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

StudyDirector: TestFacility:

Sponsor:

StudyMonitor:

Harlan StudyNumber: StudyCompletionDate: H Vryenhoef HarlanLaboratoriesLtd. ShardlowBusiness Park Shardlow Derbyshire DE722GD 

### UK PCBTFTestingConsortium c/o Greenwich Chemical Consulting, Inc. 3300 PGA Boulevard, Suite 635 Palm Beach Gardens, FL 33410 USA Mr. JohnV. Massingale Greenwich ChemicalConsulting,Inc. 3300 PGA Boulevard, Suite 635 Palm Beach Gardens, FL 33410 USA 41304731 21 April2015

Page 1of47Harlan StudyNumber: 41304731 Report Page2


## CONTENTS CONTENTS ....................................................................................................................................2 STUDY DIRECTOR STATEMENT OF GLP COMPLIANCE ....................................................4 QUALITY ASSURANCE STATEMENT......................................................................................5 SIGNATURE OF CONTRIBUTING SCIENTISTS......................................................................6 SUMMARY .................................................................................................................................... ? 

### Introduction............................................................................................................................7 Methods...............................................................................................................................7 Results..................................................................................................................................7 GENERAL INFORMATION .........................................................................................................9 Schedule.................................................................................................................................9 Deviations from Study Plan...................................................................................................9 Archiving ...............................................................................................................................9 1 INTRODUCTIONAND PURPOSE................................................................................... 10 1.1 Guidelines I

### Regulations........................................................................................ 10 2 TEST ITEM ......................................................................................................................... 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 MediaPreparation Trial....................................................... 13 3.4.2 Range-Finding Test ................................................................................. 13 3.4.3 Definitive Test......................................................................................... 14 3.4.4 Evaluations .............................................................................................. 15 3.4.5 DataAnalysis........................................................................................... 16 3.5 Validation Criteria................................................................................................. 18 4 RESULTS ............................................................................................................................ 19 4.1 Range-finding Test ................................................................................................ 19 4.2 Definitive Test....................................................................................................... 19 4.2.1 Verification ofTestConcentrations ........................................................ 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 StudyNumber: 41304731 Report Page 3


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

## LIST OF FIGURES Figure 1 Figure 2 MeanCell Densities v Time for the Definitive Test...................................................28 Inhibition ofGrowth Rate Based on 0-HourMeasured Test Concentrations ............................................................................................................29 Figure 3 Inhibition ofYieldBased on0-HourMeasured Test Concentrations ........................30 

## LIST OF APPENDICES Appendix 1 Appendix 2 Appendix 3 Appendix 4 Appendix 5 Appendix 6 Certificate ofAnalysis...........................................................................................31 Positive Control.....................................................................................................32 Culture Medium.....................................................................................................33 PreliminaryMediaPreparationTrial.....................................................................34 Analytical Investigations .......................................................................................35 Monitoring Authority Statement ofGLP Compliance ..........................................47Harlan StudyNumber: 41304731 Report Page4


## STUDYDIRECTOR STATEMENT OF GLP COMPLIANCE HarlanLaboratories Ltd., ShardlowBusiness Park, Shardlow, Derbyshire, DE72 2GD, UK Harlan StudyNumber: 41304731 Study Title: Benzene, 1-chloro-4-(trifluoromethyl)- CAS RN 98-56-6 (paraChlorobenzotrifluoride (PCBTF)): Algal Growth InhibitionTest 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 2004110/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 (MHLW, MAFF and METI). This report fully and accurately reflects the procedures used and data generated. There were no circumstances consideredto have affectedthe integrity ofthe study orthe validity ofthe data. StudyDirector: H Vryenhoef



# ..

**4~.L/**

# ....... Date-:



## .

**2~** 

## 1APR 

## 2015Harlan StudyNumber: 41304731 Report Page 5 

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

The general facilities and activities are inspected at least once a year and the results are reported to the relevantresponsibleperson 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 ofInspection Type ofInspection Phase Inspected Report Date 07 November 2014 StudyPlan N/A 07 November 2014 Verification 26 January 2015 Process

- based TestItemPreparation 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 ReportAudit N/A 12 March 2015 Verification ofStudy PlanAmendment for GLP compliance was performed during the course of the study. This statementconfirms that this reportreflects the rawdata and theprocedures followed. Quality



### Assurance:~ **\~~'C.**



## .......
**~**.................... Date: 

## 20APR 2015Harlan StudyNumber: 41304731 Report Page 6


## SIGNATURE OF CONTRIBUTING SCIENTISTS
Analytical Chemistry: DMMullee Harlan Laboratories Ltd., ShardlowBusiness Park, Shardlow, Derbyshire, DE72 2GD, UK

Date: 

## 2 1APR 2015Harlan StudyNumber: 41304731 Report Page 7


## 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 ofCommissionRegulation (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 mediapreparationtrial 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 itemundertest conditions. Following a preliminary range-finding test, Pseudokirchneriella subcapitata 

### was exposed to solutions ofthe test item at nominal 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) (five replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 

### 1 °C. The test item solutions were prepared by stirring an excess (100 mg/L) oftestitem inculture mediumusing a magnetic stirrer at arate such that a 5% vortex was formed for 24 hours. After the stirring period any undissolved test item was removed by filtration (0.2 j.lm 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 andtreatment group, using a Coulter® MultisizerParticle 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 addedto 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 ofPseudokirchneriella subcapitata to the test item gave the following results based on the 0-Hourmeasuredtest concentrations:
Response 95% Confidence No ObservedEffect Lowest Observed ECso (mg/L) Concentration (NOEC) Effect Concentration Variable Limits (mg/L) (mg!L) (LOEC) (mg/L) GrowthRate 6.2 4.8 

### - 8.1 0.84 3.7 Yield 2.2 1.8 

### - 2.6 0.84 3.7Harlan StudyNumber: 41304731 Report


## GENERALINFORMATION Schedule Experimental Starting Date: Experimental Completion Date: 

### 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 bythe Sponsor, the studyplan (general study plan and study specific supplement), all raw data (paper and electronic) and the final reportwill 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 datawill be discarded withoutcontactingthe 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. Pseudokirchneriella 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 importantregulatory 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 preparedby stirring an excess (100 mg/L) oftest item in culture medium for aperiod of24hours prior to removing any undissolved test item present by filtration (0.2

**~-tm** 

### 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 and Mixtures. OECD Series onTesting and Assessment, No. 23. OECD Publishing.Harlan StudyNumber: 41304731 Report Page ll


## 2 TESTITEM Information as provided by the Sponsor. A Certificate ofAnalysis supplied by the Sponsor is giveninAppendix 1. Identification: Physical state/ Appearance: Batch: Purity: Expirydate: Storage conditions: Intended use/ 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 inthe dark Chemical intermediateHarlan StudyNumber: 41304731 Report Page 12


## 3 MATERIALSAND METHODS 3.1 Definitions and Abbreviations Biomass:

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 overthe testperiod. GrowthRate (Average Specific The logarithmic increase in biomass during the exposure GrowthRate): period. Yield:

LOEC (Lowest Observed Effect Concentration): NOEC (No ObservedEffect 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 lowesttestconcentration at which a statistically significant inhibition ofgrowth is determinedrelative 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 constantillumination 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 withpolyurethane foam stoppers and kept under constant agitationby orbital shaker (100- 150 rrm) and constant illumination at 24 ± 1

## oc until the algal cell density was approximately 104

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



### A positive control (Harlan StudyNumber41403365) used potassium dichromate as the reference item. Details of the positive control are given in Appendix 2. The positive control was conductedbetween 14 October 2014 and 31 October 2014. 

### 3.3 TestWater The culture medium used for both the range-finding and definitive tests was the same as that used to maintainthe stockculture. Forthe 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 adjustedto pH 7.0 afterthe addition ofthe sodiumbicarbonate. 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/Lbased onthe U.S. EPA's citation ofaMerck Index ofunknownreliability. 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 itemundertest conditions (see Appendix 4). 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 in the definitive test were determined by a preliminary rangefinding test. The range-finding test was conducted by exposing Pseudokirchneriella 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 10.9litersofculture medium withthe 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 J.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-Hourmeasured concentrations of0.068, 0.60, 5.0 and 50 mg/L). The test was conducted in 250 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 was maintainedunder identical conditions butnot exposedto 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 run) 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 in this test by the 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 ofeachtest concentration was taken for chemical analysis at 0 and 72 hours in order to determine the stability ofthe test itemundertest 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 suchthat 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 J.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 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 ofalgal 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 StudyNumber: 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 testpreparations 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) until 48 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 ofthe test replicates containing treated test solutions was used only once for sampling before being discarded. Ofthe nine control replicates prepared atthe 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 whichtime samples were taken for cell density determinationthen 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 maintainedunder identical conditions butnotexposedto 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 of2 liters oftest medium with 5.2 mL ofthis algal suspension gave an initial nominal cell density of 3 x 1 03 

### cells per mL and had no significant dilution 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 rpm for 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 StudyNumber: 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 test preparation 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 0hours 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 in Appendix 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 growthrate from time t1to tn cell concentration att1 cell concentration at tn time offirst measurement time ofnth 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 thanthe measured starting value in orderto 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: I = Jlc -J..tt X 

### 100

r


### Where: 

### Ir llc llt llc



### percentage inhibition ofaverage specific growth rate 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 atthe 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:Harlan StudyNumber: 41304731 Report



### Where: percentage inhibition ofyield mean value for yield inthe 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 simplifiedmethod ofevaluating dose-effect experiments ofLitchfield and Wilcoxon (1949). 3.4.5.4 Statistical Analysis One way analysis ofvariance incorporating Bartlett's test for homogeneity ofvariance (Sokal and Rohlf, 1981) and Dunnett's multiple comparisonprocedure 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 The results ofthe test 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 section by 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 gtven m Table 1. The results showed no effect on growth atthe test concentrations of0.10 and 1.0% v/v saturated solution. However, growth was observedto be reduced at 10 and 100% v/v saturated solution. Based on this 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 pH values 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 giveninTable 4. The mean cell densities versus time for the definitive test are presented in Figure 1. Percentage inhibitionvalues are plotted againsttest concentrationinFigure 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-Hourexposureperiod. Accordinglythe following results were determined from the data:Harlan StudyNumber: 41304731

4.2.2.1 Inhibition ofGrowthRate
: 1.8 mg/L :2.9mg!L Report

ErCIO (0

- 72 h) ErC2o (0

- 72 h) ErCso (0

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

- 8.1 mg/L whereErCx is the test concentrationthatreduced growthrate byx%. 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 (Sokal and Rohlf, 1981) and Dunnett's multiple comparisonprocedure 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~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 "LowestObserved Effect Concentration" (LOEC) based ongrowthrate was 3.7 mg/L. 4.2.2.2 Inhibition ofYield :0.87mg/L : 1.2 mg/L EyCIO (0

- 72 h) EyC2o (0

- 72 h) EyCso (0

- 72 h) :2.2 mg/L; 95% confidence limits 1.8

- 2.6 mg/L Where: EyCx is thetestconcentrationthatreduced yieldbyx%. 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~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. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 3.7 mg/L. 

### 4.2.3 Validation Criteria The following data showthat 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 enhancementmustbe at leastby a factor of 16 after 72 hours. Mean cell density ofcontrol at 0hours Mean cell density ofcontrol at 72 hours 3.00 x 103 cells permL 5.96 x 105 cells permLHarlan StudyNumber: 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 mustnot 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 thatthis mustnot 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 throughoutthe test. The pH value ofthe control cultures (see Table 2) was observed to increase from pH 7.9 at 0hours 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 withinthe limits given inthe Test Guidelines. 4.2.6 Observations on TestItem 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 observedto be clear colorless solutions.Harlan StudyNumber: 41304731 Report Page 22


## 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 StatAs soc 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 andUnstable Substances. HERMAN etal. (1990) Impact ofvolatile aromatic hydrocarbons, alone and in combination, on growthofthefreshwater algaSelenastrum capricornutum.Aquatic Toxicology 18, 87- 100 LITCHFIELD, J.T. and WILCOXON, F. (1949) A Simplified Method ofEvaluating Dose-Effect Experiments. JPharmacalExp 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 andChemistry, 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. NewYork: W H FREEMAN and Company. Xlfit, ID Business Solutions Ltd.Harlan StudyNumber: 41304731 Report Page 24


## TABLES Table 1 CellDensities andPercentageInhibition ofGrowthfrom theRange-findingTest Cell Densities* (cells permL) Inhibition Values(%) Nominal Concentration (% v/v Saturated Solution) 0 Hours 72 Hours GrowthRate Yield R1 3.00£+03 5.31£+05 Control Rz 3.00£+03 5.94£+05


### - - Mean 3.00£+03 5.62£+05 R1 3.00£+03 6.40£+05 0.10 Rz 3.00£+03 5.12£+05 0 [2] Mean 3.00£+03 5.76£+05 RI 3.00£+03 5.65£+05 1.0 Rz 3.00£+03 4.63£+05 3 9 Mean 3.00£+03 5.14£+05 R1 3.00£+03 7.87£+04 10 Rz 3.00£+03 6.20£+04 40 88 Mean 3.00£+03 7.03£+04 R1 3.00£+03 3.63£+03 100 Rz 3.00£+03 2.99£+03 99 100 Mean 3.00£+03 3.31£+03

* Cell densities representthemeannumber ofcells permL calculated from the mean ofthe cell counts from 3 counts for each ofthe replicate flasks. R1and R2 = Replicates 1and 2 [Increase ingrowthcomparedto controls]Harlan StudyNumber: 41304731 Report Page 25

Table2 CellDensities and pHValues in the Definitive Test
Nominal Concentration pH CellDensities* (cells per mL) (% v/v Saturated Solution) Oh Oh 24 h 48 h 72h Rt 3.00E+03 1.31E+04 1.18E+05 5.72E+05 Rz 3.00E+03 1.16E+04 1.09E+05 5.50E+05 Control 7.9 R3 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 Rt 3.00E+03 1.22E+04 1.23E+05 6.52E+05 1.0 Rz 3.00E+03 6.10E+05 7.7 R3 3.00E+03 6.29E+05 Mean 3.00E+03 1.22E+04 1.23E+05 6.30E+05 Rt 3.00E+03 1.20E+04 8.41E+04 4.69E+05 3.2 Rz 3.00E+03 6.29E+05 7.6 R3 3.00E+03 4.96E+05 Mean 3.00E+03 1.20E+04 8.41E+04 5.32E+05 Rt 3.00E+03 6.13E+03 2.97E+04 1.30E+05 Rz 3.00E+03 1.37E+05 10 7.5 R3 3.00E+03 1.39E+05 Mean 3.00E+03 6.13E+03 2.97E+04 1.35E+05 Rt 3.00E+03 2.75E+03 5.42E+03 6.19E+03 32 Rz 3.00E+03 5.49E+03 7.5 R3 3.00E+03 6.31E+03 Mean 3.00E+03 2.75E+03 5.42E+03 5.99E+03 Rt 3.00E+03 1.55E+03 3.74E+03 5.63E+03 100 Rz 3.00E+03 4.63E+03 7.4 R3 3.00E+03 6.69E+03 Mean 3.00E+03 1.55E+03 3.74E+03 5.65E+03

* Cell densities representthe meannumber ofcellspermL calculated from the mean ofthe cell counts from 3 counts for each ofthereplicate flasks. R 1 - R3 = Replicates 1to 3 pH 72h

9.4

9.1

9.2

8.1

7.7

7.5Harlan StudyNumber: 41304731 Report Page 26

Table3 Daily Specific GrowthRates forthe Control CulturesintheDefinitiveTest Daily Specific Growth Rate (cells/mL/hour) Day0-1 Day 1-2 Day 2-3 Rt 0.062 0.092 0.066 R2 0.056 0.093 0.067 Control R3 0.062 0.089 0.075 Mean 0.060 0.091 0.069

R1 - R3 =Replicates 1to 3Harlan StudyNumber: 41304731 Report Page 27

Table 4 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* RI 0.073


### - 5.69E+05 R2 0.072 5.47E+05 Control R3 0.075


### - 6.64E+05


### - Mean 0.073 5.93E+05 SD 0.002 6.22E+04 RI 0.075 -3 6.49E+05 R2 0.074 -1 6.07E+05 1.0 R3 0.074 -1 6.26E+05 [6] Mean 0.074 -1 6.27E+05 SD 0.001 2.12E+04 RI 0.070 4 4.66E+05 R2 0.074 -1 6.26E+05 3.2 R3 0.071 3 4.93E+05 11 Mean 0.072 2 5.29E+05 SD 0.002 8.59E+04 RI 0.052 29 1.27E+05 R2 0.053 27 1.34E+05 10 R3 0.053 27 1.36E+05 78 Mean 0.053 27 1.32E+05 SD 0.001 4.67E+03 R1 0.010 86 3.19E+03 R2 0.008 89 2.49E+03 32 R3 0.010 86 3.31E+03 99 Mean 0.009 88 2.99E+03 SD 0.001 4.44E+02 R1 0.009 88 2.63E+03 R2 0.006 92 1.63E+03 100 R3 0.011 85 3.69E+03 100 Mean 0.009 88 2.65E+03 SD 0.003 1.03E+03



### * In accordance withthe OECD test guideline onlythe mean value for yieldfor eachtest concentration is calculated R1 - R3 = Replicates 1to 3 SD = StandardDeviation [Increase in growth compare to controls]Harlan StudyNumber: 41304731 Report


## FIGURES Figure 1 Mean CellDensitiesv Timeforthe DefinitiveTest
l.OOE+06

**-r--r---------------------------~---------_-** 

### __

-_-

__ -_..,+------------------------------------------_- __

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__ -_-

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**l.OOE+03+-------------------~·--------------------~·~-------------------4** 0 24 48 72 Time(Hours) Page 28Harlan StudyNumber: 41304731 Report Page29
Figure2 Inhibition ofGrowth Rate Based on 0-HourMeasured Test Concentrations

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0.1 10 100 Concentration (mg!L)Harlan StudyNumber: 41304731 Report Page 30


### Figure 3 Inhibition ofYield Based on 0-HourMeasured Test Concentrations

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### : : +: : 10 100 Concentration (mg'L)Harlan StudyNumber: 41304731 Report 

## APPENDICES Appendix 1 Certificate ofAnalysis

70W&st41)th Sire&! NewYork, NY 10018 Phone: (646)366.Q400 Fa>c (646!36f>.0595 

### CERTIFICATE OFANALYSIS ProductName: Quality: Batch Number: Test/Property

Acidity, ppm (spec. ion probe) Alkalinity. ppmas NaOH Appearance Assay,% Color(APHA) Non Volatile Residue, Wt % Specific Gravity(20C/25C) Watercontent, ppm Parachlorobenzotrifluoride-OOON SMC Spec. 900 2014·02-08 Specification 3.0 PPM MAXIMUM 10.0 PPM MAXIMUM Clear liquid, free ofsuspended material 99.00 %MINIMUM 20APHA MAXIMUM 0.0020% MAXIMUM 1.3300TO 1.3500GICC 150 PPM MAXIMUM

CERTIFIED TRUEAND CORRECT W!Jl

Approval Date:2117/2014 $peeRevision Date: Page 31

Result

1.7 0.0 Clear liquid, frflft ofsuspended material 99.77 10 0.0013 1.3340 78

Print Date: 11/5114Harlan StudyNumber: 41304731 Report Page 32
Appendix2 Positive Control A positive control (Harlan StudyNumber41403365) used potassium dichromate as the reference item atconcentrations of0.050, 0.13, 0.32, 0.80 and 2.0 mg/L. Six flasks each completely filled with testpreparation 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 ± 1 °C 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. The culture medium used was preparedwiththe addition of500mg/LNaHC03. Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results: ErCso (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 Observed Effect Concentration (NOEC) based on growthrate: No Observed Effect Concentration (NOEC) based onyield: LowestObservedEffect Concentration (LOEC) based on growthrate: Lowest ObservedEffect Concentration (LOEC) based onyield: 0.32 mg/L 0.32 mg/L 0.80 mg/L 0.80 mg/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 MgS04.1H20 14.6 mg/L K2HP04 1.044 mg/L NaHC03 15.0 mg/L H3B03 0.186 mg/L MnChAH20 0.415 mg/L ZnCh 0.00327 mg/L FeCh.6H20 0.160 mg/L CoCh.6H20 0.00143 mg/L Na2Mo04.2H20 0.00726 mg/L CuCh.2H20 0.000012 mg/L Na2EDTA.2H20 0.30 mg/L The culture medium was prepared using reverse osmosis purified deionized water*. For the purposes ofthe definitive test, additional sodium bicarbonate (500 mg/L) was added to the prepared culture mediumpriorto adjusting the pH to 7.0 with 0.1NNaOH orHCI.

*Elga Optima 15+ orElgaPurelab Option R-15 BPHarlan StudyNumber: 41304731 Report Page 34
Appendix 4 Preliminary Media Preparation Trial 1. Saturated Solution Preparation 

### A nominal amount oftest item (1

### 090 mg) was dispersed, in duplicate, in 10.9 liters ofculture medium with the aid of magnetic stirring at a rate such that a vortex was formed which represented approximately 5% ofthe 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 10000 g 

### for 30 minutes • Centrifugation at 40000 g 

### for 30 minutes • Filtration through a 0.2 J..Lm Sartorius Sartopore filter (approximately 1 liter discarded in order to pre-conditionthe filter) • Filtration through a 0.2 J..Lm Sartorius Sartopore filter (approximately 2 liters discarded in orderto pre-conditionthe filter)
2. Results Stirring Period and Treatment Concentration Found (mg/L) 24 Hours Centrifuged 10000g 19.0 24 Hours Centrifuged40000 g 11.4 24 Hours Filtered

**~** 1liter discarded 57.2 24 HoursFiltered

**~** 2 liters discarded 56.7 48 Hours Centrifuged 10000g 18.5 48 Hours Centrifuged40000 g 9.90 48 Hours Filtered

**~** 1liter discarded 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 remove the 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 100 mg/L, stirred for a period of24 hours prior to the removal of any undissolved test item by filtration through a 0.2 J..Lm 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 undertest conditions.Harlan StudyNumber: 41304731 Report Page 35
Appendix 5 AnalyticalInvestigations 

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

### 1.2 TestItem The test item is described inthe biological partofthis study. 

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

### 1.4 Analytical Procedure 1.4.1 Storage 

### The samples were analyzed onthe day ofreceipt. 1.4.2 Reagents and Solvents 

### Testmedium: as described inthe biologicalpart ofthis study. Solvents: tetrahydrofuran, Sigma-Aldrich, HPLC grade. hexane, Fisher Scientific, pesticide residue grade.Harlan StudyNumber: 41304731 Report Page 36 Analytical Investigations 1.4.3 Preparation ofCalibration Standards 

### The test item (nominal 100 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. Defined volumes ofthis solution were then diluted with hexane to obtain calibration standards in the range of0.1 to 1.0 mg/L. A second series ofcalibration standards was similarly prepared in the range of0.25 to 2.5 mg/L. These solutions were used to determine the recovery andtest sample concentrations. 1.4.4 Preparation ofLinearity Standards 

### The test item (nominal 100 mg) was dissolved in hexane (100 mL) to prepare a stock solution with a concentration of 1 000 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 oftest 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 of0.050 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: Injectionvolume Injectiontemperature: Detectortemperature: Retentiontime: HP 5890 Series II, with autosampler ECD ZB-624 (60 m x 0.32 mm i.d.;1.8 !Jm film thickness) double gooseneck nitrogen 10 psi

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

on at 1minute

250

## oc 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 1. From the calibration curve, the concentration "x" of the test item in an injected sample was calculated using the following equation:



### -b+~b **2** -4·a·(c- y) X

## =---'----------'--- (1) 2·a where x: y: a, b, c: concentrationofthetestiteminthe injected sample [ mg/L] averagepeakareaofthetestiteminthe injected sample [counts] constants The concentration of the test item in the test samples was calculated usmg the following equation:

c 

## = X ·F R/100 where c: 

### x: F: R: (2) concentrationofthetest item inthetest sample [ mg/L] concentrationofthetestiteminthe injected sample solution [mg!L] samplepreparationfactor recoveryrate [%] (Ifnot correcting for recoverythenR is takenas 1 00) The recovery rate (R) of the spiked recovery samples was calculated using the following equation: R =-c-·100 cfort where R: c: (3) recoveryrate [%] determinedconcentrationofthe testiteminthe spikedrecovery sample [mg/L] fortified concentrationofthe test iteminthe spikedrecovery sample [mg/L]Harlan StudyNumber: 41304731 Report Page 39 Analytical Investigations 

## 2 RESULTS 2.1 Range-Finding Test The analytical methodology was not validatedpriorto the analysis ofrange-finding 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 The biological control samples showed no significant interfering response attheretentiontime 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 1. The data was found to have a quadratic correlation within the calibration range of0to 52 mg/L. The R2 fit ofthe calibration curve to the datawas 0.9997, and was consideredto 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 test item inthe recovery samples are presented in Table 4. The method was considered to be sufficiently accurate and precise for the purposes ofthis test. The test sample results were notcorrectedfor recovery. 

### Limit ofQuantification The limit of quantification (LOQ) was determined as ten times the standard deviation offive synthetic controls. Usingthis methodthe LOQ was determined to be 0.0099 mg/L. 

### 2.2.2 Test Samples The results obtained for the concentration ofthe test item found inthe test samples are presented in Table 5. Typical chromatograms are shownin Figures 2 to 5.Harlan StudyNumber: 41304731 Report Page 40 AnalyticalInvestigations 

## 3 DISCUSSION The detection system was found to have acceptable correlation to the fit ofa quadratic equation. The analytical procedure was found to have acceptable recoveries oftest item in test medium. The method ofanalysis was validated andprovento be suitable for use.Harlan StudyNumber: 41304731 Report Page 41 Analytical Investigations 

## TABLES Table 1 PreparationofTest Samples
Nominal Sample Sample Concentrationof Volume FinalVolume Preparation TestItem Factor [% 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 LinearityData
ConcentrationofTestItem MeanArea [mg/L] [counts] 0 1.584 X 104 0.104 1.149 X 105 0.264 2.352 X 105 0.520 3.887 X 105

1.04 6.535 X 105 2.64 1.396x 106 5.20 2.448 X 106 10.6 4.625 X 106 26.4 1.068 X 107 52.0 2.086 X 107Harlan StudyNumber: 41304731 Report Page 42 Analytical Investigations Table3 Results for Range-Finding Samples Nominal Measured Sample Determined Concentration of Concentration Preparation Concentration Expressed as a TestItem in ofTestItem in Factor ofTestItem in percentage of Time point Range-Finding SampleVial Range-Finding the0-Hour Sample Sample Measured Concentration X F c Cnom [% 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

Table4 Results for SpikedRecovery Samples Nominal Fortified Measured Sample Determined Mean Concentration Concentration Concentration Preparation Concentration Analytical Precision of ofTestItem in ofTestItem in Factor ofTestItem in Recovery (Relative TestItem the Spiked the Sample the Spiked Rate 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
Forreporting purposes, x hasbeen calculated from c retrospectively.Harlan StudyNumber: 41304731 Report Page 43 AnalyticalInvestigations Table5 Results for Test Samples Nominal Measured Sample Determined Concentration Concentration Preparation Concentration of ofTest Item in Factor ofTestItem in Timepoint Test Item in SampleVial Test Sample Test Sample
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 Forreporting purposes, x hasbeen calculated from cretrospectively.Harlan StudyNumber: 41304731 Analytical Investigations 

## FIGURES Figure 1 Linearity

,...__

**-~** 25000000 2.

**~** 

### " 0 r;;.

**~** 20000000 .!<: 

# ..

**~** 15000000

10000000

5000000 Report

y = -259.3502x2+410,505.1239x+ 179,290.0843 R2=0.9997

0 **~------,--------r-------.-------.--------r-------,**

0 10 20 30 40 50 60 Concentration(mgl1) Page 44Harlan StudyNumber: 41304731 Report Analytical Investigations Figure 2 Chromatogram ofCalibration Solution (5 mg/L)

0 2 4 6 8 10 Minutes Figure 3 Chromatogram ofBiological Control Sample*

0 2 4 6

*Response seen determined to be less than the LOQ. 8 Minutes 10 Page 45

12 14 16

12 14 16Harlan StudyNumber: 41304731 Report Analytical Investigations 

### Figure 4 Chromatogram ofTest Sample (1.0% v/v 

### Saturated Solution)

2 4 6 8 Minutes 10 12 14 

### Figure 5 Chromatogram ofTest Sample (100% v/v 

### Saturated Solution)



### t 2000

1000

2 4 6 8 Minutes 10 12 14 Page 46

16

1000

16Harlan StudyNumber: 41304731 Report
Appendix 6 MonitoringAuthority StatementofGLP Compliance


### 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 TESTTYPE(S) Analytical/Clinical Chemistry Environmental 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. Thisstatement constitutes a Good Laboratory Practice Instrument (as defined in the UK Good Laboratory Practice Regulations 1999)
.

Dr. Andrew J. Gray Head, UK GLP Monitoring Authority

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