## , CBIC Control Number 

# ASSOCIATION MANAGEMENT SERVICES a division 

# of 

# 360598**,,~f~~~~** 1850 M StNYVSte 700,Washington, DC 20036 • Ph (20?.) 721-4100 • Fx (202) 296-8120 • ams@socmacom • www.socma.con11Ass<xiati<>nMaiJag<



### Document Control Office (7407 M) Office of Pollution Prevention and Toxics (OPPT) Environmental Protection Agency EPA East Building, Room E6428 (202-564-8930) 1201 Constitution Avenue, NW Washington, DC 20460 

### Attention: TSCA Section 4 Docket ID Number EPA-HQ-OPPT-2009-0112 ToWhom It May Concern:

## 201~

# JUN 16 AH 10: 26

June 16, 2014



### RE: NewTest Plan; Testing ofCertain High Production Volume Chemicals; Third Group ofChemicals. Federal Register 76 (204), 65385-65410, October 21, 2011. 1-Propanesulfonicacid, 2-hydroxy-3-(2-propen-1-yloxy)-, sodium salt (1 :1) (CAS No. 52556-42-0)
The COPS-1 Consortium, a cost-sharing consortium formed and managed underthe Society of Chemical Manufacturers and Affiliates (SOCMA) submits this newtest plan in response to the April28, 2014 letterfrom Paul Lewis. The consortium had submitted data previously to satisfy the testing requirements ofthe subjecttest rule for 1-propanesulfonic acid, 2-hydroxy-3-(2-propen-1-yloxy)-, sodium salt (1: 1) (CAS No. 52556-42-0), also known as COPS-1. In his letter, Dr. Lewis explained that EPA had reviewed the previous submissions and found the gene mutation and chromosomal aberration test data to be inadequate to satisfy the HPV3 test rule. The consortium agrees to repeat those studies according to the test protocols that are enclosed with this letter. Details ofthe test facility, test material, study design and methods, and interpretation of results are explained in the protocols. Members ofthe COPS-1 consortium are the following companies: AkzoNobel Surface Chemistry LLC Ashland Inc. Bimax Inc. GE Water & Process Technologies Solvay USA, Inc.

### ·' 

# ASSOCIATION adivision of 

# MANAGEMENT SERVICES 1850 M St NW,Ste 700,Washington,DC 20036 • Ph (202) 721-4100 • Fx (202) 296-8120 • ams@socma.com • www.socrna.comiAssodationManag< 

### Initiation ofthe required testing is subject to EPA's approval ofthe test plan offered in this letter. In orderto begin testing as quickly as possible to help us meetthe EPA's reporting deadline of February 16, 2015, we lookforward to Agency feedback at your earliest opportunity. Please contact the undersigned directly at 202-721-4154 or helmest@socma.com for questions or additional information.

cc: John Schaeffer Enclosures Sincerely,

C. Tucker Helmes, Ph.D. Executive Director

1. Protocol: Evaluation ofthe MutagenicActivity ofCOPS-1 in the Salmonella typhimurium 

### Reverse Mutation Assay and the Escherichia coli 

### Reverse Mutation Assay. 2. Protocol: Evaluation ofthe Ability ofCOPS-1 to Induce Chromosome Aberrations in Cultured Peripheral Human Lymphocytes.'\**:·~·** '



## PROTOCOL

Study Title 

## EVALUATION OF THE ABILITY OF COPS_1 TO INDUCE CHROMOSOMEABERRATIONS IN CULTURED PERIPHERAL HUMAN LYMPHOCYTES (ADDITIONAL EXPERIMENT TO 500681)

I.A.J. Verbaan, PhD.
Test Facility WIL Research Europe B.V. Hambakenwetering 7 5231 DO 's-Hertogenbosch The Netherlands Laboratory Project Identification



### ProjectTo be determined Substance 204240/A

- Page 1 of 11 -

Page 1of 1

### 1. CONTENTS 1. CONTENTS

......................................................................................................................................2 2. PROTOCOLAPPROVAL

.................................................................................................................3 3. INTRODUCTION

..............................................................................................................................4 3. 1. Preface

.....................................................................................................................................4 3.2. Aims ofstudy

............................................................................................................................4 3.3. Guidelines

.................................................................................................................................

### 5 3.4. Good Laboratory Practice

.........................................................................................................

### 5 

### 3.5. Quality Assurance

.....................................................................................................................

### 5 3.6. Storage and retention of records and materials

.......................................................................

### 5 4. MATERIALS AND METHODS

.........................................................................................................

### 5 

### 4.1. Test substance

.........................................................................................................................

### 5 4.1.1. Test substance information

............................................................................................

### 5 

### 4.1.2. Study specifictest substance information

......................................................................6 4.1.3. Safety precautions and disposal category
.....................................................................6 

### 4.1.4. Test substance preparation

............................................................................................

### 6 4.2. Reference substances

..............................................................................................................6 

### 4.2.1. Negative control

..............................................................................................................

### 6 4.2.2. Positive controls

.............................................................................................................6 4.3. Test system
..............................................................................................................................7 4.4. Cell culture

................................................................................................................................7 

### 4.5. Metabolic activation system
......................................................................................................7 

### 4.5.1. Preparation of59-fraction

..............................................................................................7 

### 4.5.2. Preparation of89-mix

.....................................................................................................

### 8 4.6. Study design

.............................................................................................................................

### 8 4.6.1. Dose range finding test
..................................................................................................

### 8 4.6.2. First cytogenetic assay...................................................................................................9 4.6.3. Second cytogenetic assay..............................................................................................9 

### 4.6.4. Chromosome preparation

...............................................................................................9 

### 4.6.5. Preparation ofslides

.......................................................................................................9 4.6.6. Mitotic index/dose selection for scoring ofthe cytogenetic assay..................................9 

### 4.6.7. Analysis ofslides for chromosome aberrations

............................................................ 10 4.7. Electronic data capture
...........................................................................................................10 

### 4.8. Interpretation

...........................................................................................................................10 

### 4.8.1. Acceptability ofthe assay.............................................................................................

### 10 

### 4.8.2. Data evaluation and statistical procedures

..................................................................

### 10 

### 5. REFERENCES

...............................................................................................................................11 6. DISTRIBUTION

..............................................................................................................................11

Page 2



# r .. '

### 2. PROTOCOL APPROVAL STUDY DIRECTOR:

SPONSOR: I.A.J. Verbaan, PhD.

date:

Society ofChemical Manufacturers and Affiliates Dr. C. T. Helmes

date:

Page 3

### 3. INTRODUCTION 

### 3.1. Preface Sponsor

Study Monitor Test Facility

Study Director Study Plan



### 3.2. Aims ofstudy Society ofChemical Manufacturers and Affiliates 1850 M Street, NW Suite 700 WASHINGTON, DC 20036 USA Dr. C. T. Helmes WIL Research Europe B.V. Hambakenwetering 7 5231 DD 's-Hertogenbosch The Netherlands I.A.J. Verbaan, PhD. ilona.verbaan@wilresearch.com Startweek beginning To be added Completed week beginning : To be added Proposed Reporting date To be added

The objective ofthis study is to evaluate COPS_1 for its ability to induce structural chromosome aberrations in cultured human lymphocytes (1), either in the presence orabsence ofa metabolic activation system (S9-mix). Since no correction forthe purity was applied in project 500681, this experimentwill be performed as additional projectto fulfil all criteria according to the guidelines. Background ofthe test system Whole blood samples obtained from healthy male subjects are treated with an anti-coagulant (heparin) and cultured in the presence ofa mitogen (phytohaemagglutinin). These stimulated human lymphocytes are used because they are sensitive indicators ofclastogenic activity ofa broad range of chemicals (1-5). The stimulated lymphocytes are exposed to the test substance both in the presence and absence ofa metabolic activation system (S9-mix). In combination with this metabolic activation system indirect chemical mutagens, i.e. those requiring metabolic transformation into reactive intermediates, can be tested for possible clastogenic effects in vitro. At predetermined intervals after exposure ofthe stimulated human lymphocytes to the test substance, cell division is arrested by the addition ofthe metaphase-arresting chemical colchicine. Cells are harvested, stained and metaphase cells are analysed microscopically for the presence ofstructural chromosome aberrations such as breaks, gaps, minutes, dicentrics and exchange figures. Results from cultures treated with the test substance are compared with control (vehicle) treated cultures. Chromosome aberrations are generally evaluated in the first post-exposure mitosis (i.e. 24 hours after exposure). However, since the appearance ofthe first post-exposure mitosis can be considerably delayed due to toxic insultto the cells, cells are also harvested 48 hours after exposure to coverthe interval in which maximum aberration frequency is expected. A test substance that induces a positive response in this assay is presumed to be a potential mammalian cell clastogenic agent.

Page4

### 3.3. Guidelines The study procedures described in this protocol are in compliance with the following guidelines:

- Organisation for Economic Co-operation and Development (OECD), OECD Guidelinesfor Testing ofChemicals, Guideline No. 473: In Vitro Mammalian Chromosome Aberration Test (adopted July 21, 1997).

- European Community (EC). Commission regulation (EC) No. 440/2008, Part B: Methods for the Determination ofToxicity and other health effects, Guideline 8.10: "Mutagenicity- In Vitro Mammalian Chromosome Aberration Test". Official Journal ofthe European Union No. L142, 31 May 2008. Code of Federal Regulations, Title 40- Protection of Environment. Section 799.9537 Toxic Substances Control Act (TSCA) in vitro mammalian chromosome aberration test. 

### 3.4. Good Laboratory Practice The studywill be performed in compliance with the Organization for Economic Co-operation and Development (OECD) Principles ofGood Laboratory Practice (GLP) (as revised in 1997) ENV/MC/CHEM (98) 17. 

### 3.5. Quality Assurance Study and/or process inspectionswill be performed by the WIL Research Europe Quality Assurance Unit to assure the GLP compliance ofthis study. Facility inspections are also performed at regular intervals to assure the GLP compliance ofgeneral aspects. The protocol will be inspected to confirm that it complies with GLP regulations. The reportwill be inspected to confirm thatthe methods and results accurately and completely reflect the raw data. 

### 3.6. Storage and retention of records and materials Records and materials pertaining to the study, including protocol, raw data and the final report, will be retained in theWIL Research Europe archives for a period ofat least 2 years afterfinalization ofthe report. Afterthis period, the sponsorwill be contacted to determine howthe records and materials should be handled. WIL Research Europewill retain information concerning decisions made. WIL Research Europe will retain a test substance sample until the expiry date, but no longerthan 10 years afterfinalization ofthe report. After this period the sample will be destroyed.


### 4. MATERIALS AND METHODS 4.1. Test substance The sponsor is responsible for all test substance information unless determined by WIL Research Europe B.V

.. This will be specified in the GLP compliance statement in the report. 

### 4.1.1. Test substance information Identification Synonyms Structure

Molecularformula Molecularweight Description Batch 1-Propanesulfonicacid, 2-hydroxy-3-(2-propen-1- yloxy)-, sodium salt (1:1) COPS_1 or CS-AHPS

CeH11NaOsS 218.22 Clear colourless liquid 12E1101 Page 5Purity/Composition Sodium-1-allyloxy-2- hydroxypropane sulphonate 3-allyl-1 ,2-propanediol Water Test substance storage Stability under storage conditions Expiry date CAS Number 52556-42-0 123-34-2 7732-18-5 Percent 38.64 2.26 59.1 At room temperature in the dark Stable 13 August 2013 

### 4.1.2. Study specific test substance information Purity/composition correction factor required Stability at highertemperatures Stability in vehicle Solubility in vehicle Yes Yes, maximum temperature: 80°C, maximum duration: 1 hour Water: 1year Water: 1-45% Analysis ofstability, homogeneity and concentration ofthe test substance undertest conditionswill not be performed as part ofthis study. 

### 4.1.3. Safety precautions and disposal category Safety precautions Disposal category 

### 4.1.4. Test substance preparation Gloves, goggles and face mask to ensure personnel health and safety Ill

A correction factor of

# 2i69 will be applied to correct forthe purity ofthe test substance. The test substance will be dissolved in RPMI1640 medium. COPS_1 concentrations will be prepared directly priorto use. The pH and osmolarity ofthe culture medium containing the highesttested concentration will be recorded. Test substance concentrations will be approved by the study director in the study files. 

### 4.2. Reference substances 4.2.1. Negative control The negative control will be the vehicle ofthe test substance. 

### 4.2.2. Positive controls Without metabolic activation (-S9-mix): Mitomycin C (MMC-C; CAS no. 50-07-7) will be used as a direct acting mutagen at a final concentration of0.5 and 0.75 f..lg/ml for a 3 h exposure period, 0.2 and 0.3 f..lg/ml for a 24 h exposure period and 0.1 and 0.15 f..lg/ml for a 48 h exposure period. With metabolic activation (+S9-mix): Cyclophosphamide (CP; CAS no. 50-18-0) will be used as an indirect acting mutagen, requiring metabolic activation, at a final concentration of 10 f.lg/ml for a 3 h exposure period (24 h fixation period). Solvent for positive controls Hanks' Balanced Salt Solution (HBSS) without calcium and magnesium.
Page 6 •',All reference stock solutions are stored in aliquots at S-15°C in the dark. These solutionswill be thawed immediately before use. 

### 4.3. Test system Cultured peripheral human lymphocytes are used as test system. Peripheral human lymphocytes are recommended in international guidelines (e.g. OECD, EC). Blood will be collected from healthy adult, non-smoking, male volunteers. The Average Generation Time (AGT) ofthe cells will be reported. 

### 4.4. Cell culture Blood samples Blood samples will be collected by venipuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium or lithium heparin. Immediately after blood collection lymphocyte cultures will be started. Culture medium Culture medium will consist of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calfserum, L-glutamine (2 mM), penicillin/ streptomycin (50 U/ml and 50 IJg/ml respectively) and heparin (30 U/ml). Lymphocyte cultures Whole blood (0.4 ml) treated with heparin will be added to 5 ml or4.8 ml culture medium (in the absence and presence of89-mix, respectively). Per culture 0.1 ml phytohaemagglutinin (9 mg/ml) will be added. Environmental conditions All incubations will be carried out in a humid atmosphere (80- 100%) containing 5.0 

### ± 0.5% C02 in air in the dark at 37.0 

### ± 1.0°C. Temperature and humidity will be continuously monitored throughout the experiment. The C02 percentage will be monitored once on each working day. Temporary deviations ofmaximally 2 hourfrom the temperature (in the range of34.0- 36.0°C), humidity (with a maximum of 30%) and C02 percentage (with a maximum of 1%) may occur due to opening and closing ofthe incubatordoor. Based on laboratory historical data these deviations are considered notto affectthe study integrity. 

### 4.5. Metabolic activation system Rat liver microsomal enzymes are routinely prepared from adult male Wistar rats (6), which are obtained from Charles River (Sulzfeld, Germany). 

### 4.5.1. Preparation of59-fraction The animals are housed atWIL Research Europe in a special room under standard laboratory conditions, asdescribed in the Standard Operating Procedures, and allowed to acclimatise for at least 5 days. The rats are orally dosed forthree consecutive dayswith a suspension ofphenobarbital (80 mg/kg bodyweight) and B-naphthoflavone (1 00 mg/kg body weight) in corn oil (they are denied access to food for 3 to 4 hours preceding each dosing). One day afterthe final exposure (24 h), the rats are sedated using oxygen/carbon dioxide and then killed by decapitation. Rats receive a limited quantity of food during the night before sacrifice. The livers ofthe rats are removed aseptically, and washed in cold (0°C) sterile 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1 mM NarEDTA. The livers are minced in a blender and homogenised in 3volumes of phosphate bufferwith a Potter homogeniser. The homogenate is centrifuged for 15 min at 9000 

### g. The supernatant (S9) is transferred into sterile ampules, which are stored in liquid nitrogen (-196°C) for a maximum of 1year. The S9 batch is characterised in a bacterial reverse mutation assay in Salmonella typhimuriumtester strain TA98 with the mutagens Benzo-(a)-pyrene and 2-aminoanthracene at concentrations of Page 75 IJg/plate and 1 IJg/plate, respectively. These mutagens require metabolic activation. 

### 4.5.2. Preparation of59-mix 89-mixwill be prepared immediately before use and kept on ice. 89-mix components contains per ml: 1.63 mg MgCb.6H2

### 0; 2.46 mg KCI; 1.7 mg glucose-6-phosphate; 3.4 mg NADP; 4**~mol** HEPE8. The above solution will be filter (0.22**~m)-sterilized.** To 0.5 ml 89-mix components 0.5 ml 89-fraction will be added (50% (v/v) 89-fraction) to complete the 89-mix. Metabolic activation will be achieved by adding 0.2 ml 89-mixto 5.3 ml ofa lymphocyte culture (containing 4.8 ml culture medium, 0.4 ml blood and 0.1 ml phytohaemagglutinin (9 mg/ml)). The concentration ofthe 89-fraction in the exposure medium will be 1.8% (v/v). 

### 4.6. Study design 4.6.1. Dose range finding test In orderto selectthe appropriate dose levels for the chromosome aberration test cytotoxicity data will be obtained in a dose range finding test. COP8_1 will be tested in the absence and presence of 89-mix. Lymphocytes will be cultured for48 h and thereafter exposed to selected doses ofCOP8_1 for 3 h, 24 h and 48 h in the absence of89-mix orfor 3 h in the presence of89-mix. A vehicle control will be included at each exposure time. In case COP8_1 will be difficultto dissolve in aqueous solutions, the highest dose level for the 3 h exposure time will be determined bythe solubility in the culture medium. When precipitation in the culture medium will be observed at concentrations of 100 !Jg/ml and lower, lymphocyteswill be cultured in duplicate in the absence and presence of89-mix. The appropriate vehicle and positive controls will be included. Atthe 24 and 48 h exposuretime, COP8_1 will be tested at concentrations beyond the limit of solubilityto obtain adequate toxicity data. Concentrations exceeding 5 mg/ml or 0.01 M (whichever is the lowest) will not be tested. After 3 h exposureto COP8_1 in the absence or presence of89-mix, the cells will be separated from the exposure medium by centrifugation (5 min, 365 g). The supernatantwill be removed and cells will be rinsed with 5 ml HB88. After a second centrifugation step, HB88will be removed and cells will be resuspended in 5 ml culture medium and incubated for another20- 22 h (24 h fixation time). The cells thatwill be treated for 24 h and 48 h in the absence of89-mix, will not be rinsed after exposure but will be fixed immediately (24 hand 48 h fixation time). Cytotoxicity ofCOP8_1 in the lymphocyte cultures will be determined using the mitotic index. Based on the results ofthe dose range finding test an appropriate range ofdose levelswill be chosen forthe cytogenetic assays. The highest dose levelsfor the 3 h exposure time will be determined by the solubility ofthe test substance in the culture medium. In case lymphocytes will be cultured in duplicate in the dose range finding study atthe 3 h exposure time and no toxicity is observed, these slides will be scored for chromosome aberrations and the first cytogenetic assaywill be omitted. Ifdose-related cytotoxicitywill be observed atthe 24 and 48 h exposure times, the highest dose level will be based on toxicity irrespective ofsolubility. However, the precipitate may not interferewith the scoring of chromosome aberrations. In case ofsoluble test substances that showtoxicity, the highest concentration should showan inhibition ofthe mitotic index of50% or greaterwhereas the mitotic index ofthe lowest dose level should be approximately the same as the mitotic index ofthe solvent control.

Page 8



### I

### 4.6.2. First cytogenetic assay The cytogenetic assays will be carried out as described by Evans, 1984 (2) with minor modifications. Lymphocyteswill be cultured for48 h and thereafter exposed in duplicateto selected doses of COPS_1 for 3 h in the absence and presence ofS9-mix. After 3 h exposure, the cells will be separated from the exposure medium by centrifugation (5 min, 365 g). The supernatantwill be removed and the cells will be rinsed oncewith 5 ml HBSS. After a second centrifugation step, HBSS will be removed and cells will be resuspended in 5 ml culture medium and incubated for another20 - 22 h (24 h fixation time). Appropriate vehicle and positive controls will be included in the first cytogenetic assay. 

### 4.6.3. Second cytogenetic assay Lymphocytes will be cultured for48 h and thereafter exposed in duplicate to selected doses of COPS_1 for 24 h and 48 h in the absence ofS9-mix and for 3 h in the presence ofS9-mix. After 3 htreatment, the cells exposed to the test substance in the presence of S9-mixwill be separated from the exposure medium by centrifugation (5 min, 365 g). The supernatantwill be removed and the cells will be rinsed once with 5 ml of HBSS and incubated in 5 ml culture medium for another44- 46 h (48 h fixation time). The cells thatwill be treated for 24 

### hand 48 h in the absence ofS9-mix, will not be rinsed after exposure butwill be fixed immediately after24 hand48 h (24 hand 48 h fixation time). Appropriate vehicle and positive controls will be included in the second cytogenetic assay. 

### 4.6.4. Chromosome preparation During the last 2.5 - 3 h ofthe culture period, cell division will be arrested by addition ofthe spindle poison colchicine (0.5 f.tg/ml medium). The cell cultures will be subsequently centrifuged for 5 min at 365 

### g and the supernatantwill be removed. Cells in the remaining cell pelletwill be swollen by a 5 min treatmentwith hypotonic 0.56% (w/v) potassium chloride solution at 37"C. After hypotonic treatment, cells will be fixed with 3 changes of methanol/acetic acid fixative (3: 1v/v). 

### 4.6.5. Preparation ofslides Fixed cells will be dropped onto cleaned slides, which will be first immersed in a 1:1 mixture of 96% (v/v) ethanol/etherand cleaned with a tissue. The slides will be marked with theWIL Research Europe study identification numberand group number. At leasttwo slides will be prepared per culture. Slides will be allowed to dry and thereafter stained for 10 - 30 min with 5% (v/v) Giemsa solution in tap water. Slideswill be subsequently rinsed in tapwater and allowed to dry. The dry slides will be automatically embedded in a 1:10 mixture ofxylene/pertex and mounted with a coverslip with an automated coverslipper (Leica Microsystems B.V., Rijswijk, The Netherlands). 

### 4.6.6. Mitotic index/dose selection for scoring ofthe cytogenetic assay The mitotic index ofeach culture will be determined by counting the numberofmetaphases from at least 1000 cells (with a maximum deviation of5%). Chromosomes ofmetaphase spreads will be analysed ofthose cultures with an inhibition ofthe mitotic index ofabout 50% orabove, whereas the mitotic index ofthe lowest dose levelwill be approximatelythe same asthe mitotic index ofthe solvent control. Also cultures treated with an intermediate dosewill be examined for chromosome aberrations. In case COPS_1 is not cytotoxic and/or is difficultto dissolve in aqueous solutions, the highest concentration analysed atthe 3 h exposure time will be determined by the solubility in the culture medium. Ifdose related cytotoxicity is observed, the highest concentration analysed at the 24 and 48 h continuous exposure times will be based on toxicity irrespective ofsolubility ofCOPS_1 in the culture medium. However, the extent ofprecipitation may not interfere with the scoring ofchromosome aberrations.

Page 9

### 4.6.7. Analysis ofslides for chromosome aberrations To prevent bias, all slides will be randomly coded before examination ofchromosome aberrations and scored. An adhesive label with WIL Research Europe study identification number and code will be placed overthe marked slide. One hundred metaphase chromosome spreads per culture will be examined by light microscopyfor chromosome aberrations. In case the number ofaberrant cells, gaps excluded,**is~** 25 in 50 metaphases no more metaphases will be examined. Only metaphases containing 46 ±2 centromeres (chromosomes) will be analysed. The number ofcells with aberrations and the numberofaberrations will be calculated. In case no statistically significant increasewill be observed in the cultures dosed with the lowest dose ofMMC-C, both doseswill be scored for the presence ofchromosomal aberrations. 

### 4.7. Electronic data capture Observations/measurements in the study will be recorded electronically using the following programme: REES Centron Environmental Monitoring system version SOL 2.0 (REES Scientific, Trenton, NJ, USA). Any upgrades will be approved bythe Study Director (or Principal ScientisUinvestigator) in the study files. 

### 4.8. Interpretation 4.8.1. Acceptability ofthe assay A chromosome aberration test is considered acceptable if it meets the following criteria: a) The numberofchromosome 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, onesided, p < 0.05) increase in the numberofcells with chromosome aberrations. c) A homogeneous response between the replicate cultures is observed. d) A possible precipitate present on the slides should not interfere with the scoring ofchromosome aberrations. If (one of) the acceptability criteria are not met and the Study Director decides thatthis has a critical effect on the study, the testwill be rejected and repeated. 

### 4.8.2. Data evaluation and statistical procedures The incidence ofaberrant cells (cells with one or more chromosome aberrations, inclusive or exclusive gaps) for each exposure group outside the laboratory historical control data range will be compared to that ofthe solvent control using Chi-square statistics. A test substance is considered positive (clastogenic) in the chromosome aberration test if: a) It induces a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the numberofcells with chromosome aberrations. b) A statistically significant and biologically relevant increase in the frequencies ofthe number ofcells with chromosome aberrations is observed in the absence ofa clear dose-response relationship. A test substance is considered negative (not clastogenic) in the chromosome aberration test ifnone of the tested concentrations induces a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the numberofcells with chromosome aberrations. The preceding criteria are not absolute and other modifying factors may enter into the final evaluation decision.

Page 10. 

### ' ' 

# .

5. REFERENCES 1. Evans H.J. (1976). Cytological methods for detecting chemical mutagens. In: Chemical mutagens, principles and methods fortheir detection, Vol. 4, Ed. A. Hollaender (NewYork, London, PlenumPress), 1 - 29. 2. Evans H.J. (1984). Human Peripheral Blood Lymphocytes fortheAnalysis ofChromosome Aberrations in Mutagen Tests. In: Handbook of Mutagenicity Test Procedures, Eds. B.J. Kilbey, M. Legator, W. Nichols and C. Ramel, 405-427, Elsevier Science Publishers B.V., Amsterdam. 3. Howard P.N., Bloom A.D., Krooth R.S. (1972). Chromosomal aberrations induced by N-methyi-N'- nitro-N-nitrosoguanidine in mammalian cells. In Vitro 7: 359 - 365. 4. Preston R.J., Au W., Bender M.A., Brewen J.G., Carrano A.V., Heddle J.A., McFeeA.F., WolffS., Wassom J.S. (1981). Mammalian in vivo and in vitro cytogenetic assays: A report ofthe Gene-tox Program. Mutat Res 87: 143 - 188. 5. Galloway S.M., Aardema M.J., lshidate M.,Jr., Ivett, J.L., Kirkland D.J., Morita T., Mosesso P., Sofuni T. (1994). Report from Working Group on in Vitro Tests for Chromosomal Aberrations. Mutat Res 312: 241 -261. 6. Ames B.N., McCann J. and Yamasaki, E. (1975). Methods for detecting carcinogens and mutagenswith the Salmonella/mammalian microsome mutagenicitytest. Mutat Res 31, 347- 364. 7. Morita T., Nagaki T., Fukuda 

### I. and Okumura K. (1992). Clastogenicity oflow pH to various cultured mammalian cells. Mutat Res 268: 297 - 305.


### 6. DISTRIBUTION Original: 1Copy: 1Copy: Study Director QAU/Management Sponsor

Page 11

## PROTOCOL

Study Title

# PJ(u(t~**c~:)**

EVALUATION OF THE MUTAGENIC ACTIVITY OF COPS_1 IN

THE SALMONELLA TYPHIMURIUM REVERSE MUTATION ASSAYAND THE ESCHERICHIA COLIREVERSE MUTATION ASSAY (ADDITIONAL EXPERIMENT TO 500680)

C.M. Verspeek-Rip
Test Facility WIL Research Europe B.V. Hambakenwetering 7 5231 DO 's-Hertogenbosch The Netherlands Laboratory Project Identification



### ProjectTo be determined Substance 204240/A

- Page 1 of 10 -

Page 1of 101. CONTENTS 

### 1. CONTENTS

......................................................................................................................................2 2. PROTOCOLAPPROVAL.

................................................................................................................3 3. INTRODUCTION

..............................................................................................................................4 3.1. Preface

.....................................................................................................................................4 3.2. Aims ofstudy

............................................................................................................................4 3.3. Guidelines

.................................................................................................................................4 3.4. Good Laboratory Practice

.........................................................................................................

### 5 

### 3.5. Quality Assurance

.....................................................................................................................

### 5 3.6. Storage and retention of records and materials

.......................................................................

### 5 4. MATERIALS AND METHODS

.........................................................................................................5 4.1. Test substance

.........................................................................................................................5 4.1.1. Test substance information

............................................................................................5 4.1.2. Study specifictest substance information

......................................................................5 4.1.3. Safety precautions and disposal category
.....................................................................6 4.1.4. Test substance preparation

............................................................................................6 4.2. Reference substances

..............................................................................................................6 4.2.1. Vehicle control

................................................................................................................6 4.2.2. Positive controls

.............................................................................................................6 4.3. Test system
..............................................................................................................................6 4.4. Cell culture

................................................................................................................................7 4.5. Metabolic activation system
......................................................................................................7 4.5.1. Preparation of59-fraction

..............................................................................................8 4.5.2. Preparation ofS9-mix

.....................................................................................................8 4.6. Study design

.............................................................................................................................8 4.6.1. Mutation assay ...............................................................................................................8 4.6.2. Colony counting

..............................................................................................................8 4.7. Electronic data capture
.............................................................................................................9 4.8. Interpretation

.............................................................................................................................9 4.8.1. Acceptability ofthe assay................................................................................ - ...............9 4.8.2. Data evaluation and statistical procedures

....................................................................9 5. REFERENCES

...............................................................................................................................10 6. DISTRIBUTION

..............................................................................................................................10

Page 2

### 2. PROTOCOL APPROVAL STUDY DIRECTOR:

SPONSOR: C.M. Verspeek-Rip

date:

Society ofChemical Manufacturers and**Affiliate~** Dr. C. T. Helmes

date:

Page 3

### 3. INTRODUCTION 3.1. Preface Sponsor

Study Monitor Test Facility

Study Director Study Plan



### 3.2. Aims ofstudy Society ofChemical Manufacturers and Affiliate 1850 M Street, NW Suite 700 WASHINGTON, DC 20036 USA Dr. C. T. Helmes WIL Research Europe B.V. Hambakenwetering 7 5231 DD 's-Hertogenbosch The Netherlands C.M. Verspeek-Rip ineke.verspeek@wilresearch.com Startweek beginning To be added Completed week beginning : To be added Proposed Reporting date To be added

The objective ofthis study is to evaluate the test substance for its ability to induce reverse mutations in a gene ofhistidine-requiring Salmonella typhimurium bacterial strains resulting in histidineindependent Salmonella typhimurium strains, and in a gene oftryptophan-requiring Escherichia coli bacterial strain resulting in a tryptophan-independent strain. Since no correction forthe purity was applied in project 500680, this experimentwill be performed as additional projectto fulfil all criteria according to the guidelines. Background ofthe test system The Salmonella typhimurium reverse mutation assay and the Escherichia colireverse mutation assay have been shown to be rapid and adequate indicators ofthe mutagenic activity ofa wide range of chemical compounds. The assays will be conducted in the absence and in the presence ofa metabolic system (S9-mix). The Salmonella typhimurium strains used in this study will be TA98, TA100, TA1535 and TA1537, and the Escherichia colistrain used will be WP2uvrA. The strains TA98 and TA1537 are capable ofdetecting frameshift mutagens, and the strains TA100, TA1535 and WP2 uvrA are capable ofdetecting base-pair substitution mutagens (1-5). 

### 3.3. Guidelines The study procedures described in this protocol are in compliance with the following guidelines: Organisation for Economic Co-operation and Development (OECD), OECD Guidelinesfor Testing ofChemicals; Guideline no. 471: "Genetic Toxicology: Bacterial Reverse Mutation Test". (Adopted July 21, 1997). European Community (EC). Commission regulation (EC) No. 440/2008, Part B: Methods forthe Determination ofToxicity and other health effects, Guideline B.13/14: "Mutagenicity: Reverse Mutation Test using Bacteria". Official Journal ofthe European Union No. L142, 31 May 2008. Code of Federal Regulations, Title 40- Protection of Environment. Section 799.9510 Toxic Substances Control Act (TSCA) bacterial reverse mutation test.

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### 3.4. Good Laboratory Practice The studywill be performed in compliance with the Organization for Economic Co-operation and Development (OECD) Principles ofGood Laboratory Practice (GLP) (as revised in 1997) ENV/MC/CHEM (98) 17. 

### 3.5. Quality Assurance Study and/or process inspections will be performed by the WIL Research Europe Quality Assurance Unitto assurethe GLP compliance ofthis study. Facility inspectionswill be also performed at regular intervals to assure the GLP compliance ofgeneral aspects. The protocol will be inspected to confirm that itcomplies with GLP regulations. The reportwill be inspected to confirm thatthe methods and results accurately and completely reflectthe raw data. 

### 3.6. Storage and retention of records and materials Records and materials pertaining to the study, including protocol, raw data and the final report, will be retained in the WIL Research Europe archives for a period ofat least 2 years afterfinalization ofthe report. Afterthis period, the sponsorwill be contacted to determine howthe records and materials should be handled. WIL Research Europewill retain information concerning decisions made. WIL Research Europe will retain atest substance sample until the expiry date, but no longerthan 10 years afterfinalization ofthe report. Afterthis period the sample will be destroyed. 

### 4. MATERIALS AND METHODS 4.1. Test substance The sponsor is responsible for all test substance information unless determined by WIL Research Europe B.V

.. This will be specified in the GLP compliance statement in the report. 

### 4.1.1. Test substance information Identification Synonyms Structure

Molecularformula Molecularweight Description Batch Purity/Composition Sodium-1-allyloxy-2- hydroxypropane sulphonate 3-allyl-1 ,2-propanediol Water Test substance storage Stability under storage conditions Expiry date 1-Propanesulfonicacid, 2-hydroxy-3-(2-propen-1- yloxy)-, sodium salt (1:1) COPS_1 or CS-AHPS

CeH11NaOsS 218.22 Clear colourless liquid 12E1101 CAS Number 52556-42-0 Percent 38.64 123-34-2 2.26 7732-18-5 59.1 At room temperature in the dark Stable 13 August 2013 

### 4.1.2. Study specific test substance information Purity/composition correction factor required Stability at highertemperatures Yes Yes, maximum temperature: 80°C, maximum duration: 1 hour Page 5

### Stability in vehicle Solubility in vehicle Water: 1year Water: 1-45% Analysis ofstability, homogeneity and concentration ofthe test substance undertest conditions will not be performed as part ofthis study. 

### 4.1.3. Safety precautions and disposal category Safety precautions Gloves, goggles and face mask to ensure personnel health and safety Disposal category 

### Ill 4.1.4. Test substance preparation A correction factor of**~.59** 

### will be applied to correct forthe purity ofthe test substance. The test substance will be dissolved in Milli-Q water. Test substance concentrations will be used within 4 hours after preparation. 

### 4.2. Reference substances 4.2.1. Vehicle control The vehicle ofthe test article. 

### 4.2.2. Positive controls Without metabolic activation (-S9-mix): Strain Chemical TA1535 sodium azide (SA) TA1537 ICR-191 TA98 2-nitrofluorene (NF) TA100 methylmethanesulfonate (MMS) WP2

### uvrA 4-nitroquinoline N-oxide (4-NQO) With metabolic activation (+S9-mix): Concentration/plate 5 IJg 2.5 IJg 10 IJg 650 IJg

10 IJg Solvent Saline DMSO DMSO DMSO DMSO The positive control substance used for all tester strains will be 2-aminoanthracene (2AA). The following doseswill be used: Strain Concentration/plate Amount of S9-mix 5and 10% Solvent DMSO DMSO DMSO DMSO DMSO DMSO DMSO TA1535 2.5 IJg TA1537 2.5 IJg 5% TA1537 5 IJg 10% TA98 1 IJg 5and10% TA100 1 IJg 5% TA100 2.5 IJg 10% WP2

### uvrA 10 IJg 5 and 10% Solvents for reference substances Saline

# =physiological saline DMSO

# = dimethyl sulfoxide 

### 4.3. Test system Test System Rationale
Source Salmonella typhimurium 

### bacteria and Escherichia coli

### bacteria Recommended test system in international guidelines (e.g. OECD, EC). Trinova Biochem GmbH, Germany (Master culture from Dr. Bruce N. Ames)(TA1535:2006, TA1537:2009,TA98:2006,TA100:2006)and
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### I(Master culture from The National Collections of Industrial and Marine Bacteria, Aberdeen, UK) (WP2uvrA, 2008) The characteristics ofthe different Salmonella typhimurium strains are asfollows: Strain Histidine mutation Mutation type TA1537 hisC3076 Frameshift TA98 hisD3052/R-factor* Frameshift T A1535 hisG46 Base-pair substitutions TA100 hisG46/R-factor* Base-pair substitutions *: R-factor

# =plasmid pKM101 (increases error-prone DNA repair) Each tester strain contains the following additional mutations: rfa :deep rough (defective lipopolysaccharide cellcoat) 

### gg! : mutation in the galactose metabolism chi : mutation in nitrate reductase bio : defective biotin synthesis uvrB : loss ofthe excision repair system (deletion ofthe ultraviolet-repair B gene) The Salmonella typhimurium strains are regularly checked to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants. The Escherichia coliWP2uvrA strain detects base-pair substitutions. The strain lacks an excision repair system and is sensitive to agents such as UV. The sensitivity ofthe strain to a wide variety of mutagens has been enhanced by permeabilization ofthe strain using Tris-EDTAtreatment (ref.1). The strain is regularly checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants. Stock cultures ofthe five strains are stored in liquid nitrogen (-196°C). 

### 4.4. Cell culture Preparation ofbacterial cultures Samples offrozen stock cultures ofbacteria will be transferred into enriched nutrient broth and incubated in a shaking incubator (37°C, 150 spm), until the cultures reach an optical density of 1.0 ± 0. 1 at 700 nm (109 cells/ml). Freshly grown cultures ofeach strain will be used for a test. Agar plates Agar plates 

### (0 9 em) contain 25 ml glucose agar medium. Glucose agar medium contains per liter: 18 g purified agar in Vogel-Bonner Medium E, 20 g glucose. The agar plate forthe testwith the Salmonella typhimurium strains also contains 12.5 IJg/plate biotin and 15 j.Jg/plate histidine and the agar plates forthe testwith the Escherichia colistrain contains 15 j.Jg/plate tryptophan. Top agar Milli-Q watercontaining 0.6% (w/v) bacteriological agarand 0.5% (w/v) Sodium Chloridewill be heated to dissolve the agar. Samples of3 ml top agarwill be transferred into 10 ml glass tubeswith metal caps. Top agartubes will be autoclaved for 20 min at 121 ± 3

## •c. Environmental conditions All incubations will be carried out in the dark at 37.0 ± 1.0

# ·c. The temperaturewill be continuously monitored throughoutthe experiment. Temporary deviations ofmaximally 2 hours (in the range of 34.0- 36°C and 38- 39.0°C} may occur dueto opening and closing the incubatordoor. Based on laboratory historical data these deviations are considered notto affectthe study integrity. 

### 4.5. Metabolic activation system Rat S9 homogenatewill be obtained from Trinova Biochem GmbH, Giessen, Germany and is prepared from male Sprague Dawley rats that have been intraperitoneally injected with Aroclor 1254 (500 mg/kg Page 7bodyweight). 

### 4.5.1. Preparation of59-fraction The animals are housed at WIL Research Europe in a special room under standard laboratory conditions, as described in the SOP's. The rats are orally dosed for three consecutive dayswith a suspension of phenobarbital (80 mg/kg body weight) and 1!-naphthoflavone (100 mg/kg body weight) in corn oil (they are denied access to food for3 to 4 hours preceding each dosing). One day afterthe final exposure (24 h), the rats are sedated using oxygen/carbon dioxide and then killed by decapitation. Rats receive a limited quantity offood during the night before sacrifice. The livers ofthe rats are removed aseptically, and washed in cold (0°C) sterile 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1 mM Na2-EDTA. The livers are minced in a blender and homogenised in 3 volumes of phosphate bufferwith a Potter homogeniser. The homogenate is centrifuged for 15 min at 9000 g. The supernatant (S9) is transferred into sterile ampules, which are stored in liquid nitrogen (-196°C) for a maximum of 1 year. The S9 batch is characterised with the mutagens Benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA98 at concentrations of5 IJg/plate and 1 IJg/plate, respectively. 

### 4.5.2. Preparation of59-mix 89-mix is prepared immediately before use and kept on ice. S9-mix contains per 10 ml: 30 mg NADP and 15.2 mg glucose-6-phosphate in 5.5 ml or 5.0 ml Milli-Q water (first orsecond experiment respectively); 2 ml 0.5 M sodium phosphate buffer pH 7.4; 1 ml 0.08 M MgCI2 solution; 1 ml 0.33 M KCI solution. The above solution is filter (0.22 1Jm)-sterilized. To 9.5 ml ofS9-mix components 0.5 ml 59-fraction is added (5% (v/v) 59-fraction) to complete the S9-mix in the first experimentand to 9.0 ml of89-mix components 1.0 ml 59-fraction is added (10% (v/v) 59-fraction) to completethe 89-mix in the second experiment. 

### 4.6. Study design 4.6.1. Mutation assay The test substance will be tested in triplicate in each strain at a dose level of5000 IJg/plate. COPS_1 will be tested both in the absence and presence of5 and 10% (v/v) S9-mix. The vehicle control and relevant positive controls will be concurrently tested in each strain in the presence and absence of89-mix. Top agar in top agartubes will be molten and heated to 45°C. Thefollowing solutions will be successively added to 3 ml molten top agar: 0.1 ml ofa fresh bacterial culture (109 cells/ml) ofone of the tester strains, 0.1 ml ofa dilution ofthe test substance in a suitable solvent, and either 0.5 ml S9-mix (in case ofactivation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays). The ingredientswill be mixed on a Vortex and the content ofthe top agartube will be poured onto a selective agar plate. After solidification ofthe top agar, the plates will be inverted and incubated in the dark at 37.0 ± 1.0°C for48 ±4 h. Afterthis period revertant colonies (histidine independentfor Salmonella typhimurium bacteria and tryptophan independentfor Escherichia colibacteria) will be counted. 

### 4.6.2. Colony counting The revertant colonies (histidine independent ortryptophan independent) will be counted manually if less than 40 colonies per plate are present. If more than 40 colonies are present, these can be counted automaticallywith a Biocount4000 Pro-S-colony counter. Plateswith sufficient test article precipitateto interfere with automated colony counting will be counted manually and evidence oftest article precipitate on the plates will be recorded. The condition ofthe bacterial background lawn will be evaluated, both macroscopically and microscopically by using a dissecting microscope. Page 8 

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### 4.7. Electronic data capture Observations/measurements in the study will be recorded electronically using the following programme: REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ, USA). Any upgradeswill be approved by the Study Director (or Principal Scientist/Investigator) in the study files. 

### 4.8. Interpretation 4.8.1. Acceptability ofthe assay A Salmonella typhimurium reverse mutation assay and/or Escherichia colireverse mutation assay is considered acceptable ifit meets the following criteria: a) The vehicle control data (number ofspontaneous revertants per plate) should be within the laboratory historical range for each tester strain. b) The positive control chemicals should produce responses in all tester strains, which are within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at leastthree times the concurrent vehicle control group mean. If (one of) the acceptability criteria are not met and the Study Director decides thatthis has a critical effect on the study, the testwill be rejected and repeated. In case no clear conclusion for positive ornegative result can be made an additional confirmation studywill be performed to confirm the study results. 

### 4.8.2. Data evaluation and statistical procedures No formal hypothesis testing will be done. In addition to the criteria stated below, any increase in the total number ofrevertants should be evaluated for its biological relevance including a comparison ofthe results with the historical control data range. A test substance is considered negative (not mutagenic) in the test if: a) The total numberof revertants in tester strain T A 100 is not greaterthan two (2) times the concurrent control, and the total number of revertants in tester strain TA1535, TA1537, TA98 or WP2uvrA is not greaterthan three (3) timesthe concurrent control. A test substance is considered positive (mutagenic) in the test if: a) The total number of revertants in tester strain TA100 is greaterthan two (2) times the concurrent control, orthe total number of revertants in tester strain T A1535, T A1537, TA98 or WPzuvrA is greaterthan three (3) times the concurrent control. The preceding criteria are not absolute and other modifying factors may enter into the final evaluation decision.

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### 6. REFERENCES Leonardo, J.M., Dornfeld, S.S. and Peak, M.J., 1984, Evaluation of

### E. coliK12 343 \ 113 and derived strainsfor microbial mutagenicity assays. Mutation Res., 130, 87-95. Ames, B.N., McCann, J. and Yamasaki, E., 1975, Methods for detecting carcinogens and mutagenswith the Salmonella/mammalian microsome mutagenicity test, Mutation**Res.,~.** 347- 364. ' Maron, D.M. and Ames, B.N., 1983, Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Erratum, 1983, Mutation Res., 113,533. Green, M.H.L. and Muriel, W.J., 1976, Mutagen testing using Trp+ reversion in Escherichia coli, Mutation Res., 38, 3-32. Vogel, H.J. and Bonner, D.M., 1956, Acetylornithinase ofEscherichia coli: partial purification and some properties. J. Bioi. Chern., 218,97-106.
DISTRIBUTION Original: 1Copy: 1 Copy: 1 Copy: Study Director Technical Coordinator QAU/Management Sponsor

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