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### REPORT

: Acute Toxicity t o Daphnia Magna

HLS study number: CEF0009 Version ID: Audited Draft 1 Report Issue date: 30 September 2013Huntingdon Life Sciences: CEF0009

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## Sponsor and Te st Facility Details Sponsor

Test facility Huntingdon Life Sciences Eye Research Centre Eye Suffolk IP23 7PX UKHuntingdon Life Sciences: CEF0009

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## Ta ble of Contents Sponsor and Test Facility Details

.......................................................................................... 2 Compliance with Go od Laboratory Practice

.......................................................................... 4 Quality Assurance Statement

................................................................................................ 5 Contributing Scientists

.......................................................................................................... 6 Summary

.............................................................................................................................. 7 1. Introduction

................................................................................................................... 8 2. Test Substance

............................................................................................................. 9 3. Experimental Procedure

.............................................................................................. 10 3.1 Test organism

....................................................................................................... 10 3.2 Dilution medium

.................................................................................................... 10 3.3 Test substance preparation

................................................................................... 10 3.4 Exposure conditions

.............................................................................................. 10 3.4.1 Experimental design

...................................................................................... 10 3.4.2 Test concentrations

........................................................................................ 11 3.4.3 Medium renewal

............................................................................................ 11 3.4 .4 Stability of test concentrations

....................................................................... 11 3.4.5 Environmental conditions

............................................................................... 11 3.5 Criterion of effect

................................................................................................... 11 3.6 Evaluation of data

................................................................................................ . 12 3.7 Validi ty criteria

...................................................................................................... 12 3.8 Protocol deviations

................................................................................................ 12 4. Maintenance of Records

.............................................................................................. 13 5. Results

........................................................................................................................ 14 5.1 Chemical analysis

................................................................................................ . 14 5.2 Immobility

.............................................................................................................. 14 5.3 Environmental parameters

.................................................................................... 14 6. Conclusions

................................................................................................................. 15 

## List of T ables Table 1 Measured con centrations

............................................................................. 16 Table 2 Environmental parameters

........................................................................... 17 

## List of Appendices Appendix 1 Certificate of analysis

................................................................................... 18 Appendix 2 Elendt M4 medium

....................................................................................... 19 Appendix 3 The determination of in Elendt M4 medium

................................ . 20 Appendix 4 Compliance statement

................................................................................. 30Huntingdon Life Sciences: CEF0009

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## Compliance with Good Laboratory Pra ctice
: Acute Toxicity t o Daphnia Magna The study described in this report was conducted in co mp liance with the fo llo wing Good Laboratory

Practi ce standards and I consider the data generated to be valid. The UK Good Laboratory Practi ce Regulat io ns (Statutory Instrument 1999 No. 3106, as amended by Statutory Instrument 2004 No. 994). OECD Principles of Good Laboratory Practi ce (as revised in 1997), ENV/MC/CHEM (98) 17. EC Commissio n Di

rective 2004/10/EC of 11 February 2004 (Official Journal No L 50/4 4). These principles of Good Laboratory Practice are accepted by

the Regulatory

A uthori ti es of th e United States of America and Japan on the basis of Intergovernmental Agreements.

________________________________ ___________ ________________________ Romalie Allen BSc Date Study Di rector Hunt ingdon Life SciencesHuntingdon Life Sciences: CEF0009

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## Quali ty Assurance Statement
: A cute Toxicity t o Daphnia Magna The fo llo wing inspect io ns and audits have been carried out in relation to this study: Study Phase Date(s) of Inspection Date of Reporting to Study Director and Management Protocol Audit 13 Jun 2013 13 Jun 2013 Report Audi

t 23 Sep 2013 23 Sep 2013 Process based inspections : At o r about th e time this study w as in progress inspect io ns of procedures em

ploy ed on this ty

pe of study

w ere carri ed out. T hese were conducted and reported to appro pri ate Com pany Management as indicated below: Process Based Inspections Date(s) of Inspection Date of Reporting to Management Envi ronmental

m easurements & mo nit oring/Ammo nia analysis 07 Mar 2013 07 Mar 2013 Experimental Set -up 08 Aug 2013 09 Aug 20 13 Sam pling of test media for chemical analysis 08 Aug 2013 09 Aug 2013 Dose Form

ulation 08 Aug 2013 16 Aug 2013 Study management & conduct 09 Aug 2013 16 Aug 2013 Observations 13 Sep 2013 13 Sep 2013 Sam ple Preparati on (to include sample manag ement & tracking) 27 Feb 2013 01 Mar 2013 Standard preparation & Fortificat io n procedures 05 Aug 2013

- 06 Aug 2013 07 Aug 2013 Results Processing 30 Aug 2013 30 Aug 2013 Chro mat ography 30 Aug 2013 30 Aug 2013 In addit io n, an inspect io n of the facil it y where thi s study was conducted was carried out on an annual basis. These inspect io ns were reported to Company Management.

________________________________ ___________ ________________________ T om Pullinger MRQA Date Group Manager Department of Qualit y Assurance Hunt ingdon Life SciencesHuntingdon Life Sciences: CEF0009

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## Contributing Scientist s
: A cute Toxicity t o Daphnia Magna 

### Study management Aquatic Ecotoxicology and Biodegradation Romalie Allen BSc Study Di rector 

### Expert department Environmental Analysis Simo n Tate Higher National Certificate (Applied Chemistry) Senior Study

ManagerHuntingdon Life Sciences: CEF0009

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## Summary The acute toxicit y of to Daphnia magna was assessed under static exposure condi tions. The study was conducted in accordance wit h EC Methods for Determinat io n of Ecotoxicit y, Annex to Commissio n Regulat io n (EC) No 440/2008 Part C, Method 2 “ Daphnia sp. Acute Im mo bilisat io n Test” and the OECD Guideline for Testing o f Chemicals No. 202, “ Daphnia Acute Immo bilisation Test” (2004). Groups of twenty Daphnia , less than 24 hours old, were exposed for 48 hours to , at nominal concentrations o f 0.52, 1.15, 2.54, 5.58, 12.3 and 27.0 mg/L. The test media were prepared in Elendt M4 medium b y the di rect addi ti on of the test substance to the dilution medium at nominally

27.0 m g/L and serial dilut io n thereafter

. At all concentrations, the intended exposure levels of were adequately achieved (between 82 and 96% of

thei r no minal values) and maintained during the test (between 90 and 96 % of

thei r starting values). Based on an ari thmet ic mean, t he overall measured concentrations were 0.471, 0.9 99, 2.25, 4.82, 10.9 and 24.8 mg/L and these values have been used in the calculation of study end

- points. Observations of the Daphnia in each control and test vessel were made after 24 and 48 hours

. After 48 hours no immo bilisation was observed. Based o n these findings, the 48

- hour EC 50 co uld not be calculated and must be greater than 24.8 m g/L and the no

- observed

- effect concentration’ (NOEC) must be at least 24.8 mg/L , th e maximum concentration employ ed in the study

.Huntingdon Life Sciences: CEF0009

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## 1. Introduction The obj ect ive o f th e st udy was to determine the acute toxicit y (48 hour median effect concentration

- EC 50 ) of to Daphnia magna

. The study was conducted in accordance wit h EC Methods for Determinat io n of Ecotoxicit y, Annex to Commissio n Regulat io n (EC) No 440/2008 Part C , Method 2 “ Daphnia sp. Acute Im mo bilisat io n Test” and the OECD Guideline for Testing o f Chemicals No. 202, “ Daphnia Acute Immo bilisation Test” (2004). The protocol was approved by Huntingdon Life Sciences Management and the Study Di rector on 17 June 2013. The experimental start and complet io n dates of the study were 5 July

2013 and 22 August 2013 , respectively. The study was conducted at Huntingdon Life Sciences, Eye Research Centre, Eye, Suffo lk, IP23 7PX, England. A separate physiochemical s tudy to determine the properties of (HLS study : CEF0001) indicated that the limit of so lubilit y of the test substance was 27.5 mg/L at 20oC.Huntingdon Life Sciences: CEF0009

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## 2. Te st Substance
Name Chemical name CAS No. Physical state Powder Batch number 12342 Puri ty To be included Storage Room tem perature in the dark Expi ry Date To be included Date received: 4 March 2013 Certificate of analysis: Appendix 1Huntingdon Life Sciences: CEF0009

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## 3. Experimental P rocedure 3.1 Test organism Daphnia magna (Straus) used in this study were cultured in

- house and were obtained fro m a strain originat ing fro m th e Nati onal Inst it ute f

or Applied Chemical Research (IRCHA), France. Stock cultures of Daphnia magna were maintained in glass vessels contain ing approximately 0.5 to 0.8 lit res of Elendt M4 culture medium in a temperature

- controlled laboratory at no minally 20  2  C. A photoperi od of 16 hours light : 8 hours dark was maintained, with peri ods of

subdued lighting at the beginning and end of each light phase. The culture medium was renewed three times each week. Cul tures were fed daily

wi th a suspensi on

of the unicellular green algae, Pseudokirchneriella subcapitata , to provide no minally 0.1 to 0.2 mg carbon per daphnid, per day, except during th e init ial three days when a slight ly lo wer ration was given. Cul ture condi

ti ons ensure that th e stock animals reproduce by parthenogenesis. The day b ef ore the start of the study, all juvenile Daphnia were removed fro m th e laboratory cul tures. The following morning, j uveniles produced by the gravid (egg

- bearing) adult Daphnia were removed fro m th e cul ture vessels and held in a separate holding vessel; these animals, which were less than 24 hours old, were used in the test. 

### 3.2 Dilution medium The test organi sms were maintained and the tests conducted in Elendt M4 medium ( Appendix 2 ). The medium was prepared in deio nised, reverse osmosis water. 

### 3.3 Test substance preparation The method of preparation used during the definit ive test was based on the results of a range finding test. The test substance (54.2 mg) was ground into a smooth pourable paste on an agate mortar wi th the dropwi se addit io n of Elendt M4 medium . The paste was then added to a volumetric flask (2 L) containing Elendt M4 med ium (1 7 00 mL), whi ch was then shaken before being made up to volume and st irred f or ca . one hour until no particulate matter remained. The preparati on was then filtered (0.45 μm pore size) to provide an aqueous solut io n wi th a no minal concentration of 27.0 m g/L ; this was eit her used direct ly at t he highest test concentration or a liquots (454, 454, 455, 453 and 452 mL) were serially diluted in vo lu metric flasks (1 L) containing Elendt M4 med ium to provide test media with nominal concentrations of 1 2.3, 5.58, 2.54, 1.15 or 0 .52 m g/L. 

### 3.4 Exposure conditions 3.4.1 Experimental design A range finding test was fo llo wed by a definit ive test wi th six test concentrations, pl us a dilut io n med ium control

group .Huntingdon Life Sciences: CEF0009

11 Twent y Daphnia , f

our replicates of five animals per vess el , were exposed in each control and test group. The first instar Daphnia were placed in groups of five, at random into 120 m L glass di shes containing 1 2 0 m

L o f med ium to give a loading of 24 mL medium per organism. The dishes were loosely covered. 3.4.2 Test concentrations The range finding test emplo yed no minal test concentrations of 0.1, 1, 10 and 25 mg/L. After 48 hours, th ere was 90% immo bilisat io n of the Daphnia at25 mg/ L , and 50% at 10 m g/L, wi th no immobilisat io n observed as th e other test groups. Ba sed on these results, the definit ive test emp lo yed no minal concentrations o f 0.52, 1.15, 2.54, 5.58, 12.3 and 27.0 mg/L ; t he highest concentration em ploy ed approximated to the limit of so lubilit y fo r the test substance

. 3.4.3 Medium renewal Daphnia were exposed to the test or control conditio ns fo r a period of 48 hours wi

thout renewal of

test media. 3.4.4 Stability of test concentrations The test concentrations of were measured using liquid chromatography wi th tandem m ass spectrometric detection (LC

- MS/MS, see Appendix 3 ). At the start of the definit ive test, two samples ( 10 mL) were taken from the freshly

- prepared control and test media and each 10 m

L sample was added to a vial containing water:acetonitrile:formic acid (95:5:0 .1). After 48 hours, the contents of the test vessels fro m each group were pooled and further samples were taken for analysis. On each occasio n, one of the samples was analysed and the other was stored in a freezer in case further analysis was required. 3.4.5 Environmental conditions During the definit ive test, continuous monitoring of the medium in an addit io nal vessel indicated that temperatures ranged between 20.0 and 20.7  C, confirming that the test area was maintained to wi

thin  1  C. A photoperiod of 16 hours light : 8 hours dark was maintained, with periods of subdued lighting at the beginning and end of each light phase. No supplementary

aeration was em

ploy ed and no feed was given during the exposure period. The tem perature, pH and dissolved oxygen le vels of control and test media were recorded at th e start and at the end of the study . The total h ardness and al

kalini ty of the dilut io n med ium was me asured before use. 

### 3.5 Criterion of effect Daphnia were considered to be immo bile if they were unable to swim wi thin approximately 15 seconds fo llo wing gent le agitat io n of the test vessel.Huntingdon Life Sciences: CEF0009

12 The numbers o f mo bile, immo bile and float ing Daphnia were counted approximately 24 and 48 hours after the start of the study

. 

### 3.6 Evaluation of data The “no observed effect conce nt ration” (NOEC) was derived by direct inspect io n of the data on the immo bilit y of the animals. An incidence rate of more than 10% was considered to be significant. 

### 3.7 Validity c riteria For the study

to be valid, the number of float ing and/or immo bile Daphni a in the control group m ust not be exceed 10% at the end of the test and the concentrations of dissolved oxy gen must be > 3 mg/L at the end of the test. 

### 3.8 Protocol deviations There were no protocol deviat io ns .Huntingdon Life Sciences: CEF0009

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## 4. Ma intenance of Records This report has been co mp iled fro m original data, which will be stored, together with a copy of the final report, a copy

of the protocol

and amendments in Hunt ingdon Life Sciences’ Archives, Huntingdon Life Sciences Ltd, Eye, Suffol k, UK. Records will be retained for a minimum p eri od of at least one year from the date of issue of th e final report. At the end of the one year retention peri od the Sponsor will be contacted and advice sought on the future requirements. Under no circumstances will any i tem be di scarded wi thout the S ponsor’s knowledge. Hunt ingdon Life Sciences will retain the Qualit y Assurance records relevant to this study f or twenty y ears, and a copy

of the final report in its archives indefinitely

.Huntingdon Life Sciences: CEF0009

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## 5. Results 5.1 Chemical analysis The results of chemical analysis are g iven in Table 1 and example chromatograms are illustrated in Appendix 3
. Concentrations are expressed in terms of the measured levels o f
At all concentrations, the intended exposure lev els of were adequately achieved (between 82 and 96% of

thei r no minal values) and maintained during the test (between 90 and 96 % of

thei r starting values). Based on an ari thmet ic mean, t he overall measured concentrations were 0.471, 0.999, 2.25, 4. 82, 10.9 and 24.8 mg/L and these values have been used in the calculation of study end

- points. 

### 5.2 Immobility Observations of the Daphnia in each control and test vessel were made after 24 and 48 hours

. After 48 hours , no immo bilisat io n or adverse effects o n the daphnia were observed. Based on these findings, the 48- hour EC 50 co uld not be calculated and must be greater than 24.8 m g/L and the no

- observed

- effect concentration’ (NOEC) must be at least 24.8 mg/L the maximum concentration employ ed in the test. 

### 5.3 E nvironmental parameters The measurements of water qualit y (tem perature, pH, di ssolved oxy gen, total hardness and al kalini ty ) are summarised in Table 2 ; they remained wi

thin acceptable limits during the study

. At the start and end of

the test, the test media were colourless at all concentrations.Huntingdon Life Sciences: CEF0009

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## 6. Conclusions

was found not to be acutely toxi c to Daphnia magna when d issolved in water to a maximum measured concentration of 24.8 mg/L under the condi tions of this study . T hus t he 48

- hour EC 50 could not be calculated and must be greater than 24.8 mg/L and the no

observed

- effect concentration’ (NOEC) must be at least 24.8 mg/L the maximum employ ed concentration in the test.Huntingdon Life Sciences: CEF0009

16 Table 1 Measured concentra tions Nominal concentration (mg/L ) Measured concentrations (mg/L ) Overall Mean* 0 hours %N 48 hours %N % ti Control nd

- nd

- - nd 0.52 0.493 95 0.448 86 91 0.471 1.15 1.05 91 0.947 82 90 0.999 2.54 2.30 91 2.20 87 96 2.25 5.58 4.97 89 4.66 84 94 4.82 12.3 11.10 90 10.7 87 96 10.9 27.0 25.9 96 23.6 87 91 24.8
nd N one detected (less than the limit of detection of 0.04 mg/L) %N Mean measured concentration expressed as a percentage of the nominal concentration % ti M ean mea sured concentration after 48 hours expressed as a percentage of th e mean starting concentration * Arithmetic meanHuntingdon Life Sciences: CEF0009

17 Table 2 Environmental parameters Exposure conc.

(mg/L) Temperature (°C) pH Dissolved oxygen (%ASV) N ominal M easured 0 h 48 h 0 h 48 h 0 h 48 h Control nd 20.1 20.0 7.64 7.89 100 98 0.52 0.471 20.2 19.9 7.65 7.90 101 98 1.15 0.999 20.2 19.6 7.68 7.89 100 98 2.54 2.25 20.1 19.6 7.69 7.87 101 97 5.58 4.82 20.2 19.8 7.70 7.87 100 97 12.3 10 .9 20.3 19.7 7.67 7.84 100 97 27.0 24.8 20.4 19.7 7.62 7.88 97 98
nd N one detected (less than the limit of detection of 0.04 mg/L) ASV Air saturation value (100% ASV = 9.09 mgO 2/L at 20oC)

. The total hardness and alkalinity of the batch of Elendt M4 medium used were 245 and 60 mg/L as CaCO 3 , respectively. Continuous monitoring of an additional vessel containing dilution medium = 20.0 to 20. 7 ° C.Huntingdon Life Sciences: CEF0009

18 Appendix 1 Certificate of analysis To be included w

hen suppliedHuntingdon Life Sciences: CEF0009

19 Appe ndix 2 Elendt M4 medium 1. Micro elements mg/L H 3 BO 3 2.86 MnCl 2 .4H 2 O 0.36 LiCl 0.31 RbCl 0.071 SrCl 2 .6H 2 O 0.152 NaBr 0.016 Na2 MoO 4 .2H2 O 0.063 CuCl 2 .2H 2 O 0.017 ZnCl 2 0.013 CoCl 2 .6H 2 O 0.010 KI 0.0033 Na2 .SeO 3 0.0022 NH 4 VO 3 0.00058 Fe
- EDTA so lut io n 3.50
2. Macro nutri ents mg/L CaCl 2 .2H 2 O 294 MgSO 4 .7H 2 O 123 KCl 5.80 NaHCO 3 64.8
3. Buffer nutrients mg/L Na2 .Si O 3 .9H 2 O 10 .0 NaNO 3 0.274 KH 2 PO 4 0.143 K 2 HPO 4 0.184
4. Vi tamins mg/L Thiamine hydrochloride 0.075 Cyanocobalamine (B12) 0.0010 Bi otin 0.00075 The above analyt ical

grade reagents are dissolved in deio nised, reverse osmosis water.Huntingdon Life Sciences: CEF0009

20 Appendix 3 The determination of in E lendt M4 medium 

## 1. Method 1.1 General principle Test sol ut io ns were diluted with water:acetonitrile:formic acid (95:5:0.1 v:v:v) pri or to quant it ation by liquid chromatography wi th tandem m ass spectrometric detection system (LC

- MS/MS). Materials Grade Actonitrile HPLC Ammonium formate AR Formic acid AR Methanol HPLC Water HPLC 1.1.1 Test article fortifying and calibration solutions Some standard sol

utions used in this study

have been shared wit h other GLP studies being performed f

or the same sponsor. Thi s is considered to have no impact on the integrit y or validit y of

this study

. A weighed amount of was di ssolved in acetonitrile to produce a 1 mg/mL stock standard solut io n. The stock standard was diluted progressively wit h acetoni tr ile to give fo rtificat io n standard solut io ns. The appropriate fortificat io n standard so lut io n was further diluted with water:acetoni trile:formic acid (95:5:0.1 v:v:v) to produce a series of calibrat io n sol ut io ns in the range 0.1 to 5 ng/mL. 1.1.2 Ty pical preparation of reagents Preparati on of water:acetonitrile:formic acid (95:5:0.1 v:v:v) : Acetonitrile (5 0 mL) and formic acid (1 mL) w ere thoroughly mixed with water ( 95 0 mL) pri or to use. Preparati on of

water:methano l: fo rmic acid (90:10:0.1 v:v:v) + 0.01M ammo nium formate: Water (900 mL) was thoroughly mixed wit h met hano l (100 mL), formic acid (1 mL) and ammo nium formate (0.63 g) prior to use. Preparati on of

0.1% formic acid in methano l: Methano l (1000 mL) was thoroughly mixed with formic acid (1 mL) prior to use. NOTE

– to prepare different volumes of the above reagents, the ratio of the individual com ponents was maintained.Huntingdon Life Sciences: CEF0009

21 

### 1.2 Procedure An aliquot (10 mL) of the sample was taken and fortified if required. Water:acetonitrile:formic acid (95:5:0.1 v:v:v) (10 mL) was added and mixed well . The sample was diluted by

a factor of 200 wi

th water:acetonitrile:formic acid (95:5:0.1 v:v:v)

. Any further dilut io ns, where required, were performed with water:acetoni tri le:formic acid (95:5:0.1 v:v:v) pri or to quanti tation by L C

- MS/MS. 1.2.1 Instrumentation LC - MS/MS conditions Instrument: AB Sciex API 4000 Data system: Analyst v.1.4.2 Ioni sat io n mo de: Ionspray posi tive Ion moni toring details: MRM m/z 186>57 Col umn: Pheno me nex aqua C 18 (5 cm  2 mm) Mobile phase A: Water :methano l: fo rmic acid (90:10:0.1 v:v:v) + 0.01M ammo nium formate Mobile phase B: Methano l + 0.1% formic acid (by v ol ume) Pum p programme: 60:40 A:B isocratic Cycle time: 8 minutes Flow rate: 0.3 m L/min Inject io n vo lu me: 20 μL Retention t ime: Approximately 1
. 5 minutes LOQ: 0. 1 m g/L LOD: 0.1 ng/m L (equivalent to 0.04 mg/L in aqueous media)Huntingdon Life Sciences: CEF0009

22 1.2.2 Calculation of results Test samples were quant ified using the fo llo wing equation: Residue found (mg/L) = 1000 1 x D x M 1 x x Where x (residue concentration in fin al so lut io n) was calculated using the linear regression:
y = m x + c where x (concentration in ng/mL) = y  c m c = int ercept m = slope y = peak area of sample M = matri

x concentration (mL/mL) D = dilut io n factorHuntingdon Life Sciences: CEF0009

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## 2.0 Results and Discussion 2.1 Calibration The chro mat ographi c response to was shown to be linear over the range of concentrations 0.1 to 5 ng/mL. Ty pical calibrat io n data is presented in Table 1 and Figure 1. Ty pical chromatograms of calibrat io n standards are presented in Figures 2 and 3. 

### 2.2 Validation The methodology

f or the determinat io n of residues in Elendt M4 was validated at 0.1 m g/L and 3 0 m

g/L. The validat io n data are presented in Table 2 and summarised in the fo llo wing table: Fortification level (mg/L) Mean recovery (%) CV (%) 0.1 96 3.2 3 0 98 2.7 ( 97 ) ( 3.1 ) ( ) O verall values 

### 2.3 Limit of quantitation (LOQ) The limit of quant it ation is defined as the lowest fortificat io n level at which acceptable recovery data are obtained. The valid ation of the methodology

f or the determination of residues of in Elendt M4 , demonstrated that i t can be accurately determined at a limit of quant it ation (LOQ) of 0.1 m g/L. 

### 2.4 Limit of detection (LOD) The limit of detection of the method is defined as the value of the lowest calibrat io n standard chro mat ographed that gave a peak height to baseline no ise ratio  3. For this study the limit of detection was 0.1 ng/m

L (equivalent to 0. 04 m

g/L in Elendt M4 ). 

### 2.5 Samples Sam ples o f Elendt M4 from the test were analysed in suitably sized batches alo ng with control samples fortified wit h which acted as procedural recovery samples. Procedural

recoveri es performed on control Elendt M4 were in the range 70 to 110% confirming the validit y of the methodo lo gy

on each day

of analysis. Ty

pical chromatograms of untreated, untreated fortified wit h and treated sam ples are presented in Figures 4 to 7.Huntingdon Life Sciences: CEF0009

24 Table 1 Ty pical standard calibration for by LC - MS/MS

Standard concentration (ng/mL ) Peak area

0 0 0.1 1.16562e+004 0.2 1.75401e+004 0.4 3.42392e+004 0.8 6.23427e+004 1 7.10164e+004 2 1.38658e+005 3 1.96590e+005 4 2.82324e+005 5 3.47633e+005 Calibration curve: y

= 68349.6 x + 3423.15 (batch 4 ) r = 0.99 92 Where x = concentration y = peak area r = regression coefficientHuntingdon Life Sciences: CEF0009

25 Table 2 Validation of in Elendt M4 by LC - MS/MS

Fortification level Replicate Analyte Detected Recovery Mean Standard Deviation Relative

Standard Deviation Unit: mg/L Unit: mg/L % % % % Control A ND
- Control B ND
- 0.1 A 0.0913 91 0.1 B 0.0979 98 96 3.0 3.2 0.1 C 0.0983 98 0.1 D 0.0942 94 0.1 E 0.0969 97 30 A 28.2 94 30 B 29.9 100 98 2.7 2.7 30 C 30.0 100 30 D 29.0 97 30 E 29.9 100 Ov erall mean (%) 97 Overall standard deviation (%) 3.0 Overall relative standard deviation (%) 3.1 General Notes: ND

– Not detected (<LOD of 0.04 mg/L) Control aquatic media sample used

– 13/00/A2758 Data system: Analyst v.1.4.2Huntingdon Life Sciences: CEF0009

26 Figure 1 Ty pical standard chemical calibration for by

LC - MS/MS

cef0009_b4.rdb ( : "Linear" Regression ("No" weighting): y = 68349.6 x + 3423.15 (r = 0.9991629)

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 Concentration, ng/mL 0.0 5.0e4 1.0e5 1.5e5 2.0e5 2.5e5 3.0e5 3.5e5Area, countsHuntingdon Life Sciences: CEF0009

27 Figure 2 Ty pical chromatogram of a 0.1 ng/mL calibration

standard

Sample Name: "Cal 0.1 ng/mL" Sample ID: "" File: "CEF0009_B407.wiff" Peak Name: " Mass(es): "186.0/57.0 amu" Comment: "" Annotation: "" Sample Index: 1 Sample Type: Standard Concentration: 0.100 ng/mL Calculated Conc: 0.120 ng/mL Acq. Date: 20/08/2013 Acq. Time: 20:24:35 Modified: Yes Proc. Algorithm: IntelliQuan - IQA Min. Peak Height:1000.00 cps Min. Peak Width: 0.00 sec Smoothing Width: 9 points RT Window: 6.00 sec Expected RT: 1.46 min Use Relative RT: No Int. Type: Manual Retention Time: 1.46 min Area: 1.16562e+004 counts Height: 1.74e+003 cps Start Time: 1.33 min End Time: 1.67 min

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 Time, min 0.0 2000.0 4000.0 6000.0 8000.0 1.0e4 1.2e4 1.4e4 1.6e4 1.8e4 2.0e4 2.2e4 2.4e4 2.6e4 2.8e4 3.0e4 3.2e4 3.4e4 3.6e4 3.8e4 4.0e4 4.2e4 4.4e4 4.6e4 4.8e4 5.0e4 5.2e4Intensity, cps

1.46 1.51 1.70 1.88 5.40 4.34 2.05 5.71 5.03 4.04 1.08 4.45 0.32 2.91

0.88 4.91 3.02 0.75 7.68 2.82 6.13 1.15 6.46 6.62 3.69 5.79 2.59 5.20 3.26 7.42 0.06 7.35 6.88 7.88

Figure 3 Ty pical chromatogram of a 5 ng/mL calibration

standard

Sample Name: "Cal 5 ng/mL" Sample ID: "" File: "CEF0009_B414.wiff" Peak Name: " Mass(es): "186.0/57.0 amu" Comment: "" Annotation: "" Sample Index: 1 Sample Type: Standard Concentration: 5.00 ng/mL Calculated Conc: 5.04 ng/mL Acq. Date: 20/08/2013 Acq. Time: 21:16:35 Modified: Yes Proc. Algorithm: IntelliQuan - IQA Min. Peak Height:1000.00 cps Min. Peak Width: 0.00 sec Smoothing Width: 9 points RT Window: 6.00 sec Expected RT: 1.46 min Use Relative RT: No Int. Type: Manual Retention Time: 1.45 min Area: 3.47633e+005 counts Height: 5.23e+004 cps Start Time: 1.26 min End Time: 2.13 min

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 Time, min 0.0 2000.0 4000.0 6000.0 8000.0 1.0e4 1.2e4 1.4e4 1.6e4 1.8e4 2.0e4 2.2e4 2.4e4 2.6e4 2.8e4 3.0e4 3.2e4 3.4e4 3.6e4 3.8e4 4.0e4 4.2e4 4.4e4 4.6e4 4.8e4 5.0e4 5.2e4Intensity, cps 1.45

1.83 0.83 2.67 5.51 5.13 3.06 4.80 2.46 0.33 6.64 0.15 4.18 3.73 2.96 1.16 3.15 7.14 6.75 0.41 5.96 3.65 7.83 7.25 4.36 6.44 7.45 5.65Huntingdon Life Sciences: CEF0009

28 Figure 4 Chromatogram of an untreated Elendt M4 sample (analytical

identification 13/00/A3 279 Con)

Sample Name: "A3279 Con" Sample ID: "" File: "CEF0009_B413.wiff" Peak Name: " " Mass(es): "186.0/57.0 amu" Comment: "" Annotation: "" Sample Index: 1 Sample Type: Unknown Concentration: N/A Calculated Conc: 0.00 ng/mL Acq. Date: 20/08/2013 Acq. Time: 21:07:54 Modified: No

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 Time, min 0.0 2000.0 4000.0 6000.0 8000.0 1.0e4 1.2e4 1.4e4 1.6e4 1.8e4 2.0e4 2.2e4 2.4e4 2.6e4 2.8e4 3.0e4 3.2e4 3.4e4 3.6e4 3.8e4 4.0e4 4.2e4 4.4e4 4.6e4 4.8e4 5.0e4 5.2e4Intensity, cps

1.45 1.50 5.55 5.18 0.47 2.82 3.57 4.24 2.11 7.65 4.53 7.16 3.97 2.45 4.83 7.90 0.06 1.77 0.37 6.136.00

1.04 3.08

1.29 3.52 6.69 7.32 3.68 7.02 2.51 6.20 0.81 5.73 5.37 6.41

Figure 5 Chromatogram of an untreated Elendt M4 sample fortified with at 10 m g/L (analytical identification 13/00/A3 279 F 10 )

Sample Name: "A3279 F10" Sample ID: "" File: "CEF0009_B415.wiff" Peak Name: " Mass(es): "186.0/57.0 amu" Comment: "" Annotation: "" Sample Index: 1 Sample Type: Unknown Concentration: N/A Calculated Conc: 27.3 ng/mL Acq. Date: 20/08/2013 Acq. Time: 21:25:16 Modified: Yes Proc. Algorithm: IntelliQuan - IQA Min. Peak Height:1000.00 cps Min. Peak Width: 0.00 sec Smoothing Width: 9 points RT Window: 6.00 sec Expected RT: 1.46 min Use Relative RT: No Int. Type: Manual Retention Time: 1.47 min Area: 1.90161e+005 counts Height: 3.05e+004 cps Start Time: 1.34 min End Time: 1.81 min

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 Time, min 0.0 2000.0 4000.0 6000.0 8000.0 1.0e4 1.2e4 1.4e4 1.6e4 1.8e4 2.0e4 2.2e4 2.4e4 2.6e4 2.8e4 3.0e4 3.2e4 3.4e4 3.6e4 3.8e4 4.0e4 4.2e4 4.4e4 4.6e4 4.8e4 5.0e4 5.2e4Intensity, cps 1.47Huntingdon Life Sciences: CEF0009

29 Figure 6 Chromatogram of a test sample at 2.54 m g/L of on

Day 0 (analytical identification 13/ CEF /A3 344 )

Sample Name: "A3344" Sample ID: "" File: "CEF0009_B411.wiff" Peak Name: " " Mass(es): "186.0/57.0 amu" Comment: "" Annotation: "" Sample Index: 1 Sample Type: Unknown Concentration: N/A Calculated Conc: 5.76 ng/mL Acq. Date: 20/08/2013 Acq. Time: 20:50:34 Modified: No Proc. Algorithm: IntelliQuan - IQA Min. Peak Height:1000.00 cps Min. Peak Width: 0.00 sec Smoothing Width: 9 points RT Window: 6.00 sec Expected RT: 1.46 min Use Relative RT: No Int. Type: Valley Retention Time: 1.46 min Area: 2.00137e+005 counts Height: 3.18e+004 cps Start Time: 1.32 min End Time: 1.81 min

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 Time, min 0.0 2000.0 4000.0 6000.0 8000.0 1.0e4 1.2e4 1.4e4 1.6e4 1.8e4 2.0e4 2.2e4 2.4e4 2.6e4 2.8e4 3.0e4 3.2e4 3.4e4 3.6e4 3.8e4 4.0e4 4.2e4 4.4e4 4.6e4 4.8e4 5.0e4 5.2e4Intensity, cps 1.46

Figure 7 Chromatogram of a test sample at 2.54 m g/L of on

Day 2 (analytical identification 13/ CEF /A3 394 )

Sample Name: "A3394" Sample ID: "" File: "CEF0009_B518.wiff" Peak Name: " Mass(es): "186.0/57.0 amu" Comment: "" Annotation: "" Sample Index: 1 Sample Type: Unknown Concentration: N/A Calculated Conc: 5.49 ng/mL Acq. Date: 22/08/2013 Acq. Time: 18:05:33 Modified: No Proc. Algorithm: IntelliQuan - IQA Min. Peak Height:1000.00 cps Min. Peak Width: 0.00 sec Smoothing Width: 9 points RT Window: 6.00 sec Expected RT: 1.21 min Use Relative RT: No Int. Type: Valley Retention Time: 1.21 min Area: 2.11214e+005 counts Height: 3.20e+004 cps Start Time: 0.997 min End Time: 1.60 min

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 Time, min 0.0 2000.0 4000.0 6000.0 8000.0 1.0e4 1.2e4 1.4e4 1.6e4 1.8e4 2.0e4 2.2e4 2.4e4 2.6e4 2.8e4 3.0e4 3.2e4 3.4e4 3.6e4 3.8e4 4.0e4 4.2e4 4.4e4 4.6e4 4.8e4 5.0e4 5.2e4 5.4e4 5.6e4 5.8e4Intensity, cps 1.21

1.42Huntingdon Life Sciences: CEF0009

30 Appendix 4 Compliance statement