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Working for a better future

REPORT

: Acute Toxicity to Daphnia Magna

HLS study number: CEF0009

Version ID: Audited Draft 1 Report

Issue date: 30 September 2013
Huntingdon Life Sciences: CEF0009

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Sponsor and Test Facility Details

Sponsor

Test facility Huntingdon Life Sciences

Eye Research Centre

Eye

Suffolk

IP23 7PX

UK
Huntingdon Life Sciences: CEF0009

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Table of Contents

Sponsor and Test Facility Details.......................................................................................... 2

Compliance with Good 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 Validity 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 Tables

Table 1 Measured concentrations.............................................................................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 .................................................................................30
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Compliance with Good Laboratory Practice

: Acute Toxicity to Daphnia Magna

The study described in this report was conducted in compliance with the following Good 

Laboratory Practice standards and I consider the data generated to be valid.

The UK Good Laboratory Practice Regulations (Statutory Instrument 1999 No. 

3106, as amended by Statutory Instrument 2004 No. 994).

OECD Principles of Good Laboratory Practice (as revised in 1997), 

ENV/MC/CHEM (98) 17.

EC Commission Directive 2004/10/EC of 11 February 2004 (Official Journal No L 

50/44).

These principles of Good Laboratory Practice are accepted by the Regulatory Authorities of

the United States of America and Japan on the basis of Intergovernmental Agreements.

___________________________________________ ________________________

Romalie Allen BSc Date

Study Director

Huntingdon Life Sciences 
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Quality Assurance Statement

: Acute Toxicity to Daphnia Magna

The following inspections 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 Audit 23 Sep 2013 23 Sep 2013

Process based inspections: At or about the time this study was in progress inspections of

procedures employed on this type of study were carried out. These were conducted and 

reported to appropriate Company Management as indicated below:

 

Process Based Inspections Date(s) of Inspection Date of Reporting to 

Management

Environmental measurements & 

monitoring/Ammonia analysis 

07 Mar 2013 07 Mar 2013

Experimental Set-up 08 Aug 2013 09 Aug 2013

Sampling of test media for 

chemical analysis 

08 Aug 2013 09 Aug 2013

Dose Formulation 08 Aug 2013 16 Aug 2013

Study management & conduct 09 Aug 2013 16 Aug 2013

Observations 13 Sep 2013 13 Sep 2013

Sample Preparation (to include 

sample management & tracking)

27 Feb 2013 01 Mar 2013

Standard preparation & 

Fortification procedures 

05 Aug 2013-06 Aug 2013 07 Aug 2013

Results Processing 30 Aug 2013 30 Aug 2013

Chromatography 30 Aug 2013 30 Aug 2013

In addition, an inspection of the facility where this study was conducted was carried out on an 

annual basis. These inspections were reported to Company Management.

___________________________________________ ________________________

Tom Pullinger MRQA Date

Group Manager

Department of Quality Assurance

Huntingdon Life Sciences 
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Contributing Scientists

: Acute Toxicity to Daphnia Magna

Study management 

Aquatic Ecotoxicology and Biodegradation

Romalie Allen BSc

Study Director

Expert department

Environmental Analysis

Simon Tate Higher National Certificate (Applied Chemistry)

Senior Study Manager
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Summary

The acute toxicity of to Daphnia magna was assessed under static exposure 

conditions.

The study was conducted in accordance with EC Methods for Determination of Ecotoxicity, 

Annex to Commission Regulation (EC) No 440/2008 Part C, Method 2 “Daphnia sp. Acute 

Immobilisation Test” and the OECD Guideline for Testing of Chemicals No. 202, “Daphnia

Acute Immobilisation Test” (2004).

Groups of twenty Daphnia, less than 24 hours old, were exposed for 48 hours to , 

at nominal concentrations of 0.52, 1.15, 2.54, 5.58, 12.3 and 27.0 mg/L. The test media were 

prepared in Elendt M4 medium by the direct addition of the test substance to the dilution 

medium at nominally 27.0 mg/L and serial dilution thereafter. 

At all concentrations, the intended exposure levels of were adequately achieved 

(between 82 and 96% of their nominal values) and maintained during the test (between 90 

and 96% of their starting values). Based on an arithmetic mean, the 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.

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

After 48 hours no immobilisation was observed.

Based on these findings, the 48-hour EC50 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 concentration employed in the study.
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1. Introduction

The objective of the study was to determine the acute toxicity (48 hour median effect 

concentration - EC50) of to Daphnia magna.

The study was conducted in accordance with EC Methods for Determination of Ecotoxicity, 

Annex to Commission Regulation (EC) No 440/2008 Part C, Method 2 “Daphnia sp. Acute 

Immobilisation Test” and the OECD Guideline for Testing of Chemicals No. 202, “Daphnia

Acute Immobilisation Test” (2004).

The protocol was approved by Huntingdon Life Sciences Management and the Study

Director on 17 June 2013.

The experimental start and completion 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, Suffolk, 

IP23 7PX, England.

A separate physiochemical study to determine the properties of (HLS study: 

CEF0001) indicated that the limit of solubility of the test substance was 27.5 mg/L at 20oC.
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2. Test Substance

Name

Chemical name

CAS No.

Physical state Powder

Batch number 12342

Purity To be included 

Storage Room temperature in the dark

Expiry Date To be included 

Date received: 4 March 2013

Certificate of analysis: Appendix 1
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3. Experimental Procedure

3.1 Test organism

Daphnia magna (Straus) used in this study were cultured in-house and were obtained from a 

strain originating from the National Institute for Applied Chemical Research (IRCHA), 

France.

Stock cultures of Daphnia magna were maintained in glass vessels containing approximately 

0.5 to 0.8 litres of Elendt M4 culture medium in a temperature-controlled laboratory at 

nominally 20  2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. The culture 

medium was renewed three times each week.

Cultures were fed daily with a suspension of the unicellular green algae, Pseudokirchneriella 

subcapitata, to provide nominally 0.1 to 0.2 mg carbon per daphnid, per day, except during 

the initial three days when a slightly lower ration was given. Culture conditions ensure that 

the stock animals reproduce by parthenogenesis.

The day before the start of the study, all juvenile Daphnia were removed from the laboratory

cultures. The following morning, juveniles produced by the gravid (egg-bearing) adult 

Daphnia were removed from the culture 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 organisms were maintained and the tests conducted in Elendt M4 medium 

(Appendix 2). The medium was prepared in deionised, reverse osmosis water.

3.3 Test substance preparation

The method of preparation used during the definitive 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 

with the dropwise addition of Elendt M4 medium. The paste was then added to a volumetric 

flask (2 L) containing Elendt M4 medium (1700 mL), which was then shaken before being 

made up to volume and stirred for ca. one hour until no particulate matter remained. The 

preparation was then filtered (0.45 μm pore size) to provide an aqueous solution with a 

nominal concentration of 27.0 mg/L; this was either used directly at the highest test 

concentration or aliquots (454, 454, 455, 453 and 452 mL) were serially diluted in volumetric 

flasks (1 L) containing Elendt M4 medium to provide test media with nominal concentrations 

of 12.3, 5.58, 2.54, 1.15 or 0.52 mg/L.

3.4 Exposure conditions

3.4.1 Experimental design

A range finding test was followed by a definitive test with six test concentrations, plus a 

dilution medium control group.
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Twenty Daphnia, four replicates of five animals per vessel, were exposed in each control and 

test group.

The first instar Daphnia were placed in groups of five, at random into 120 mL glass dishes 

containing 120 mL of medium to give a loading of 24 mL medium per organism. The dishes 

were loosely covered. 

3.4.2 Test concentrations

The range finding test employed nominal test concentrations of 0.1, 1, 10 and 25 mg/L. After 

48 hours, there was 90% immobilisation of the Daphnia at 25 mg/L, and 50% at 10 mg/L, 

with no immobilisation observed as the other test groups. Based on these results, the 

definitive test employed nominal concentrations of 0.52, 1.15, 2.54, 5.58, 12.3 and 

27.0 mg/L; the highest concentration employed approximated to the limit of solubility for the 

test substance.

3.4.3 Medium renewal

Daphnia were exposed to the test or control conditions for a period of 48 hours without 

renewal of test media.

3.4.4 Stability of test concentrations

The test concentrations of were measured using liquid chromatography with

tandem mass spectrometric detection (LC-MS/MS, see Appendix 3). At the start of the 

definitive test, two samples (10 mL) were taken from the freshly-prepared control and test 

media and each 10 mL sample was added to a vial containing water:acetonitrile:formic acid 

(95:5:0.1). After 48 hours, the contents of the test vessels from each group were pooled and 

further samples were taken for analysis. 

On each occasion, 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 definitive test, continuous monitoring of the medium in an additional vessel 

indicated that temperatures ranged between 20.0 and 20.7C, confirming that the test area 

was maintained to within  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 employed and no feed was given during the exposure period.

The temperature, pH and dissolved oxygen levels of control and test media were recorded at 

the start and at the end of the study. The total hardness and alkalinity of the dilution medium 

was measured before use.

3.5 Criterion of effect

Daphnia were considered to be immobile if they were unable to swim within approximately

15 seconds following gentle agitation of the test vessel. 
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The numbers of mobile, immobile and floating Daphnia were counted approximately 24 and 

48 hours after the start of the study.

3.6 Evaluation of data

The “no observed effect concentration” (NOEC) was derived by direct inspection of the data 

on the immobility of the animals. An incidence rate of more than 10% was considered to be 

significant.

3.7 Validity criteria

For the study to be valid, the number of floating and/or immobile Daphnia in the control 

group must not be exceed 10% at the end of the test and the concentrations of dissolved 

oxygen must be > 3 mg/L at the end of the test.

3.8 Protocol deviations

There were no protocol deviations.
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4. Maintenance of Records

This report has been compiled from original data, which will be stored, together with a copy

of the final report, a copy of the protocol and amendments in Huntingdon Life Sciences’ 

Archives, Huntingdon Life Sciences Ltd, Eye, Suffolk, UK.

Records will be retained for a minimum period of at least one year from the date of issue of 

the final report. At the end of the one year retention period the Sponsor will be contacted and 

advice sought on the future requirements. Under no circumstances will any item be discarded 

without the Sponsor’s knowledge.

Huntingdon Life Sciences will retain the Quality Assurance records relevant to this study for 

twenty years, and a copy of the final report in its archives indefinitely.
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5. Results

5.1 Chemical analysis 

The results of chemical analysis are given in Table 1 and example chromatograms are 

illustrated in Appendix 3. Concentrations are expressed in terms of the measured levels of 

At all concentrations, the intended exposure levels of were adequately achieved 

(between 82 and 96% of their nominal values) and maintained during the test (between 90 

and 96% of their starting values). Based on an arithmetic mean, the 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 immobilisation or adverse effects on the daphnia were observed.

Based on these findings, the 48-hour EC50 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 concentration employed in the test. 

5.3 Environmental parameters

The measurements of water quality (temperature, pH, dissolved oxygen, total hardness and 

alkalinity) are summarised in Table 2; they remained within acceptable limits during the 

study. 

At the start and end of the test, the test media were colourless at all concentrations.
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6. Conclusions

 was found not to be acutely toxic to Daphnia magna when dissolved in water to a 

maximum measured concentration of 24.8 mg/L under the conditions of this study. Thus the 

48-hour EC50 could not be calculated and must be greater than 24.8 mg/L and the noobserved-effect concentration’ (NOEC) must be at least 24.8 mg/L the maximum employed 

concentration in the test.
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Table 1 Measured concentrations

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 None 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 Mean measured concentration after 48 hours expressed as a percentage of the mean starting 

concentration

* Arithmetic mean
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Table 2 Environmental parameters

Exposure conc. 

(mg/L) Temperature (°C) pH Dissolved oxygen(%ASV)

Nominal Measured 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 None detected (less than the limit of detection of 0.04 mg/L)

ASV Air saturation value (100% ASV = 9.09 mgO2/L at 20oC).

The total hardness and alkalinity of the batch of Elendt M4 medium used were 245 and 60 mg/L as CaCO3, 

respectively.

Continuous monitoring of an additional vessel containing dilution medium = 20.0 to 20.7°C.
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Appendix 1 Certificate of analysis

To be included when supplied
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Appendix 2 Elendt M4 medium

1. Micro elements mg/L

H3BO3 2.86

MnCl2.4H2O 0.36

LiCl 0.31

RbCl 0.071

SrCl2.6H2O 0.152

NaBr 0.016

Na2MoO4.2H2O 0.063

CuCl2.2H2O 0.017

ZnCl2 0.013

CoCl2.6H2O 0.010

KI 0.0033

Na2.SeO3 0.0022

NH4VO3 0.00058

Fe-EDTA solution 3.50

2. Macro nutrients mg/L

CaCl2.2H2O 294

MgSO4.7H2O 123

KCl 5.80

NaHCO3 64.8

3. Buffer nutrients mg/L

Na2.SiO3.9H2O 10.0

NaNO3 0.274

KH2PO4 0.143

K2HPO4 0.184

4. Vitamins mg/L

Thiamine hydrochloride 0.075

Cyanocobalamine (B12) 0.0010

Biotin 0.00075

The above analytical grade reagents are dissolved in deionised, reverse osmosis water.
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Appendix 3 The determination of in Elendt M4 medium

1. Method

1.1 General principle

Test solutions were diluted with water:acetonitrile:formic acid (95:5:0.1 v:v:v) prior to 

quantitation by liquid chromatography with tandem mass 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 solutions used in this study have been shared with other GLP studies being 

performed for the same sponsor. This is considered to have no impact on the integrity or 

validity of this study.

A weighed amount of was dissolved in acetonitrile to produce a 1 mg/mL stock 

standard solution. The stock standard was diluted progressively with acetonitrile to give 

fortification standard solutions. The appropriate fortification standard solution was further 

diluted with water:acetonitrile:formic acid (95:5:0.1 v:v:v) to produce a series of calibration 

solutions in the range 0.1 to 5 ng/mL.

1.1.2 Typical preparation of reagents

Preparation of water:acetonitrile:formic acid (95:5:0.1 v:v:v):

Acetonitrile (50 mL) and formic acid (1 mL) were thoroughly mixed with water (950 mL) 

prior to use.

Preparation of water:methanol:formic acid (90:10:0.1 v:v:v) + 0.01M ammonium formate:

Water (900 mL) was thoroughly mixed with methanol (100 mL), formic acid (1 mL) and 

ammonium formate (0.63 g) prior to use.

Preparation of 0.1% formic acid in methanol:

Methanol (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 

components was maintained.
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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 with water:acetonitrile:formic acid (95:5:0.1 v:v:v). 

Any further dilutions, where required, were performed with water:acetonitrile:formic acid 

(95:5:0.1 v:v:v) prior to quantitation by LC-MS/MS.

1.2.1 Instrumentation

LC-MS/MS conditions

Instrument: AB Sciex API 4000

Data system: Analyst v.1.4.2

Ionisation mode: Ionspray positive

Ion monitoring details: MRM m/z 186>57

Column: Phenomenex aqua C18 (5 cm  2 mm)

Mobile phase A: Water:methanol:formic acid (90:10:0.1 v:v:v) + 0.01M 

ammonium formate

Mobile phase B: Methanol + 0.1% formic acid (by volume)

Pump programme: 60:40 A:B isocratic

Cycle time: 8 minutes

Flow rate: 0.3 mL/min

Injection volume: 20 μL

Retention time: Approximately 1.5 minutes

LOQ: 0.1 mg/L

LOD: 0.1 ng/mL (equivalent to 0.04 mg/L in aqueous media)
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1.2.2 Calculation of results

Test samples were quantified using the following equation:

Residue found (mg/L) = 

1000

1

 x D x 

M

1

x x 

Where x (residue concentration in final solution) was calculated using the linear regression:

y = m x + c where x (concentration in ng/mL) = 

y  c

m

c = intercept

m = slope

y = peak area of sample

M = matrix concentration (mL/mL)

D = dilution factor
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2.0 Results and Discussion

2.1 Calibration

The chromatographic response to was shown to be linear over the range of 

concentrations 0.1 to 5 ng/mL. Typical calibration data is presented in Table 1 and Figure 1. 

Typical chromatograms of calibration standards are presented in Figures 2 and 3.

2.2 Validation

The methodology for the determination of residues in Elendt M4 was validated at 

0.1 mg/L and 30 mg/L. The validation data are presented in Table 2 and summarised in the 

following table:

Fortification level

(mg/L) Mean recovery (%) CV (%)

0.1 96 3.2

30 98 2.7

(97) (3.1)

( ) Overall values

2.3 Limit of quantitation (LOQ)

The limit of quantitation is defined as the lowest fortification level at which acceptable 

recovery data are obtained. The validation of the methodology for the determination of 

residues of in Elendt M4, demonstrated that it can be accurately determined at a 

limit of quantitation (LOQ) of 0.1 mg/L.

2.4 Limit of detection (LOD)

The limit of detection of the method is defined as the value of the lowest calibration standard 

chromatographed that gave a peak height to baseline noise ratio 3. For this study the limit 

of detection was 0.1 ng/mL (equivalent to 0.04 mg/L in Elendt M4).

2.5 Samples

Samples of Elendt M4 from the test were analysed in suitably sized batches along with 

control samples fortified with which acted as procedural recovery samples. 

Procedural recoveries performed on control Elendt M4 were in the range 70 to 110% 

confirming the validity of the methodology on each day of analysis. Typical chromatograms 

of untreated, untreated fortified with and treated samples are presented in 

Figures 4 to 7.
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Table 1 Typical 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 = 0.9992

Where x = concentration

y = peak area

r = regression coefficient
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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

Overall 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.1.4.2
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Figure 1 Typical 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

3.5e5

Area, counts
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Figure 2 Typical 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

5.2e4

Intensity, cps

1.46

1.51

0.06 0.32 0.75 0.88 1.08 1.15 1.70 1.882.05 2.59 2.82 2.91 3.02 3.26 3.69 4.04 4.34 4.45 4.91 5.035.20 5.40 5.71 5.79 6.13 6.46 6.62 6.88 7.35 7.42 7.68 7.88

Figure 3 Typical 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

5.2e4

Intensity, cps

1.45

1.83 0.15 0.33 0.41 0.83 1.16 2.46 2.672.96 3.06 3.15 3.65 3.73 4.18 4.36 4.80 5.13 5.51 5.65 5.96 6.44 6.64 6.75 7.14 7.25 7.45 7.83
Huntingdon Life Sciences: CEF0009

28

Figure 4 Chromatogram of an untreated Elendt M4 sample (analytical 

identification 13/00/A3279 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

5.2e4

Intensity, cps

0.06 0.37 0.47 0.81 1.04 1.291.451.50 1.77 2.11 2.45 2.51 2.82 3.08 3.523.57 3.68 3.97 4.24 4.53 4.83 5.18 5.37 5.55 5.73 6.136.00 6.20 6.416.69 7.02 7.16 7.32 7.65 7.90

Figure 5 Chromatogram of an untreated Elendt M4 sample fortified with 

at 10 mg/L (analytical identification 13/00/A3279 F10)

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

5.2e4

Intensity, cps

1.47
Huntingdon Life Sciences: CEF0009

29

Figure 6 Chromatogram of a test sample at 2.54 mg/L of on 

Day 0 (analytical identification 13/CEF/A3344)

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

5.2e4

Intensity, cps

1.46

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

Day 2 (analytical identification 13/CEF/A3394)

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

5.8e4

Intensity, cps

1.21

1.42
Huntingdon Life Sciences: CEF0009

30

Appendix 4 Compliance statement