, CBIC Control Number 

ASSOCIATION 

MANAGEMENT 

SERVICES 

a division of 360598 

,,~f~~~~ 1850 M St NYV Ste 700, Washington, DC 20036 • Ph (20?.) 721-4100 • Fx (202) 296-8120 • ams@socma com • www.socma.con11Ass<xiati<>nMaiJag< 

Document Control Office (7 407 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 

To Whom It May Concern: 

201~ JUN 16 AH 10: 26 

June 16, 2014 

RE: New Test Plan; Testing of Certain High Production Volume Chemicals; Third 

Group of Chemicals. Federal Register 76 (204), 65385-65410, October 21, 2011. 

1-Propanesulfonic acid, 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 under the 

Society of Chemical Manufacturers and Affiliates (SOCMA) submits this new test plan in 

response to the April28, 2014 letter from Paul Lewis. The consortium had submitted data 

previously to satisfy the testing requirements of the subject test 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 HPV 3 test rule. 

The consortium agrees to repeat those studies according to the test protocols that are 

enclosed with this letter. Details of the test facility, test material, study design and methods, and 

interpretation of results are explained in the protocols. 

Members of the COPS-1 consortium are the following companies: 

AkzoNobel Surface Chemistry LLC 

Ashland Inc. 

Bimax Inc. 

GE Water & Process Technologies 

Solvay USA, Inc. 
·' ASSOCIATION a division 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 of the required testing is subject to EPA's approval of the test plan offered in 

this letter. In order to begin testing as quickly as possible to help us meet the EPA's reporting 

deadline of February 16, 2015, we look forward 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 of the Mutagenic Activity of COPS-1 in the Salmonella typhimurium 

Reverse Mutation Assay and the Escherichia coli Reverse Mutation Assay. 

2. Protocol: Evaluation of the Ability of COPS-1 to Induce Chromosome Aberrations in 

Cultured Peripheral Human Lymphocytes. 
'\ :·~· 

' 

PROTOCOL 

Study Title 

EVALUATION OF THE ABILITY OF 

COPS_1 TO INDUCE CHROMOSOME ABERRATIONS 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 

Project To be determined 

Substance 204240/A 

- Page 1 of 11 -

Page 1 of 1 
1. CONTENTS 

1. CONTENTS ...................................................................................................................................... 2 

2. PROTOCOL APPROVAL ................................................................................................................. 3 

3. INTRODUCTION .............................................................................................................................. 4 

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

3.2. Aims of study ............................................................................................................................ 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 specific test 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 of 59-fraction .............................................................................................. 7 

4.5.2. Preparation of 89-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 of slides ....................................................................................................... 9 

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

4.6. 7. Analysis of slides for chromosome aberrations ............................................................ 1 0 

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

4.8. Interpretation ........................................................................................................................... 10 

4.8.1. Acceptability of the assay ............................................................................................. 1 0 

4.8.2. Data evaluation and statistical procedures .................................................................. 1 0 

5. REFERENCES ............................................................................................................................... 11 

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

Page 2 

r 

.. 

' 
2. PROTOCOL APPROVAL 

STUDY DIRECTOR: 

SPONSOR: 

I.A.J. Verbaan, PhD. 

date: 

Society of Chemical 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 of study 

Society of Chemical 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 

Start week beginning To be added 

Completed week beginning : To be added 

Proposed Reporting date To be added 

The objective of this study is to evaluate COPS_1 for its ability to induce structural chromosome 

aberrations in cultured human lymphocytes (1 ), either in the presence or absence of a metabolic 

activation system (S9-mix). Since no correction for the purity was applied in project 500681, this 

experiment will be performed as additional project to fulfil all criteria according to the guidelines. 

Background of the test system 

Whole blood samples obtained from healthy male subjects are treated with an anti-coagulant (heparin) 

and cultured in the presence of a mitogen (phytohaemagglutinin). These stimulated human 

lymphocytes are used because they are sensitive indicators of clastogenic activity of a broad range of 

chemicals (1-5). 

The stimulated lymphocytes are exposed to the test substance both in the presence and absence of a 

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 of the stimulated human lymphocytes to the test substance, 

cell division is arrested by the addition of the metaphase-arresting chemical colchicine. Cells are 

harvested, stained and metaphase cells are analysed microscopically for the presence of structural 

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 of the first post-exposure mitosis can be considerably 

delayed due to toxic insult to the cells, cells are also harvested 48 hours after exposure to cover the 

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 Guidelines for Testing 

of Chemicals, 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 of Toxicity and other health effects, Guideline 8.10: "Mutagenicity- In Vitro 

Mammalian Chromosome Aberration Test". Official Journal of the European Union No. L 142, 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 study will be performed in compliance with the Organization for Economic Co-operation and 

Development (OECD) Principles of Good 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 

Unit to assure the GLP compliance of this study. Facility inspections are also performed at regular 

intervals to assure the GLP compliance of general aspects. 

The protocol will be inspected to confirm that it complies with GLP regulations. The report will be 

inspected to confirm that the 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 the WIL Research Europe archives for a period of at least 2 years after finalization of the 

report. After this period, the sponsor will be contacted to determine how the records and materials 

should be handled. WIL Research Europe will retain information concerning decisions made. 

WIL Research Europe will retain a test substance sample until the expiry date, but no longer than 10 

years after finalization of the 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 

Molecular formula 

Molecular weight 

Description 

Batch 

1-Propanesulfonic acid, 2-hydroxy-3-(2-propen-1-

yloxy)-, sodium salt (1:1) 

COPS_1 or CS-AHPS 

CeH11NaOsS 

218.22 

Clear colourless liquid 

12E1101 

Page 5 
Purity/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 higher temperatures 

Stability in vehicle 

Solubility in vehicle 

Yes 

Yes, maximum temperature: 80°C, 

maximum duration: 1 hour 

Water: 1 year 

Water: 1-45% 

Analysis of stability, homogeneity and concentration of the test substance under test conditions will not 

be performed as part of this 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 for the purity of the test substance. 

The test substance will be dissolved in RPMI1640 medium. COPS_1 concentrations will be prepared 

directly prior to use. The pH and osmolarity of the culture medium containing the highest tested 

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 of the 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 of 0.5 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 solutions will 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) of the 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 calf serum, 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 or 4.8 ml culture medium (in the 

absence and presence of 89-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- 1 00%) 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 

of maximally 2 hour from the temperature (in the range of 34.0- 36.0°C), humidity (with a maximum of 

30%) and C02 percentage (with a maximum of 1%) may occur due to opening and closing of the 

incubator door. Based on laboratory historical data these deviations are considered not to affect the 

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 of 59-fraction 

The animals are housed at WIL Research Europe in a special room under standard laboratory 

conditions, as described in the Standard Operating Procedures, and allowed to acclimatise for at least 

5 days. The rats are orally dosed for three consecutive days with a suspension of phenobarbital (80 

mg/kg body weight) 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 after the 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 of the 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 3 volumes of phosphate buffer with 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 in a bacterial reverse mutation assay in Salmonella typhimurium tester 

strain TA98 with the mutagens Benzo-(a)-pyrene and 2-aminoanthracene at concentrations of 

Page 7 
5 IJg/plate and 1 IJg/plate, respectively. These mutagens require metabolic activation. 

4.5.2. Preparation of 59-mix 

89-mix will be prepared immediately before use and kept on ice. 89-mix components contains per ml: 

1.63 mg MgCb.6H20; 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-mix to 5.3 ml of a lymphocyte culture 

(containing 4.8 ml culture medium, 0.4 ml blood and 0.1 ml phytohaemagglutinin (9 mg/ml)). The 

concentration of the 89-fraction in the exposure medium will be 1.8% (v/v). 

4.6. Study design 

4.6.1. Dose range finding test 

In order to select the 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 for 48 h and thereafter exposed to selected doses of COP8_1 

for 3 h, 24 h and 48 h in the absence of 89-mix or for 3 h in the presence of 89-mix. A vehicle control 

will be included at each exposure time. 

In case COP8_1 will be difficult to dissolve in aqueous solutions, the highest dose level for the 

3 h exposure time will be determined by the solubility in the culture medium. When precipitation in the 

culture medium will be observed at concentrations of 100 !Jg/ml and lower, lymphocytes will be 

cultured in duplicate in the absence and presence of 89-mix. The appropriate vehicle and positive 

controls will be included. 

At the 24 and 48 h exposure time, COP8_1 will be tested at concentrations beyond the limit of 

solubility to obtain adequate toxicity data. Concentrations exceeding 5 mg/ml or 0.01 M (whichever is 

the lowest) will not be tested. 

After 3 h exposure to COP8_1 in the absence or presence of 89-mix, the cells will be separated from 

the exposure medium by centrifugation (5 min, 365 g). The supernatant will be removed and cells will 

be rinsed with 5 ml HB88. After a second centrifugation step, HB88 will be removed and cells will be 

resuspended in 5 ml culture medium and incubated for another 20- 22 h (24 h fixation time). The cells 

that will be treated for 24 h and 48 h in the absence of 89-mix, will not be rinsed after exposure but will 

be fixed immediately (24 hand 48 h fixation time). 

Cytotoxicity of COP8_1 in the lymphocyte cultures will be determined using the mitotic index. 

Based on the results of the dose range finding test an appropriate range of dose levels will be chosen 

for the cytogenetic assays. The highest dose levels for the 3 h exposure time will be determined by the 

solubility of the test substance in the culture medium. In case lymphocytes will be cultured in duplicate 

in the dose range finding study at the 3 h exposure time and no toxicity is observed, these slides will 

be scored for chromosome aberrations and the first cytogenetic assay will be omitted. If dose-related 

cytotoxicity will be observed at the 24 and 48 h exposure times, the highest dose level will be based 

on toxicity irrespective of solubility. However, the precipitate may not interfere with the scoring of 

chromosome aberrations. In case of soluble test substances that show toxicity, the highest 

concentration should show an inhibition of the mitotic index of 50% or greater whereas the mitotic 

index of the lowest dose level should be approximately the same as the mitotic index of the 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. 

Lymphocytes will be cultured for 48 h and thereafter exposed in duplicate to selected doses of 

COPS_1 for 3 h in the absence and presence of S9-mix. After 3 h exposure, the cells will be 

separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant will be 

removed and the cells will be rinsed once with 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 another 20 -

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 for 48 h and thereafter exposed in duplicate to selected doses of 

COPS_1 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 treatment, the cells exposed to the test substance in the presence of S9-mix will be separated from 

the exposure medium by centrifugation (5 min, 365 g). The supernatant will be removed and the cells 

will be rinsed once with 5 ml of HBSS and incubated in 5 ml culture medium for another 44- 46 h (48 h 

fixation time). The cells that will be treated for 24 h and 48 h in the absence of S9-mix, will not be 

rinsed after exposure but will be fixed immediately after 24 hand 48 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 of the culture period, cell division will be arrested by addition of the spindle 

poison colchicine (0.5 f.tg/ml medium). The cell cultures will be subsequently centrifuged for 5 min at 

365 g and the supernatant will be removed. Cells in the remaining cell pellet will be swollen by a 5 min 

treatment with 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: 1 v/v). 

4.6.5. Preparation of slides 

Fixed cells will be dropped onto cleaned slides, which will be first immersed in a 1:1 mixture of 

96% (v/v) ethanol/ether and cleaned with a tissue. The slides will be marked with the WIL Research 

Europe study identification number and group number. At least two 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. Slides will be subsequently rinsed in tap water and allowed to dry. The dry slides will be 

automatically embedded in a 1:1 0 mixture of xylene/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 of the cytogenetic assay 

The mitotic index of each culture will be determined by counting the number of metaphases from at 

least 1000 cells (with a maximum deviation of 5%). Chromosomes of metaphase spreads will be 

analysed of those cultures with an inhibition of the mitotic index of about 50% or above, whereas the 

mitotic index of the lowest dose level will be approximately the same as the mitotic index of the solvent 

control. Also cultures treated with an intermediate dose will be examined for chromosome aberrations. 

In case COPS_1 is not cytotoxic and/or is difficult to dissolve in aqueous solutions, the highest 

concentration analysed at the 3 h exposure time will be determined by the solubility in the culture 

medium. If dose related cytotoxicity is observed, the highest concentration analysed at the 24 and 48 

h continuous exposure times will be based on toxicity irrespective of solubility of COPS_1 in the 

culture medium. However, the extent of precipitation may not interfere with the scoring of chromosome 

aberrations. 

Page 9 
4.6. 7. Analysis of slides for chromosome aberrations 

To prevent bias, all slides will be randomly coded before examination of chromosome aberrations and 

scored. An adhesive label with WIL Research Europe study identification number and code will be 

placed over the marked slide. One hundred metaphase chromosome spreads per culture will be 

examined by light microscopy for chromosome aberrations. In case the number of aberrant 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 of cells with aberrations 

and the number of aberrations will be calculated. In case no statistically significant increase will be 

observed in the cultures dosed with the lowest dose of MMC-C, both doses will be scored for the 

presence of chromosomal 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 by the Study Director (or Principal ScientisUinvestigator) in the study 

files. 

4.8. Interpretation 

4.8.1. Acceptability of the assay 

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, onesided, p < 0.05) increase in the number of cells 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 of chromosome 

aberrations. 

If (one of) the acceptability criteria are not met and the Study Director decides that this has a critical 

effect on the study, the test will be rejected and repeated. 

4.8.2. Data evaluation and statistical procedures 

The incidence of aberrant 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 of the 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 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 is observed in the absence of a clear dose-response relationship. 

A test substance is considered negative (not clastogenic) in the chromosome aberration test if none of 

the tested concentrations induces a statistically significant (Chi-square test, one-sided, p < 0.05) 

increase in the number of cells 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 for their detection, Vol. 4, Ed. A. Hollaender (New York, 

London, PlenumPress), 1 - 29. 

2. Evans H.J. (1984). Human Peripheral Blood Lymphocytes for the Analysis of Chromosome 

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., McFee A. F., Wolff S., 

Wassom J.S. (1981 ). Mammalian in vivo and in vitro cytogenetic assays: A report of the 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 

mutagens with the Salmonella/mammalian microsome mutagenicity test. Mutat Res 31, 347-

364. 

7. Morita T., Nagaki T., Fukuda I. and Okumura K. (1992). Clastogenicity of low pH to various 

cultured mammalian cells. Mutat Res 268: 297 - 305. 

6. DISTRIBUTION 

Original: 

1 Copy: 

1 Copy: 

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 

ASSAY AND THE ESCHERICHIA COLI REVERSE 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 

Project To be determined 

Substance 204240/A 

- Page 1 of 1 0 -

Page 1 of 10 
1. CONTENTS 

1. CONTENTS ...................................................................................................................................... 2 

2. PROTOCOL APPROVAL. ................................................................................................................ 3 

3. INTRODUCTION .............................................................................................................................. 4 

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

3.2. Aims of study ............................................................................................................................ 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 specific test 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 of 59-fraction .............................................................................................. 8 

4.5.2. Preparation of S9-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 of the 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 of Chemical 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 of study 

Society of Chemical 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 

Start week beginning To be added 

Completed week beginning : To be added 

Proposed Reporting date To be added 

The objective of this study is to evaluate the test substance for its ability to induce reverse mutations in 

a gene of histidine-requiring Salmonella typhimurium bacterial strains resulting in histidineindependent Salmonella typhimurium strains, and in a gene of tryptophan-requiring Escherichia coli 

bacterial strain resulting in a tryptophan-independent strain. Since no correction for the purity was 

applied in project 500680, this experiment will be performed as additional project to fulfil all criteria 

according to the guidelines. 

Background of the test system 

The Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay 

have been shown to be rapid and adequate indicators of the mutagenic activity of a wide range of 

chemical compounds. 

The assays will be conducted in the absence and in the presence of a metabolic system (S9-mix). The 

Salmonella typhimurium strains used in this study will be TA98, TA 1 00, TA 1535 and TA 1537, and the 

Escherichia coli strain used will be WP2uvrA. The strains TA98 and TA1537 are capable of detecting 

frameshift mutagens, and the strains TA 1 00, TA 1535 and WP2uvrA are capable of detecting 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 Guidelines for 

Testing of Chemicals; 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 for the 

Determination of Toxicity and other health effects, Guideline B.13/14: "Mutagenicity: Reverse 

Mutation Test using Bacteria". Official Journal of the European Union No. L 142, 31 May 2008. 

Code of Federal Regulations, Title 40- Protection of Environment. Section 799.9510 Toxic 

Substances Control Act (TSCA) bacterial reverse mutation test. 

Page4 
3.4. Good Laboratory Practice 

The study will be performed in compliance with the Organization for Economic Co-operation and 

Development (OECD) Principles of Good 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 

Unit to assure the GLP compliance of this study. Facility inspections will be also performed at regular 

intervals to assure the GLP compliance of general aspects. 

The protocol will be inspected to confirm that it complies with GLP regulations. The report will be 

inspected to confirm that the 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 the WIL Research Europe archives for a period of at least 2 years after finalization of the 

report. After this period, the sponsor will be contacted to determine how the records and materials 

should be handled. WIL Research Europe will retain information concerning decisions made. 

WIL Research Europe will retain a test substance sample until the expiry date, but no longer than 

10 years after finalization of the 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 

Molecular formula 

Molecular weight 

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-Propanesulfonic acid, 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 higher temperatures 

Yes 

Yes, maximum temperature: 80°C, 

maximum duration: 1 hour 

Page 5 
Stability in vehicle 

Solubility in vehicle 

Water: 1 year 

Water: 1-45% 

Analysis of stability, homogeneity and concentration of the test substance under test conditions will not 

be performed as part of this 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 for the purity of the 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 of the 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) 

WP2uvrA 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 doses will be used: 

Strain Concentration/plate Amount of S9-mix 

5 and 10% 

Solvent 

DMSO 

DMSO 

DMSO 

DMSO 

DMSO 

DMSO 

DMSO 

TA 1535 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% 

WP2uvrA 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 

Page 6 

I 
(Master culture from The National Collections of Industrial and Marine 

Bacteria, Aberdeen, UK) (WP2uvrA, 2008) 

The characteristics of the different Salmonella typhimurium strains are as follows: 

Strain Histidine mutation Mutation type 

TA1537 hisC3076 Frameshift 

TA98 hisD3052/R-factor* Frameshift 

T A 1535 hisG46 Base-pair substitutions 

TA 100 hisG46/R-factor* Base-pair substitutions 

*: R-factor = plasmid pKM1 01 (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 of the excision repair system (deletion of the 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 coli WP2uvrA strain detects base-pair substitutions. The strain lacks an excision 

repair system and is sensitive to agents such as UV. The sensitivity of the strain to a wide variety of 

mutagens has been enhanced by permeabilization of the strain using Tris-EDTA treatment (ref.1 ). The 

strain is regularly checked to confirm the tryptophan-requirement, UV-sensitivity and the number of 

spontaneous revertants. 

Stock cultures of the five strains are stored in liquid nitrogen (-196°C). 

4.4. Cell culture 

Preparation of bacterial cultures 

Samples of frozen stock cultures of bacteria 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 of each 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 for the test with the 

Salmonella typhimurium strains also contains 12.5 IJg/plate biotin and 15 j.Jg/plate histidine and the 

agar plates for the test with the Escherichia coli strain contains 15 j.Jg/plate tryptophan. 

Top agar 

Milli-Q water containing 0.6% (w/v) bacteriological agar and 0.5% (w/v) Sodium Chloride will be heated 

to dissolve the agar. Samples of 3 ml top agar will be transferred into 10 ml glass tubes with metal 

caps. Top agar tubes 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 temperature will be continuously 

monitored throughout the experiment. Temporary deviations of maximally 2 hours (in the range of 

34.0- 36°C and 38- 39.0°C} may occur due to opening and closing the incubator door. Based on 

laboratory historical data these deviations are considered not to affect the study integrity. 

4.5. Metabolic activation system 

Rat S9 homogenate will 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 7 
body weight). 

4.5.1. Preparation of 59-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 days with 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 for 3 to 4 hours preceding each dosing). One day after the 

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 of the 

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 buffer with 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 of 5 IJg/plate and 

1 IJg/plate, respectively. 

4.5.2. Preparation of 59-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 or second 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 of S9-mix components 0.5 ml 

59-fraction is added (5% (v/v) 59-fraction) to complete the S9-mix in the first experiment and to 9.0 ml 

of 89-mix components 1.0 ml 59-fraction is added (1 0% (v/v) 59-fraction) to complete the 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 of 5000 IJg/plate. COPS_1 

will be tested both in the absence and presence of 5 and 1 0% (v/v) S9-mix. 

The vehicle control and relevant positive controls will be concurrently tested in each strain in the 

presence and absence of 89-mix. 

Top agar in top agar tubes will be molten and heated to 45°C. The following solutions will be 

successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (1 09 cells/ml) of one of 

the tester strains, 0.1 ml of a dilution of the test substance in a suitable solvent, and either 0.5 ml 

S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation 

assays). The ingredients will be mixed on a Vortex and the content of the top agar tube will be poured 

onto a selective agar plate. After solidification of the top agar, the plates will be inverted and incubated 

in the dark at 37.0 ± 1.0°C for 48 ± 4 h. After this period revertant colonies (histidine independent for 

Salmonella typhimurium bacteria and tryptophan independent for Escherichia coli bacteria) will be 

counted. 

4.6.2. Colony counting 

The revertant colonies (histidine independent or tryptophan 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 automatically with a Biocount 4000 Pro-S-colony counter. Plates with sufficient test article 

precipitate to interfere with automated colony counting will be counted manually and evidence of test 

article precipitate on the plates will be recorded. The condition of the bacterial background lawn will be 

evaluated, both macroscopically and microscopically by using a dissecting microscope. 

Page 8 

.. . ' 
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 upgrades will be approved by the Study Director (or Principal Scientist/Investigator) in the study 

files. 

4.8. Interpretation 

4.8.1. Acceptability of the assay 

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 data (number of spontaneous revertants per plate) should be within the 

laboratory historical range for each tester strain. 

b) The positive control chemicals should produce responses in all tester strains, which are within the 

laboratory historical range documented for each positive control substance. Furthermore, the 

mean plate count should be at least three times the concurrent vehicle control group mean. 

If (one of) the acceptability criteria are not met and the Study Director decides that this has a critical 

effect on the study, the test will be rejected and repeated. 

In case no clear conclusion for positive or negative result can be made an additional confirmation 

study will 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 of revertants should be 

evaluated for its biological relevance including a comparison of the results with the historical control 

data range. 

A test substance is considered negative (not mutagenic) in the test if: 

a) The total number of revertants in tester strain T A 1 00 is not greater than two (2) times the 

concurrent control, and the total number of revertants in tester strain TA1535, TA1537, TA98 or 

WP2uvrA is not greater than three (3) times the concurrent control. 

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 strain T A 1535, T A 1537, T A98 or WPzuvrA is 

greater than three (3) times the concurrent control. 

The preceding criteria are not absolute and other modifying factors may enter into the final evaluation 

decision. 

Page 9 
5. 

2 

3 

4 

5 

6. 

REFERENCES 

Leonardo, J.M., Dornfeld, S.S. and Peak, M.J., 1984, Evaluation of E. coli K12 343 \ 113 and 

derived strains for microbial mutagenicity assays. Mutation Res., 130, 87-95. 

Ames, B.N., McCann, J. and Yamasaki, E., 1975, Methods for detecting carcinogens and 

mutagens with 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 of Escherichia coli: partial purification and 

some properties. J. Bioi. Chern., 218,97-106. 

DISTRIBUTION 

Original: 

1 Copy: 

1 Copy: 

1 Copy: 

Study Director 

Technical Coordinator 

QAU/Management 

Sponsor 

Page 10 

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