SRC TR-15-054

HPV3 TEST RULE DATA ADEQUACY REVIEW (FINAL):

GENE MUTATION AND CHROMOSOMAL ABERRATIONS 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 FG007.4.009.006

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, 2015
Final HPV3 Test Rule Data Adequacy Review (September 2015) CASRN 52556-42-0

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TABLE OF CONTENTS

A. SOURCE MATERIAL USED FOR ASSESSMENT .............................................................. 1

B. EVALUATING DATA ADEQUACY ..................................................................................... 2

C. SUMMARY OF DATA ADEQUACY FOR TESTING REQUIRED BY TEST RULE ........ 2

D. DATA FOR SIDS ENDPOINTS.............................................................................................. 3

D-1. Genetic Toxicity – Gene Mutation...................................................................................... 3

D-2. Genetic Toxicity – Chromosomal Aberrations.................................................................. 8

E. DATA ADEQUACY SUMMARY TABLE........................................................................... 14
Final HPV3 Test Rule Data Adequacy Review (September 2015) CASRN 52556-42-0

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A. SOURCE MATERIAL USED FOR ASSESSMENT

This document evaluates a bacterial reverse mutation test and a chromosomal aberrations 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 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 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 Kow < 4.2 or Test Group 2 [chronic toxicity to Daphnia and toxicity to algae 

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

E2 (chromosomal aberrations or micronucleus test), and F1 (combined repeated-dose toxicity 

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

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

to aquatic organisms.

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 bacterial 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 Affiliates (SOCMA), Washington, DC.

 

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

letter from the COPS-1 Consortium to EPA dated August 29, 2012 available online at http://www.regulations.gov/

#!documentDetail;D=EPA-HQ-OPPT-2009-0112-0118 as of September 10, 2015. 

2 These data 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=EPAHQ-OPPT-2009-0112-0303 as of September 10, 2015.
Final HPV3 Test Rule Data Adequacy Review (September 2015) CASRN 52556-42-0

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

evaluated in this report. 

B. EVALUATING DATA ADEQUACY

The submitted toxicological data 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 test rule requirements, study details were compared to current standard test 

guidelines to establish whether the study met current testing requirements. If a study was 

determined to be inadequate, a basis for that conclusion is supplied. A table summarizing data 

availability/adequacy for each 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 chromosomal aberrations (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=EPA-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 requirements for toxicity testing of CASRN 

52556-42-0.

 

1 These data 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=EPAHQ-OPPT-2009-0112-0303 as of September 10, 2015.
Final HPV3 Test Rule Data Adequacy Review (September 2015) CASRN 52556-42-0

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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 (VerspeekRip, 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-AHPS1; commercial product that 

contains 40.3 wt. % 1-propanesulfonic acid, 2-hydroxy-3-(2-propen-1-yloxy)-, sodium 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 preparation of concentrations based on weight of active ingredient (a.i.) per liter, a

correction factor of 2.48 was used to correct for 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, Aberdeen, UK)

Metabolic activation: With (Aroclor 1254-induced rat liver S9 microsomal fraction) and 

without

 

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

CASRN 52556-42-0. 
Final HPV3 Test Rule Data Adequacy Review (September 2015) CASRN 52556-42-0

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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 (without 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 stock 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:

“Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth... and 

incubated in a shaking incubator (37°C, 150 spm) until the cultures reached an optical density of

1.0 ± 0.1 at 700 nm (109cells/ml). Freshly grown cultures of each strain were used for [all 

tests].” [Verspeek-Rip 2015, p. 9]

“Agar plates (9 cm) containing 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 the agar was dissolved. The top agar was then transferred to 

glass tubes and autoclaved for 20 minutes. For each test, top agar was melted by heating to 45 ± 

2°C and the following solutions were successively added to 3 mL of molten top agar: 0.1 mL 

fresh bacterial culture (109cells/mL), 0.1 mL of diluted test substance preparation in Milli-Q 

water, and either 0.5 mL S9-mix (for assays with S9 metabolic activation) or 0.5 mL 0.1 M 

phosphate buffer (for assays without S9 metabolic activation). The ingredients were mixed by 

vortexing and melted top agar was poured onto each plate. After solidification, the plates were 

inverted and the bacteria were incubated at 37.0 ± 1.0°C in the dark for 48 ± 4 hours.
Final HPV3 Test Rule Data Adequacy Review (September 2015) CASRN 52556-42-0

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Rat Liver S9 Metabolic Activation System:

S9 homogenate (rat liver microsomal enzyme mix), obtained from Trinova Biochem GmbH, was

prepared from male Sprague Dawley rats that were injected intraperitoneally with Aroclor 1254 

(500 mg/kg-bw). Before use, all S9 batches were characterized with benzo-(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 toxicity 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 typhimurium 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 test with 

Salmonella typhimurium strain TA100 and Escherichia coli strain WP2 uvrA, it was used as a 

first definitive mutagenicity test for these two strains. In Experiment 2, all five bacterial strains 

were tested in triplicate at concentrations of 0 (solvent control), 492, 878, 1568, 2800, and 5000 

μg a.i./plate, with and without 10% (v/v) S-9 mix.
Final HPV3 Test Rule Data Adequacy Review (September 2015) CASRN 52556-42-0

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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 automatically 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 

background 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 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 follow up 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 follow up 

experiment.” [Verspeek-Rip 2015, 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 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 

extend 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, pp. 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 definitive 
Final HPV3 Test Rule Data Adequacy Review (September 2015) CASRN 52556-42-0

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tests. Test substance precipitation was not seen on any of the plates. The test substance was not 

cytotoxic at any test concentration in either the absence or presence of metabolic activation. 

“The negative and strain-specific positive control values were within the laboratory historical 

control data ranges indicating that the test conditions were adequate and that the metabolic 

activation system functioned properly.” [Verspeek-Rip 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.

CONCLUSIONS

The test substance did not induce gene mutations in bacteria either with or without metabolic 

activation under the conditions of this study.

STUDY RELIABILITY

[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 Netherlands; Laboratory Project No. 508230. Sponsor: Society of 

Chemical Manufacturers and Affiliates (SOCMA), Washington, DC. 

Text in quotes is taken directly from Verspeek-Rip (2015).
Final HPV3 Test Rule Data Adequacy Review (September 2015) CASRN 52556-42-0

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D-2. Genetic Toxicity – Chromosomal Aberrations

Summary of endpoint: Adequate 

Study 1: Chromosomal aberrations 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-AHPS1; commercial product that 

contains 40.3 wt. % 1-propanesulfonic acid, 2-hydroxy-3-(2-propen-1-yloxy)-, sodium 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 preparation of concentrations expressed as μg active ingredient (a.i.)/mL, a

correction factor of 2.48 was used to correct for the presence of 40.3% CASRN 52556-42-0 in 

the commercial product, COPS_1.

METHOD

Method/guideline followed: OECD 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 (23-32 years old); average generation time of cells: 12.8-12.9 hours.

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

fraction) and without 

 

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

CASRN 52556-42-0. 
Final HPV3 Test Rule Data Adequacy Review (September 2015) CASRN 52556-42-0

9

Concentrations tested1: 

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 Assay:

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 μg 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 solvent 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 concentrations 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 male 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 activation 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 MgCl2.6H2O...; 2.46 mg KCl...; 1.7 mg glucose-6-

phosphate...; 3.4 mg NADP...; [and] 4 μmol HEPES... The above solution was filter (0.22 

μm)-sterilized. To 0.5 ml S9-mix components 0.5 ml S9-fraction [S9 homogenate] was added 

(50% (v/v) S9-fraction) to complete the S9-mix.” [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. 
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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 fetal calf serum, L-glutamine (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 volume 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 incubations 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% CO2 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 CO2 percentage was monitored once on 

each working day. Temporary deviations from the temperature, humidity and CO2 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.” [Verbaan 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 medium, 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 HPV3 Test Rule Data Adequacy Review (September 2015) CASRN 52556-42-0

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and incubated in culture medium for 20 to 22 hours (post-exposure incubation period) after 

which the cells were centrifuged and the cell pellet was fixed for determination of the mitotic 

index. The cells 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 end of the exposure period after which 

the cell pellet was fixed for mitotic index determination.

Definitive Test Procedure:

In the definitive chromosomal aberrations assays, 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. 

Concentrations 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 

centrifugation... The supernatant was removed and the cells were rinsed once with 

5 ml HBSS. After a second centrifugation step, HBSS was removed and cells were resuspended 

in 5 ml culture medium and incubated for another 20–22 h [post-exposure incubation period].” 

[Verbaan 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 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. After 3 h exposure, the cells exposed to [the test substance] in the 

presence of S9-mix were separated from the exposure medium by centrifugation... The 

supernatant was removed and the cells were rinsed once with 5 ml of HBSS and incubated in 5 

ml culture medium for another 44–46 h [post-exposure incubation period]. The cells that were 

treated for 24 h and 48 h in the absence of S9-mix were not rinsed after exposure but were fixed 

immediately after 24 h and 48 h...” [Verbaan 2015, p. 11] After completion of these treatment 

protocols, cultures were centrifuged and the cell pellet was fixed for determination of mitotic 

index and chromosomal aberrations.

Chromosome and Slide Preparation:

Lymphocytes 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, cells 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. At least two slides 

per culture were prepared. Slides were dried, stained with 5% (v/v) buffered 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 metaphases from 

at least 1000 cells (with a maximum deviation of 5%).” [Verbaan 2015, p. 12]
Final HPV3 Test Rule Data Adequacy Review (September 2015) CASRN 52556-42-0

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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 analysed. The 

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

p. 12]

Criteria for Positive Results: 

“A test substance is considered positive (clastogenic) in the chromosome aberration test if: 

a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) 

increase in the number of cells with chromosome aberrations.

b) A statistically significant and biologically relevant increase in the frequencies of the 

number of cells with chromosome aberrations was observed in the absence of a clear 

dose-response relationship.” [Verbaan 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 substances should produce a statistically significant (Chi-square test, 

one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.

c) A homogenous response between the replicate cultures is observed.

d) A possible precipitate present on the slides should not interfere with the scoring of 

chromosome aberrations.” [Verbaan 2015, p. 12] 

RESULTS

Genotoxicity result: Negative

Cytotoxic concentration: The toxicity of CASRN 52556-42-0 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./mL) 

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 assays

(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 HPV3 Test Rule Data Adequacy Review (September 2015) CASRN 52556-42-0

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The test substance did not induce an increase in the number of cells with chromosomal

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 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 2015, pp. 14-15]

CONCLUSIONS

Under the conditions of this test, the test substance did not induce chromosomal aberrations in 

human lymphocytes in vitro.

STUDY RELIABILITY

[1] Reliable without restrictions. 

REFERENCE

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 Affiliates 

(SOCMA), Washington, DC.

Text in quotes is taken directly from Verbaan (2015).
Final HPV3 Test Rule Data Adequacy Review (September 2015) CASRN 52556-42-0

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E. DATA ADEQUACY SUMMARY TABLE 

Endpoint Study Information 

Available

Acceptable Data Gap

Aquatic Effects

Acute Toxicity to Fish Yes Yes No

Acute Toxicity to Aquatic Invertebrates Yes Yes No

Toxicity to Aquatic Plants Yes Yes No

Health Effects

Acute Toxicity Yes No No1

Repeated-Dose Toxicity Yes Yes No

Gene Mutation Yes 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 52556-42-0 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.