REPORT 

++++ 

£NVIGO 

D-erythro-Hex-2-enonic acid, .gamma.-lactone, monosodium 

salt 

[CAS RN 6381-77-7]: 

Daphnia sp., 48-Hour Acute Immobilization Test 

Study Director: 

Test Facility: 

Sponsor: 

Study Monitor: 

Study Number: 

Study Completion Date: 

D Sacker 

Envigo Research Limited 

Shardlow Business Park 

Shardlow 

Derbyshire 

DE72 2GD 

UK 

Univar USA Inc. 

17425 NE Union Hill Road 

Redmond 

WA 98052 

UNITED STATES OF AMERICA 

Greenwich Chemical Consulting, Inc. 

3300 PGA Boulevard, Suite 635 

Palm Beach 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 of37 
Study Number: 4140117 4 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 of Test Concentrations ........................................................ 14 

4.2.2 Immobilization Data ................................................................................ 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 ............................................................................................................................... 21 
Study Number: 41401174 Report Page 3 

LIST OF TABLES 

Table 1 Cumulative Immobilization Data and Observations in the in the 

Range-finding Test ...................................................................................................... 18 

Table 2 Cumulative Immobilization Data and Observations in the 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 of Analysis ........................................................................................... 21 

Reconstituted Water- Elendt M7 Medium ........................................................... 22 

Positive Control ..................................................................................................... 23 

Reconstituted Water - ISO Medium ..................................................................... 24 

Analytical Investigations ....................................................................................... 25 

Monitoring Authority Statement of GLP Compliance .......................................... 3 7 
Study Number: 41401174 Report Page4 

STUDY DIRECTOR STATEMENT OF GLP COMPLIANCE 

Envigo Research Limited, Shardlow Business Park, Shardlow, Derbyshire, DE72 2GD, UK 

Study Number: 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 of Directives 2004/9/EC and 2004/1 0/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 affected the integrity of the study or the validity of the data. 

Study Director: D Sacker 

...... ~ .. 

Date: 1 2 JAN 2016 
Study Number: 4140117 4 Report Page 5 

QUALITY ASSURANCE STATEMENT 

Study Number: 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 the relevant responsible person and management. 

Study-related procedures 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 of QA Inspections relevant Study 

Director and Test 

Facility Management 

Date of Inspection Type of Inspection Phase Inspected Report Date 

10 April2014 Study Plan NIA 10 April2014 

Verification 

27 October 2015 Process - based Test Item Preparation 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 that this report reflects the raw data and the procedures followed. 

Quality Assurance: L ~ ( c_G-·S 

.............. ~.C1. .. ~ ............... . Date: 

1 2 JAN 2016 
Study Number: 4140117 4 Report Page 6 

SUMMARY 

Introduction 

A study was performed to assess the acute toxicity of the 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 of Commission 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 of the test item at a concentration of 100 mg/L for 48 hours at a 

temperature of 21 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 of the test preparations at 0, 24 and 48 hours showed measured test concentrations to 

range from 88% to 93% of nominal concentration and so the results are based on nominal test 

concentrations. 

Exposure of Daphnia magna to the test item gave EC50 values of greater than 100 mg/L. The No 

Observed Effect Concentration was 1 00 mg/L. 

It was considered unnecessary and unrealistic to test at concentrations in excess of 1 00 mg/L. 
Study Number: 4140117 4 Report 

GENERAL INFORMATION 

Schedule 

Experimental Starting Date: 

Experimental Completion Date: 

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 

chargeable to the Sponsor. 

No data will be discarded without contacting the Sponsor to obtain their written consent. 
Study Number: 4140117 4 Report Page 8 

1 INTRODUCTION AND PURPOSE 

This report contains a description of the methods used and results obtained during a study to 

investigate the acute toxicity of the test item to Daphnia magna. 

Daphnia magna is a freshwater invertebrate representative of a wide variety of natural habitats, 

and can therefore be considered as an important non-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 of Chemicals (April2004) No 202 "Daphnia sp., Acute 

Immobilisation Test" 

• Method C.2 of Commission Regulation (EC) No. 440/2008 
Study Number: 41401174 Report Page 9 

2 TESTITEM 

Information as provided by the Sponsor. A Certificate of Analysis supplied by the Sponsor is 

given in Appendix 1. 

Identification: 

Physical state/ Appearance: 

Batch: 

Purity: 

Expiry Date: 

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 in the dark 
Study Number: 4140117 4 Report Page 10 

3 MATERIALS AND 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 of the test container are considered to be 

immobile. 

The calculated concentration of test item which results in 50% 

immobilization within the exposure period. 

The lowest test concentration at which a significant toxic effect 

on the test organisms is observed within the 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 (test water 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 

transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus 

subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction 

was by parthenogenesis. Gravid adults were isolated the day before initiation of the 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 contaminant that would affect the integrity or outcome of the study. 

A positive control (Study Number 41502271) used potassium dichromate as the reference item. 

Details of the 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 both the range-finding and definitive tests is defined 

in Appendix 4. 
Study Number: 41401174 Report Page 11 

3.4 Procedure 

Due to the potentially light sensitive nature of the test item, all test item preparation was 

performed under 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 of nominal test concentrations 

of 10 and 100 mg/L under continuous flow-through conditions. 

Nominal amounts of test 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 of 0.25 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 maintained under identical conditions but not exposed to the test item. 

A sample of each test concentration was taken for chemical analysis at 0 and 48 hours in order to 

determine the stability of the test item under test conditions. In order to ensure stability of the 

test item, a volume of glacial acetic acid (5 mL) was added to each sample bottle before addition 

of the test sample. 

3.4.2 Definitive Test 

Based on the results of the 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 of test item (12 g) was dissolved in deionized reverse osmosis water and the 

volume adjusted to 1 liter 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 of the 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 if they 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 of Test 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 if necessary. In order to ensure stability of the test item, a volume of glacial acetic acid 

(5 mL) was added to each sample bottle before addition ofthe test sample. 

The method of analysis, recovery and test preparation analyses are described in Appendix 5. 

3.4.4 Statistical Analysis 

An estimate of the EC5o values was given by inspection of the 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 (TOXRA T). 

3.5 Validation Criteria 

The results of the test are considered valid if the following performance criteria are met: 

• No more than 10% of the control daphnids show immobilization or other signs of disease 

or stress (e.g. discoloration or unusual behavior such as trapping at the surface water). 

• The dissolved oxygen concentration at the end of the test is equal to or greater than 

3 mg/L in the control and test vessels. 
Study Number: 4140117 4 Report Page 14 

4 RESULTS 

4.1 Range-finding Test 

Cumulative immobilization data and other observations from the exposure of Daphnia magna to 

the test item during the range-finding test are given in Table 1. 

No immobilization was 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 of the test preparations at 0 hours (see Appendix 5) showed measured test 

concentrations to range from 79% to 92% of nominal concentrations. At 48 hours the measured 

test concentrations ranged from 89% to 98% of nominal, indicating that the test item was stable 

under test conditions. 

4.2 Definitive Test 

4.2.1 Verification of Test Concentrations 

Analysis of the test preparations at 0, 24 and 48 hours (see Appendix 5) showed measured test 

concentrations to range from 88% to 93% of nominal concentrations and so the results are based 

on nominal test concentrations. 

4.2.2 Immobilization Data 

Cumulative immobilization data and other observations from the exposure of Daphnia magna to 

the test item during the definitive test are given in Table 2. 

The No Observed Effect Concentration after 48 hours exposure was 100 mg/L. 

It was considered unnecessary 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 of the two immobilized daphnia in control 

replicate 1, all daphnia in the control were observed to be normal at 48 hours. This observation 

was considered not to have adversely affected the results of the test. 

4.2.4 Validation Criteria 

The test was considered to be valid given that no more than 10% of the control daphnids showed 

immobilization or other signs of disease or stress and that the oxygen concentration at the end of 

the test was equal to or greater than 3 mg/L in the control and test vessels. 

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 or pH. 

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. 
Study Number: 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 of greater 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 2001-2010 
Study Number: 41401174 Report Page 18 

TABLES 

Table 1 Cumulative Immobilization Data and Observations in the in the Range-finding 

Test 

Observations (Initial Population: 10 Per Replicate) 

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, considered no adverse effect as all daphnia were accounted for at 48 hours 
Study Number: 41401174 Report 

Table 2 Cumulative Immobilization Data and Observations in the Definitive Test 

Nominal Cumulative Immobilized Daphnia Concentration (Initial Poll_ulation: 5 Per Replicate) (mg/L) 

Rt R2 R3 Itt Total % 

Control 0 0 0 0 0 0 

100 0 1 0 0 1 5 

Nominal Cumulative Immobilized Daphnia Concentration 

(mg/L) (Initial Population: 5 Per Replicate) 

Rt R2 R3 Itt Total % 

Control 2 0 0 0 2 10 

100 1 2 0 1 4 20 

R1 - ~ =Replicates 1 to 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 

4N 
Study Number: 41401174 Report Page 20 

Table 3 Water Quality Measurements 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 1 
Study Number: 41401174 Report 

APPENDICES 

Appendix 1 Certificate of Analysis 

Sodium Erythorbate ~cc Grade ttoch.r 

Cla&EPOI 

Lot Number: 

-..otlonO.W. 

~luallan Dl .. : 

ao ...... Code: 

Fectary: 

Procluot c_, 

Prut~l"f' 

1\pp.a,;;•~· 

lder!tlflet~Cion 

Aas&y 

Het"Y Melillo (H Pbl 

t~'!:ail'~u'"'nt FCC)· 

Las& on drying 

·3JCatate 

GS&E 5092 

14031314 

Mer 28,2014 

Mar. 28,2018 

01D-081·01 

DaWig PRhn. Doing, Jlllngx~ Chine 1182511 (25kpC8tlon) 

::"r;'J;."'fm·ma)... 

-:-::l5"'r!'.- -n~ •• 

Sp~iflc ~~!!!!~_l!!l.;o.~---·-· ·--. 

P ~sse~: te";l ....~t.d· ... gip.,. 

QuntVI\R 

j·~~~,-,,n~ 

c::.nr~.-;rr;. 

CrJ<:font 

99..19· 

9.[). 

c~,nf-ofm 

~~q_-

lJH"•"~>l 

·p~~-.N .. !·~Qill!ll-ll(ii~-.rc:(~ 

i-!tV"~.-: :1"(~ i,'\.J.\ >1P.O~i:-,~'1S 

:r~;-~;w,!f, .... :r.~· 

Page 21 
Study Number: 41401174 Report 

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 B12) 

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 pH of the prepared media was 7.9 ± 0.3 and stored at approximately 21 °C. 

Page 22 
Study Number: 41401174 Report Page 23 

Appendix 3 Positive Control 

A positive control (Study Number 41502271) used potassium dichromate as the reference item at 

concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L. 

Exposure conditions for the positive control were similar to those in the definitive test. 

Analysis of the immobilization data by the pro bit analysis using the linear maximum likelihood 

regression method at 24 and 48 hours using the ToxRat Professional computer software package 

based on the nominal test concentrations gave the following results: 

No Observed Lowest Observed 

Time Point ECso 95% Confidence Limits Effect Effect 

(Hours) (mg/L) (mg/L) Concentration Concentration 

(NOEC) (mg/L) (LOEC) (mg/L) 

24 0.90 Not possible 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. 
Study Number: 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 water had a pH of7.8 ± 0.2 adjusted (if necessary) 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 of250 mg/L as CaC03. 
Study Number: 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 of a single peak. 

The method was developed and implemented by the Department of Analytical Services, Envigo 

Research Limited, Shardlow, UK. 

1.2 Test Item 

The test item is described in the biological part of this 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 on the day of receipt. 

1.4.2 Reagents and Solvents 

Water: prepared using an ELGA Pure Lab Option R-15 water purification system. 

Test medium: as described in the biological part of this study. 

Solvents: 

Reagents: 

acetonitrile, Fisher Scientific, HPLC grade. 

tetrabutylammonium, Sigma-Aldrich, 2: 99.0% 

acetic acid, Fisher Scientific, analytical reagent 
Study Number: 4140117 4 Report Page 26 

Analytical Investigations 

1.4.3 Preparation of Calibration 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 of this solution 

was then diluted with 5% (v/v) acetic acid in water to obtain calibration standards in the range 

of 5 to 100 mg/L. A second series of calibration standards was similarly prepared in the range of 

10 to 125 mg/L. These solutions were used to determine the recovery and test sample 

concentrations. 

1.4.4 Preparation of Linearity 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 of this solution 

was then diluted with 5% (v/v) acetic acid in water to obtain calibration standards in the range 

of 5 to 100 mg/L. A second series of calibration standards was similarly prepared in the range of 

10 to 125 mg/L. These standards were used to evaluate the linearity ofthe analytical system. 

1.4.5 Preparation of Spiked Recovery Samples 

To demonstrate the validity of the analytical procedure, volumes of test medium were spiked 

with the test item and the recovery was assessed. The test item (nominal 100 mg) was initially 

dissolved in 5% (v/v) acetic acid in test medium to prepare a stock solution with a concentration 

of 1000 mg/L. A defined volume of this 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 of the test item (synthetic control) was also analyzed. 

1.4.6 Preparation of Test Samples 

The test samples were analyzed on the day of receipt. An aliquot of acetic acid (5 mL) was 

added to the test samples (95 mL). See Table 1 for more information on preparation volumes. 
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 mm id.) 

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 

Injection volume 

Flow rate 1 mL/minute 

Column temperature 20 oc 

Autosampler temperature 

Detection wavelength 280nm 

Retention time approximately 3 minutes 

1.4.8 Calculations 

A calibration curve was prepared using a quadratic function of the 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 of the test item in the injected sample [mg/L] 

average peak area ofthe test item in the injected sample [counts] 

* TBAB solution= 4 g oftetrabutylammonium bromide in 200 mL acetonitrile 
Study Number: 4140117 4 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 of the test item in the test sample [ mg/L] 

concentration of the test item in the injected sample solution [mg/L] 

sample preparation factor 

recovery rate [%] (If not correcting for recovery then R is taken as 1 00) 

The recovery rate (R) of the spiked recovery samples was calculated usmg the following 

equation: 

R =-c-·100 

cfort 

where R: 

c: 

Cfort: 

(3) 

recovery rate [%] 

determined concentration of the test item in the spiked recovery sample [ mg!L] 

fortified concentration of the test item in the spiked recovery sample [ mg/L] 

The percentage of nominal concentration of the test item in the test samples was calculated using 

the following equation: 

%nominal= _c_ ·100 

cnom 

(4) 

where Cnom: nominal concentration in the test sample [mg/L] 
Study Number: 41401174 Report Page 29 

Analytical Investigations 

2 RESULTS 

2.1 Range-Finding Test 

The analytical methodology was not validated prior to the analysis of range-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 of the Analytical Method 

Specificity 

The biological control samples and an analyzed analytical blank showed no significant 

interfering response at the retention time of the test item. The standard solutions contained a 

peak specific for the test item whose area changed accordingly with known concentration; hence 

the specificity of the method by retention time was confirmed. 

Linearity 

The calibration data for the calibration standards of the test item is given in Table 2 and Figure 1. 

The data was found to have a quadratic correlation within the calibration range of 0 to 132 mg/L. 

The R2 fit of the 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 of the test item in the 

recovery samples are presented in Table 4. 

The method was considered to be sufficiently accurate and precise for the purposes of this test. 

The test sample results were not corrected for recovery. 

Limit of Quantification 

The limit of quantification (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 of the test item found in the test samples are presented 

in Table 5. 

Typical chromatograms are shown in Figures 2 to 4. 
Study Number: 41401174 Report Page 30 

Analytical Investigations 

3 DISCUSSION 

The detection system was found to have acceptable correlation to the fit of a quadratic equation. 

The analytical procedure was found to have acceptable recoveries of test item in test medium. 

The method of analysis was validated and proven to be suitable for use. 
Study Number: 4140117 4 Report Page 31 

Analytical Investigations 

TABLES 

Table 1 Preparation of Test Samples 

Nominal Sample Final Nominal 

Concentration of Test Sample Volume Final Volume Preparation Concentration of Test 

Item Factor Item 

[mg/L] [mL] [mL] F [mg/L] 

Control 95 100 1.1 -

100 95 100 1.1 95 

not applicable 

Table 2 Calibration Standard Data 

Concentration of Test Item 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 106 
Study Number: 4140117 4 Report Page 32 

Analytical Investigations 

Table 3 Results for Range-Finding Samples 

Determined 

Nominal Sample Concentration %of Nominal Timepoint Concentration Preparation of Test 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 of Test Item in Preparation of Test 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 <10 
Study Number: 4140117 4 Report 

Analytical Investigations 

Table 5 Results for Test Samples in the Definitive Test 

LOQ 

Nominal Timepoint Concentration 

Cnom 

[hours] [mg/L] 

0 Control 

100 

24 Control 

100 

48 Control 

100 

Limit of Quantification 

not applicable 

Sample Determined 

Concentration Preparation of Test 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 

%of Nominal 

Concentration 

[%] 

-

93 

-

92 

-

88 
Study Number: 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) 
Study Number: 4140117 4 

Analytical Investigations 

Report 

Figure 2 Chromatogram of Calibration Solution (100 mg/L) 

Figure 3 Chromatogram of Biological Control Sample, 0 Hour* 

* assessed as less than the limit of quantification 

Page 35 
Study Number: 4140117 4 Report Page 36 

Analytical Investigations 

Figure 4 Chromatogram of Test Sample (100 mg/L), 0 Hour 
Study Number: 41401174 Report 

Appendix 6 Monitoring Authority Statement of GLP 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, UK GLP Monitoring Authority 

Page 37 

On 21 September 2015 the legal entity Harlan Laboratories Ltd. was renamed as En vi go Research Limited. No 

other changes to the legal entity have occurred.