## REPORT 

# ++++ £NVIGO D-erythro-Hex-2-enonic acid, .gamma.-lactone, monosodium salt [CAS RN 6381-77-7]: Daphnia sp., 48-HourAcute Immobilization Test

StudyDirector: TestFacility:

Sponsor:

StudyMonitor:

StudyNumber: Study CompletionDate: D Sacker Envigo ResearchLimited ShardlowBusiness Park Shardlow Derbyshire DE72 2GD 

### UK UnivarUSA Inc. 17425 NEUnion Hill Road Redmond WA 98052 UNITED STATES OF AMERICA Greenwich Chemical Consulting,Inc. 3300 PGA Boulevard, Suite 635 PalmBeach Gardens FL33410 UNITED STATES OF AMERICA 41401174 12 January 2016

On 21 September 2015 the legal entity Harlan Laboratories Ltd was renamed as Envigo Research Limited. No other changes to the legal entity have occurred. Page 1 of37StudyNumber: 41401174 Report Page 2


## CONTENTS CONTENTS ....................................................................................................................................2 STUDY DIRECTOR STATEMENT OF GLP COMPLIANCE ....................................................4 QUALITY ASSURANCE STATEMENT......................................................................................5 SUMMARY ....................................................................................................................................6 Introduction............................................................................................................................ 6 Methods...............................................................................................................................6 Results..................................................................................................................................6 GENERAL INFORMATION .........................................................................................................7 Schedule.................................................................................................................................7 Deviations from Study Plan...................................................................................................7 Archiving ...............................................................................................................................7 1 INTRODUCTION AND PURPOSE.....................................................................................7 1.1 Guidelines I Regulations.......................................................................................... 8 2 TEST ITEM ...........................................................................................................................9 3 MATERIALS AND METHODS......................................................................................... 10 3.1 Definitions and Abbreviations............................................................................... 10 3.2 Test System............................................................................................................ 10 3.3 Test Water.............................................................................................................. 10 3.4 Procedure ............................................................................................................... 11 3.4.1 Range-finding Test .................................................................................. 11 3.4.2 Definitive Test......................................................................................... 11 3.4.3 Evaluations .............................................................................................. 12 3.4.4 Statistical Analysis .................................................................................. 13 3.5 Validation Criteria................................................................................................. 13 4 RESULTS ............................................................................................................................ 14 4.1 Range-finding Test ................................................................................................ 14 4.2 Definitive Test ....................................................................................................... 14 4.2.1 Verification ofTest Concentrations ........................................................ 14 4.2.2 ImmobilizationData................................................................................ 14 4.2.3 Sub-Lethal Effects ................................................................................... 14 4.2.4 Validation Criteria................................................................................... 15 4.2.5 Water Quality Criteria............................................................................. 15 4.2.6 Observations on Test Item Solubility...................................................... 15 5 CONCLUSION.................................................................................................................... 16 6 REFERENCES .................................................................................................................... 17 TABLES........................................................................................................................................ 18 APPENDICES...............................................................................................................................21Study Number: 41401174 Report Page 3


## LIST OF TABLES Table 1 Cumulative Immobilization Data and Observations inthe inthe Range-finding Test...................................................................................................... 18 Table 2 Cumulative Immobilization Data and Observations inthe Definitive Test.............................................................................................................................. 19 Table 3 Water Quality Measurements in the Definitive Test ..................................................20 

## LIST OF APPENDICES Appendix 1 Appendix 2 Appendix 3 Appendix 4 Appendix 5 Appendix 6 Certificate ofAnalysis ...........................................................................................21 Reconstituted Water- Elendt M7 Medium...........................................................22 Positive Control .....................................................................................................23 Reconstituted Water

- ISO Medium .....................................................................24 Analytical Investigations .......................................................................................25 Monitoring Authority Statement ofGLP Compliance ..........................................37StudyNumber: 41401174 Report Page4


## STUDYDIRECTOR STATEMENT OF GLP COMPLIANCE Envigo Research Limited, Shardlow Business Park, Shardlow, Derbyshire, DE72 2GD, UK StudyNumber: 41401174 Study Title: D-erythro-Hex-2-enonic acid, .gamma.-lactone, monosodium salt [CAS RN 6381-77-7]: Daphnia sp., 48-Hour Acute Immobilization Test This study was performed in compliance with UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)). These Regulations are in accordance with GLP standards published as OECD Principles on Good Laboratory Practice (revised 1997, ENV/MC/CHEM(98)17); and are in accordance with, and implement, the requirements ofDirectives 2004/9/EC and 2004/10/EC. These principles are compatible with Good Laboratory Practice regulations specified by regulatory authorities throughout the European Community, the United States (EPA and FDA), and Japan (MHLW, MAFF and METI). This report fully and accurately reflects the procedures used and data generated. There were no circumstances consideredto have affected the integrity ofthe study orthe validity ofthe data. Study Director: D Sacker



### ......

# ~.. Date: 

### 1 2 JAN 2016StudyNumber: 41401174 Report Page 5 

## QUALITYASSURANCE STATEMENT StudyNumber: 41401174 Study Title: D-erythro-Hex-2-enonic acid, .gamma.-lactone, monosodium salt [CAS RN 6381-77-7]: Daphnia sp., 48-Hour Acute Immobilization Test
The general facilities and activities are inspected at least once a year and the results are reported to therelevantresponsible person and management. Study-relatedprocedures conducted at the test facility were audited and inspected. The details of these audits and inspections are givenbelow. Reported to the Dates and Types ofQAInspections relevant Study Director and Test Facility Management Date ofInspection Type ofInspection Phase Inspected Report Date 10 April2014 StudyPlan NIA 10 April2014 Verification 27 October 2015 Process

- based Test ItemPreparation 27 October 2015 27 October 2015 Process

- based Test System 27 October 2015 Preparation and Exposure 12 November 2015 Process

- based Assessment of 12 November 2015 Response 15 October 2015 Process

- Based Chemical Analysis 15 October 2015 06 January 2016 Report Audit N/A 06 January 2016 Verification of Study Plan Amendments for GLP compliance was performed during the course ofthe study. This statement confirms thatthis report reflects the raw data andthe procedures followed. QualityAssurance: 

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## 1 2 JAN 2016StudyNumber: 41401174 Report Page 6


## 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 ofCommissionRegulation (EC) No. 440/2008. 

### Methods Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution ofthe test item at a concentration of 100 mg/L for 48 hours at a temperature of21 to 22 °C under continuous flow-through test conditions. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours. 

### Results Analysis ofthe test preparations at 0, 24 and 48 hours showed measured test concentrations to range from 88% to 93% ofnominal concentration and so the results are based on nominal test concentrations. Exposure ofDaphniamagnato the test item gave EC50 values ofgreater than 100 mg/L. The No ObservedEffect Concentration was 1 00 mg/L. Itwas considered unnecessary and unrealistic to test at concentrations inexcess of 1 00 mg/L.StudyNumber: 41401174 Report


## GENERALINFORMATION Schedule Experimental Starting Date: Experimental CompletionDate: 

### Deviations from Study Plan 22 September 2014 29 October 2015

There were no deviations (unplanned changes) from the study plan. 

### Archiving Page 7

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 Envigo Research Limited, Shardlow, UK archives for five years after which instructions will be sought as to further retention or disposal. Further retention or return of the data will be chargeableto the Sponsor. No datawill be discarded without contacting the Sponsorto obtaintheir written consent.StudyNumber: 41401174 Report Page 8


## 1 INTRODUCTIONAND PURPOSE This report contains a description ofthe methods used and results obtained during a study to investigate the acute toxicity ofthe test item to 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 Immobilisation Test" • Method C.2 ofCommissionRegulation (EC) No. 440/2008StudyNumber: 41401174 Report Page 9


## 2 TESTITEM Information as provided by the Sponsor. A Certificate ofAnalysis supplied by the Sponsor is given inAppendix 1. Identification: Physical state/ Appearance: Batch: Purity: ExpiryDate: Storage Conditions: D-erythro-Hex-2-enonic acid,
.gamma.-lactone, monosodium salt [CAS RN 6381-77-7] White granular solid 14031314 99.2% 23 March 2016 Room temperature inthe darkStudyNumber: 41401174 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 System 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 oftest item which results in 50% immobilizationwithinthe exposure period. The lowesttest concentration at which a significanttoxic effect onthe test organisms is observed withinthe exposure period. The highest test concentration at which no significant toxic effect on the test organisms is observed within the exposure period. Comprises test item treatments and control (testwater only).

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 2) in a temperature controlled room at approximately 20 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transitionperiods. 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 affectthe integrity or outcome ofthe study. A positive control (Study Number 41502271) used potassium dichromate as the reference item. Details ofthe positive control are given in Appendix 3. The positive control was conducted between 07 September 2015 and 23 September 2015. 

### 3.3 Test Water Reconstituted water (ISO medium) used for boththe range-finding and definitive tests is defined in Appendix 4.StudyNumber: 41401174 Report Page 11



### 3.4 Procedure Due to the potentially light sensitive nature of the test item, all test item preparation was performedunder non-actinic lighting I shielded from the light.
3.4.1 Range-finding Test The test concentration to be used in the definitive test was determined by a preliminary rangefinding test. In the range-finding test Daphnia magna were exposed to a series ofnominal test concentrations of 10 and 100 mg/L under continuous flow-through conditions. Nominal amounts oftest item (1.2 and 12 g) were dissolved in deionized reverse osmosis water and the volume adjusted to 1 liter to give the 1.2 and 12 g/L stock concentrations. The stock solutions were sonicated for 5 minutes and inverted several times to ensure adequate mixing and homogeneity. Dilution water was dosed at a flow rate of 30 mL per minute using Watson Marlow 505S peristaltic pump and the stock solutions at a flow rate of0.25 mL per minute using a Watson Marlow 205U peristaltic pump. The dilution water and stock solutions were combined in separate mixing vessels and delivered to the test vessels to give the required test concentrations of 1 0 and 1 00 mg/L. In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at approximately 20 °C in darkness. Glass test vessels (150 mL beakers) with stainless steel mesh sides were placed in a tank retaining approximately 5 liters of test solution. The control group was maintainedunder identical conditions but not exposed to the test item. A sample ofeachtest concentration was taken for chemical analysis at 0 and 48 hours in order to determine the stability ofthe test item under test conditions. In order to ensure stability ofthe test item, a volume ofglacial acetic acid (5 mL) was added to each sample bottle before addition ofthe test sample. 3.4.2 Definitive Test Based on the results ofthe range-finding test a "Limit test" was conducted at a concentration of 100 mg/L to confirm that at the maximum concentration given in the OECD/EC Test Guidelines no immobilization or adverse reactions to exposure were observed.Study Number: 41401174 Report Page 12

3.4.2.1 Experimental Preparation A nominal amount oftest item (12 g) was dissolved in deionized reverse osmosis water and the volume adjusted to 1liter to give the 12 g/L stock solution. The stock solution was sonicated for 5 minutes and inverted several times to ensure adequate mixing and homogeneity. Dilution water was dosed at a flow rate of 30 mL per minute using Watson Marlow 505S peristaltic pump and the stock solution at a flow rate of 0.25 mL per minute using a Watson Marlow 205U peristaltic pump. The dilution water and stock solution were combined in a mixing vessel and delivered to the test vessels to give the required test concentration of 100 mg/L.
3.4.2.2 Exposure Conditions Continuous flow-through test conditions were employed in the test in an effort to maintain dissolved test item concentrations. As in the range-finding test glass test vessels (150 mL beakers) with stainless steel mesh sides were placed in a tank retaining approximately 5 liters of test solution. 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 approximately 20

## oc in the dark. 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 exposed to the test item. Dynamic test conditions were employed in the test in an effort to maintain dissolved test item concentrations. The stock solution was freshly prepared daily and the flow rates adjusted as necessary. 3.4.3 Evaluations 3.4.3.1 Test Organism Observations Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that daphnia were considered to be immobilized ifthey were unable to swim within 15 seconds after gentle agitation. 3.4.3.2 Water Quality Criteria Water temperature was recorded daily throughout the 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 Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.Study Number: 41401174 Report Page 13

3.4.3.3 Verification ofTest Concentrations Samples were taken from the control and the 1 00 mg/L test group for analysis every 24 hours throughout the test. Duplicate samples were taken at each occasion and stored frozen for further analysis ifnecessary. In order to ensure stability ofthe test item, a volume ofglacial acetic acid (5 mL) was added to each sample bottle before addition ofthetest sample. The method ofanalysis, recovery and testpreparation analyses are described inAppendix 5.
3.4.4 StatisticalAnalysis An estimate ofthe EC5ovalues was given by inspection ofthe immobilization data. The No Observed Effect Concentration (NOEC) at 24 and 48 hours was calculated using the Fisher's Exact Binomial Test with Bonferroni correction using the ToxRat Professional computer software package (TOXRAT). 

### 3.5 Validation Criteria The results ofthe test are considered valid ifthe following performance criteria are met:
• No more than 10% ofthe control daphnids show immobilization or other signs ofdisease or stress (e.g. discoloration orunusual behavior such as trapping atthe surface water).
• The dissolved oxygen concentration at the end of the test is equal to or greater than 3 mg/L inthe control and test vessels.StudyNumber: 41401174 Report Page 14


## 4 RESULTS 4.1 Range-finding Test Cumulative immobilization data and other observations from the exposure ofDaphnia magna to the test item during the range-finding test are given in Table 1. No immobilizationwas observed at the test concentrations of 10 and 100 mg/L. Based on this information, a single test concentration of four replicates, of 1 00 mg/L was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that at the maximum test concentration given in the OECD/EC Test Guidelines no significant immobilization or adverse reactions to exposure were observed. Chemical analysis ofthe test preparations at 0 hours (see Appendix 5) showed measured test concentrations to range from 79% to 92% ofnominal concentrations. At 48 hours the measured test concentrations ranged from 89% to 98% ofnominal, indicating that the test item was stable under test conditions. 

### 4.2 Definitive Test 4.2.1 Verification ofTest Concentrations Analysis ofthe test preparations at 0, 24 and 48 hours (see Appendix 5) showed measured test concentrations to range from 88% to 93% ofnominal concentrations and so the results are based onnominal test concentrations.
4.2.2 Immobilization Data Cumulative immobilization data and other observations from the exposure ofDaphnia magna to the testitem during the definitive test are given in Table 2. The No Observed Effect Concentration after48 hours exposure was 100 mg/L. Itwas consideredunnecessary and unrealistic to test at concentrations in excess of 1 00 mg/L.
4.2.3 Sub-Lethal Effects Sub-lethal effects of exposure were observed in the 100 mg/L test concentration. These responses were covered with debris and reduced mobility (see Table 2).Study Number: 41401174 Report Page 15

A single daphnia in control replicate 2 was observed to be trapped at the surface at the observation at 24 hours, however, with the exception ofthe two immobilized daphnia in control replicate 1, all daphnia in the control were observed to be normal at 48 hours. This observation was considered notto have adversely affectedthe results ofthe test. 4.2.4 Validation Criteria The test was considered to be valid given that no more than 10% ofthe control daphnids showed immobilization or other signs ofdisease or stress and that the oxygen concentration at the end of the test was equal to or greaterthan 3 mg/L inthe control and testvessels.
4.2.5 Water Quality Criteria The results of the water quality measurements are given in Table 3. Temperature was maintained at 21 to 22 

## oc throughout the test, while there were no treatment related differences for oxygen concentration orpH.
4.2.6 Observations on Test Item Solubility At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.StudyNumber: 41401174 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 EC50 value ofgreater than 100 mg/L. The No Observed Effect Concentration was 1 00 mg/L.Study Number: 41401174 Report Page 17 

# 6 REFERENCES TOXRAT PROFESSIONAL VERSION 2.10.06. TOXRAT® SOLUTIONS GMBH 2001-2010StudyNumber: 41401174 Report Page 18


## TABLES Table 1 CumulativeImmobilization Data and Observations in thein theRange-finding Test Observations (Initial Population: 10 PerReplicate) Nominal 24 Hours 48 Hours Concentration (mg/L) Cumulative Cumulative Immobilized Observations Immobilized Observations

Daphnia Daphnia Control 0 9N* 0 ION IO 0 ION 0 ION IOO 0 ION 0 ION

N =No sub-lethal effects observed 

### *Only 9 daphnia were observed, consideredno adverse effect as all daphnia were accounted for at 48 hoursStudyNumber: 41401174 Report

Table2 CumulativeImmobilizationData and Observations in theDefinitive Test
Nominal CumulativeImmobilized Daphnia Concentration (Initial Poll_ulation: 5 PerReplicate) (mg/L) Rt R2 R3 Itt Total % Control 0 0 0 0 0 0

100 0 1 0 0 1 5

Nominal CumulativeImmobilized Daphnia Concentration (mg/L) (Initial Population: 5 PerReplicate) Rt R2 R3 Itt Total % Control 2 0 0 0 2 10

100 1 2 0 1 4 20

R1 -

**~** =Replicates 1to 4 N =No sub-lethal effects observed R = Reduced mobility C = Covered with debris T = Trapped at surface 24 Hours Observations Rt R2 R3 5N 4N, IT 5N 4N, lR 3N, lR 5N
48 Hours Observations Rt R2 R3 3N 5N 5N 3N, lR 2N, lR 4N, lC Page 19

R4 5N 5N

R4 5N 4NStudyNumber: 41401174 Report Page 20

Table3 Water QualityMeasurements in the Definitive Test
Nominal 0 Hours 24 Hours 48 Hours Concentration (mg/L) pH mgOiL T°C T"C pH mgOiL T°C Control Rl 8.1 8.9 21 21 8.1 8.8 21

100 Rl 8.0 8.4 21 21 8.0 8.5 22

R1 =Replicate 1StudyNumber: 41401174 Report


## APPENDICES Appendix 1 Certificate ofAnalysis

Sodium Erythorbate

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Appendix 2 Reconstituted Water - Elendt M7 Medium

Ingredient H3B03 MnCbAH20 LiCl RbCl SrCb.6H20 NaBr Na2Mo04.2H20 CuCb.2H20 

### ZnCh CoCb.6H20 KI Na2Se03 NH4V03 Na2EDTA.2H20 FeS04.7H20 CaCb.2H20 NaHC03 MgS04.7H20 Na2Si03.9H20 KCI NaN03 K2HP04 KH2P04 Thiamine hydrochloride Cyanocobalamine (vitamin B 12) D(+) biotin (vitamin H) Final Concentration (mg/L) 0.715 0.090 0.077 0.018 0.038 0.004 0.016 0.004 0.013 0.010 0.0033 0.0022 0.00058 0.625 0.249 293.8 64.8 123.3 10 5.8 0.274 0.184 0.143 0.075 0.0010 0.00075
The pHofthe prepared mediawas 7.9 ± 0.3 and stored atapproximately 21 °C. Page 22StudyNumber: 41401174 Report Page 23
Appendix3 Positive Control A positive control (StudyNumber 41502271) 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 in the definitive test. Analysis ofthe immobilization data by the probit analysis using the linear maximum likelihood regression method at 24 and 48 hours using the ToxRat Professional computer software package based onthe nominal test concentrations gave the following results:

No Observed Lowest Observed TimePoint ECso 95% ConfidenceLimits Effect Effect

(Hours) (mg/L) (mg/L) Concentration Concentration (NOEC) (mg/L) (LOEC) (mg/L) 24 0.90 Notpossible to determine 0.56 1.0 48 0.54 0.47 

### - 0.63 0.32 0.56 The No Observed Effect Concentration is based upon equal to or less than 10% immobilization at this concentration. The results from the positive control with potassium dichromate were within the normal range for this reference item.StudyNumber: 41401174 Report
Appendix 4 Reconstituted Water- ISO Medium

Ingredient CaCb.2H20 MgS04.1H20 NaHC03 KCl Final Concentration (mg/L) 294 123 65 5.8 Page 24

The reconstituted waterhad a pH of7.8 ± 0.2 adjusted (ifnecessary) withNaOH or HCl and was aerateduntil the dissolved oxygen concentrationwas approximately air-saturation value. Thereconstituted waterhad anapproximate theoreticaltotal hardness of250mg/L as CaC03.StudyNumber: 41401174 Report Page 25
Appendix 5 Analytical Investigations 

## 1 RESULTS 1.1 Range-Finding Test The test item concentration in the test samples was determined by high performance liquid chromatography with UV detection (HPLC/UV) using an external standard. The test item gave a chromatographic profile consisting ofa single peak. The method was developed and implemented by the Department ofAnalytical Services, Envigo Research Limited, Shardlow, UK. 

### 1.2 Test Item 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 samples were analyzed onthe day ofreceipt.
1.4.2 Reagents and Solvents Water: preparedusing an ELGA Pure Lab Option R-15 waterpurification system. Testmedium: as described inthe biological part ofthis study. Solvents: Reagents: acetonitrile, Fisher Scientific, HPLC grade. tetrabutylammonium, Sigma-Aldrich, 2: 99.0% acetic acid, Fisher Scientific, analytical reagentStudyNumber: 41401174 Report Page 26 Analytical Investigations 1.4.3 Preparation ofCalibration Standards The test item (nominal 1000mg) was dissolved in 5% (v/v) acetic acid in water (100 mL) to prepare a stock solution with a concentration of 1000 mg/L. A defined volume ofthis solution was then diluted with 5% (v/v) acetic acid in water to obtain calibration standards in the range of5to 100 mg/L. A second series ofcalibration standards was similarly prepared inthe range of 10 to 125 mg/L. These solutions were used to determine the recovery and test sample concentrations.
1.4.4 Preparation ofLinearity Standards The test item (nominal 1000 mg) was dissolved in 5% (v/v) acetic acid in water (100 mL) to prepare a stock solution with a concentration of 1000 mg/L. A defined volume ofthis solution was then diluted with 5% (v/v) acetic acid in water to obtain calibration standards in the range of5to 100 mg/L. A second series ofcalibration standards was similarly prepared inthe range of 10 to 125 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 the recovery was assessed. The test item (nominal 100 mg) was initially dissolved in 5% (v/v) acetic acid in test medium to prepare a stock solution with a concentration of 1000 mg/L. A defined volume ofthis stock solution was diluted with 5% (v/v) acetic acid in test medium to obtain spiked recovery samples at a concentration of 100 mg/L. Five replicates were prepared and subjected to the same treatment as the test samples. In addition, test medium without the addition ofthe testitem (synthetic control) was also analyzed.
1.4.6 Preparation ofTest Samples The test samples were analyzed on the day ofreceipt. An aliquot of acetic acid (5 mL) was added to the test samples (95 mL). See Table 1for more information onpreparationvolumes.Study Number: 41401174 Report Page 27 Analytical Investigations 1.4.7 Instrumental Parameters HPLC System Agilent 1200 Series Column: Gemini, C18, 3 1-1m (100 x 4.6 mmid.) Mobile phase Mobile Phase A: water+ TBAB solution* (90:10 v/v) Mobile Phase B: acetonitrile+ TBAB solution* (90:10 v/v) Gradient Time %A %B (mins) 0 100 0 5 0 100 5.1 100 0 10 100 0 Injectionvolume Flowrate 1mL/minute Columntemperature 20

## oc Autosamplertemperature Detectionwavelength 280nm Retentiontime approximately 3 minutes

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)
where x: y: 2·a concentration ofthe test item inthe injected sample [mg/L] average peak area ofthetest item inthe injected sample [counts]

* TBAB solution=4 g oftetrabutylammonium bromide in 200 mL acetonitrileStudyNumber: 41401174 Report Page 28 Analytical Investigations
The concentration of the test item in the test samples was calculated usmg the following equation:

c X .p R/100 where c: 

### x: F: R: (2) concentration ofthe test item inthe test sample [mg/L] concentrationofthe testiteminthe injected sample solution [mg/L] samplepreparation factor recoveryrate [%] (Ifnotcorrecting for recovery then R istaken as 100)
The recovery rate (R) of the spiked recovery samples was calculated usmg the following equation:
R =-c-·100 cfort where R: c: Cfort: (3)
recoveryrate [%] determined concentrationofthetestitem inthe spikedrecovery sample [ mg!L] fortified concentration ofthe testitem inthe spikedrecovery sample [ mg/L]
The percentage ofnominal concentration ofthe test item inthe test samples was calculated using the following equation: %nominal=_c_ ·100
cnom (4)
where Cnom: nominal concentrationinthe test sample [mg/L]StudyNumber: 41401174 Report Page 29 Analytical Investigations 

## 2 RESULTS 2.1 Range-Finding Test The analytical methodology was not validated prior to 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 and an analyzed analytical blank showed no significant interfering response at the retention time ofthe 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 retentiontime 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 calibrationrange of0to 132 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 ofQuantification The limit ofquantification (LOQ) was determined by calculating the sample concentration that gave a peak equivalent to ten times the baseline noise. Using this method the LOQ was determined to be 2.4 mg/L.
2.2.2 Test Samples The results obtained for the concentration ofthe test item found in the test samples are presented in Table 5. Typical chromatograms are shown inFigures 2to 4.StudyNumber: 41401174 Report Page 30 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 and proven to be suitable for use.StudyNumber: 41401174 Report Page 31 Analytical Investigations 

## TABLES Table 1 Preparation ofTest Samples Nominal Sample FinalNominal Concentration ofTest SampleVolume FinalVolume Preparation Concentration ofTest Item Factor Item [mg/L] [mL] [mL] F [mg/L] Control 95 100 1.1


### -

100 95 100 1.1 95
not applicable
Table2 Calibration StandardData

Concentration ofTestItem Mean Area [mg/L] [counts] 0 0 5.05 1.861 X 105 10.6 4.472 X 105 26.5 1.242 X 106 50.5 2.645 X 106 101 5.965 X 106 132 8.022 X 106Study Number: 41401174 Report Page 32 Analytical Investigations Table3 Results for Range-Finding Samples
Determined Nominal Sample Concentration %ofNominal Timepoint Concentration Preparation ofTest Item in Concentration Factor Range-Finding Sample

Cnom F c [hours] [mg!L] [mg/L] [%] 0 10 1.l 7.94 79 100 1.l 92.4 92 48 10 1.l 8.91 89 100 1.l 98.3 98
Table 4 Results for Spiked Recovery Samples
Nominal Fortified Determined Mean Precision Concentration Concentration Sample Concentration Analytical (Relative of ofTest Item in Preparation ofTest Item in Recovery Standard Test Item the Spiked Factor the Spiked Rate Deviation of Sample Sample Recovery)
Cnom Cfort F c R [mg/L] [mg/L]

. [mg/L] [%] [%] 1.l 101 1.l 101 100 101 1.l 101 100 0.29 1.1 102 1.1 101 Acceptance Target 80-120 <10Study Number: 41401174 Report Analytical Investigations Table 5 Results for Test Samples intheDefinitive Test

LOQ Nominal Timepoint Concentration

Cnom [hours] [mg/L] 0 Control 100 24 Control 100 48 Control 100 Limit ofQuantification not applicable Sample Determined Concentration Preparation ofTest Item in Factor Test Sample F c [mg/L] 1.1 <LOQ 1.1 93.5 1.1 <LOQ 1.1 92.5 1.1 <LOQ 1.1 88.5 Page 33

%ofNominal Concentration

[%]



### - 93



### - 92



### - 88StudyNumber: 41401174 Analytical Investigations 

## FIGURES Figure 1 Calibration Plot

**~** ·a 9oooooo t:l

........

**~** < 8000000

**~** &:! 7000000 6000000 5000000 4000000 3000000 Report

y =

### n. 7609x2+5l,565.5I61x- II7,lBO.one; R"=0.9997

60 

### so Page 34

100 120 140 Go:acentratim.(m,g.•L)StudyNumber: 41401174 Analytical Investigations Report
Figure 2 Chromatogram ofCalibration Solution (100 mg/L)

Figure 3 Chromatogram ofBiological Control Sample, 0 Hour*

* assessed as less thanthe limit ofquantification Page 35StudyNumber: 41401174 Report Page 36 Analytical Investigations Figure 4 Chromatogram ofTest Sample (100 mg/L), 0 HourStudyNumber: 41401174 Report
Appendix 6 MonitoringAuthority Statement ofGLP Compliance 

## i

# oli) 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

Envigo Research Limited Shardlow Business Park London Road Shardlow Derbyshire DE722GD Note: This statement has been issued following a company name change.

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

Toxicology

17 June 2015 An inspection for compliance with the Principles of Good Laboratory Practice was carried out at the above test facility as part of the UK Good Laboratory Practice Compliance Monitoring Programme. This statement confirms that, on the date of issue, the UK Good Laboratory Practice Monitoring Authority were satisfied that the above test facility was operating in compliance with the OECD Principles of Good Laboratory Practice. This statement constitutes a Good Laboratory Practice Instrument (as defined in the UK Good Laboratory Practice Regulations 1999}

. 

# • Dr. Andrew J. Gray Head, UKGLP Monitoring Authority Page 37

On21 September 2015 the legal entity HarlanLaboratories Ltd. was renamed as Envigo Research Limited. No other changes to the legal entity have occurred.