## REPORT


## Daphnia sp., 48-HourAcute Immobilization Test

StudyDirector: TestFacility:

Sponsor:

Harlan StudyNumber: Study Completion Date: SParr 

### HarlanLaboratories Ltd. Shardlow Business Park Shardlow Derbyshire DE722GD UK



### 41206434
26 April2013

Page 1of41Harlan StudyNumber: 41206434 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
......................................................................................................... 8 Schedule
................................................................................................................................. 8 Archiving
...........................................................................................,................................... 8 1 INTRODUCTION AND PURPOSE
.....................................................................................9 1.1 Guidelines IRegulations
..........................................................................................9 2 TEST ITEM
...........................................................................................................................9 3 MATERIALS AND METHODS
......................................................................................... 10 3.1 Definitions and Abbreviations
............................................................................... 10 3.2 Test Species
........................................................................................................... 10 3.3 TestWater
.............................................................................................................. 10 3.4 Procedure
............................................................................................................... 11 3.4.1 Range-finding Test
.................................................................................. 11 3.4.2 Definitive Test
......................................................................................... 11 4 RESULTS
............................................................................................................................ 14 4.1 Range-finding Test
................................................................................................ 14 4.2 Definitive Test
....................................................................................................... 14 4.2.1 Immobilization Data
................................................................................ 14 4.2.2 Observations on Test Item Solubility
...................................................... 15 4.2.3 Physico-Chemical Measurements
........................................................... 15 4.2.4 Verification ofTest Concentrations
........................................................ 15 5 CONCLUSION
.................................................................................................................... 16 6 REFERENCES
.................................................................................................................... 17 TABLES
........................................................................................................................................ 18 FIGURES
......................................................................................................................................20 APPENDICES
...............................................................................................................................22 TABLES
........................................................................................................................................34 FIGURES
......................................................................................................................................37 

## LIST OF TABLES Table 1 Table 2 Cumulative ImmobilizationDatainthe Range-finding Test..
.................................... 18 Cumulative Immobilization Datainthe Definitive Test..
........................................... 19Harlan StudyNumber: 41206434 Report Page 3


## LIST OF FIGURES Figure 1 Figure 2 Concentration-Response Curve after 24 Hours
..........................................................20 Concentration-Response Curve after 48 Hours
..........................................................21 

## LIST OF APPENDICES Appendix 1 Appendix 2 Appendix 3 Appendix 4 Appendix 5 Appendix 6 Reconstituted Water- Elendt M7 Medium
...........................................................22 Positive Control
.....................................................................................................24 Reconstituted Water
..............................................................................................25 Analytical Investigations
.......................................................................................26 Physico-Chemical Measurements
..........................................................................40 Monitoring Authority Statement ofGLP Compliance
..........................................41Harlan StudyNumber: 41206434 Report Page4


## STUDYDIRECTOR STATEMENT OF GLP COMPLIANCE Harlan Laboratories Ltd., Shardlow Business Park, Shardlow, Derbyshire, DE72 2GD, UK
Harlan StudyNumber: 41206434 Study Title: Daphnia sp., 48-Hour Acute ImmobilizationTest 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 (MHLW, MAFF and METI). This report fully and accurately reflects the procedures used and data generated. There were no circumstances considered to have affectedthe integrity ofthe study orthe validity ofthe data. Study Director: SParr

# ········~··········· Date: 

### 2 6 APR ?013Harlan StudyNumber: 41206434 Report Page 5 

## QUALITYASSURANCE STATEMENT Harlan StudyNumber: 41206434 StudyTitle: : Daphnia sp., 48-Hour Acute ImmobilizationTest

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 at the test facility were audited and inspected. The details of these audits and inspections are given below. Reported to the Dates and Types ofQAInspections relevant Study Director and Test Facility Management Date ofInspection Type ofInspection Phase Inspected Report Date 19 November 2012 General Study Plan N/A 19 November 2012 Verification 15 January 2013 Process

- based Test Item Preparation 15 January 2013 10 January 2013 Process

- based Test System 10 January 2013 Preparation 15 January 2013 Process

- based Exposure 15 January 2013 10 January 2013 Process

- based Assessment of 10 January 2013 Response 11 January 2013 Process

- based Chemical Analysis 11 January 2013 09 April 2013 ReportAudit N/A 09 April 2013 The complete study plan (including Study Specific Supplement) was verified for GLP compliance at report audit. This statement confirms thatthis report reflects the rawdata and the procedures followed. Quality Assurance:

# .L~....

# ~:-:.~.~.. Date: 

## 2 4 APR 2013Harlan StudyNumber: 41206434 Report Page 6


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

# ····~············~·~············Z~13 Date:Harlan StudyNumber: 41206434 Report Page 7


## SUMMARY Introduction A study was performed to assess the acute toxicity ofthe test item to Daphnia magna. 

### The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia 

### sp., Acute Immobilisation Test" referenced as Method C.2 ofCommission Regulation (EC) No. 440/2008. 

### Methods Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution ofthetest item at concentrations of 1.0, 3.2, 10,32 and 100 mg/L for 48 hours at a temperature of20 °C to 21 °C under static test conditions. The numbers of immobilized Daphnia 

### were recorded after 24 and 48 hours. 

### Results The 48-Hour ECso for the test item to Daphnia magna 

### based on nominal test concentrations was 8.5 mg/L with 95% confidence limits of 6.6

- 11 mg/L. The Lowest Observed Effect Concentration was considered to be 10 mg/L. The No Observed Effect Concentration was 3.2 mg/L. Analysis ofthe test preparations at 0 and 48 hours showed measured test concentrations to be near nominal and so the results are based on nominal test concentrations only.Harlan StudyNumber: 41206434 Report


## GENERALINFORMATION Schedule Experimental Starting Date: Experimental CompletionDate: 

### Archiving 11 December 2012 07 February 2013 Page 8

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 in the 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 Sponsor to obtaintheir written consent.Harlan StudyNumber: 41206434 Report Page9


## 1 INTRODUCTIONAND PURPOSE This report contains a description ofthe methods used and results obtained during a study to investigate the acute toxicity ofthe test itemto Daphnia magna. Daphnia magna is a freshwater invertebrate representative ofa wide variety ofnatural habitats, and cantherefore be considered as an importantnon-target organism in freshwater ecosystems. 

### 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 ofChemicals (April2004)No 202 "Daphnia sp., Acute ImmobilisationTest" • Method C.2 ofCommissionRegulation (EC) No. 440/2008 

## 2 TESTITEM The integrity ofthe supplied data relating to the identity, purity and stability ofthe test item is the responsibility ofthe Sponsor. Identification: Description: Batch: Purity: Expiry Date: Storage Conditions:Harlan StudyNumber: 41206434 Report Page 10


## 3 MATERIALSAND METHODS 3.1 Definitions and Abbreviations Immobilization:
ECso: LOEC (Lowest Observed _Effect Concentration): NOEC (No Observed_Effect Concentration): Treatment:


### 3.2 Test Species Those organisms which are not able to swim within 15 seconds after gentle agitation ofthe test container are considered to be immobile. The calculated concentration oftestitem which results in 50% immobilization withinthe exposure period. The lowesttest concentration at which a significant toxic effect onthe test organisms is observed within the exposure period. The highest test concentration or loading rate at which no significanttoxic effect onthe test organisms is observed within the exposure period. Comprises test itemtreatments, control (test water only) and solvent control (ifapplicable).

The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures. Adult Daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium (see Appendix 1) in a temperature controlled room at approximately 20 °C. The lighting cycle was controlled to give a 16 hours light and 8hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture ofalgal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation ofthe test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminantthat would affect the integrity or outcome ofthe study. A positive control (Harlan Study Number: 41203341) used potassium dichromate as the reference item. Details ofthe positive control are given in Appendix 2. The positive control was conductedbetween 6 June 2012 and 8 June 2012. 

### 3.3 TestWater Reconstituted water used for boththe range-finding and definitive tests is defined inAppendix 3.Harlan StudyNumber: 41206434 Report Page II



### 3.4 Procedure 3.4.1 Range-finding Test The test concentrations to be used in the definitive test were determined by a preliminary range-finding test. In the range-finding test Daphnia magna were exposed to a series ofnominal test concentrations of0.10, 1.0, 10 and 100 mg/L. The test item was dissolved directly in reconstituted water. An amount oftestitem (100 mg) was dissolved in reconstituted water and the volume adjusted to 1 liter to give the 100 mg/L test concentration. Serial dilutions in reconstituted water were then performed from the 100 mg/L test concentration to give the 0.10, 1.0 and 10 mg/L test concentrations. Eachtest concentration was inverted several times to ensure adequate mixing and homogeneity. In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at 20 °C to 21 °C with a photoperiod of 16 hours light and 8hours darkness for a period of48 hours with 20 minute dawn and dusk transitionperiods. Each 250 mL test and control vessel contained 200 mL oftest media and was covered to reduce evaporation. After 24 and 48 hours the number ofimmobilized Daphnia magna were recorded. The control group was maintained under identical conditions but not exposed to the test item. A sample ofeach test concentration was taken for chemical analysis at 0 and 48 hours in orderto determine the stability of the test item under test conditions. All samples were stored at approximately -20 °C prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.


### 3.4.2 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 mg/L. 3.4.2.1 Experimental Preparation Forthe purpose ofthe definitive testthe test item was dissolved directly inreconstituted water. An amount oftestitem (500 mg) was dissolved in reconstituted water and the volume adjusted to 5 liters to give the 100 mg/L test concentration. Aliquots (20, 64, 200 and 640 mL) of the 100 mg/L test concentration were then each separately added to a final volume of 2 liters ofHarlan StudyNumber: 41206434 Report Page 12



### reconstituted water to give the further test concentrations of 1.0, 3.2, 10 and 32 mg/L respectively. Each prepared concentration was 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 and 48 hours (see Appendix 4).
3.4.2.2 Exposure Conditions As in the range-finding test 250 mL glass jars containing approximately 200 mL of test preparation were used. At the start ofthe test 5 daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at 20 °C to 21 °C with a photoperiod of 16 hours light and 8hours darkness with 20 minute dawn and dusk transition periods with a light intensity ranging from 651 to 669 lux. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated. The control group was maintained under identical conditions but not exposedto the test item. The test preparations were not renewed during the exposure period. Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start ofexposure. The criterion ofeffect used was that Daphnia 

### were considered to be immobilized ifthey were unable to swim for approximately 15 seconds after gentle agitation.
3.4.2.3 Physico-Chemical Measurements Water temperature and light intensity were recorded daily throughoutthe test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test. The pH and dissolved oxygen concentration were measured using a Hach HQ30d Flexi handheld meter whilstthe temperature was measured using a Hanna Instruments HI 93510 digital thermometer.
3.4.2.4 Verification ofTest Concentrations Water samples were taken from the control and each test group (replicates 

### Rt**-~pooled)** 

### at 0 and 48 hours for quantitative analysis. 0 hour samples were stored at approximately -20 °C priorto analysis withthe 48 hour samples. Duplicate samples were taken and stored at approximately -20 °C for further analysis if necessary.Harlan StudyNumber: 41206434 Report Page 13

Samples at the No ObservedEffect Concentration and above only were analyzed. The method ofanalysis, recovery andtestpreparation analyses are described in Appendix 4.
3.4.2.5 Evaluation ofData The EC5

### o value and associated confidence limits at 24 hours were calculated by the maximum-likelihood probit method (Finney, 1971) and at 48 hours by the trimmed Spearman-Karber method (Hamilton 

### et al, 1977) using the ToxCalc computer software package (ToxCalc, 1999) Probit analysis is used where two ormore partial responses to exposure are shown. When only one partial response is shownthe trimmed Spearman-Karber method is appropriate.Harlan StudyNumber: 41206434 Report Page 14


## 4 RESULTS 4.1 Range-finding Test Cumulative immobilization data from the exposure ofDaphnia magnato the test item during the range-finding test are given in Table 1. No immobilization was observed at the test concentrations of 0.10 and 1.0 mg/L. However, immobilization was observed at 10 and 100 mg/L. Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L were selected for the definitive test. Chemical analysis ofthe 1.0, 10 and 100 mg/L test preparations at 0 hours (see Appendix 4) showed measured concentrations of 1.2, 12 and 128 mg/L respectively, and at 48 hours showed measured concentrations of 1.2, 12 and 118 mg/L respectively, indicating that the test item was stable undertest conditions. 

### 4.2 Definitive Test 4.2.1 Immobilization Data Cumulative immobilization data from the exposure ofDaphnia magna to the test item during the definitive test are given in Table 2. The relationship between percentage immobilization and concentration at 24 and 48 hours is given in Figure 1and Figure 2. Analysis ofthe immobilization data by the the probit method (Finney, 1971) at 24 hours and by the trimmed Spearman-Karber method (Hamilton et a!, 1977) at 48 hours based on the nominal test concentrations gave the following results: Time (h) ECso (mg/L) 95% Confidence limits (mg/L) 24 43 29-74 48 8.5 

### 6.6-11 The No Observed Effect Concentrations after 24 and 48 hours exposure were 10 and 3.2 mg/L respectively. The Lowest Observed Effect Concentration (LOEC) after 24 an4 48 hours exposure were considered to be 32 and 10 mg/L respectively. · Due to the unsuitable nature ofthe data it was not possible to calculate the slope and error of response curve at 24 or48 hours.Harlan StudyNumber: 41206434 Report Page 15



### 4.2.2 Observations on Test Item Solubility 

### The testpreparations were observed to be clear colorless solutions for the duration ofthe test. 4.2.3 Physico-ChemicalMeasurements 

### The results ofthe physico-chemical measurements are given in Appendix 5. Temperature was maintained at 20 °C to 21 °C throughout the test, while there were no treatment related differences for oxygen concentration orpH.
4.2.4 Verification ofTest Concentrations 

### Analysis of the test preparations at 0 and 48 hours (see Appendix 4) showed measured test concentrations to be near nominal and so it was considered justifiable to calculate the EC50 

### values in terms ofthe nominal test concentrations only.Harlan StudyNumber: 41206434 Report Page 16


## 5 CONCLUSION The acute toxicity of the test item to the freshwater invertebrate Daphnia magna 

### has been investigated and gave a 48-Hour EC5o 

### value of 8.5 mg/L with 95% confidence limits of 6.6

- 11 mg/L. The Lowest Observed Effect Concentration was considered to be 10 mg/L. The No Observed Effect Concentration at48 hours was 3.2 mg/L.Harlan StudyNumber: 41206434 Report Page 17 

# 6 REFERENCES FINNEY, D J (1971) Statistical Method in Biological Assay. London : Griffin and Company Ltd. HAMILTON, M A, RUSSO, R C AND THURSTON, R V (1977) Trimmed Spearman-Karber Method for Estimating Median Lethal Concentration in Toxicity Bioassays. Environ Sci Technol 11' 714-719. ToxCalc Version 5.0.23C (1999), Tidepool Scientific Software, McKinleyville, CA 95519, USA.Harlan StudyNumber: 41206434 Report Page 18 

# TABLES Table 1 CumulativeImmobilization Datain theRange-findingTest Nominal CumulativeImmobilized

### Daphnia Concentration (InitialPopulation: 10 PerReplicate) {mg/L) 24 Hours 48 Hours Control 0 0

0.10 0 0

1.0 0 0 10 4 4 100 8 10Harlan StudyNumber: 41206434 Report Page 19

Table 2 CumulativeImmobilization Datain theDefinitive Test CumulativeImmobilized Daphnia Nominal (InitialPopulation: 5 Per Replicate) Concentration (mg/L) 24 Hours 48 Hours Rt R2 RJ**~** Total % Rt R2 R3 R4 Total % Control 0 0 0 0 0 0 0 0 0 0 0 0 1.0 0 0 0 0 0 0 0 0 0 0 0 0 3.2 0 0 0 0 0 0 0 0 0 0 0 0 10 0 0 0 2 2 10 1 5 3 4 13 65 32 4 2 1 3 10 50 5 5 5 5 20 100 100 4 2 5 3 14 70 5 5 5 5 20 100

R1 -**~** = Replicates 1to 4Harlan StudyNumber: 41206434 Report Page 20


## FIGURES Figure 1 Concentration-Response Curve after 24 Hours

99.9

99

90**-~** l; §< Q 70
"0 <I) 50**~** :E 0 8 8 

# - 30**~** 10

0.1
0.01 6-----------o-----'---'-....;_J--------------' 1 10 100 Concentration (mg/L)Harlan StudyNumber: 41206434 Report Page 21
Figure 2 Concentration-Response Curve after 48 Hours

99.9

99

90**~** ·;;:: .J:: 70 §< Q "0 <U 50**~** :.0 0 

### s s 

# - 30 '$. 10

0.1**0.01~----------~~----------~----------------~--~--~** 1 10 100 Concentration (mg/L)Harlan StudyNumber: 41206434 Report


## APPENDICES Appendix 1 Reconstituted Water- Elendt M7 Medium

Solution (I) H3B03 (II) MnChAH20 

### (III)**~iCl** (IV) RbCl (V) SrCh.6H20 (VI) NaBr (VII) Na2Mo04.2H20 (VIII) CuCh.2H20

(IX) ZnCh (X) CoCh.6H20 

### (XI) Kl (XII) Na2Se03 

### (XIII) NH4V03 (XIV) Na2EDTA.2H20 FeS04.7H20 Concentration of stock solution (mg/L) 57190 7210 6120 1420 3040 320 1260 335 260 200 65 43.8 11.5 5000 1991 Page 22

An aliquot (dependant onthe volume ofmedium required) ofeach stock solution was added to a final volume of deionized reverse osmosis water to give stock solution A and stored at approximately 21 °C.Harlan StudyNumber: 41206434 Reconstituted Water- ElendtM7 Macro Nutrient StockSolutions

Solution

(XV) CaCb.2H20 (XVI) NaHC03 (XVII) MgS04.1H20 (XVIII) Na2Si03.9H20

(XIX) KCl (XX) NaN03 (XXI) K2HP04 (XXII) KH2P04
Vitamin Nutrients

Solution (XXIII) Thiamine hydrochloride Cyanocobalamine (vitamin B12) D(+) biotin (vitamin H) Report

Concentration of stock solution (giL) 293.80

64.80 246.60

50.00 58.00

2.74 1.84 1.43

Concentration of stock solution (mg/L) 750

10

7.5 Page 23

The final medium was prepared by adding an aliquot ofstock solution A along with aliquots of each individual Macro Nutrient Stock Solution and an aliquot of the vitamin nutrient to the required amount (final volume) ofdeionized reverse osmosis water. The pH oftheprepared mediawas 8.0 ± 0.2 and stored at approximately 21 °C.Harlan StudyNumber: 41206434 Report Page 24
Appendix 2 Positive Control A positive control (Harlan Study Number: 41203341) used potassium dichromate as the reference item at concentrations of0.32, 0.56, 1.0, 1.8 and 3.2 mg/L. Exposure conditions for the positive control were similarto those inthe definitive test. Analysis ofthe immobilization data by the geometric mean method at 24 hours and the trimmed Spearman-Karber method (Hamilton 

### et a! 1977*) at 48 hours based on the nominal test concentrations gave the following results: Time (h) 

### ECso (mg/L) 95% Confidence limits (mg!L) 24 0.75 0.56 

# - 1.0 48 0.45 0.42


## - 0.48 The No Observed Effect Concentrations after 24 and 48 hours were 0.56 and 0.32 mg/L respectively. The No Observed Effect Concentration is based upon zero immobilization at this concentration. The results from the positive control with potassium dichromate were within the normal range for this reference item.

* HAMILTON, M A, RUSSO, R C AND THURSTON, R V (1977) Trimmed Spearman-Karber Method for Estimating Median Lethal Concentration in Toxicity Bioassays. Environ Sci Technol11, 714-719.Harlan StudyNumber: 41206434
Appendix 3 Reconstituted Water i) Stock Solutions a) b) c) d) CaCh.2H20 MgS04.7H20 NaHC03 KCl ii) Preparation Report

11.76 g/L 4.93 g/L 2.59 giL 0.23 g/L Page 25

An aliquot (25 mL) of each of solutions a-d was added to each liter (final volume) of deionized water with a conductivity of<5 flS cm- 1• The reconstituted water had a pH of 7.8 ± 0.2 adjusted (ifnecessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value. The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaC03.Harlan StudyNumber: 41206434 Report Page 26


### Appendix 4 Analytical Investigations 

## CONTENTS CONTENTS
..................................................................................................................................26 1 MATERIALS AND METHODS
.........................................................................................27 1.1 Introduction
...........................................................................................................27 1.2 Test Item
................................................................................................................27 1.3 Analytical Standard
...............................................................................................27 1.4 Analytical Procedure
.............................................................................................27 1.4.1 Storage
.....................................................................................................27 1.4.2 Reagents and Solvents
.............................................................................27 1.4.3 Preparation ofCalibration Standards
.......................................................28 1.4.4 Preparation ofLinearity Standards
..........................................................28 1.4.5 Preparation ofSpiked Recovery Samples
...............................................28 1.4.6 Preparation ofTestSamples
....................................................................28 1.4.7 Instrumental Parameters
..........................................................................28 1.4.8 Calculations
.............................................................................................30 2 RESULTS
............................................................................................................................32 2.1 Range-Finding Test
...............................................................................................32 2.2 Definitive Test
.......................................................................................................32 2.2.1 Validation ofthe Analytical Method
.......................................................32 2.2.2 Test Samples
............................................................................................32 3 DISCUSSION
......................................................................................................................33 TABLES
........................................................................................................................................34 Table 1 Preparation ofTest Samples
.................................................................................34 Table 2 Calibration Standard Data
....................................................................................34 Table 3 Results for Range-Finding Samples
.....................................................................35 Table 4 Results for Spiked Recovery Samples
..................................................................35 Table 5 Results for Test Samples
......................................................................................36 FIGURES
......................................................................................................................................37 Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Calibration Plot
......................................................................................................37 Chromatogram ofCalibration Solution (1.0 mg/L)
............................................... 38 Chromatogram ofBiological Control Sample
.......................................................38 Chromatogram ofTest Sample (3.2 mg/L)
...........................................................39 Chromatogram ofTest Sample (100 mg/L)
..........................................................39Harlan StudyNumber: 41206434 Report Page 27 Analytical Investigations 

## 1 MATERIALSAND METHODS 1.1 Introduction The test item concentration in the test samples was determined by direct injection to an electrospray mass spectrometer (MS) using an external standard. The method was developed by the Department ofAnalytical Services, Harlan Laboratories Ltd, Shardlow, UK. 

### 1.2 TestItem The test item is described in the biological part ofthis 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 0 hour samples were stored at approximately -20 °C prior to analysis, whilst the 48 hour samples were analyzed onthe day ofreceipt.
1.4.2 Reagents and Solvents 

### Water: water, Romil, UpS ultra-gradient grade. Test medium: as described inthe biological part ofthis study. Solvents: methanol, Fisher Scientific, HPLC grade. Reagents: formic acid, Fisher Scientific, analytical reagent grade.Harlan StudyNumber: 41206434 Report Page 28 Analytical Investigations 1.4.3 Preparation ofCalibration Standards 

### The test item (nominal100 mg) was dissolved in (50:50 v/v) methanol:test medium (100 mL) to prepare a stock solution with a concentration of 1000 mg/L. This stock solution was further diluted with (50:50 v/v) methanol:test medium to produce a solution of 10 mg/L. Defined volumes of this solution were then diluted with (50:50 v/v) methanol:test medium to obtain calibration standards in the range of0.10 to 1.0 mg/L. A second series ofcalibration standards was similarly prepared in the range of0.30 to 1.5 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 (50:50 v/v) methanol:test medium (100 mL) to prepare a stock solution with a concentration of 1000 mg/L. This stock solution was further diluted with (50:50 v/v) methanol:test medium to produce a solution of 10 mg/L. Defined volumes of this solution were then diluted with (50:50 v/v) methanol:test medium to obtain calibration standards in the range of0.10 to 1.0 mg/L. A second series ofcalibration standards was similarly prepared in the range of0.30 to 1.5 mg/L. These standards were used to evaluate the linearity ofthe 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 its recovery was assessed. The test item (nominal 100 mg) was initially dissolved in (50:50 v/v) methanol:test medium to prepare a stock solution with a concentration of 1000 mg/L. This stock solution was further diluted with (50:50 v/v) methanol:test medium to produce a solution of 100 mg/L. A defined volume ofthis stock solution was diluted with test medium to obtain spiked recovery samples at a concentration of 1.0 mg/L. Five replicates were prepared and subjected to the same treatment as the test samples. In addition, test medium withoutthe addition ofthe test item (synthetic control) was also analyzed.
1.4.6 Preparation ofTest Samples 

### The 0 hour test samples were thawed with the aid ofsonication, whilst the 48 hour samples were analyzed on the day of receipt. They were initially diluted with methanol and then further diluted into the calibrationrange with (50:50 v/v) methanol:test medium as shown in Table 1.Harlan StudyNumber: 41206434 Report Analytical Investigations 

### 1.4.7 Instrumental Parameters Autosampler: Pump: Detector: Column: Mobile phase: Flowrate: Injectionvolume: Ionizationmode: Polarity: Capillary voltage: Gas temperature: Nebulizerpressure: Fragmentorvoltage: Drying gas flow: Data acquisitionmode: SIM ion: Agilent 1200 Series Agilent 1200 Series Agilent 1200 Series MSD
no column water:methanol:formic acid (500:500:0.5 v/v/v) 0.5 mL/minute 

### 10**~L** ESI positive 1000V 350

## oc 40psi 100V 10 L/min Single IonMonitoring (SIM) Page 29Harlan StudyNumber: 41206434 Report Page 30 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: concentrationofthetest itemininjected sample [mg/L] averagepeakareaofthe test item ininjected sample [counts] constants

The concentration of the test item in the test samples was calculated using the following equation:



### c 

## = X 

### ·F R/100 where c: x: F: R: (2) concentrationofthe test iteminthe test sample [mg/L] concentrationofthetestiteminthe injected sample solution [mg!L] samplepreparationfactor recoveryrate[%] (Ifnot correcting for recoverythenR is takenas 100)

The recovery rate (R) of the spiked recovery samples was calculated using the following equation: R

### =~·100 cfort where R: c: (3)
recoveryrate [%] determined concentration ofthe testiteminthe spikedrecovery sample [mg/L] fortified concentration ofthe test item inthe spikedrecovery sample [mg!L]Harlan StudyNumber: 41206434 Report Page 31 Analytical Investigations

The percentage ofnominal concentration ofthe testitem inthe test samples was calculatedusing the following equation: %nominal=_c_·100 cnom (4) where Cnom: nominal concentration in the test sample [mg/L]Harlan StudyNumber: 41206434 Report Page 32 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 specifically over concentration range to be used in the definitive test. The results for the range-finding test samples are summarized in Table 3. 

### 2.2 Definitive Test 2.2.1 Validation ofthe Analytical Method 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 of 0to 1.84 mg/L. The R2 fit ofthe calibration curve to 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 test item in the 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 not corrected for recovery. 

### Limit ofQuantitation (LOQ) The limit ofquantitation (LOQ) was determined by calculating the concentration equivalent to ten times the standard deviation of five synthetic controls. Using this method the limit of quantitationwas determined to be 0.053 mg/L.


### 2.2.2 Test Samples The results obtained for the concentration ofthe test item found in the test samples aie presented in Table 5. Typical chromatograms are shown in Figures 2to 5.Harlan StudyNumber: 41206434 Report Page 33 Analytical Investigations 

## 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 andproven to be suitable for use.Harlan Study Number: 41206434 Report Page 34 Analytical Investigations 

## TABLES Table 1 Preparation ofTest Samples
Nominal Sample Sample FinalNominal Concentration of Volume FinalVolume Preparation Concentration ofTest Test Item Factor Item

[mg/L] [mL] [mL] F [mg/L] Control 5 10 2 

## - 3.2 5 10, then 4 0.80 5--710 10 5 10, then 10 1.0 4--720 32 5 10, then 40 0.80 1--720 100 5 10, then 100 1.0 2--7100
not applicable


### Table 2 Calibration StandardData
Concentration ofTestItem Area [mg/L] [counts] 0 2.245 X 104 0.0950 3.166 X 104 0.368 5.370 X 104 0.475 6.281 X 104 0.858 9.225 X 104 0.950 9.711 X 104 1.838 1.607 X 105Harlan StudyNumber: 41206434 Report Page 35 Analytical Investigations Table 3 Results for Range-Finding Samples Nominal Measured Sample Determined Concentration of Concentration Preparation Concentration Test Item in ofTestItem in Factor ofTestItem in %ofNominal Time Point Range-Finding Sample Vial Range-Finding Concentration Sample Sample
X F c Cnom [hours] [mg!L] [mg/L] [mg/L] [%] 0 1.0 0.595 2 1.19 119 10 1.17 10 11.7 117 100 1.28 100 128 128 48 1.0 0.595 2 1.19 119 10 1.20 10 12.0 120 100 1.18 100 118 118

Table4 Results for Spiked Recovery Samples Nominal Fortified Measured Sample Determined Analytical Concentration Concentration Concentration Preparation Concentration Recovery Precision of ofTest Item in ofTest Item in Factor ofTest Item 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] [%] [%]

1.0 0.948 0.362 2 0.724 0.406 2 0.811 0.397 2 0.794 80 5.8



# . 0.354 2 0.707 0.378 2 0.756 Acceptance Target 80-120 <10 LOQ Limit ofQuantitation Forreporting purposes, x has been calculated from c retrospectively.Harlan StudyNumber: 41206434 Report Analytical Investigations Table 5 Results for Test Samples Nominal Measured Sample Concentration Concentration Preparation TimePoint

[hours] 0

48

LOQ of ofTestItem in TestItem in SampleVial Test Sample
Cnom X [mg/L] [mg/L] Control <LOQ

3.2 0.770 10 0.945 32 0.770 100 1.00 Control <LOQ

3.2 0.735 10 0.958 32 0.793 100 0.962 Limit ofQuantitation not applicable Forreporting purposes, x has been calculated from c retrospectively. Factor

F
2 4 10 40 100 2 4 10 40 100 Page 36

Determined Concentration ofTestItem in %ofNominal

Test Sample Concentration

c [mg/L] [%] <LOQ 

## -

3.08 96 9.45 95 30.8 96 100 100 <LOQ 

## -

2.94 92 9.58 96 31.7 99 96.2 96Harlan StudyNumber: 41206434 Analytical Investigations 

## FIGURES Figure 1 Calibration Plot 

### 2 180000 ·;:: 2 160000 co I!! <( 140000 -"" co (J) 120000 a.. 100000 80000 60000 40000 20000 0 0.00 Report

y

### = -5.106E+03x2 + 8.408E+04x + 2.326E+04 R

2

### = 9.997E-01

0.50 1.00 Page 37

1.50 2.00

Concentration (mg/L)Harlan StudyNumber: 41206434 Report Page 38 Analytical Investigations 

### Figure 2 Chromatogram ofCalibration Solution (1.0 mg!L)

Figure 3 Chromatogram ofBiological Control Sample*

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 min

• Background interference level ofthe response. Nottest item.Harlan StudyNumber: 41206434 Report Page 39 Analytical Investigations Figure 4 Chromatogram ofTest Sample (3.2 mg/L)

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 min Figure 5 Chromatogram ofTest Sample (100 mg/L)

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 minHarlan StudyNumber: 41206434 Report


### Appendix 5 Physico-ChemicalMeasurements
Nominal 0 Hours 24 Hours 48 Hours Concentration (mg/L) pH mg %ASV* T°C T"C pH mg 0 2/L 0 2/L RI 8.0 9.1 100 20 21 8.1 8.8 Control Rz 8.0 9.0 99 20 21 8.1 8.7 R3 8.0 9.0 99 20 21 8.1 8.7**~** 8.0 8.9 98 20 21 8.1 8.6 R1 8.0 8.9 98 20 21 8.1 8.7 1.0 Rz 8.0 8.9 98 20 21 8.1 8.6 R3 8.0 8.9 98 20 21 8.1 8.6**~** 8.0 8.9 98 20 21 8.1 8.6 R1 8.0 8.9 98 20 21 8.1 8.6 3.2 Rz 8.0 8.9 98 20 21 8.1 8.6 R3 8.0 8.9 98 20 21 8.1 8.6**~** 8.0 8.9 98 20 21 8.0 8.6 R1 8.0 8.9 98 20 21 8.0 8.7 10 Rz 8.0 8.9 98 20 21 8.0 8.7 R3 8.0 8.9 98 20 21 8.0 8.7**~** 8.0 8.9 98 20 21 8.0 8.7 RI 8.0 8.9 98 20 21 8.0 8.8 32 Rz 7.9 8.9 98 20 21 8.0 8.7 R3 7.9 8.9 98 20 21 8.0 8.7**~** 7.9 8.9 98 20 21 8.0 8.7 RI 7.9 8.9 98 20 21 8.0 8.7 100 Rz 7.8 8.9 98 20 21 8.0 8.7 R3 7.8 8.9 98 20 21 8.0 8.7**~** 7.8 8.9 98 20 21 8.0 8.7

* ASV = Dissolved oxygen concentration expressed as apercentage ofAir Saturation Value R1 - Rt = Replicates 1to 4 %ASV* 99 98 98 97 98 97 97 97 97 97 97 97 98 98 98 98 99 98 98 98 98 98 98 98 Page 40

T°C

21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21Harlan Study Number: 41206434 Report
Appendix 6 MonitoringAuthority Statement ofGLP 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 Ltd Shardlow Business Park London Road Shardlow Derby m:722GD

DATE OF INSPECTION

10 July2012 TEST TYPE(S) Analytical/Clinical Chemistry Environmental Toxicity Environmental Fate Mutagenicity Phys/Chem. Tests Toxicology

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 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).

Dr. Andrew J. Gray Head, UK GLP Monitoring Authority Page 41