ van Otterdijk 2013, p. 19]Final HPV 3 Test Rule Data Adequacy Review (April 2014) CASRN 52556 - 42 - 0

26 RESULTS Endpoint value(s): Systemic Toxicity: LOAEL ( female, systemic toxicity) = 300 mg test substance /kg

- bw/day (= 11 6 mg a.i./kg

bw/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./kg

bw/day)
. Reproductive Toxicity: LOAEL (reproductive toxicity) = 1 00 mg test substance /kg

- bw/day (= 38.6 mg a.i./kg

- bw/day), based on reduc tions in the mean number s 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./kg

bw/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 s howed t hat the formulations (nominal doses of 100, 300, and 1000 mg test substance /kg

- bw/day) were within 99

– 1 0 2% 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 /kg

bw/day group was euthanized before scheduled necropsy due to tot al 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 s train 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 anal yses of females in the mid

- dose group (300 mg test substance /kg

- bw/day) during the post

coital period and lactation were based on the mean value for the two pregnant females in thisFinal HPV 3 Test Rule Data Adequacy Review (April 2014) CASRN 52556 - 42 - 0

27 group. At 300 mg test substance /kg

- bw/day , mean female body weights wer e significantly (p < 0.01) reduced on post

- coitum days 17 and 20 by 16 % and 24 %, respectively, relat ive to controls. Mean f emale 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 f ood consumption during this time period (po st
- coitum day intervals 14

– 17 and 17

– 20) was s ignificantly reduced at 300 mg test substance /kg

- bw/day by 3 0% (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 /kg

bw/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 ther efore 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 find i ng 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 reduc ed 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 < 0.0 1) 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 testFinal HPV 3 Test Rule Data Adequacy Review (April 2014) CASRN 52556 - 42 - 0

28 substance /kg

- bw/day groups by 4 .0 % (p < 0.05) and 5 .1 % (p < 0.01) , re spectively , 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 2 6 % , respectively, relative to the control group . Relative liver weights of females were significantly reduced at 300 and 1000 m g test substance /kg

- bw/day (p < 0.05 and p < 0.01, respectively) by 1 5 % and 2 1 % , 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 6 5 % , respectively, relative to controls. Relative thymus weights were significantly (p < 0.01) increased in females at 300 and 1000 mg test substance /kg

bw/day by 9 5 % and 7 7 % , 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, rela tive 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. 0 5) 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.” [ va n 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 ind ex 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 f emales in the 0, 100, 300, and 1000 mg test substance /kg

bw/day treatment groups , respectively, which delivered 9, 8, 0, and 0 litters, respectively. TheFinal HPV 3 Test Rule Data Adequacy Review (April 2014) CASRN 52556 - 42 - 0

29 mean number of c orpora lutea per dam in the 100 and 300 mg test substance /kg

- bw/day groups was decreased by 22% and 25%, respectively, relat ive to the control group
. The mean number of i mplantation 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
. The refore, the EPA reviewer considers the reduced numbers of corpora lutea and implantation sites in the low

- a nd 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 an d 1 implantation site, delivered a single pup which died on lactation day 5. A nother female in the same group did not deliver a litter and had one corpora lutea and one implantation site. Only t wo 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 ; o ne female had 20 corpora lutea and 1 implantation site, while the other had 2 corpora lutea and 2 implantation sites. No pregnan cies 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 m a de 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 Of fspring: 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 /kg

bw/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/missin g pups since the mortality incidences remained within the range considered normal for pups of this age.” [ van Otterdijk 2013, p. 25]Final HPV 3 Test Rule Data Adequacy Review (April 2014) CASRN 52556 - 42 - 0

30 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 , an d 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] F 1 Offspring Necropsy: Findings in p ups 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 ( 11 6 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./kg

bw/day). Reproductive Toxicity: The LOAEL for reproductive toxicity is 1 00 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
. The NOAEL for reproductive toxicity is not established
. Developmental Toxicity: The LOAEL for developmental toxicity is 1 00 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. 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 HPV 3 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 a vailable 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 Otter dijk (2013).Final HPV 3 Test Rule Data Adequacy Review (April 2014) CASRN 52556 - 42 - 0

32 D - 6 . Genetic Toxicity

– Gene Mutation Summary of endpoint: Not a dequate Study 1: Mutagenicity study in Salmonella typhimurium and Escherichia coli ( Verspeek Rip , 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) Th is study is : Adequate Not Adequate The study is not adequate due to the following deficienc y : 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 ci ted 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 s ubstance (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

- AHPS 1 ; 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 s alt (1:1) (CASRN 52556

- 42

- 0), 2.26% 3

- allyl

1,2

- 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 E PA 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 HPV 3 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 ( p henobarbital 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. T he t est c oncentrations 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 ofFinal HPV 3 Test Rule Data Adequacy Review (April 2014) CASRN 52556 - 42 - 0

34 1.0 ± 0.1 at 700 nm (10 9 cells/m l ). Freshly grown cultures of each strain were used for [all tests].” [ Verspeek

- Rip 2013, p. 9]

“ Agar plates (9 cm) contained 25 m l 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/pla te biotin
... and 15 μg/plate histidine
... and the agar plates for the test with the Escherichia coli strain contained 15 μg/p late tryptophan
... ” [ Verspeek

- Rip 2013, p. 9] To p 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. T he t op agar was then transferred to glass tubes and autoclaved for 20 minutes
. For each test, top agar was melted by heating to 45°C an d the following solutions were successively added to 3 mL of molten top agar: 0.1 mL fresh bacterial culture (10 9 cells/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 ea ch 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 homogenate s w ere 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 B enzo - (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 uvrAFinal HPV 3 Test Rule Data Adequacy Review (April 2014) CASRN 52556 - 42 - 0

35 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

- Aminoanthrac ene 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 dimeth y lsulfoxide 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 te st 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

- mix 1

. 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 Assessme nt: “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 e valuated, 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 procedu ral error .Final HPV 3 Test Rule Data Adequacy Review (April 2014) CASRN 52556 - 42 - 0

36 The test substance was considered to be toxic if there was a reduction in the bacterial background lawn and/or a biologically relevant decreas e in the mean number of revertant colonies per plate. Criteria for Pos i tive 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 chemic als 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 re sult: 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 f ive strains tested in the range

- finding or definitive tests . Test substance p recipitat ion was not seen on any of the plates. The test substance was not cytotoxic at any test concentration i n 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. 1 3 ] 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. InFinal HPV 3 Test Rule Data Adequacy Review (April 2014) CASRN 52556 - 42 - 0

37 addition, each positive control mean plate count wa s 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 d id 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 ( O ECD TG 471 and TSCA 799.9510) and did not produce an increased number of revertant colonies , cytotoxicity, or precipitation during the test
. Also, t est 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. D ue 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 re sults a s the strain was tested at four higher concentrations and no toxicity, precipitation, or increase in revertant colonies w as observed. Addit i onally, assays at 100 μg test substance /plate in the absence of S9

- mix were conducted in triplicate in the range

- fi nding 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/#!documentDeta il;D=EPA

- HQ

- OPPT

- 2009

- 0112

- 0146 as of April 18 , 2014
. Text in quotes is taken directly from Verspeek

- Rip (2013).Final HPV 3 Test Rule Data Adequacy Review (April 2014) CASRN 52556 - 42 - 0

38 D - 7 . Genetic Toxicity

– Chromosomal Aberrations Summary of endpoint: Not a dequate Study 1: Chromosom e aberration assay in human lymphocytes ( Verbaan , 20 13 ) 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 lymph ocytes (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 abe rrations 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 c omposition. TEST SUBSTANCE Identity: Bimax Product CS

- AHPS 1 ; 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

- allyl

1,2

- 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 T est ( in vi tro ) 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 HPV 3 Test Rule Data Adequacy Review (April 2014) CASRN 52556 - 42 - 0

39 Year study performed: 20 13 Test species: Human peripheral blood lymphocytes ; obtained from healthy , non - smoking adult males ; average generation time: 12.9

– 13.5 hours
. Metabolic activation: With ( p henobarbital 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 a pproximate ly 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 subst ance /mL With S 9 : 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 Chi

square 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 ) w ere recorded.Final HPV 3 Test Rule Data Adequacy Review (April 2014) CASRN 52556 - 42 - 0

40 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 med ium 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% CO 2 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 CO 2 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 st ored 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 th e exposure medium was used. “The S9 batch was characterized in a bacterial reverse mutation assay in Salmonella typhimurium tester strain TA98 with the mutagens B enzo - (a)

- pyrene ... and 2

- aminoanthracene
... at concentrations of 5 μg/plate and 1 μg/plate, respe ctively. 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 : Mit omycin 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 substanc e.Final HPV 3 Test Rule Data Adequacy Review (April 2014) CASRN 52556 - 42 - 0

41 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 tota l of 24 hours (exposure + post

- exposure incubation period) had elapsed. The c ells 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 t he test substance was det ermined from the mitotic index by counting the number of metaph ases 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, t he 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 m l culture medium and incuba ted 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 m L of HBSS and incubated in 5 m L 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 rins ed 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 xyl ene/pertex. All slides were randomly coded for evaluation.Final HPV 3 Test Rule Data Adequacy Review (April 2014) CASRN 52556 - 42 - 0

42 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 met a phases containing 46 ± 2 centromeres (chromosomes) were analyzed. The num ber of cells with aberrations and the number of aberrations were calculated.” [ Verbaan 2013, p. 12] Criteria for Pos i tive 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, on e - 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 aberratio ns was observed in the absence of a clear dose

- response relationship. ” [ Ver baan 2013, p. 1 3 ] Acceptability Criteria: “A chromosome aberration test is considered acceptable if it meets the following criteria: a) The number of chromosome aberrations found i n the solvent control cultures should reasonably be wit h in the laboratory historical control data range
... b) The positive control substances should produce a statistically si gnificant (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 chromosom al 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 conce ntration during the definitive chromosomal aberrations assay. No precipitation of the test substance in the culture medium was observed
.Final HPV 3 Test Rule Data Adequacy Review (April 2014) CASRN 52556 - 42 - 0

43 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, an d 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).” [ Ver baan 2013, p. 14] CONCLUSIONS Under the conditions of this test, t he test substance d id 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., whi chever 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 co ncentrations 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., T he 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 , 201 4
.Final HPV 3 Test Rule Data Adequacy Review (April 2014) CASRN 52556 - 42 - 0

44 Text in quotes is taken directly from Ver baan (2013).Final HPV 3 Test Rule Data Adequacy Review (April 2014) CASRN 52556 - 42 - 0

45 E . DATA ADEQ U ACY 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 No 1 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 HPV 3 Test Rule Data Adequacy Review (April 2014) CASRN 52556 - 42 - 0

46 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 e ach 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)” 1 and 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 LD 50 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 LD 50 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 reg arding 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 ranke d 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 maximu m 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” st ates: “... The following definitions also apply to this test guideline.” and “ LD 50 (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 LD 50 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 HPV 3 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 limi t 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 - sect ion (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: “LD 50 and CI”. Summary of Quoted Material: A primary objective of an acute toxicit y study is to determine a discrete LD 50 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 LD 50 is greater than the limit dose; if the LD 50 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 li mit 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 T echnical Report No. TR

- 12

- 092.Final HPV 3 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 ( O ECD 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”, i tem (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 shou ld 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 adequa tely 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 HPV 3 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 HPV 3 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., whi chever 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 co ncentrations 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 concentrat ion 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”, Su b - 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 sho uld 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 m itotic index, (all greater than 50%)... For relatively non

- cytotoxic compounds the maximum concentration should be 5 μ[l]/ml 1 , 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 HPV 3 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 te sted 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; Laborato ry Project No. 500681. Sponsor: Society of Chemical Manufacturers and Affiliates (SOCMA), Washington, DC. Study report available online at http://www.regulatio ns.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.