### April23, 2015 GREENWICH CHEMICAL CONSULTING, 

### 3300 PGA BouLEVARD • SuiTE 

### 635

PALM BEACH GARDENS, 

### FL 33410 TELEPHONE 561.578.8110 FACS!Mli.E 561.578.8120 CBIC Control Number

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION ByFederalExpress



### OPPTDocument Control Office EPA EastBuilding RoomE6428 U.S. EnvironmentalProtection Agency 1201 Constitution Ave., NW Washington, DC 20004 Attention: DocketID Number: EPA-HQ-OPPT-2009-0112 40 CFR 799.5089

Re: 

### Submission ofthe Final Test Report for the Algae Study Re-conducted by the PCBTF Testing Consortium under the TSCA Section 4 Test Rule on the Testing of Certain High Production Volume Chemicals: Third Group of Chemicals Benzene, 1-chloro-4-(trifluoromethyl)- CAS RN 98-56-6 (para-Chlorobenzotrifluoride (PCBTF)) Docket ID Number: EPA-HQ-OPPT-2009-0112 40 CFR799.5089

Dear Sir/Madam: On behalf of the PCBTF Testing Consortium, Greenwich Chemical Consulting, Inc. (Greenwich Chemical Consulting) submits to the U.S. Environmental Protection AgencyU.S. EnvironmentalProtectionAgency April23, 2015 

### Page2 CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION



### (EPA or the Agency) the flnal test report for the algae study re-conducted on the chemical substance, benzene, 1-chloro-4-(trifluoromethyl)- [CAS RN 98-56-6] which is subject to a Toxic Substances Control Act (TSCA) Section 4 test rule.' Pot purposes of this letter, the chemical substance, benzene, 1-chloro-4-(trifluoromethyl)-, is herein referred to as, 

### PCBIF. ThePCBTFTesting Consortium submits this testreportin accordancewith this test rule and the regulations set forth at 40 CFR799.5089. I. General Background 

### Greenwich Chemical Consulting functions and operates as the panel manager for and authorizedrepresentative ofthePCBTFTestingConsortium. Therefore, Greenwich Chemical Consulting submits this letter and the enclosed final test report on behalf of the PCBTF Testing Consortium's 15 member companies identified below:2 

### • • • • • 

### • • • • 

### • • • • 

### • 

### • Brenntag NorthAmerica, Inc
. Chemsynergy, Inc
. China Specialty Chemical Company, Ltd
. EMCO Chemical Distributors, Inc. Greenchem Industries LLC Kowa American Cotporation MakhteshimAgan ofNorthAmerica, Inc
. MC International, LLC' Moris Technology Center, LLC Nexeo Solutions, LLC Silver Pern Chemical, Inc. Special Materials Company Superior Oil Company, Inc. Univar USA, Inc
. Valspar Corporation This fmal test rule addresses 15 high production volume (HPV) chemicals that went unsponsored under theAgency's voluntaryHPVChemical Challenge Program. One ofthe 15 chemicals identified in theruleis PCBTF. Thefinal rule identifies the following two (2) studies onPCBTF:
• Aquatic ToxicityTesting: toxicity to plants (algae)) • Ready Biodegradation ThePCBTF Testing Consortiumconductedthese tests andtimely submitted fmal testreports to the Agencyinits letter datedApril18, 2013. Based onEPA's review ofthe algae testreport,

1 76 Fed. Reg. 65385; October 21,2011. 2 See Attachment A for the addresses ofmember companies. 3 .NIC International, LLC operates the entity, lvliami Chemical, Inc., as a DBA. For purposes ofU.S. Customs and TSCA, 1v1C International, LLC is the importer.U.S. Environmental Protection Agency April23, 2015 Page 3 CONTAINSNO CONFIDENTIAL BUSINESS INFORMATION

however, the Agency concluded that the study was inadequate for purposes of the TSCA Section 4 test rule and must be re-conducted. The Agency's conclusions were set forth in its letter ofSeptember 26, 2013. The algae study was re-conducted as described below. As required under 40 CFR 790.50 and 799.5089, the PCBTF Testing Consortium timely submittedto EPA the study plan for a new algae test on the compoundwhichwas developed by the group's new contract lab, Harlan Laboratories Ltd. (Harlan Laboratories). As part of the development of this study plan, the PCBTF Testing Consortium provided to Harlan Laboratories the Agency's comments ofSeptember 26, 2013. HarlanLaboratories developed this studyplan takinginto account andincotporatingthese comments and also addressing the technical difficulties associated with testing PCBTF (i.e., poor water solubility and high volatility). This studyplanwas submittedto theAgency onJanuary 9, 2014 (ahead ofthe revisedJanuaty 12, 2014 deadline). In its response dated March 6, 2014, EPA determined that the submitted studyplanwas adequate with certain stipulations and modifications. In addition, EPA granted the PCBTFTesting Consortium's request to again use OECD testguideline 201!Thereafter, the PCBTF Testing Consortium worked with Harlan Laboratories to modify the study plan toincorporate all ofthe Agency's stipulations andmodifications. Attherequestofthe PCBTF Testing Consortium, additionalguidance was subsequentlyrequested from EPA ontheuse of serial dilutions alongwith otherminormatters. TheAgencyprovidedthis gnidance in its letter dated October 2, 2014. The PCBTF Testing Consortium subsequently finalized the study plan with Harlan Laboratories, and the testing program began in November 2014. The final test report was issued onApril21, 2015 andis included herein ahead oftl1e April30, 2015 deadline. II. Submission Deadlines for the StudyPlan and FinalTest Report Inits September 26, 2013 determination letter, EPA stated that a new study plan for the algae test must be submitted to the Agency by November 12, 2013 and the final report submitted by October 12, 2014. The Agency subsequently granted the PCBTF Testing Consortium's extension request dated November 6, 2013 for a delay to these dates by which the study plan would be submitted byJanuaty 12, 2014 and final report submitted by December 12, 2014. This requestwas granted by EPAin its response dated November 12, 2013. Because of several technical issues, the PCBTF Testing Consortium requested an additional extensioninits letter dated November4, 2014 for the submission oftl1e final test report. This date was April30, 2015 which was granted by EPAin its response dated November 10,2014.

4 In the final TSCA Section 4 test mle, EPA specified the use ofanAS'Dvf test guideline for the algae study. For both the flrst and second algae tests, the PCBTF Testing Consortium requested that the corresponding OECD test guidelines 201 be used in the conduct ofthe algae. The Agency granted this request in both cases.U.S. Environmental Protection Agency Apri123, 2015 Page4
III. List ofFinalTest Report CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION



### Consistent with the final TSCA Section 4 test rule, the following final test report is included herein. 

### Benzem, 1-dJ!otv4-(t1ifluolvmetby!)- CAS RN 98-56-6 (para-Cb!ombenzotiif!uOiide (PCBTJ')): Algal Gmwtb Inhibition Test IV. Submission ofRobust Summaries through HPVIS 

### In the fmal test rule, EPA requests, but does not require, that a robust summary also be included for each test identified in the rule consistent with the Agency's 

### Draft Guidance 011 

### DevelopingRobustSummaries. TheAgencypreviously signaledto thePCBTFTesting Consortium that the submission of these robust summaries was unnecessary. This is acceptable to the group. If 

### EPA decides at a later date that it wishes to receive such robust summaries, the PCBTF Testing Consortium will provide them. 

## * * 

## *
The PCBTF Testing Consortium thanks the Agency for its consideration in this matter. For all questions and comments concerning this matter, please use the contactinformation given below. Mr. JohnV. Massingale Greenwich Chemical Consulting, Inc. 3300 PGA Boulevard, Suite 635 PahnBeach Gardens, FL 33410 Telephone: (561) 578-8110 Facsimile: (561) 578-8120 E-mail: john@grcci.com Sincerely,
JohnV. Massingale PrincipalU.S. Environmental Ptotectjon Agency April23, 2015 PageS



### cc: Mr. P. Lewis- U.S. EPA 

### Mr. M. Matteisen

- U.S. EPA Ms. H. 

### Njo- U.S. EPA Mr.

## J. Schaeffer- U.S. EPA PCBTF Testing Consortium

Enclosure CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION

### AttachmentA- Addresses ofMember Companies
Compan;y: Address Brenntag North America, Inc. 5083 Pottsville Pike Reading, PA 19605 Chemsynergy, Inc. 1100 Burloak Drive Suite 101 Burlington, ON L7L 6B2 Canada China Specialty Chemical Company, Ltd. UnitA (RM 9) 3/F Cheong Sun Tower 116-118 WingLok Street Sheung Wan HongKong EMCO Chemical Distributors, Inc. 8601 95th Street PleasantPrairie, WI 53158 Greenchem Industries, LLC 222 Clematis Street WestPalm Beach, FL 33401 KowaAmerican Corp. 55 East 59th Street 19°' Floor NewYork, NY 10022 Makhteshim Agan ofNorth America, Inc. 171 Madison Avenue Room 1605 NewYork, NY 10016 MC International, LLC 2 NE 40"' Street Suite 501 Miami, FL 33137 Moris Technology Center,LLC 9 Ilene Court, Unit #2 Hillsborough, Nj 08844 Nexeo Solutions, LLC Nexeo Solutions, ILC 3 Waterway Square Place Suite 1000 TheWoodlands, TX 77380 Silver Fern Chemical Inc. 2226 Queen AnneAvenue North Seattle,WA 98109 Special Materials Company 70 West 40th Street 2nd Floor NewYork, NY 10018 Superior Oil Company, Inc. 1402 North CapitolA venue Suite 100 Indianapolis, IN 46202 Univar USA Inc. 17425 NE Union Hill Road Redmond, WA 98052 Valspar Corporation PO Box 1461 Minneapolis, JVIN 55440

## REPORT 

# harlan®



# .... Benzene, 1-chloro-4-(trifluoromethyl)- CAS RN 98-56-6 (para-Chlorobenzotrifluoride (PCBTF)): Algal Growth Inhibition Test

StudyDirector: TestFacility:

Sponsor:

StudyMonitor:

Harlan StudyNumber: Study Completion Date: HVryenhoef Harlan LaboratoriesLtd. ShardlowBusiness Park Shardlow Derbyshire DE722GD UK PCBTFTesting Consortium c/o Greenwich Chemical Consulting, Inc. 3300 PGA Boulevard, Suite 635 Palm Beach Gardens, FL 33410 USA Mr. John V. Massingale Greenwich Chemical Consulting, Inc. 3300 PGA Boulevard, Suite 635 PalmBeach Gardens, FL 33410 USA 41304731 21 Apri12015

Page 1 of47Harlan Study Number: 41304731 Report Page2


## CONTENTS CONTENTS
....................................................................................................................................2 STUDY DIRECTOR STATEMENT OF GLP COMPLIANCE
...................................................

### .4 QUALITY ASSURANCE STATEMENT
......................................................................................5 SIGNATURE OF CONTRIBUTING SCIENTISTS
......................................................................6 SUMMARY
....................................................................................................................................7 Introduction
............................................................................................................................7 Methods
...............................................................................................................................7 Results
..................................................................................................................................7 GENERAL INFORMATION
.........................................................................................................9 Schedule
.................................................................................................................................9 Deviations from Study Plan
................................................................................................... 9 

### Archiving
............................................................................................................................... 9 

### l INTRODUCTION AND PURPOSE
................................................................................... 10 1.1 Guidelines 

### IRegulations
........................................................................................ 10 2 TESTITEM
......................................................................................................................... 11 3 MATERIALS AND METHODS
......................................................................................... 12 3.1 Definitions and Abbreviations
............................................................................... 12 3.2 Test System
............................................................................................................ 12 3.3 Test Water
.............................................................................................................. 13 3.4 Procedure
............................................................................................................... 13 3.4.1 Preliminary Media Preparation Trial
....................................................... 13 3.4.2 Range-Finding Test
................................................................................. 13 3.4.3 Definitive Test
......................................................................................... 14 3.4.4 Evaluations
.............................................................................................. 15 3.4.5 Data Analysis
........................................................................................... 16 3.5 Validation Criteria
................................................................................................. 18 4 RESULTS
............................................................................................................................ 19 4.1 Range-fmdingTest
................................................................................................ 19 4.2 Definitive Test
....................................................................................................... 19 4.2.1 Verification ofTest Concentrations
........................................................ 19 4.2.2 Growth Data
............................................................................................ 19 4.2.3 Validation Criteria
...................................................................................20 4.2.4 Observations on Cultures
........................................................................21 4.2.5 Water Quality Criteria
.............................................................................21 4.2.6 Observations onTest Item Solubility
......................................................21 5 CONCLUSION
....................................................................................................................22 6 REFERENCES
.................................................................................................................... 23 TABLES
........................................................................................................................................24 FIGURES
......................................................................................................................................28 APPENDICES
............................................................................................................................... 31Harlan StudyNwnber: 41304731 Report Page3


## LIST OF TABLES Table 1 Cell Densities and Percentage Inhibition ofGrowthfrom the Rangefinding Test
.................................................................................................................24 Cell Densities and pHValues intheDefinitive Test..
................................................25 Table 2 Table 3 Daily Specific Growth Rates for the Control Cultures intheDefinitive Test.
.............................................................................................................................26 Table 4 InhibitionofGrowth Rate and Yield inthe Definitive Test..
.....................................27 

## LIST OF FIGURES Figure 1 Figure 2 Mean Cell Densities v Time for the Definitive Test..
.................................................28 Inhibition ofGrowthRate Based on0-HourMeasured Test Concentrations
............................................................................................................29 Figure 3 Inhibition ofYield Based on 0-Hour Measured Test Concentrations
........................ 30 

## LIST OF APPENDICES Appendix 1 Appendix 2 Appendix 3 Appendix4 Appendix 5 Appendix 6 Certificate ofAnalysis
........................................................................................... 31 Positive Control
..................................................................................................... 32 Culture Medium
.....................................................................................................33 Preliminary MediaPreparationTrial
.....................................................................34 Analytical Investigations
.......................................................................................3 5 Monitoring Authority Statement ofGLP Compliance
..........................................47Harlan StudyNumber: 41304731 Report Page4


## STUDYDIRECTOR STATEMENT OF GLP COMPLIANCE Harlan Laboratories Ltd., ShardlowBusiness Park, Shardlow, Derbyshire, DE72 2GD, UK
Harlan StudyNumber: 41304731 StudyTitle: Benzene, 1-chloro-4-(trifluoromethyl)- CAS RN 98-56-6 (paraChlorobenzotrifluoride (PCBTF)): Algal GrowthInhibition Test This study was performed in compliance with UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)). These Regulations are in accordance with GLP standards published as OECD Principles on Good Laboratory Practice (revised 1997, ENV/MC/CHEM(98)17); and are in accordance with, and implement, the requirements ofDirectives 2004/9/EC and 2004/10/EC. These principles are compatible with Good Laboratory Practice regulations specified by regulatory authorities throughout the European Community, the United States (EPA and FDA), and Japan (MHL W, MAFF andMETI).
This report fully and accurately reflects the procedures used and data generated. There were no circumstances consideredto have affected the integrity ofthe study orthe validity ofthe data. Study Director: HVryenhoef

# ·-~~4

# ....... Da · 

## 2J APR 2015Harlan Study Number: 41304731 Report PageS 

## QUALITYASSURANCE STATEMENT Harlan StudyNumber: 41304731 Study Title: Benzene, 1-chloro-4-(trifluoromethyl)- CAS RN 98-56-6 (paraChlorobenzotrifluoride (PCBTF)): Algal Growth InhibitionTest

The general facilities and activities are inspected at least once a year and the results are reported to the relevantresponsible person and management. Study-relatedprocedures conducted atthe test facility were audited and inspected. The details of these audits and inspections are givenbelow. Reported to the relevant Study Dates and Types ofQAInspections Director and Test Facility Management Date oflnspection Type oflnspection Phase Inspected Report Date 07November 2014 StudyPlan N/A 07 November 2014 Verification 26 January 2015 Process

- based Test Item Preparation 26 January 2015 06 January 2015 Process

- based Test SystemPreparation 06 January 2015 and Exposure 06 January 2015 Process

- based Assessment ofResponse 06 January 2015 20 January 2015 Process

- based Chemical Analysis 20 January 2015 12 March 2015 Report Audit N/A 12 March 2015 Verification ofStudy Plan Amendment for GLP compliance was performed during the course of the study. This statement confirms that this report reflects the raw data and theprocedures followed. Quality**Assurance:~ \,C,.~'c.** 



## ....

## ".~.

## ,,\,

... "

## ..::::::::.""

.. ".

# ·'·


# ...... Date: 

## 20 APR 2015Harlan StudyNumber: 41304731 Report


## SIGNATURE OF CONTRIBUTING SCIENTISTS
Analytical Chemistry: DMMullee HarlanLaboratories Ltd., Shardlow Business Park, Shardlow, Derbyshire, DE72 2GD, UK

# ··············~············ Date: 

## 2 I APR 

## 2015 Page 6Harlan StudyNumber: 41304731 Report Page7


## SUMMARY Introduction A study was performed to assess the effect of the test item on the growth ofthe green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 ofCommission Regulation (EC) No 761/2009. 

### Methods Information provided by the Sponsor indicated the water solubility ofthe test item to be in the range of29

- 34 mg/L. A preliminary media preparationtrial indicated that a dissolved test item concentration of approximately 57 mg/L was obtained from a saturated solution method of preparationindicatingthis to be the limit ofwater solubility ofthis itemunder test conditions. Following a preliminary range-finding test, Pseudokirchneriel/a subcapitata was exposed to solutions ofthe test item at nominal concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution (equivalent to 0-Hour measured concentrations of0.37, 0.84, 3.7, 16 and 51 mg/L) (five replicate flasks per concentration) for 72 hours, under constant illumination and shaldng at a temperature of 24 ± 1 °C. The test item solutions were prepared by stirring an excess (100 mg/L) oftest item in culture medium using a magnetic stirrer at a rate suchthat a 5% vortex was formed for 24 hours. After the stirring period any undissolved test item was removed by filtration (0.2**~-tm** Sartorius Sartopore filter, first approximate 2 liters discarded in order to precondition the filter) to produce a 100% v/v saturated solution ofthe test item (equivalent to a 0-Hour measured concentration of51 mg/L). This saturated solution was then further diluted as necessary, to provide the remaining test groups. Samples ofthe algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter. Due to the volatile nature ofthe test item, testing was conducted in completely filled (i.e., no headspace), stoppered test vessels in order to minimize possible losses due to volatilization. Following the recommendations of published data (Herman et al 1990) in order to prevent inhibition ofgrowth due to the restriction ofgaseous exchange, additional sodium bicarbonate was added to the culture mediumto provide a source ofcarbon dioxide for algal growth. 

### Results Analysis ofthe test preparations at 0 hours showed measured test concentrations of0.37, 0.84, 3.7, 16 to 51 mg/L were obtained from the 1.0, 3.2, 10, 32 and 100% v/v saturated solution test preparations respectively. There was no significant change inthe measured concentrations at 24, 48 and 72 hours (85% to 98% ofthe 0-Hour measured concentrations) and so the results are based on 0-Hour measured test concentrations only.Harlan StudyNumber: 41304731 Report Page 8
Exposure of

### Pseudokirchneriella subcapitata to the test item gave the following results based on the 0-Hour measured test concentrations:
Response 95% Confidence No Observed Effect Lowest Observed ECso(mg/L) Concentration (NOEC) Effect Concentration Variable Limits (mg/L) (mg/L) (LOEC) (mg/L) Growth Rate 6.2 4.8 - 8.1 0.84 3.7 Yield 2.2 1.8 

### - 2.6 0.84 3.7Harlan Study Number: 41304731 Report


## GENERALINFORMATION Schedule Experimental Starting Date: Experimental CompletionDate: 

### Deviations from Study Plan 29 December 2014 23 January 2015

There were no deviations (unplanned changes) from the studyplan. 

### Archiving Page 9

Unless instructed otherwise by the Sponsor, the study plan (general study plan and study specific supplement), all raw data (paper and electronic) and the final report will be retained inthe Harlan Laboratories Ltd, Shardlow, UK archives for five years after which instructions will be sought as to further retention or disposal. Further retention or return ofthe data will be chargeable to the Sponsor. No data will be discarded without contacting the Sponsorto obtaintheir written consent.Harlan StudyNumber: 41304731 Report Page 10


## 1 INTRODUCTIONAND PURPOSE This report contains a description ofthe methods used and results obtained during a study to investigate the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. Pseudokirchneriel/a subcapitata is a freshwater unicellular alga, representative of primary producers found in natural waters and can therefore be considered as an important non-target organism in freshwater ecosystems. In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification ofthe standard method for the preparation ofaqueous media was performed. An approach endorsed by several important regulatory authorities inthe EU and elsewhere (ECETOC, 1996 and OECD, 2000), is to expose organisms to a saturated solution of the test item in cases where the test item is ofhigh purity and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, a saturated solution was prepared by stirring an excess (100 mg/L) oftest item in culture medium for a period of24 hours prior to removing any undissolved test item present by filtration (0.2 !lm Sartorius Sartopore, first approximate 2 liters discarded in order to pre-condition the filter) to give a saturated solution ofthe test item. 

### 1.1 Guidelines I

### Regulations This study was designed to be compatible with the procedures indicated by the following internationally accepted guidelines and recommendations:
• OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009 • OECD (2000) Guidance Document on Aquatic Toxicity Testing ofDifficult Substances andMixtures. OECD Series on Testing and Assessment, No. 23. OECD Publishing.Harlan Study Number: 41304731 Report Page 11


## 2 TESTITEM Information as provided by the Sponsor. A Certificate ofAnalysis supplied by the Sponsor is given inAppendix I. Identification: Physical state/ Appearance: Batch: Purity: Expirydate: Storage conditions: Intendeduse/ Application: Benzene, 1-chloro-4-(trifluoromethyl)- CAS RN 98-56-6 (para-Chlorobenzotrifluoride (PCBTF)) Clear colorless liquid 2014-02-08 99.77% 17 February 2016 Room temperature in the dark Chemical intermediateHarlan StudyNumber: 41304731 Report Page 12


## 3 MATERIALSAND METHODS 3.1 Definitions and Abbreviations Biomass: 

### ECx:
Growth: Measurement variable (algal cell density) as surrogate measure for algal biomass. The calculated concentration oftest item that results in an x% reduction of the respective growth parameter relative to the control within a stated exposure period. The increase inbiomass over the test period. Growth Rate (Average Specific The logarithmic increase in biomass during the exposure GrowthRate): period. Yield:

LOEC (Lowest Observed Effect Concentration): NOEC (No Observed Effect Concentration): Treatment: Section-by-Section Growth Rate: 

### 3.2 Test System The value of the measurement variable at the end of the exposure period minus the measurement variable's value at the start of the exposure period, calculated to express biomass increase during the test. The lowest test concentration at which a statistically significant inhibition ofgrowth is detetmined relative to the control. The highest test concentration at which no statistically significant inhibition of growth is determined relative to the control. Comprises test item treatments and control (test medium only). Specific growth rate for each day during the course ofthe test (days 0-1, 1-2 and 2-3).

The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures ofPseudokirchneriella subcapitata were obtained from the Culture Collection ofAlgae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium (Section 3.3). The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1 °C. Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 103 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitationby orbital shaker (100

- 150 7m) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 1 04

- 10 cells/mL.Harlan StudyNumber: 41304731 Report Page 13

A positive control (Harlan Study Number 41403365) used potassium dichromate as the reference item. Details of the positive control are given in Appendix 2. The positive control was conducted between 14 October 2014 and 31 October 2014. 

### 3.3 Test Water The culture medium used for both the range-fmding and defmitive tests was the same as that used to maintainthe stock culture. For the purposes oftesting an additional 500 mg!L ofsodium bicarbonate was added to counteract the increase in pH due to algal growth in an enclosed system (Herman et a! 1990). At the request ofthe Sponsor the pH ofthe culture medium was adjusted to pH 7.0 after the addition ofthe sodium bicarbonate. The culture medium is defined inAppendix 3. 

### 3.4 Procedure 3.4.1 Preliminary Media Preparation Trial Information provided by the Sponsor indicated the water solubility ofthe test item to be in the range of29- 34 mg/L based onthe U.S. EPA's citation ofa Merck Index ofunknown reliability. Based on this information the test item was categorized as being a 'difficult substance' as defined by the OECD Guidance Document on Aquatic Toxicity Testing ofDifficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility ofthe test item undertest conditions (see Appendix4).
3.4.2 Range-Finding Test The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration ofapproximately 57 mg/L could be obtained using a saturated solutionmethod ofpreparation. The test concentrations to be used inthe definitive test were determined by a preliminary rangefinding test. The range-finding test was conducted by exposing Pseudokirchneriel/a subcapitata cells to a series ofnominal test concentrations of0.10, 1.0, 10 and 100% v/v saturated solution (equivalent to 0-Hour measured concentrations of0.067, 0.60, 5.0 and 50 mg!L) for a period of 72 hours. A nominal amount oftest item (1090 mg) was dispersed in 10.9liters ofculture medium with the aid ofmagnetic stirring at a rate such that a 5% vortex was formed for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 !lm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from thisHarlan StudyNumber: 41304731 Report Page 14

saturated solutionto give further stock solutions of 10, 1.0 and 0.10% v/v saturated solution. An aliquot (900 mL) ofeach ofthe stock solutions was separately inoculated with algal suspension (2.7 mL) to give the required test concentrations ofO.lO, 1.0, 10 and 100% v/v saturated solution (equivalentto 0-Hour measured concentrations of0.068, 0.60, 5.0 and 50 mg/L). The test was conducted in250 mL glass conical flasks each completely filled (i.e., no headspace) with testpreparation and sealed with a ground glass stopper to reduce evaporation and minimize losses due to the test items volatile nature. Two replicate flasks were used for each control and test concentration. The control group wasmaintained under identical conditions but not exposed to the testitem. The flasks were sealed with ground glass stoppers and incubated (INFORS Multitron® Version 2 incubator) at 24 ± 1

## oc under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380

- 730 nm) and constantly shaken at approximately 150 rpm for 72 hours. Previous experience gained has shown that the use of ground glass stoppers to seal test vessels is effective in attaining an airtight seal, thus preveting losses oftest item through volatility. This was demonstrated inthis test bythe analytical results obtained at 0 and 72 hours. After 72 hours the cell density ofeach flask was determined using a Coulter® Multisizer Particle Counter. A sample ofeach test concentration was takenfor chemical analysis at 0 and 72 hours in order to determine the stability ofthe test item under test conditions.
3.4.3 Definitive Test Based on the results ofthe range-finding test the following test concentrations were assigned to the definitive test: 1.0, 3.2, 10,32 and 100% v/v saturated solution.
3.4.3.1 Experimental Preparation A nominal amount oftest item (1100 mg) was dispersed in 11 liters ofculture medium with the aid ofmagnetic stirring at a rate such that a 5% vortex was formed for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2**~-tm** Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give stock solutions of 32, 10, 3.2 and 1.0% v/v saturated solution. An aliquot (2 liters) ofeach ofthe stock solutions was separately inoculated with 5.2 mL of algal suspension to give the required test concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution (equivalentto 0-Hour measured concentrations of0.37, 0.84, 3.7, 16 and 51 mg/L).Harlan Study Number: 41304731 Report Page 15

The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity. The concentration and stability ofthe test item in the test preparations were verified by chemical analysis at 0, 24, 48 and 72 hours (see Appendix 5).
3.4.3.2 Exposure Conditions As in the range-finding test 250 mL glass conical flasks each completely filled (i.e., no headspace) with test solution were used. Ofthe five test replicates prepared at the start ofthe test: (a) one replicate was kept sealed (with no prior sampling) until 24 hours, at which time samples were taken for both cell density determination and chemical analysis; this replicate was then discarded; (b) one test replicate was kept sealed (with no prior sampling) until48 hours, at which time samples were taken for both cell density determination and chemical analysis; this replicate was then discarded; (c) three test replicates were kept sealed (with no prior sampling) until 72 hours, at which time samples were taken for both cell density determination and chemical analysis. Therefore, each of the test replicates containing treated test solutions was used only once for samplingbefore being discarded. Ofthe nine control replicates prepared at the start ofthe test: (a) three replicates were kept sealed (with no prior sampling) until 24 hours, at which time samples were taken for cell density determination then discarded; (b) three replicates were kept sealed (with no prior sampling) until 48 hours, at which time samples were taken for cell density determination then discarded; and (c) three replicates were kept sealed (with no prior sampling) until 72 hours, at which time samples were takenfor both cell density determination and chemical analysis. The control group was maintained under identical conditions but not exposed to the test item. Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 1.16 x 106 cells per mL. Inoculation of2liters oftest medium with 5.2 mL ofthis algal suspension gave an initial nominal cell density of 3 x 103 cells per mL and had no significantdilution effect onthe final test concentration. The flasks were sealed with ground glass stoppers and incubated (INFORS Multitron® Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380

- 730 nm) and constantly shaken at approximately 150 rpmfor 72 hours.


### 3.4.4 Evaluations 3.4.4.1 Test Organism Observations The 0-Hour cell density was determined by electronic particle counting (Coulter® Multisizer Particle Counter) ofthe pre-culture and division by the dilution factor. Samples were taken atHarlan Study Number: 41304731 Report Page 16
24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. To determine the potential effect ofthe test item on the appearance ofalgal cells, a sample was removed from each test and control culture (replicates pooled) at the end ofthe test. The shape and size ofthe algal cells was inspected microscopically and any abnormalities recorded.
3.4.4.2 Water Quality Criteria The pH ofthe control and each testpreparation was determined at initiation ofthe test and after 72 hours exposure. The pH was measured using a Hach HQ30d Flexi handheld meter. The temperature within the incubator was recorded daily.
3.4.4.3 Verification ofTest Concentrations Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis ifnecessary. A sample ofthe vessel sacrificed for cell density determination at 24 and 48 hours was taken for chemical analysis. The method ofanalysis, recovery and test solution analyses are described inAppendix 5.


### 3.4.5 Data Analysis 3.4.5.1 Comparison ofGrowth Rates The average specific growth rate for a specified period is calculated as the logarithmic increase inbiomass from the equation:

Where:


## = average specific growth rate from time t1 to tn cell concentration at t1 cell concentration attn time offirst measurement time ofn'h measurementHarlan StudyNumber: 41304731 Report Page 17

The average specific growth rate over the test duration was calculated for each replicate control and test item vessel using the nominally inoculated cell concentration as the starting value rather thanthemeasured starting value in order to increase the precision ofthe calculation. In addition the section by section specific growth rate (days 0-1, 1-2 and 2-3) was calculated for the control cultures and the results examined in order to determine whether the growth rate remained constant. Percentage inhibition ofgrowth rate for each replicate test item vessel was calculated using the following equation:

Where: I, J.lc J.lt percentage inhibition ofaverage specific growthrate mean average specific growthrate for the control cultures average specific growth rate for the test culture
3.4.5.2 Comparison ofYield Yield is calculated as the increase in biomass over the exposure period using the following equation:

Where: y 

### No Nn yield cell concentration atthe start ofthe test cell concentration at the end ofthe test For each test concentration and control the mean value for yield along with the standard deviation was calculated. Percentage inhibition of yield was calculated using the following equation:

I = yHarlan StudyNumber: 41304731 Report

Where: percentage inhibition ofyield mean value for yield in the control group meanvalue for yield for the treatment group 3.4.5.3 Determination ofECxValues Page 18

For each individual test vessel (mean values for yield), percentage inhibition (arithmetic axis) was plotted against test concentration (logarithmic axis) and a line fitted by computerized interpolation using the Xlfit software package (IDBS). ECx values were then determined from the equationfor the fitted line. Where appropriate 95% confidence limits for the EC50 values were calculated, using the simplified method ofevaluating dose-effect experiments ofLitchfield and Wilcoxon (1949).
3.4.5.4 Statistical Analysis One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the growth rate and yield data after 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer softwarepackage (SAS, 1999

- 2001). 

### 3.5 Validation Criteria Theresults ofthetest are considered valid ifthe following performance criteria are met:
• The cell concentration of the control cultures must increase by a factor of at least 16 overthe testperiod.
• The mean ofthe coefficients ofvariation ofthe sectionby section daily growth rates in the control cultures during the course of the test (days 0-1, 1-2 and 2-3) must not exceed 35%.
• The coefficient of variation of the average specific growth rate in replicate control cultures must not exceed 7%.Harlan Study Number: 41304731 Report Page 19


## 4 RESULTS 4.1 Range-finding Test The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test item during the range-finding test are given in Table 1. The results showed no effect on growth atthe test concentrations of0.10 and 1.0% v/v saturated solution. However, growth was observed to be reduced at I0 and I00% v/v saturated solution. Based onthis information test concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution were selected for the definitive test. Chemical analysis ofthe test preparations at 0 hours (see Appendix 5) showed measured test concentrations to range from 0.067 to 50 mg/L whilst concentrations in the range of 0.059 to 48 mg/L were observed at 72 hours (74% to 97% ofthe 0-Hour measured concentrations). 

### 4.2 Definitive Test 4.2.1 Verification ofTest Concentrations Analysis of the test preparations at 0 hours (see Appendix 5) showed measured test concentrations of 0.37, 0.84, 3.7, 16 to 51 mg/L were obtained from the 1.0, 3.2, 10, 32 and 100% v/v saturated solution test preparations respectively
.. There was no significant change in the measured concentrations at 24, 48 and 72 hours (85% to 98% of the 0-Hour measured concentrations) and so the results are based on 0-Hour measured test concentrations only as recommended in Section 39 ofthe OECD Test Guideline No. 201.


### 4.2.2 Growth Data Cell density values determined at each sampling time and pHvalues at 0 and 72 hours are given in Table 2. Daily specific growth rates for the control cultures are given in Table 3. Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4. The mean cell densities versus time for the definitive test are presented in Figure I. Percentage inhibition values are plotted against test concentration in Figure 2 and Figure 3. From the data given in Tables 2 and 4, it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were affected by the presence ofthe test item overthe 72-Hourexposure period. Accordinglythe following results were determined from the data:Harlan StudyNumber: 41304731



### 4.2.2.1 Inhibition ofGrowth Rate :1.8mg/L : 2.9mg/L Report

E,C10 (0- 

### 72 h) 

### E,C2o (0

- 72 h) E,Cso (0

- 72 h) : 6.2 mg/L; 95% confidence limits 4.8

- 8.1 mg/L where E,C, is the test concentration that reduced growthrate by x%. Page 20



### Statistical analysis of the growth rate data was carried out for the control and all test concentrations using one way analysis ofvariance incorporating Bartlett's test for homogeneity ofvariance (Sokai and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955). There were no statistically significant differences between the control, 0.37 and 0.84 mg/L test concentrations (P:1:0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 0.84 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 3.7 mg/L.
4.2.2.2 Inhibition ofYield :0.87 mg/L :1.2mg/L 

### EyCw (0- 

### 72 h) 

### EyC2o (0

- 72 

### h) EyCso (0 - 

### 72 h) 

### :2.2 mg/L; 95% confidence limits 1.8-2.6mg/L Where: 

### EyCx is the test concentrationthatreduced yield by x%. Statistical analysis of the yield data was carried out as in Section 4.2.2.1. There were no statistically significant differences between the control, 0.37 and 0.84 mg/L test concentrations (P:1:0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 0.84 mg/L. Correspmidingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 3.7 mg/L.


### 4.2.3 Validation Criteria The following data show that the cell concentration ofthe control cultures increased by a factor of 190 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at leastby a factor of 16 after 72 hours. Mean cell density ofcontrol at 0 hours Mean cell density ofcontrol at 72 hours 3.00 x 103 

### cells per mL 5.96 x 105 

### cellsper mLHarlan Study Number: 41304731 Report Page 21

The mean coefficient of variation for section by section specific growth rate for the control cultures was 22% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%. The coefficient ofvariation for average specific growth rate for the control cultures over the test period (0

- 72 h) was 2% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.


### 4.2.4 Observations on Cultures All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 0.37, 0.84, 16 and 51 mg/L, however some enlarged/swollen cells were observed to be present in the test cultures at 3.7 mg/L.


### 4.2.5 Water Quality Criteria The pH values ofthe control and each test preparation are given in Table 2. Temperature was maintained at 24 ± 1°C throughout the test. The pH value of the control cultures (see Table 2) was observed to increase from pH 7.9 at 0 hours to pH 9.4 at 72 hours. The pH deviation in the control cultures was equal to 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.


### 4.2.6 Observations on Test Item Solubility At the start ofthe test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control, 0.37 and 0.84 mg/L test cultures were observed to be green dispersions. The 3.7 mg/L test cultures were observed to be pale green dispersions whilst the 16 and 51 mg/L test cultures were observed to be clear colorless solutions.Harlan StudyNumber: 41304731 Report Page22


## 5 CONCLUSION The effect of the test item on the growth of Pseudokirchneriella subcapitata 

### has been investigated over a 72-Hour period and based on the 0-Hour measured test concentrations gave the following results:
Response 95% Confidence No Observed Effect Lowest Observed ECso (mg/L) Concentration (NOEC) Effect Concentration Variable Limits (mg/L) (mg/L) (LOEC) (mg/L) Growth Rate 6.2 4.8 

### - 8.1 0.84 3.7 Yield 2.2 1.8 - 2.6 0.84 3.7Harlan StudyNumber: 41304731 Report Page 23


## 6 REFERENCES DUNNETT, C.W. (1955) A Multiple Comparison Procedure for Comparing Several Treatments With a Control. JAm StatAssoc 50, 1096-1121. ENVIRONMENT DIRECTORATE, ORGANISATION FOR ECONOMIC CO-OPERATION AND DEVELOPMENT (OECD) (2000) Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. EUROPEAN CENTRE FOR ECOTOXICOLOGY AND TOXICOLOGY OF CHEMICALS (ECETOC) Monograph No. 26 (1996) Aquatic Toxicity Testing of Sparingly Soluble, Volatile and Unstable Substances. HERMAN et al. (1990) Impact ofvolatile aromatic hydrocarbons, alone and in combination, on growth ofthe freshwater algaSelenastrum capricornutum.Aquatic Toxicology 18, 87

- 100 LITCHFIELD, J.T. and WILCOXON, F. (1949) A Simplified Method ofEvaluating Dose-Effect Experiments. J PharmacalExp Ther 96,99-113. MAYER, P., NYHOLM, N., VERBRUGGEN, E. M. J., HERMENS, J. L. M. and TOLLS, J. (2000) Algal growth inhibition test in filled, closed bottles for volatile and sorptive materials. Environmental Toxicology and Chemistry, 19, pp. 2551-2556. SAS/STATProprietary Software Release 8.02 (1999

- 2001), SAS Institute Inc, Cary, NC, USA. SOKAL, R.R. and ROHLF, F.J. (1981) Biometry. New York: W H FREEMAN and Company. Xlfit, ID Business Solutions Ltd.Harlan StudyNumber: 41304731 Report Page 24 

# TABLES Table 1 CellDensities andPercentageInhibition ofGrowthfrom the Range-findingTest Cell Densities* (cells permL) Inhibition Values(%) Nominal Concentration (% v/v Saturated Solution) OHours 72Hours Growth Rate Yield R, 3.00E+03 5.31E+05 Control R, 3.00E+03 5.94E+05 

## - - Mean 3.00E+03 5.62E+05 R, 3.00E+03 6.40E+05 0.!0 R, 3.00E+03 5.12E+05 0 [2] Mean 3.00E+03 5.76E+05 R, 3.00E+03 5.65E+05 1.0 R, 3.00E+03 4.63E+05 3 9 Mean 3.00E+03 5.14E+05 R, 3.00E+03 7.87E+04 !0 R, 3.00E+03 6.20E+04 40 88 Mean 3.00E+03 7.03E+04 R, 3.00E+03 3.63E+03 100 R, 3.00E+03 2.99E+03 99 100 Mean 3.00E+03 3.31E+03

* Cell densities representthe meannumber ofcells permL calculatedfrom the mean ofthe cell counts from 3 counts for each ofthereplicate flasks. R1 andR2**~Replicates** 1 and 2 [Increase in growthcompared to controls]Harlan StudyNumber: 41304731 Report Page 25



### Table2 Cell Densities and pH Valnes in the Defmitive Test
Nominal Concentration pH CellDensities* (cells per mL) (% v/v Saturated Solution) Oh Oh 24 h 48 h 72b R, 3.00E+03 1.31E+04 1.18E+05 5.72E+05 R, 3.00E+03 1.16E+04 1.09E+05 5.50E+05 Control 7.9 R, 3.00E+03 1.31E+04 1.11E+05 6.67E+05 Mean 3.00E+03 1.26E+04 1.13E+05 5.96E+05 R, 3.00E+03 1.22E+04 1.23E+05 6.52E+05 1.0 R, 7.7 3.00E+03 6.10E+05 R, 3.00E+03 6.29E+05 Mean 3.00E+03 1.22E+04 1.23E+05 6.30E+05 R, 3.00E+03 1.20E+04 8.41E+04 4.69E+05 3.2 R, 3.00E+03 6.29E+05 7.6 R, 3.00E+03 4.96E+05 Mean 3.00E+03 1.20E+04 8.41E+04 5.32E+05 R, 3.00E+03 6.13E+03 2.97E+04 1.30E+05 R, 3.00E+03 1.37E+05 10 7.5 R, 3.00E+03 1.39E+05 Mean 3.00E+03 6.13E+03 2.97E+04 1.35E+05 R, 3.00E+03 2.75E+03 5.42E+03 6.19E+03 32 R, 7.5 3.00E+03 5.49E+03 R, 3.00E+03 6.31E+03 Mean 3.00E+03 2.75E+03 5.42E+03 5.99E+03 R, 3.00E+03 1.55E+03 3.74E+03 5.63E+03 100 R, 3.00E+03 4.63E+03 7.4 R, 3.00E+03 6.69E+03 Mean 3.00E+03 1.55E+03 3.74E+03 5.65E+03

* Cell densities representthe mean number ofcells per mL calculatedfrom the mean ofthe cell counts from 3 counts for each ofthereplicate flasks. R1 - R,**~** Replicates I to 3 pH 72h

9.4

9.1

9.2

8.1

7.7

7.5Harlan Study Number: 41304731 Report Page 26



### Table 3 Daily Specific Growth Rates for the Control Cultures in the Definitive Test Daily Specific Growth Rate (cells/mL/hour) Day0-1 Day 1-2 Day2-3 R1 0.062 0.092 0.066 Control R, 0.056 0.093 0.067 R, 0.062 0.089 0,075 Mean 0.060 0.091 0.069

R1 - R 3 =Replicates I to 3Harlan StudyNumber: 41304731 Report Page 27

Table4 Inhibition ofGrowth Rate and Yield in the Definitive Test
Nominal Concentration Growth Rate (cells/mL/hour) Yield (cells/mL) (% v/v Saturated Solution) 0-72h % Inhibition 0-72h %Inhibition* R, 0.073 

## - 5.69E+05 R, 0.072 5.47E+05 Control R, 0,075 

## - 6.64E+05 

### - Mean 0.073 5.93E+05 SD 0.002 6.22E+04 R, O.D75 -3 6.49E+05 R, 0.074 -I 6.07E+05 1.0 R, 0.074 -I 6.26E+05 [6] Mean 0.074 -l 6.27E+05 SD 0.001 2.12E+04 R, 0.070 4 4.66E+05 R, 0.074 -I 6.26E+05 3.2 R, 0.071 3 4.93E+05 II Mean 0.072 2 5.29E+05 SD 0.002 8.59E+04 R, 0.052 29 1.27E+05 R, 0.053 27 1.34E+05 10 R, 0.053 27 1.36E+05 78 Mean 0.053 27 1.32E+05 SD 0.001 4.67E+03 R, 0.010 86 3.19E+03 R, 0.008 89 2.49E+03 32 R, 0.010 86 3.31E+03 99 Mean 0.009 88 2.99E+03 SD 0.001 4.44E+02 R, 0.009 88 2.63E+03 R, 0.006 92 1.63E+03 100 R, 0.011 85 3.69E+03 100 Mean 0.009 88 2.65E+03 SD 0.003 1.03E+03



### * In accordance with the OECD test guideline only the mean value for yield for each test concentrationis calculated Rr- R3=Replicates 1to 3 SD**~** Standard Deviation [Increase in growth compare to controls]Harlan StudyNumber: 41304731 Report Page28


## FIGURES Figure 1 Mean Cell Densities v Time for the Definitive Test**LOOE+06l~;::====:::;:=:::::::::r-:::::::::::::::::::::::::::::::::::::l=•**

# ::::::::::::::::::::::::::::::::::::=1 ------------+-------------------------------------1·-------------------------------------

--- ---.....- Control 

## -------------l..--------------------------------------1-------------------------------------- ' I

- -A- 0.37mg/I 

## ------------t-------------------------------------r·------------------------------------ ------------t-------------------------------------+-----------------------------------;:

------ 0.84 mg/ 

## -----------+-----------------------------------+------------------------------,/'- --8- 3.7mg!L • , /.**------------+-------------------------------------1--------------------------7~**

## -- ----

--- 16mgiL 1 I /. I I /.

--- - -e- 51 mg!L ------------T·-----------------------------·-------1-·------------------r. --- ----------- 1 ! // I I / I j / 

## -------------------------------------+------------------------------------+---------..- --- 1 

## I // I I / I ' / I 1/ i / I I /

# :::::::::::::::::::::::::::::::::::::!:::::::::::::::::::::::::::::7t:+::::::::::::::::::::::;:z~::::: l.OOE+OS 

## -------------------------------------+---------------------------- --- --**---------------------~---------------**

# :::::::::::::::::::::::::::::::::::::i:::::::::::::::::::::~t::

# __

# ::::t::::::::::;.t::::::::::::::::::: ! b 

## I // -------------------------------------+-----------------· -- ·------------j"·---/L..•..•••.••••••••.••••••••.•• I ' / 

## -------------------------------------+------------- - -----------------:h=------------------------------------ 1 / I I / I 

## -------------------------------------+------**-----------------~.::.**

## ______**~--------------------------------------** 1 / I I / I I // ! / 

# ' / 

# ' I // i



# -------------------------

# ----~~~===t~~~~;~=~=~~==::::::::::::::::::::::t=====~~~~~~==~=~~~==========~:~~:=~~~**-------------------~~---~~~~~~~~:~::t~~~~~~~~~~~~~~~~~~~:~~~~~:~:~~~~:~t~~~~~~~:::::::~~::::::~:~~~::··-----** 

# _

..... ' 

# -- ---------::;-....-<.---------t·------------------------------- --i·---------------·-·::.·::::--""'"--------

.......... I ' --- 

## ------.....**-::::-:;;~.::::.**

## _________________t------------------- --------------;*-..:::,.....""'.::::
__________________________



## ::--=------------------------------- ---**--------------------;;~-..:,_-:_**

## ___l-------------------------------------

................ ' // !

...... I / I



## ______________:-_::.::-....._____________**t--------?'.t::'.:::'.~--------------------~--------------------------------------** 

## ......__ i // !

- ......**~.....** I 

## : I l.OOTI+04

: I 

### ! ! l.OOE-Hl3 -1----------+-----------1------------1 0 24 48 72 Time(Hours)Harlan StudyNumber: 41304731 Report Page 29


### Figure 2 Inhibition ofGrowth RateBased on 0-HourMeasured Test Concentrations 100 90 80 70 60**~** 

## :E 50 

# :a .s 40 'if. 30 20 10 : : : : i l 

### i: : : : " "' ' ; : : ::::: i 

### ! i :,i :,: :,: :_: :,: : i i iii Groj"'hll!'te j

## -!-+~!!! t 

# r---r; T1T!T 1,_ -i·',_

# -t __**--~.:,_** 

## 1
..**,_-~·',_-i·',_-** jjjjjjjj :!!"'" 

## + ' ' ' ' ' ' ' ' --------!,
_______ ,,----:_ ..---·.:.

### ·-.~.**--.!.--.~.**_!.'· -.' ••----

### ------.~.**-----~.:.---** 

# -·------,-·-rT·rr·r·rr :--TTTrr : : ::::: : i _._:__ 

### ij !,_!,!___ __i,
_____

### j l : ! l l:
o o o o ooo o I o ...--.---**------------------,---~,---~,,--~,,-':,-':,·',,- --T--1··~-T·r-fT""••••••··r·---··r···:·**  

## ;_ !. 

## 1.**~-·** 

## :. 1. · · 

# : : : : : : : : : : __L.LUJ

--..

:---·_;--·_;--.h.'-.t-.1·----------
..**~------.r---·.:·** 

### --:.·--..**:-~-** 

### :. t.:.· ..... 

### i i 

### iii ' i i 

### i i iii 1 

### i i

### --~-J**+-~-~~- --~--~-~-~-~-**  -------·-;,·--**--._;---~.r--~.:--:.~--.r-.r-.r~.r----------·.~------r.**----.! : : : : :

' '.· .·' .·.' .·' .·' .•

### : ' '' '' ' : : : I:::: : I : ' : :! :: ----------J------r----,L---J---t--:--r-r-:-----------r------:-----1-- --r-+-:--:--J--- 

## -------r,,-- -+

## --1·--r--t·n·i-

, : ,: ,i ,: ,: ·.: : •• i, ,i : ,: ,i : : : \:: : : : : : : : : : : • i,
.

### .l,
__ L_ !,

### !,-~.·-- : 1 1 

## i i i 

## i -------·-t·-----r--·t·t·t··r-r-rt--------_ ., __ r_---- · ---- . :-. -.- :·- 

### --------r.--- 

# ·r·T-TTTTT

: i 

### i iii i \

-- ' i f j 

## iii : : : ! l i i**-----~-----\----~--~--~-~-~-:--** ,---,--r---.·-r·r--.--**--------~--** --T·--,_.---r,--,r·',.-',:T i : i i 

### i::: !, i, '.· i, 1, l, !, : : : ::::: : : : :: i**---~-----j**____J__J
_ _Lj_j_J_



## ----t-

## -----i

## ---+-·j--1--r-r 

### -1·

## + --- --1---.! -

### -r-1--r-r1---

---- 

### : : : : : : " ' i 

### i i 111 

### i 1 l 

### i ! l 1 ! i: 

### o -- 

### --!-- 

## !-- · · 

## :----r,, --__,f-·
__ 

## ,:--·_,:-+__ ,f',:,-·,:,·-:
__ ,; 1 1 : 

# --r-rTrfr·------- -10 L--.L_--'-_L_LL.Ll.J.L_
_ _L__L---'-.L..l..LLLL----'--'--'-_j_JU-J.-'J O.l 10 100 Concentration (mg!L)Harlan StudyNumber: 41304731 Report


### Figure 3 100 90 80 70 60 

### =**:~** 50

# ~ 40 

### .._ 30 20 10 0 -10

0.1 

### Inhibition ofYield Based on 0-HourMeasured Test Concentrations

' ' ' '' '' ' ' ' '' '' ' ' ' '' '' '



# __yiel1···••I••·I·"I·I·'·I•ri ···-·····1, 1,.· 

# t. o;·F_·_·_·_·_·_·_·_:,,_
..•.-.•_-_-_:_!,:,.·-·_
.. ___ ',:,,
.. _--_·._!,:,,.•.-.-_i,:,,_·····:,,,i:_·:_:,:,,.····j':,,···· ------··--r--

### ---~----l

# ..**L-l.~**

# .**.l.l~----** 

# ····**---~---···** 

# ···j···

### j
__

### J.· j

## .J..l.L·· : i l i 

### i iii i i : 

### i i ! 

### i 

### i!! : : : : : :::: : : : : : :::' 

### ---------r------r----r---:---r-1-++-:-----------l·--- --:----l--+--:--t--:--H-----------l------t----1---t--t -r,_·t,_·t,_· 

## i i i i i i

# lii i i i 

# l i ij i j j 

## i**--------+----+---~·--!---H--}--}--!------------!- --~---~---!--i--!--!+---------+-----4----i---l--i--~-i-i-** **~** j j 

### i ! 

### iii j : j j j j j 

### ii i i j j j j j j : : : : : :::: : : : :::: : : : : : ::: 

# --------r----r--r-rrrnr -r 

# r--:-**-~-rrrrr---------r·----r-·r-rTr1T ---------+------~---+-+-+++++---**  

## -----!,- ---f- -1,**+-·H+~- --~------~----i---t---1--H+**  

### ! 

## i ! r ! 

## i i J i , . . . r 

# !!

## !! ! : 1 1 l 

# i: i

---- --+--**---~---+--:---f-+-~-H---** -------+-----+----:--++-1+-:+---------+- --+----J---f---1--f··-l-1· i i i i i iii i i i i i i iii i i i i i l i! i i i i i : i:!' i i i i i iii i i i i i i iii**---------~------~----~---:---,'--:-- '-:------------:-------:----",-··{·-~·-f·-:--:-1·----------1-------:-----:---t---J--f-·1-{-**  

### i i i i : 

### i!! i i ! 

### i 

### ! !

### ii i 

### i i ! i i : : ' ' : :::: ' : : : :::: : : ::: 

## r 

# rJ1rrnr
c 

# T c

# T 1

# T1F ,,.,

# nn
10 100 Concentration (mgfL) Page 30Harlan StudyNumber: 41304731 Report


## APPENDICES Appendix 1 Certificate ofAnalysis

70 We$t40th**Slre~lt** New York, NY 10018 Phone; (648) 366·0400 Fa)(: (646) 366-0595 

### CERTIFICATE OF ANALYSIS Product Name: Quality: Batch Number: TutiProperty

Acidity, ppm (spec. ion probe) Alkalinity, ppm as NaOH Appearance Assay,% Color (APHA} Non Volatile Residue. Wt. % Specific Gravity (20C/25C) Water content, ppm ParaohlorobenzotriDuorlde-900N SMC Spec. 900 2014·02-08 Speclflcatton 3.0 PPM MAXIMUM 10.0 PPM MAXIMUM Cffioar liquid, free ofsuspended material 99.00 % MINIMUM 20 APHA MAXIMUM 0.0020 % MAXIMUM 1.3300 TO 1.3500 GICC 150 PPM MAXIMUM

CERTIFIED TRUEAND CORRECT 

### W!Jl

ApprovalDate: 2/17/2014 GMO**~·~1-'o"** Qato: Page 31

Rllult

1.7 0.0 Clear liquid, frefl ofau11panded material 99.77 10 0.00.13 1.3340 78

Print Date: 1115/14Harlan StudyNumber: 41304731 Report Page 32


### Appendix 2 Positive Control A positive control (Harlan Study Number 41403365) used potassium dichromate as the reference item at concentrations of0.050, 0.13, 0.32, 0.80 and 2.0 mg/L. Six flasks each completely filled with test preparation were used for the control, and three flasks each completely filled with test preparation were used for each test concentration. The flasks were sealed with ground glass stoppers and incubated at 24 ± I 

## oc under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380

- 730 run) and constantly shaken at approximately 150 rpm for 72 hours. The culture medium used was preparedwiththe addition of500 mg!L NaHC03. Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results: E,Cso (0

- 72 h) EyCso (0

- 72 h) 1.2 mg/L; 95% confidence limits 0.97- 1.4 mg/L 0.52 mg/L; 95% confidence limits 0.45-0.60 mg/L No ObservedEffect Concentration (NOEC) based on growth rate: No ObservedEffect Concentration (NOEC) based on yield: Lowest Observed Effect Concentration (LOEC) based on growth rate: Lowest Observed Effect Concentration (LOEC) based onyield: 0.32mg/L 0.32 mg/L 0.80 mg/L 0.80mg!L The results from the positive control with potassium dichromate were within the normal ranges for this reference item.Harlan StudyNumber: 41304731 Report Page 33
Appendix3 Culture Medium NaN03 25.5 mg/L MgCh.6H20 12.16 mg/L CaCh.2H20 4.41 mg/L MgS04.7H20 14.6 mg/L K2HP04 1.044 mg/L NaHC03 15.0 mg/L H,BO, 0.186 mg/L MnChAH20 0.415 mg/L ZnCh 0.00327 mg/L FeCb.6H20 0.160 mg/L CoCh.6H2 0 0.00143 mg/L Na2Mo04.2H20 0.00726 mg/L CuCh.2H20 0.000012 mg/L Na 2 EDTA.2H20 0.30 mg/L The culture medium was prepared using reverse osmosis purified deionized water*. For the purposes of the definitive test, additional sodium bicarbonate (500 mg/L) was added to the prepared culture mediumprior to adjusting the pH to 7.0 with 0.1NNaOH or HCI.

• E1ga Optima 15+ orElgaPurelab Option R-15 BPHarlan Study Number: 41304731 Report Page 34


### Appendix 4 Preliminary Media Preparation Trial 1. Saturated Solution Preparation A nominal amount oftest item (I090 mg) was dispersed, in duplicate, in I0.9 liters ofculture medium with the aid of magnetic stirring at a rate such that a vortex was formed which represented approximately 5% of the overall media column height for periods of either 24 or 48 hours. After stirring samples were taken for chemical analysis after the following pretreatments:
• Centrifugation at I0000 g for 30 minutes • Centrifugation at 40000 g for 30 minutes • Filtration through a 0.2 11m Sartorius Sartopore filter (approximately I liter discarded in order to pre-condition the filter) • Filtration through a 0.2 Jlffi Sartorius Sartopore filter (approximately 2 liters discarded in order to pre-condition the filter)


### 2. Results Stirring Period and Treatment Concentration Found (mg/L) 24 Hours Centrifuged 10000g 19.0 24 Hours Centrifuged40000 g 11.4 24 HoursFiltered- I liter discarded 57.2 24 Hours Filtered**~** 2 liters discarded 56.7 48 Hours Centrifuged I0000 g 18.5 48 Hours Centrifuged40000 g 9.90 48 Hours**Filtered~** lliterdiscarded 54.2 48 Hours Filtered**~** 2 liters discarded 59.0


### 3. Discussion The results obtained indicated that significantly higher dissolved test item concentrations were obtained following removal ofany undissolved test item by filtration rather than centrifugation. This was considered to be due to losses oftest item through volatility during the time taken to removethe supernatant after centrifugation. Based on this information the test item was prepared using a saturated solution method of preparation at an initial loading rate of I00 mg/L, stirred for a period of 24 hours prior to the removal of any undissolved test item by filtration through a 0.2 f!m Sartorius Sartopore filter (first approximate 2 liters discarded) to give a nominal test concentration of approximately 57 mg/L, the limit ofwater solubility ofthis Test item under test conditions.Harlan StudyNumber: 41304731 Report Page 35


### Appendix**~** 

### Analytical Investigations 

## 1 MATERIALS AND METHODS 1.1 Introduction The test item concentration in the test samples was determined by gas chromatography (GC) using au external staudard. The test item gave a chromatographic profile consisting of a single peak. The method was developed by the Department ofAnalytical Services, Harlan Laboratories Ltd, Shardlow, UK. 

### 1.2 TestItem Thetest item is described inthe biological part ofthis study. 

### 1.3 Analytical Standard The test item described in the biological part of this study was also used as the aualytical staudard. 

### 1.4 Analytical Procedure 1.4.1 Storage The samples were aualyzed onthe day ofreceipt.


### 1.4.2 Reagents and Solvents
Testmedium: as described inthe biologicalpart ofthis study. Solvents: tetrahydrofuran, Sigma-Aldrich, HPLC grade. hexaue, Fisher Scientific, pesticideresidue grade.Harlan StudyNumber: 41304731 Report Page 36 Analytical Investigations 

### 1.4.3 Preparation ofCalibration Standards 

### The test item (nominal!OO mg) was dissolved in hexane (100 mL) to prepare a stock solution with a concentration of 1000 mg/L. This stock solution was further diluted with hexane to produce a solution of50 mg/L. Defmed volumes ofthis solution were then diluted with hexane to obtain calibration standards in the range of 0.1 to 1.0 mg/L. A second series of calibration standards was similarly prepared in the range of0.25 to 2.5 mg/L. These solutions were used to determine the recovery and test sample concentrations.
1.4.4 Preparation ofLinearity Standards 

### The test item (nominal100 mg) was dissolved in hexane (100 mL) to prepare a stock solution with a concentration of I000 mg/L. Defined volumes ofthis solution were then diluted with hexane to obtain calibration standards in the range of 0.1 to 50 mg/L. A second series of calibration standards was similarly prepared in the range of0.25 to 25 mg/L. These solutions were used to determine the test sample concentrations and to evaluate the linearity of the analytical system.
1.4.5 Preparation ofSpiked Recovery Samples 

### To demonstrate the validity ofthe analytical procedure, volumes of test medium were spiked with the test item and the recovery was assessed. The test item (nominal 100 mg) was initially dissolved intetrahydrofuran to prepare a stock solution with a concentration of 1000 mg/L. This stock solution was further diluted with tetrahydrofuran to produce a stock solution of50 mg/L. A defined volume ofthis stock solution was diluted with test medium to obtain spiked recovery samples at a concentration of 0.050 mg/L. Five replicates were prepared and subjected to the same treatment as the test samples. In

### addition, test medium without the addition ofthe test item (synthetic control) was also analyzed.
1.4.6 Preparation ofTest Samples 

### The test samples were analyzed on the day ofreceipt. The test samples were extracted directly with hexane in the same bottle. An aliquot ofthe hexane layer was taken for analysis. See Table 1for more information onpreparation volumes.Harlan StudyNumber: 41304731 Report Analytical Investigations 

### 1.4.7 Instrumental Parameters Gas chromatograph: Detector: Column:
Inletliner: Carrier gas: Pressure: Oventemperature Program:
Injectionmode:
Inletpurgetime: Injection volume Injectiontemperature: Detectortemperature: Retention time: HP 5890 Series II, with autosampler ECD ZB-624 (60 m x 0.32 mmi.d.;1.8 J.lm film thickness) double gooseneck nitrogen 10 psi

oven: 40 °C, for 0 minutes with 15 °C/minute to 220 °C, for 5 minutes splitless

onat 1minute

150 °C 250 °C approximately 14.8 minutes Page 37Harlan StudyNumber: 41304731 Report Page 38 Analytical Investigations 1.4.8 Calculations A calibration curve was prepared using a quadratic function ofthe form shown in equation (1). The calibration data is presented in Table 2 and Figure I. From the calibration curve, the concentration "x" of the test item in an injected sample was calculated using the following equation:

x=

### -b+~b**-4·a·(c-y)** 2·a (1)
where x: y: 

### a, b, 

### c: concentration ofthe test item inthe injected sample [ mg/L] average peak area ofthe test item inthe injected sample [counts] constants The concentration of the test item in the test samples was calculated using the following equation:
X c =--·F R/100 where c: x: 

### F: R: (2) concentration ofthe test item inthe test sample [ mg/L] concentration ofthe testiteminthe injected sample solution [mg/L] samplepreparation factor recovery rate[%] (Ifnot correcting for recovery then R is taken as 100) The recovery rate (R) of the spiked recovery samples was calculated using the following equation: R =-c-·100 0 rort where R: 

### c: (3)
recovery rate [%] determined concentrationofthe test item inthe spikedrecovery sample [mg/L] fortified concentration ofthe test iteminthe spiked recovery sample [mg/L]Harlan StudyNumber: 41304731 Report Page39 Analytical Investigations 

## 2 RESULTS 2.1 Range-Finding Test The analytical methodology was not validatedpriorto the analysis ofrange-:fmding samples; full validation was later conducted at a suitable concentration. The results for the range-finding test samples are summarized in Table 3. 

### 2.2 Definitive Test 2.2.1 Validation ofthe Analytical Method Specificity Thebiological control samples showed no significantinterfering response atthe retention time of the test item. The standard solutions contained a peak specific for the test item whose area changed accordingly with known concentration, hence the specificity ofthe method by retention time was confirmed. Linearity The calibration data for the calibration standards ofthe test item is given in Table 2 and Figure I. The data was found to have a quadratic correlation within the calibration range of0 to 52 mg/1. TheR2 fit ofthe calibration curveto the data was 0.9997, and was considered to be acceptable. Accuracy (Recovery) and Precision A set of recovery samples accurately fortified at a relevant concentration of test item was prepared five-fold and analyzed. The results obtained for the concentration ofthe testiteminthe recovery samples are presented inTable4. The method was considered to be sufficiently accurate and precise for the purposes ofthis test. The test sample results were notcorrected for recovery. Limit ofQuantification The limit of quantification (LOQ) was determined as ten times the standard deviation of five synthetic controls. Usingthis method the LOQ was determined to be 0.0099 mg/L.
2.2.2 Test Samples The results obtained for the concentration ofthe test item found in the test samples are presented in Table 5. Typical chromatograms are showninFigures 2 to 5.Harlan StudyNumber: 41304731 Report Page 40 Analytical Investigations 

## 3 DISCUSSION The detection system was found to have acceptable cotTelation to the fit ofa quadratic equation. The analytical procedure was found to have acceptable recoveries oftest item in test medium. Themethod ofanalysis was validated and proven to be suitable for use.Harlan StudyNumber: 41304731 Report Page41 AnalyticalInvestigations 

# TABLES Table 1 Preparation ofTest Samples
Nominal Sample Sample Final Volume Preparation Concentration of Volume Factor TestItem [% v/v Saturated [mL] [mL] F Solution]

Control 50 10 0.2 1.0 50 10 0.2 3.2 50 10 0.2 10 50 10 0.2 32 50 100 2 100 50 100 2


### Table2 Linearity Data
Concentration ofTest Item Mean Area [mg/L] (counts] 0 1.584 X 10 4 0.104 1.149 X 105 0.264 2.352 X 105 0.520 3.887x 105

1.04 6.535 X 105 2.64 1.396 X 106 5.20 2.448 X 106 10.6 4.625 X 106 26.4 1.068x 107 52.0 2.086 X 107Harlan StudyNumber: 41304731 Report Page42 AnalyticalInvestigations 

### Table 3 Results for Range-Finding Samples Nominal Measured Sample Determined Concentration of Concentration Preparation Concentration Expressed as a Test Item in ofTest Item in Factor ofTest Item in percentage of Timepoint Range-Finding SampleVial Range-Finding the 0-Hour Sample Measured Sample Concentration X F c Cnorn [%v/v [hours] Saturated [mg/L] [mg!L] (%) Solution] 0 0.10 0.335 0.2 0.0669 1.0 3.01 0.2 0.602 NA 10 25.0 0.2 5.00 100 24.9 2.0 49.8 72 0.10 0.293 0.2 0.0585 87 1.0 2.22 0.2 0.444 74 10 20.3 0.2 4.06 81 100 24.1 2.0 48.1 97



### Table 4 Results for Spiked Recovery Samples
Nominal Fortified Measured Sample Determined Mean Analytical Concentration Concentration Concentration Preparation Concentration Recovery Precision of ofTestItem in ofTestItem in Factor ofTestItem in Rate (Relative Test Item the Spiked the Sample the Spiked Standard Sample Vial Sample Deviation of Recovery) Cnom Crort X F c R [mg!L] [mg/L] [mg!L] [mg/L] [%] [%] 0.286 0.2 0.0572 0.285 0.2 0.0569 0.050 0.0529 0.279 0.2 0.0557 107 1.4 0.283 0.2 0.0566 0.277 0.2 0.0554 Acceptance Target 80-120 <10
For reporting purposes, x has been calculated from cretrospectively.Harlan StudyNumber: 41304731 Report Page43 Analytical fuvestigations 

### TableS Results for Test Samples
Nominal Measured Sample Determined Concentration Concentration Preparation Concentration of ofTest Item in Factor ofTest Item in Time point TestItem in SampleVial Test Sample TestSample

Cnom X F c [%v/v [hours] Saturated [mg/L] [mg/L] Solution] 0 Control <LOQ 0.2 <LOQ 1.0 1.86 0.2 0.372 3.2 4.22 0.2 0.843 10 18.5 0.2 3.69 32 7.80 2 15.6 100 25.4 2 50.7 24 1.0 1.59 0.2 0.318 3.2 4.15 0.2 0.830 10 17.5 0.2 3.50 32 7.55 2 15.1 100 24.8 2 49.6 48 1.0 1.72 0.2 0.344 3.2 4.05 0.2 0.809 10 17.0 0.2 3.39 32 7.50 2 15.0 100 24.6 2 49.2 72 Control <LOQ 0.2 <LOQ 1.0 1.66 0.2 0.332 3.2 4.07 0.2 0.814 10 17.3 0.2 3.46 32 7.55 2 15.1 100 24.6 2 49.2 LOQ Limit ofQuantification For reporting purposes, x has been calculated from cretrospectively.Harlan Study Number: 41304731 Analytical Investigations 

## FIGURES Figure 1 Linearity**~** 25000000 2-
120000000 

# £
15000000

10000000

5000000 Report

y = -259.3502x2+41 0,505.1239x+ 179,290.0843**R~=0.9997 0~----~----~----~----~----~----.**  0 10 20 30 40 50 60 Concentmtion (mgll) Page44Harlan StudyNumber: 41304731 Report Page 45 Analytical Investigations 

### Figure2 Chromatogram ofCalibration Solution (5 mg/L)**1ooo,----..;;: 19** **:ch;:;oi,-; ,--~,---~---~---~-** **--~---~---~-T1ooo**

-**$U.~dard** a s mg-1] 

# 800 ____**:~:~-~~~~h,i ~~:~**  

# ________!__ 

# r·····-**------~--** 

# --r-**--~------**

# ~ ------ 800

600

-- 000 

## '"

200
... -------:.**---------~ ----------~**  -------------, --------------,--- 

## '" 

# 0 , ' 0 10 

## " Minutes 

### Figure 3 Chromatogram ofBiological Control Sample'**1000y----:.:.-:,.c:h:c:oi-:1--~, ---~----~---~---~---~---~-,1000** 

- et14G024 control ! Retentlo!J Time '

# ··············~**·············~** J 800

-

800 --------------L--

400

------ -------+ ------- ------· ---------------**-------------~--**

200 --------------,

0 0 

## ' ' ' ' ' ' ' ' ' ' ' ' ' 

### -- _,___ -----------r-- ····j------------ 

## ' 0 Minutes

•Response seen determined to be less than the LOQ. 000

----------------- ---------··<··-- 400
------j --------------c- 

## ---r-- 200

0 

## " "Harlan StudyNumber: 41304731 Report Page46 Analytical Investigations 

### Figure 4 Chromatogram ofTest Sample (1.0% v/v Saturated Solution)**1000~~~~.~** ••**~.~**

••**~,~~~.,,,:,: --~** 

- oU$9026 1.0**Ratentlo~** Time**~~~~~~--r1000** 

## '" .. ···-f·-**----·······-~** -------------r·· ---------------, --------------r -------------,------ 800

600 

# !

<00

200

------- --------------: --------------L



### --r 

## -------------!--------------:

Minutes**~ ~** 

### -----:------------TFigure5 Chromatogram ofTest Sample (100% v/v Saturated Solution) 600

400

200**<aoo~~~~=~~~~~~~~~~~~~~~~-** ..**~~~~~~~~~~~~T4ooo** Slgllar 1

- et1lf9029100 Retentiof Tima

3000 

# ------------· _____. _._'
___.*_.___.,___._._**~**

2000 ----·----------- ---·-- ------------"**-------------~-- ---------~----**  ---------

## ·--

-

1000

-----

0 2**-------------~** --------------L ··•·•·••••••••



## • ' Minutes 

## " " 3000

2000 

## ""



# IHarlan Study Number: 41304731 Report


### Appendix 6 MonitoringAuthority Statement ofGLP Compliance 

# {

nH) Department**~** 

### ofHealth
THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM GOOD LABORATORY PRACTICE

STATEMENT OF COMPLIANCE IN ACCORDANCE WITH DIRECTIVE 2004/9/EC
TEST FACILITY

Harlan Laboratories Limited Shardlow Business Park London Road Shardlow Derbyshire DE722GD

DATE OF INSPECTION TEST TYPE(S) Analytical/Clinical Chemistry Environmantal Fate Environmental Toxicity Phys.Chem. Testing Mutagenicity

Toxicology

12to 14 March 2014 An Inspection for compliance with the Principles of Good Laboratory Practice was carried out at the above test facility as part of the UK Good Laboratory Practice Compliance Monitoring Programme. This statement confirms that, on the date of Issue, the UK Good Laboratory Practice Monitoring Authority were satisfied that the above test facility was operating in compliance with the OECD Principles of Good Laboratory Practice. This statement constitutes a Good Laboratory PractiCe Instrument (as defined in the UK Good Laboratory Practice Regulations 1999).

### ~C::~>:sL;<. ,,.

## I 1¥ Dr. Andrew J. Gray Head, UK GLP Monitoring Authority Page47