SRC TR

- 15

- 054

HPV3 TEST RULE DATA ADEQUACY R EVIEW (FINAL ) : GENE MUTATION AND CH ROMOSOMAL ABERRATION S TESTS

1

- Propanesulfonic acid, 2

- hydroxy

- 3

- (2

- propen

- 1

- yloxy)

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

- 42

- 0

Prepared by:

Christopher Janson , William 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

Cynthia Bowie , Project Officer David Brooks, Work Assignment Manager Amy Benson, Alternate Work Assignment Manager

September 10 , 2015Final HPV 3 Test Rule Data Adequacy Review ( September 2015

) 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

................................ ................................ .............................. 3

D

- 1. Genetic Toxicity

– Gene Mutation

................................ ................................ ...................... 3

D

- 2. Genetic Toxicity

– Chromosomal Aberrations

................................ ................................ .. 8
E. DATA ADEQUACY SUMMARY TABLE

................................ ................................ ........... 14Final HPV 3 Test Rule Data Adequacy Review (September 2015) CASRN 52556

- 42

- 0

1 A. SOURCE MATERIAL USED FOR ASSESSMENT

This d ocument evaluates a bacterial reverse mutation test and a chromosom al aberration s test of 1

- propanesulfonic acid, 2

- hydroxy

- 3

- (2

- propen

- 1

- yloxy)

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

- 42

- 0) submitted in July 2015 by the COPS

- 1 Consortium, a cost

- sharing consortium formed and managed under the Society of Chemical Manufacturers and Affiliates (SOCMA) , to EPA in response to the final test rule, “Testing of Certain High Production Volume Chemica ls; 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 September 10 , 2015).

The HPV3 test rule requires the following toxicological testing for CASRN 52556

- 42

- 0: C1 (consisting of Test Group 1 [acute toxicity to fish, acute toxicity to Daphnia , and toxicity to algae tests ] if log K ow < 4.2 or Test Group 2 [chronic toxicity to Daphnia and toxicity to algae
test s ] if log K ow ≥ 4.2), D (acute mammalian toxicity test), E1 (bacterial reverse mutation test), E2 (chromosom al aberration s or micronucleus test), and F1 (combined repeated

- do se 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.

Data previously submitted by the COPS

- 1 Consortium for the required genotoxicity (E1 and E2) endpoints (submission dated August 28, 2013; available online at http://www.regulations.gov/ #!documentDetail;D=EPA

- HQ

- OPPT

- 2009

- 0112

- 0146 as of September 10 , 2015 ) were not considered adequate to satisfy the bacterial reverse mutation (E1) and chromosomal aberrations (E2) testing requirements because the highest concentration of active ingredient tested in each study was substantially lower than the maximum concentration recommended by the corresponding test guidelines, no test substance

- related cytotoxicity or precipitation was observed, and no evidence of genotoxicity was observed. 2

The bacteri al reverse mutation test and the chromosomal aberrations test were repeated and the resulting study reports were submitted by the COPS

- 1 Consortium in July 2015:

 Verspeek

- Rip, C.M. 2015. Evaluation of the mutagenic activity of COPS_1 in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay. WIL Research Europe B.V., The Netherlands; Laboratory Project No. 508230. Sponsor: Society of Chemical Manufacturers and Affiliates (SOCMA), Washington, DC.

 Verbaan, I.A .J. 2015. Evaluation of the ability of COPS_1 to induce chromosome aberrations in cultured peripheral human lymphocytes. WIL Research Europe B.V., The Netherlands; Laboratory Project No. 508229. Sponsor: Society of Chemical Manufacturers and Affiliate s (SOCMA), Washington, DC.

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 online at http://www.regulations.gov/ #!doc umentDetail;D=EPA

- HQ

- OPPT

- 2009

- 0112

- 0118 as of September 10 , 2015

. 2 These d ata were reviewed in the following document: “ HPV3 Test Rule Data Adequacy Review : 1

- Propanesulfonic acid, 2

- hydroxy

- 3

- (2

- propen

- 1

- yloxy)

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

- 42

- 0”, SRC Technical Report #14

- 005, dated April 18, 2014 , available online at http://www.regulations.gov/#!documentDetail;D=EPA

HQ

- OPPT

- 2009

- 0112

- 0303 as of September 10 , 2015

.Final HPV 3 Test Rule Data Adequacy Review (September 2015) CASRN 52556

- 42

- 0

2 These newly submitted data are evaluated for adequacy herein. No other toxicity data are evaluated in this report.

B. EVALUATING DATA ADEQUACY

The submitted toxicological data are evaluated for adequacy in Section D. Ea ch 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 test rule requirements , study detail s were compared to current standard test guidelines to establish whether the study met current testing requirements. If a study was determined to be inadequate, a basis for that conclusion is supplied. A table summarizing data availability/adequacy for e ach toxicological SIDS endpoint is included in Section E of this document.

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

Evaluation of Data Submitted in July 2015 for Genotoxicity Testing Requirements for CASRN 52556

- 42

- 0: Data submitted by the COPS

- 1 Consortium in July 2015 are adequate to satisfy the bacterial reverse mutation (E1) and chromosom al aberration s (E2) testing requirements for CASRN 52556

- 42

- 0 promulgated in the final HPV3 test rule.

Other HPV3 Test Rule Toxicity Testing Requirements for CASRN 52556

- 42

- 0: In a previous submission (dated August 28, 2013; available online at http://www.regulations.gov/ #!documentDetail;D=EP A

- HQ

- OPPT

- 2009

- 0112

- 0146 as of September 10 , 2015 ) 1

, the COPS

- 1 Consortium provided adequate data to satisfy the acute toxicity to fish (component of C1), acute toxicity to Daphnia (component of C1), toxicity to algae (component of C1), and combined repeated

- dose/reproductive/developmental toxicity (F1) testing requirements. Although no adequate data were submitted for the acute mammalian toxicity (D) testing requirement , EPA does not currently consider acute mammalian toxicity testing to be necessary given that adequate data are available for the repeated

- dose toxicity endpoint.

Summary of Data Adequacy for Toxicological Endpoints: There are no outstanding HPV3 test rule requireme nts for toxicity testing of CASRN 52556

- 42

- 0

.

1 These d ata were reviewed in the following document: “ HPV3 Test Rule Data Adequacy Review : 1

- Propanesulfonic acid, 2

- hydroxy

- 3

- (2

- propen

- 1

- yloxy)

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

- 42

- 0”, SRC Technical Report #14

- 005, dated April 18, 2014 , availab le online at http://www.regulations.gov/#!documentDetail;D=EPA

HQ

- OPPT

- 2009

- 0112

- 0303 as of September 10 , 2015

.Final HPV 3 Test Rule Data Adequacy Review (September 2015) CASRN 52556

- 42

- 0

3 D. DATA FOR SIDS ENDPOINTS

D

- 1

. Genetic Toxicity

– Gene Mutation

Summary of endpoint: Adequate

Study 1: Mutagenicity study in Salmonella typhimurium and Escherichia coli (Verspeek

Rip, 2015)

Title: Evaluation of the mutagenic activity of COPS_1 in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: COPS_1

, also referred to as Bimax Product CS

- AHPS 1 ; commer cial product that contains 40.3 wt. % 1

- propanesulfonic acid, 2

- hydroxy

- 3

- (2

- propen

- 1

- yloxy)

- , sodiu m salt (1:1) (CASRN 52556

- 42

- 0) in water

.

Remarks: Clear colorless liquid; Batch No. 14L1103; Composition: 40.3 wt % 1

- propanesulfonic acid, 2

- hydroxy

- 3

- (2

- propen

- 1

- yloxy)

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

- 42

- 0) in water. For preparat ion of concentrations based on weight of active ingredient (a.i.) per l iter, a correction factor of 2.48 was used to correct f or the presence of 40.3% CASRN 52556

- 42

- 0 in the commercial product, COPS_1

.

METHOD

Method/guideline followed: OECD TG 471 (1997) , Method B.13/14 of Commission Regulation (EC) No. 440/2008, and OPPTS 870.5100 (1998)

Test type: Bacterial Reverse Mutation Test

GLP compliant?: Yes

Year study performed: 2015

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, Ab erdeen, UK)

Metabolic activation: With ( Aroclor 1254

- induced rat liver S9 microsomal fraction) and without

1 E PA previously accepted the submitter’s request to test Bimax Product CS

- AHPS in the toxicity tests required for CASRN 52556

- 42

- 0.Final HPV 3 Test Rule Data Adequacy Review (September 2015) CASRN 52556

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- 0

4 Concentrations tested: Range

- Finding Toxicity Test Concentrations: TA100 and WP2 uvrA (without and with 5% S9): 0 (solvent control)

, 1.7, 5.4, 17, 52, 164, 512, 1600, and 5000 μg a.i. /plate

Definitive Mutagenicity Assay Concentrations: Experiment 1: TA98, TA1535, and TA1537 (withou t and with 5% S9): 0 (solvent control)

, 52, 164, 512, 1600, and 5000 μg a.i. /plate Experiment 2: TA98, TA100, TA1535, TA1537, and WP2 uvrA (without and with 10% S9): 0 (solvent control)

, 492, 878, 1568, 2800, and 5000 μg a.i. /plate

Solvent: Milli

- Q water

Statistical methods used: No formal statistical analyses were conducted.

Test method/conditions remarks: Preparation of Test Concentrations: A stock solution (124 mg/mL) of the test substance was prepared by dissolving the test substance in Milli

- Q water. Test concentrations were prepared by diluting the st ock solution in Milli

- Q water and were used within 2 hours of preparation. The test concentrations selected for the mutagenicity assay were based on the results of a range

- finding toxicity test in Salmonella typhimurium strain TA100 and Escherichia coli strain WP2 uvrA.

Bacterial Cultures and Plating: “Sa mples of frozen stock cultures of bacteria were transferred into enriched nutrient broth... and incubated in a shaking incubator (37°C, 150 spm) until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (10 9 cells/ml). Freshly grown cultures of e ach strain were used for [all tests].” [Verspeek

- Rip 2015 , p. 9]

“Agar plates (9 cm) contain ing 25 ml glucose agar medium [were used]

. Glucose agar medium contained [the following] per liter: 18 g purified agar... in Vogel

- Bonner Medium E [and] 20 g glucose... The agar plates for the test with the Salmonella typhimurium strains also contained 12.5 μg/plate biotin... and 15 μg/plate histidine... and the agar plates for the test with the Escherichia coli strain contained 15 μg/plate tryptophan...” [Verspeek

- Rip 2015 , p. 9]

Top agar was prepared by heating Milli

- Q water containing 0.6% (w/v) bacteriological agar and 0.5% (w/v) sodium chloride until t he agar was dissolved. The top agar was then transferred to glass tubes and autoclaved for 20 minutes. For each test, top agar was melted by heating to 45 ± 2 °C and 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 ingredie nts were mixed by vortexing and melted top agar was poured onto each plate. After solidification, the plates were inverted and the bacteria were incubated at 37.0 ± 1.0°C in the dark for 48 ± 4 hours.Final HPV 3 Test Rule Data Adequacy Review (September 2015) CASRN 52556

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- 0

5 Rat Liver S9 M etabolic Activation System : S9 homogenate (rat liver microsomal enzyme mix) , obtained from Trinova Biochem GmbH, was prepared from m ale Sprague Dawley rats that were injected intraperitoneally with Aroclor 1254 (500 mg/kg

- bw ). Before use, all S9 batches were characterized with b enzo

- (a )

- pyrene and 2

- aminoanthracene

– mutagens which require metabolic activation

– at concentrations of 5 and 2.5 μg/plate, respectively, in Salmonella typhimurium strain TA100. S9 mix was prepared with either 5% ( v/v ) S9 homogenate (for the range

- finding tox icity test and first mutagenicity test) or 10% ( v/v ) S9 homogenate (for the second mutagenicity test) immediately prior to use and kept on ice.

Positive Controls: The following positive controls were evaluated in the absence of S9

- mix: TA1535 : Sodium azide ( 5 μg/plate ) TA1537 : ICR

- 191 ( 2.5 μg/plate ) TA98 : 2

- Nitrofluorene ( 10 μg/plate ) TA100 : Methylmethanesulfonate ( 650 μg/plate ) WP2 uvrA : 4

- Nitroquinoline N

- oxide ( 10 μg/plate )

2

- Aminoanthracene was used as the positive control for all strains in the presence of S9

- mix

. The following concentrations were used : TA1535 : 2.5 μg/plate with 5% and 10% S9 TA1537 : 2.5 μg/plate with 5% S9; 5 μg/plate with 10% S9 TA98 : 1 μg/plate with 5% and 10% S9 TA100 : 1 μg/plate with 5% S9; 2 μg/plate with 10 % S9 WP2 uvrA : 15 μg/plate with 5% and 10% S9

Solvents for the positive controls were saline for sodium azide and dimethylsulfoxide for all other chemicals.

Range

- Finding Toxicity Test Conditions : Salmonella typhimurium strain TA100 and Escherichia coli strain WP2 uvrA were tested in triplicate at concentrations of 0 (solvent control), 1.7, 5.4, 17, 52, 164, 512, 1600, and 5000 μg a.i. /plate, with and without 5% (v/v) S9

- mix.

Definitive Mutagenicity Test Conditions : In Experiment 1, Salmonella typhimur ium strains TA98, TA1535, and TA1537 were tested in triplicate at concentrations of 0 (solvent control), 52, 164, 512, 1600, and 5000 μg a.i./plate, with and without 5% (v/v) S9

- mix

. Since no toxicity was observed in the range

- finding tes t with Salmonella typhimurium strain TA100 and Escherichia coli strain WP2 uvrA , it was used as a first definitive mutagenicity test for these two strains. I n Experiment 2, all five bacterial strains were tested in triplicate at concentrations of 0 (solvent cont rol), 492, 878, 1568, 2800, and 5000 μg a.i./plate, with and without 10% (v/v) S

- 9 mix

.Final HPV 3 Test Rule Data Adequacy Review (September 2015) CASRN 52556

- 42

- 0

6 Mutagenicity and Toxicity Assessment: At the end of the incubation period of the range

- finding and definitive studies , each plate was examined for evidence of test substance

- induced toxicity (reduction of the background lawn, increase in the size of microcolonies, and reduction in revertant colonies per plate) and determination of the number of revertant colonies per plate.

“The revertant colonies were counted aut omatically with the Sorceror Colony Counter. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually. Evidence of test article precipitate on the plates and the condition of the bacterial backgrou nd lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.” [Verspeek

- Rip 2015, p. 10 ]

The test substance was considered to be toxic if there was a reduction in the bacterial background lawn and/or a biologically relevant decrease in the mean number of revertant colonies per plate.

Criteria for Positive Results: “A test substance is considered positive (mutagenic) in the test if: a) The total number of revertants in tester strain TA100 is greate r 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 follow up experiment is performed when a positive re sponse is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.” [Verspeek

- Rip 2015 , p. 11]

Acceptability Criteria: “A Salmonella typhimurium reverse mutation assay and/or Escherichia col i reverse mutation assay is considered acceptable if it meets the following criteria: a) The vehicle control and positive control plates from each tester strain (with or without S9

- mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at WIL Research Europe. b) 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

ex ten d to 5 mg/plate. c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.” [Verspeek

- Rip 2015 , p p

. 10

- 11]

RESULTS

Genotoxicity result: Negative with and without metabolic activation

Cytotoxic concentration (if applicable): No cytotoxicity was observed at the concentrations tested with or without activation.

Results remarks: The test substance showed no indication of induction of mutations with or without metabolic activation in any of the five strains tested in the range

- finding or definitiveFinal HPV 3 Test Rule Data Adequacy Review (September 2015) CASRN 52556

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- 0

7 tests. Test substance precipitation was not seen on any of the plates. The test substance was not cytotoxic at any test c oncentration in either the absence or presence of metabolic activation. “The negative and strain

- specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the meta bolic activation system functioned prope rly.” [Verspeek

- Rip 2015, p. 12 ]

All three acceptability criteria were satisfied during the assay:

 The vehicle control and positive control data were within the laboratory historical control range for each tester strain.

 Each tester strain was exposed to test substance concentrations extending to 5 mg a.i./plate.

 No plates were lost through contamination or some other unforeseen event.

CONCL USIONS

The test substance did not induce gene mutations in bacteria either with or without metabolic activation under the conditions of this study.

STUDY RELIABILITY

[1 ] Reliable without restrictions.

REFERENCE

Verspeek

- Rip, C.M. 2015

. Evaluation of the mutagenic activity of COPS_1 in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay

. WIL Research Europe B.V., The Netherland s; Laboratory Project No. 508230

. Sponsor: Society of Chemical Ma nufacturers and Affiliates (SOCMA), Washington, DC.

Text in quotes is taken directly from Verspeek

- Rip (2015 ).Final HPV 3 Test Rule Data Adequacy Review (September 2015) CASRN 52556

- 42

- 0

8 D

- 2

. Genetic Toxicity

– Chromosomal Aberrations

Summary of endpoint: Adequate

Study 1: Chromosom al aberration s assay in human lymphocytes (Verbaan, 2015)

Title: Evaluation of the ability of COPS_1 to induce chromosome aberrations in cultured peripheral human lymphocytes

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: COPS_1 , also referred to as Bimax Product CS

- AHPS 1 ; commer cial product that contains 40.3 wt. % 1

- propanesulfonic acid, 2

- hydroxy

- 3

- (2

- propen

- 1

- yloxy)

- , sodiu m salt (1:1) (CASRN 52556

- 42

- 0) in water

.

Remarks: Clear col orless liquid; Batch No. 14L1103 ; Composition: 40.3 wt. % 1

- propanesulfonic acid, 2

- hydroxy

- 3

- (2

- propen

- 1

- yloxy)

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

- 42

- 0) in water. For preparation of co ncentrations expressed as μg ac tive ingredient (a.i.)/mL , a correction factor of 2.48 was used to correct f or the presence of 40.3% CASRN 52556

- 42

- 0 in the commercial product, COPS_1

.

METHOD

Method/guideline followed: OE CD TG 473 (2014), Method B.10 of Commission Regulation (EC) No. 440/2008, and TSCA 799.9537

Test type: Mammalian Chromosome Aberration Test ( in vitro )

GLP compliant?: Yes

Year study performed: 2015

Test species: Human peripheral blood lymphocytes; obtained from healthy , non

- smoking adult males ( 2 3

- 32 years old) ; av erage generation time of cells : 12.8

- 12.9 hours.

Metabolic activation: With (phenobarbital and β

- naphthoflavone

- induced rat S9 microsomal fraction) and without

1 E PA previously accepted the submitter’s request to test Bimax Product CS

- AHPS in the toxicity tests required for CASRN 525 56

- 42

- 0.Final HPV 3 Test Rule Data Adequacy Review (September 2015) CASRN 52556

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- 0

9 Concentrations tested 1 : Range

- Finding Assay : Without S9 ( 3

- or 24

- h exposure, cell harvest at 24 h ) : 52, 164, 512, 1600, and 2182 μg a.i. /mL Without S9 ( 48

- h exposure, cell harvest at 48 h ) : 52, 164, 512, 1600, and 2182 μg a.i. /mL With S9 ( 3

- h exposure, cell harvest at 24 h ) : 52, 164, 512, 1600, and 2182 μg a.i. /mL

Definitive Assa y : Experiment 1: Without S9 ( 3

- h exposure, cell harvest at 24 h ) : 512, 1600, and 2182 μg a.i. /mL With S9 ( 3

- h exposure, cell harvest at 24 h ) : 512, 1600, and 2182 μ g a.i. /mL

Experiment 2: Without S9 ( 24

- h exposure, cell harvest at 24 h ) : 512, 1600, and 2182 μg a.i. /mL Without S9 ( 48

- h exposure, cell harvest at 48 h ) : 512, 1600, and 2182 μg a.i. /mL With S9 ( 3

- h exposure, cell harvest at 48 h ) : 512, 1600, and 2182 μg a.i. /mL

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 so lvent control using Chi

- square statistics.” [Verbaan 2015, pp. 12

- 13 ] A one

- tailed Chi

- square test was used, with a significance level of 0.05.

Vehicle: Culture medium

Test method/conditions remarks: Preparation of Test Concentrations: Test concen trations were prepared by dissolving the test substance in culture medium. Test solutions were used within 2.5 hours of preparation.

Rat Liver S9 Metabolic Activation System : S9 homogenate (rat liver microsomal enzyme mix), obtained from Trinova Biochem GmbH, was prepared from m ale Sprague Dawley rats that were orally administered a suspension of phenobarbital (80 mg/kg

- bw) and β

- naphthoflavone (100 mg/kg

- bw). The study report did not indicate if the S9 homogenate was tested for metabolic activat ion potency prior to further use.

Preparation of S9

- Mix:

“ S9

- mix was prepared immediately before use and kept on ice. S9

- mix components contained per ml physiological saline: 1.63 mg MgCl 2 .6H 2 O

... ; 2.46 mg KCl

... ; 1.7 mg glucose

- 6

- phos phate

... ; 3.4 mg NADP

... ; [and] 4 μmol HEPES

... The above solution was filter (0.22 μm)

- sterilized. To 0.5 ml S9

- m ix components 0.5 ml S9

- fraction [ S9 homogenate] was added (50% (v/v) S9

- fraction) to complete the S9

- mi x. ” [Verbaan 2015 , p. 10 ]

1 The maximum concentration tested in this study was 2182 μg a.i./mL, i.e., 2182 μg CASRN 52556

- 42

- 0/mL. Given that the molecular weight of CASRN 52556

- 42

- 0 is 218.22 g/mole, this maximum test concentration corresponds to 0.01 M CASRN 52556

- 42

- 0. The use of 2182 μg CASRN 52556

- 42

- 0/mL (i.e., 0.01 M CASRN 52556

- 42

- 0) as the maximum test concentration is compatible with test guidelines

.Final HPV 3 Test Rule Data Adequacy Review (September 2015) CASRN 52556

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- 0

10 Blood Collection and Lymphocyte Cultures : By venipuncture, blood samples from three healthy, non

- smoking , adult male volunteers, ages 23, 26, and 32 , were collected into sterile vessels containing the anticoagulant sodium heparin.

For each lymphocyte culture, a whole blood sample (0.4 mL) was incubated in culture medium [ RPMI 1640 medium containing 20% (v/v) heat

- inactivated f eta l calf serum , L

- glut amine (2 mM)

, penicillin (50 U/mL), streptomycin (50 μg/mL )

, and heparin (30 U/mL) ] that was supplemented with 0.1 mL (9 mg/mL) of the mitogen phytohemagglutinin. The v olume of culture medium to which whole blood was added was 4.8 mL for a culture to which 0.2 mL S9

- mix was to be added at 48 hours and 5 mL for a culture that was not to receive S9

- mix.

“ All incu bations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80

– 100% (actual range 53

– 91%), containing 5.0 ± 0.5% CO 2 in air in the dark at 37.0 ± 1.0 ̊C (actual range 34.7

– 37.3 ̊C). Temperature and humidity were continuously monitored throughout the experiment. The CO 2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO 2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity. ” [Verba an 2015 , p. 10]

After approximately 48 hours of mitogenic stimulation with phytohemagglutinin, duplicate cultures were further incubated at 37° C 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. “ Metabolic activation was achieved by adding 0.2 ml S9

- mix to 5.3 ml of a lymphocyte culture (containing 4.8 ml culture me dium, 0.4 ml blood

, and 0.1 ml (9 mg/ml) phytohaemagglutinin). The concentration of the S9

- fraction in the exposure medium was 1.8% (v/v). ” [Verbaan 2015, p. 10]

Positive and Negative Controls: Mitomycin C was used as the positive control in the absence of S9

- mix . The following concentrations were used : 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

Cyclophosphamide ( 10 μg/mL ) was used as the positive control in the presence of S9

- mix

. The solvent for all positive controls was Hanks’ Balanced Salt Solution (HBSS) without calcium and magnesium.

Negative controls for the range

- finding test and definitive chromosomal aberrations assay consisted of culture medium without the test substance.

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), 52, 164, 512, 1600

, or 2182 μg a.i. /mL. After 3 hours of exposure in the absence or presence of S9

- mix, the cells were separated from the exposure medium by centrifugation , rinsed with HBSS,Final HPV 3 Test Rule Data Adequacy Review (September 2015) CASRN 52556

- 42

- 0

11 and incubated in culture medium for 20 to 22 hours (post

- exposure incubation period ) after which the c ells were centrifuged and the cell pellet was fixed for determination of the mitotic index. The c ells that were exposed for 24 or 48 hours in the absence of S9

- mix were not rinsed after exposure but were immediately centrifuged at the e nd of the exposure period after which the cell pellet was fixed for mitotic index determination

. Definitiv e Test Procedure: In the definitive chromosomal aberrations assay s , 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), 512, 1600 , or 2182 μg a.i. /mL. C oncentration s selected for Experiments 1 and 2 were based on the results of the range

- finding
test.

Experiment 1 : “Lymphocytes were cultured for 48 ± 2 h and thereafter exposed in duplicate to selected [concentrations of the test substance] for 3 h in the absence and presence of S9

- mix. After 3 h exposure, the cells were separated from the exposure medium by
centr ifugation... The supernatant was removed and the cells were rinsed once with 5 ml HBSS. After a second centrifugation step, HBSS was removed and cells were resuspended in 5 ml culture medium and incubated for another 20

– 22 h [post

- exposure incubation perio d].” [ Verbaan 2015, pp. 11 ] Thereafter, the cultures were centrifuged and the cell pellet was fixed for determination of mitotic index and chromosomal aberrations.

Experiment 2: “Lymphocytes were cultured for 48 ± 2 h and thereafter exposed in duplicat e 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. After 3 h exposure, the cells exposed to [the test substance] in the presence of S9

- mix were separated from the exposure medium by centrifugation... The supernatant was removed and the cells were rinsed once with 5 ml of HBSS and incubated in 5 ml culture medium for another 44

– 46 h [post

- exposure incubation period]. The cells that were treated for 24 h and 48 h in the absen ce of S9

- mix were not rinsed after exposure but were fixed immediately after 24 h and 48 h...” [ Verbaan 2015, p. 11 ] After completion of these treatment protocols, cultures were centrifuged and the cell pellet was fixed for determination of mitotic index an d chromosomal aberrations.

Chromosome and Slide Preparation: Lymphocyte s were arrested in mitosis by addition of colchicine (0.5 μg/mL culture medium) at 2.5 to 3 hours before scheduled harvest. After centrifugation to obtain a cell pellet, c ells were treated with hypotonic 0.56% (w/v) potassium chloride solution at 37°C and fixed with methanol/acetic acid (3:1 v/v mixture)

. Fixed cells were dropped on slides

. A t least two slides per culture were prepared

. S lides were dried, stained with 5% (v/v) buf fered Giemsa solution

, rinsed in water, dried, embedded in a 1:10 mixture of xylene/pertex , coverslipped, and randomly coded for evaluation.

Mitotic Index Determination: “The mitotic index of each culture was determined by counting the number of metap hases from at least 100 0 cells (with a maximum deviation of 5%).” [Verbaa n 2015 , p. 12]Final HPV 3 Test Rule Data Adequacy Review (September 2015) CASRN 52556

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12 Analysis of Slides for Chromosomal Aberrations: All test concentrations in the definitive chromosomal aberrations assays (512, 1600, and 2182
μg a.i./mL) were scored for chromosomal aberrations. “One hundred metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. In case the number of aberrant cells, gaps excluded, was ≥ 25 in 50 metaphases, no more metaphases were examined. Only metaphases containing 46 ± 2 centromeres (chromosomes) were analy s ed

. The number of cells with aberrations and the number of aberrations were calculated.” [ Verbaan 2015

, p. 12]

Criteria for Positive Results: “A test s ubstance is considered positive (clastogenic) in the chromosome aberration test if: a) It induced a dose

- related statistically significant (Chi

- square test, one

- sided, p < 0.05) increase in the number of cells with chromosome aberrations. b) A statistically s ignificant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose

- response relationship.” [Verbaan 2015, p. 12 ]

Acceptability Criteria: “A chromosome aberration test is considered acceptable if it meets the following criteria: a) The number of chromosome aberrations found in the solvent control cultures should reasonably be within the laboratory historical control data range... b) The positive control subst ances should produce a statistically significant (Chi

- square test,

one

- sided, p < 0.05) increase in the number of cells with chromosome aberrations. c) A homogenous response between the replicate cultures is observed. d) A possible precipitate present on the s lides should not interfere with the scoring of chromosome aberrations.” [ Verbaan 2015 , p. 12 ]

RESULTS

Genotoxicity result: Negative

Cytotoxic concentration : The toxicity of CASRN 52556

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- 0 to the lymphocyte cultures was assessed by evaluating effects on the mitotic index. For all conditions tested in the range

- finding and definitive tests, the mitotic index in cultures at the highest concentration ( 2182 μg a.i. /m L)

was ≥ 60% of control in the absence of metabolic activation and ≥ 84% of control in the presence of metabolic activation.

Results remarks: Although some evidence of cytotoxicity was observed in the range

- finding and definitive tests , a biologically significant (> 50% ) reduction in mitotic index was not observed at any test concentration during the definitive chromosomal aberrations assay s (maximum inhibition of the mitotic index found among all tests was 40% in the absence of metabolic activation at the high concentration in the range

- finding test )

. No precipitation of the test substance in the culture medium was observed.Final HPV 3 Test Rule Data Adequacy Review (September 2015) CASRN 52556

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13 The test substance did not induce an increase in the number of cells with c hromosomal aberrations in the presence or absence of metabolic activation and did not induce an increase in the number of polyploid cells or cells with endoreduplicated chromosomes.

“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 e ndoreduplicated 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 ab errant cells. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9

- mix) functioned properly.” [Verbaan 2015, p p . 14

- 15 ]

CONCLUSIONS

Under the conditions of this test, the test substance did no t induce chromosomal aberrations in human lymphocytes in vitro

.

STUDY RELIABILITY

[1 ] Reliable without restriction s

.

REFERENCE

Ve rbaan, I.A.J. 2015

. Evaluation of the ability of COPS_1 to induce chromosome aberrations in cultured peripheral human lymphocytes

. WIL Research Europe B.V., The Netherland s; Laboratory Project No. 508229

. Sponsor: Society of Chemical Manufacturers and Affiliates (SOCMA), Washington, DC.

Text in quotes is t aken directly from Verbaan (2015 ).Final HPV 3 Test Rule Data Adequacy Review (September 2015) CASRN 52556

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14 E. DATA ADEQUA CY 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 Yes No Chromosomal Aberrations Yes Yes No Reproductive Toxicity Yes Yes No Developmental Toxicity Yes Yes No

1 Although no adequate data are available for CASRN 52556

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- 0 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.