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STUDY SPONSOR:

41204330.doc/JS ACUTE ORALTOXICITY IN THE RAT

- FIXED DOSE METHOD PROJECT NUMBER: 41204330

AUTHOR:

TEST FACILITY: A Pooles

Harlan Laboratories Ltd Shardlow Business Park Shardlow Derbyshire DE72 2GD UK Telephone: +44 (0) 1332 792896 Facsimile: +44 (0) 1332 799018PROJECT NUMBER: 41204330 PAGE2 QUALITYASSURANCE REPORT This study type is classed as short-term. Inspection of the routine and repetitive procedures that constitute the study is carried out as a continuous process designed to encompassthe majorphases ofthis studytype at leastonce everythree months. In addition, general facilities are inspected at least once a year and the results are reported to management. This report has been audited by the Quality Assurance Unit, and is considered to be an accurate account ofthe data generated and ofthe proceduresfollowed. In each case, the outcome of QA evaluation is reported to the Study Director and Management on the day of evaluation. Audits of study documentation, and process inspections appropriatetothetype and schedule ofthis studywere as follows: 14August 2012 Study Plan ComplianceAudit 03 July2012 Test Item Preparation 05 July2012 Animal Preparation 05 July2012 Dosing 05 July2012 Assessment of Response 11 July2012 Necropsy 

### § 26 October2012 Draft ReportAudit 

### § Date ofQASignature Final ReportAudit 

### § Evaluation specifictothis study

# ~-.h.~~~---·················· 14NOV 2012 DATE:
................................................ Forthe QualityAssurance Unit*

*Authorised QASignatures: SeniorAudit Staff: J G Riley BSc (Hons) MRQA, J M Crowther MIScT MRQA, GWren ONC MRQA, S Bevan BSc (Hons) MRQA, L Blaney MRQAPROJECT NUMBER: 41204330 PAGE3
GLP COMPLIANCE STATEMENT 

### With the exception noted below the work described was performed in compliance with UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI1999/3106 as amended by Sl2004/0994)). These Regulations are in accordance with GLP standards published as OECD Principles on Good Laboratory Practice (revised 1997, ENV/MC/CHEM(98)17); and are in accordance with, and implement, the requirements ofDirectives 2004/9/EC and 2004/10/EC. No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within two hours ofbeing applied to the test system; it is assumed that the formulation was stable for this duration. This exception is considered notto affectthe purpose or integrityofthe study. This reportfully and accurately reflects the procedures used and data generated
.



# ...................A.:
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# PC?<?.~...................................... DATE: .........•.

# ~.\'.'.\.~............................ A Pooles BA (Hons) Study Director

This report may be presented in final form as a digital (pdf} document. Such documents are prepared byscanning the paperoriginal,

and are considered ofequivalent integrityand authenticityto versions produced by optical photocopy. However, in all casesthe hand-signed paperoriginal, held in secure archives, is thedefinitive document.PROJECT NUMBER: 41204330 PAGE4 

### CONTENTS QUALITYASSURANCE REPORT 2 GLP COMPLIANCE STATEMENT 3 CONTENTS 4 SUMMARY 5 1. INTRODUCTION 7 2. TEST ITEM AND EXPERIMENTAL PREPARATION 7 2.1 Description, Identification and Storage Conditions 7 2.2 Experimental Preparation 8 

### 3. METHODS 8 3.1 Animals and Animal Husbandry 8 3.2 Procedure 9 3.3 Evaluation ofData 10 

### 4. ARCHIVES 10 5. RESULTS 11 5.1 Dose Level- 3334 mg/kg (equivalentto 2000 mg active ingredient/kg bodyweight) 11 5.2 Dose Level- 500 mg/kg (equivalentto 300 mg active ingredient/kg bodyweight) 11 

### 6. CONCLUSION 12 Table 1 Individual Clinical Observations and Mortality Data- 3334 mg/kg 13 Table 2 Individual Bodyweights and Bodyweight Changes- 3334 mg/kg 14 Table 3 Necropsy Findings- 3334 mg/kg 15 Table 4 Individual Clinical Observations and Mortality Data- 500 mg/kg 16 Table 5 Individual Bodyweights and Bodyweight Changes- 500 mg/kg 17 Table 6 Individual Necropsy Findings- 500 mg/kg 18 Appendix 1 FlowChartforthe Sighting Test 19 Appendix 2 Flow Chartforthe Main Test 20 Appendix 3 Statement ofGLP Compliance in Accordance with Directive 2004/9/EC 21PROJECT NUMBER: 41204330

ACUTE ORALTOXICITY IN THE RAT

- FIXED DOSE METHOD SUMMARY PAGES

Introduction. The study was performed to assess the acute oral toxicity ofthe test item in theWistarstrain rat. The method was designed to be compatible with the following:

• OECD Guidelines for Testing of Chemicals No 420 "Acute Oral Toxicity- Fixed Dose Method" (2001)

• Method 81 

### bisAcute Toxicity (Oral) ofCommission Regulation (EC) No. 440/2008 Method. Following a sighting test at dose levels of 3334 mg/kg and 500 mg/kg (equivalent to 2000 and 300 mg active ingredient/kg bodyweight, respectively), a further group of four fasted females was given a single oral dose of test item, as a solution in distilled water, at a dose level of 500 mg/kg bodyweight. Clinical signs and bodyweight development were monitored during the study. All animals were subjected to gross necropsy. Mortality. The animal treated at a dose level of 3334 mg/kg (equivalent to 2000 mg active ingredient/kg bodyweight) was killed for humane reasons, four hours after dosing, dueto the occurrence ofclinical signs oftoxicity that exceeded the severity limit setforth in the UK Home Office Project Licence. There were no deaths at a dose level of 500 mg/kg (equivalentto 300 mg active ingredient/kg bodyweight). Clinical Observations. Signs ofsystemic toxicity noted in the animal treated at a dose level of 3334 mg/kg (equivalent to 2000 mg active ingredient/kg bodyweight) were hunched posture, ataxia, lethargy, prostration, decreased respiratory rate, laboured respiration, hypothermia, pallor ofthe extremities, loss of righting reflex and emaciation. There were no signs of systemic toxicity at a dose level of 500 mg/kg (equivalent to 300 mg active ingredient/kg bodyweight). Bodyweight. Surviving animals showed expected gains in bodyweight.PROJECT NUMBER: 41204330 PAGES 

### Necropsy. Abnormalities noted at necropsy of the animal treated at a dose level of 3334 mg/kg (equivalent to 2000 mg active ingredient/kg bodyweight) were pale kidneys, coloured liquid present and raised limiting ridge in the stomach. No abnormalities were noted at necropsy ofanimals treated at a dose level of500 mg/kg (equivalent to 300 mg active ingredient/kg bodyweight). 

### Conclusion. The acute oral median lethal dose (LD50) of the test item in the female Wistar strain rat was estimated to be in the range of500 - 3334 mg/kg bodyweight (equivalent to 300-2000 mg active ingredient/kg bodyweight) (Globally Harmonised Classification System- Category4).PROJECT NUMBER: 41204330



### ACUTE ORALTOXICITY IN THE RAT

- FIXED DOSE METHOD 1. INTRODUCTION PAGE?

The study was performed to assess the acute oral toxicity ofthe test item in the Wistar strain rat. The method was designed to be compatible with thefollowing:
• OECD Guidelines for Testing of Chemicals No 420 "Acute Oral Toxicity - Fixed Dose Method" (2001)
• Method 81 

### bisAcuteToxicity (Oral) ofCommission Regulation (EC) No. 440/2008 The test system was chosen because the rat has been shown to be a suitable model for this type ofstudy and is recommended in the test method. The results ofthe study are believed to be ofvalue in predicting the likelytoxicity ofthetest item to man. The studywas performed between 04 September2012 and 11 October2012. 

### 2. TEST ITEM AND EXPERIMENTAL PREPARATION 2.1 Description, Identification and StorageConditions Sponsor's identification Description Batch number Purity Date received Expirydate Storage conditions The integrity ofsupplied data relating to the identity, purity and stability ofthe test item is the responsibility ofthe Sponsor.PROJECT NUMBER: 41204330 PAGES 

### 2.2 Experimental Preparation Forthe purpose ofthe studythe test item formulations were adjusted forthe purity ofthe test item (60%). For the 3334 mg/kg (equivalent to 2000 mg active ingredient/kg bodyweight) dose level the test item was used as supplied. The specific gravity was determined and used to calculate the appropriate dose volume for the required dose level. For the 500 mg/kg (equivalent to 300 mg active ingredient/kg bodyweight) dose level thetest item was freshly prepared, as required, as asolution in distilled water. The test item was formulated within two hours of being applied to the test system. It is assumed thattheformulation was stableforthis duration. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement. 

### 3. METHODS 3.1 Animals andAnimal Husbandry Female Wistar (RccHanTM:WIST) strain rats were supplied by Harlan Laboratories UK Ltd., Oxon, UK. On receipt the animals were randomly allocated to cages. The females were nulliparous and non-pregnant. After an acclimatisation period ofat least five days the animals were selected at random and given a number unique within the study by indelible ink-marking on the tail and a numberwritten on a cage card. Atthe start ofthe studythe animals were eight to twelve weeks ofage. The bodyweight variation did not exceed ±20% ofthe bodyweight ofthe initially dosed animal. The animals were housed in groups of up to four in suspended solid-floor polypropylene cages furnished with woodflakes. With the exception of an overnight fast immediately before dosing and for approximately three to four hours after dosing, free access to mains drinking water and food (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study. The diet, drinking water and bedding were routinely analysed and were considered not to contain any contaminants that would reasonably be expected to affect the purpose or integrity ofthe study. The temperature and relative humiditywere set to achieve limits of 19 to 25°C and 30 to 70% respectively. Any occasional deviations from these targets were considered not toPROJECT NUMBER: 41204330 PAGE9 have affected the purpose or integrity ofthe study. The rate ofairexchangewas at least fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness. The animals were provided with environmental enrichment items which were considered notto contain any contaminant ofa level that might have affected the purpose or integrity ofthe study. 

### 3.2 Procedure In the absence ofdata regarding the toxicity ofthe test item, 500 mg/kg was chosen as the starting dose. A single animal was treated as follows: Dose Level Concentration Dose Volume Numberof Rats

(mg/kg) (mg/ml) (ml/kg) Female

500® 50 10 1 In the absence of toxicity at a dose level of 500 mg/kg (equivalent to 300 mg active ingredient/kg bodyweight), an additional animal was treated as follows: Dose Level Dose Volume Numberof Rats

(mg/kg) Specific Gravity (ml/kg) Female 3334E9 1.132 2.95 1 Due to mortality at a dose level of 3334 mg/kg (equivalent to 2000 mg active ingredient/kg bodyweight), an additional group ofanimalswastreated as follows: Dose Level Concentration DoseVolume NumberofRats

(mg/kg) (mg/ml) (ml/kg) Female

500® 50 10 4 A total offive animalswere thereforetreated at a dose level of500 mg/kg in the study.

®

## = Equivalentto 300 mg active ingredient/kg bodyweight E9 

## = Equivalentto 2000 mg active ingredient/kg bodyweightPROJECT NUMBER: 41204330 PAGE10 All animals were dosed once only by gavage, using a metal cannula attached to a graduated syringe. The volume administered to each animal was calculated according to the fasted bodyweight at the time of dosing. Treatment of animals was sequential. Sufficient time was allowed between each dose level to confirm the survival of the previously dosed animals. Clinical observations were made %, 1, 2, and 4 hours after dosing and then daily for up to fourteen days. Morbidity and mortality checks were madetwice daily. Individual bodyweights were recorded on Day 0 (the day of dosing) and on Days 7 and 

### 14 orat death. At the end of the observation period the surv1v1ng animals were killed by cervical dislocation. All animals were subjected to gross necropsy. This consisted ofan external examination and opening ofthe abdominal and thoracic cavities. The appearance ofany macroscopic abnormalities was recorded. No tissues were retained. 

### 3.3 Evaluation ofData The test item will be classified according to Annex 3 ofthe OECD Guidelines for Testing ofChemicals No. 

### 420 "Acute Oral Toxicity- Fixed Dose Method" (adopted 

### 17 December 

### 2001) as shown in the Flow Chart in Appendix

### 2. Evaluation of data included identification ofthe number of animals that died during the study (orthat were killed for humane reasons), and determination ofthe nature, severity, onset and duration of the toxic effects. If possible, the signs of evident toxicity were described. Evident toxicity refers to the toxic effects of sufficient severity that administration ofthe next higher dose level could result in development of severe signs of toxicity and probable mortality. Effects on bodyweights and abnormalities noted at necropsywere also identified. Using the mortality data obtained, an estimate of the acute oral median lethal dose (LD5o) ofthetest item was made. 4. ARCHIVES Unless instructed otherwise by the Sponsor, all original data and the final report will be retained in the Harlan Laboratories Ltd, Shardlow, UK archives forfive years, afterwhich instructions will be sought as to further retention ordisposal.PROJECT NUMBER: 41204330 PAGE 11 

## 5. RESULTS 5.1 Dose Level - 3334 mg/kg (equivalentto2000 mgactive ingredient/kg bodyweight) Individual clinical observations and mortalitydata are given in Table 1. 

### 5.1.1 Mortality The animal was killed for humane reasons, four hours after dosing, due to the occurrence ofclinical signs oftoxicity that exceeded the severity limit set forth in the UK Home Office Project Licence. 

### 5.1.2 Clinical Observations Signs of systemic toxicity noted were hunched posture, ataxia, lethargy, prostration, decreased respiratory rate, laboured respiration, hypothermia, pallor ofthe extremities, emaciation and loss ofrighting reflex. 

### 5.1.3 Bodyweight The bodyweight on Day 0and atdeath are given in Table 2. 

### 5.1.4 Necropsy Necropsyfindings are given in Table 3. Abnormalities noted at necropsy were pale kidneys, coloured liquid present and raised limiting ridge in the stomach. 

### 5.2 Dose Level - 500 mg/kg (equivalentto300 mg active ingredient/kg bodyweight) Based on the results at a dose level of 3334 mg/kg (equivalent to 2000 mg active ingredient/kg bodyweight), a dose level of 500 mg/kg bodyweight (equivalent to 300 mg active ingredient/kg bodyweight) was investigated. Individual clinical observations and mortalitydata are given in Table4.PROJECT NUMBER: 41204330 PAGE12 

### 5.2.1 Mortality Therewere no deaths. 5.2.2 Clinical Observations No signs ofsystemictoxicitywere noted during the observation period. 5.2.3 Bodyweight Individual bodyweights and bodyweight changes are given in Table 5. All animals showed expected gains in bodyweight overthe observation period. 5.2.4 Necropsy Individual necropsyfindings are given in Table 6. No abnormalitieswere noted at necropsy. 6. CONCLUSION The acute oral median lethal dose (LD50) 

### ofthe test item in the female Wistar strain rat was estimated to be in the range of500- 3334 mg/kg bodyweight (equivalent to 300-2000 mg active ingredient/kg bodyweight) (Globally Harmonised Classification System- Category4).ACUTE ORAL TOXICITY IN THE RAT- FIXED DOSE METHOD Table 1 Individual Clinical Observations and Mortality Data - 3334 mg/kge Effects Noted After Dosing Effects Noted During Period After Dosing Dose Animal (Hours) (Days) Level Number mg/kg and Sex % 1 2 4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 3334$ 2-0 0 H HAL PrRdRIHo Female EmERrX*

EB = Equivalentto 2000 mg active ingredient/kg bodyweight 0= No signs ofsystemictoxicity H = Hunched posture A= Ataxia L = Lethargy Pr= Prostration Rd = Decreased respiratory rate Rl = Laboured respiration Ho = Hypothermia E = Pallorofthe extremities Em = Emaciation Rr = Loss of righting reflex X*= Animal killed for humane reasons due to the occurrence ofclinical signs oftoxicity that exceeded the severity limit set forth in the UK Home Office Pro·ect Licence PROJECT NUMBER: 41204330 PAGE13ACUTE ORALTOXICITY IN THE RAT- FIXED DOSE METHOD 

### Table 2 Individual Bodyweightsand BodyweightChanges - 3334 mg/kgEB
Dose Level Animal Number Bodyweight (g) at Day Bodyweight (g) mg/kg and Sex 0 7 14 at Death 3334EB 2-0 Female 165 

## - - 153

ED= Equivalentto 2000 mg active ingredient/kg bodyweight Animal dead PROJECT NUMBER: 41204330 Bodyweight Gain (g) During Week 1 2



## - -

PAGE14ACUTE ORALTOXICITY IN THE RAT- FIXED DOSE METHOD Table3 Necropsy Findings - 3334 mg/kg(D Dose Level Animal Number Time of Death Macroscopic Observations mg/kg and Sex Kidneys: pale 3334$ 2-0 Female Humanely killed Day 0 Stomach: colourless liquid present raised limiting ridge

EB = Equivalentto 2000 mg active ingredient/kg bodyweight PROJECT NUMBER: 41204330 PAGE15ACUTE ORALTOXICITY IN THE RAT- FIXED DOSE METHOD 

### Table 4 Individual Clinical Observationsand Mortality Data - 500 mg/kg® Dose Animal Effects Noted After Dosing Effects Noted During Period After Dosing Level Number (Hours) (Days) mg/kg and Sex**~** 1 2 4 1 

### 2 3 4 5 6 7 8 9 10 11 12 13 14 1-0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Female

3-0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Female 500® 3-1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Female

3-2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Female

3-3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Female

®

## = Equivalentto 300 mg active ingredient/kg bodyweight 0= No signs ofsystemictoxicity PROJECT NUMBER: 41204330 PAGE16ACUTE ORALTOXICITY IN THE RAT- FIXED DOSE METHOD 

### Table 5 Individual Bodyweightsand BodyweightChanges - 500 mg/kg®
Dose Level Animal Number Bodyweight (g) at Day BodyweightGain (g) During Week mg/kg and Sex 0 7 14 1 2 1-0 Female 164 186 196 22 10 3-0 Female 152 169 183 17 14 500® 3-1 Female 162 186 207 24 21 3-2 Female 164 185 202 21 17 3-3 Female 152 176 190 24 14

® = Equivalentto 300 mg active ingredient/kg bodyweight PROJECT NUMBER: 41204330 PAGE17ACUTE ORALTOXICITY IN THE RAT- FIXED DOSE METHOD 

### Table 6 Individual Necropsy Findings - 500 mg/kg® Dose Level Animal Number Time of Death MacroscopicObservations mg/kg and Sex 1-0 Female Killed Day 14 Noabnormalities detected 3-0 Female Killed Day 14 No abnormalities detected 500® 3-1 Female Killed Day 14 No abnormalities detected 3-2 Female Killed Day 14 No abnormalities detected 3-3 Female Killed Day 14 No abnormalities detected

®

## = Equivalentto 300 mg active ingredienUkg bodyweight

PROJECT NUMBER: 41204330 PAGE18Appendix 1 ACUTE ORALTOXICITY IN THE RAT- FIXED DOSE METHOD Flow Chartforthe Sighting Test

Evidenttoxicity No deaths orevidenttoxicity • *foroutcome**~** at 5 mg/kgthere is an optional supplementary procedureto confirm the GHS classification

• outcome. differsfrom the OECD guidelinewhich states 'NoToxicity'. This has been amended to clarifythe dosing procedure intended in the guideline
PROJECT NUMBER: 41204330 PAGE19Appendix 2 ACUTE ORALTOXICITY IN THE RAT- FIXED DOSE METHOD Flow Chartforthe Main Test**~2** deaths**~1** with evidenttoxicityand/or :51 death No deaths orevidenttoxicity • Group size The 5 animals in each main study groupwill include any animal tested atthat dose level in the sighting study *Animal welfare override Ifthis dose level caused death in the sighting study, then no further animalswill be tested. Go directlyto**outcome~** • outcome. differsfrom the OECD guidelinewhich states 'No Toxicity'. This has been amended to clarifythe dosing procedure intended in the guideline PROJECT NUMBER: 41204330 PAGE 20PROJECT NUMBER: 41204330 PAGE 21 

### Appendix 3 Statement of GLP Compliance in Accordance with Directive 2004/9/EC

OFINSPECTION 10JW,2012**Ani~rora:m~~ ~rii'IAd**  out**~t COO'Ii)l~l~**  Monitoring Pl'rn-J"i'U'li"'A**~uaim~** Cbemh*y**~~Toxic~** l!nvnmrmmtal Fa**M~tdty Ph~Tutm**  Toxk;,olO&J

Thl!s**$m~t ~**  tl'l-, oo I»**et~&** of inoo,**tl'l~** Good**ll~ Pm~**  Morlitcmng**tn~t ~** **~~** t"t**t~c~iity** M!ifll nMmtirlt1in Pm:at$0@,**~li~.~Pm~I~(&~I:W~rtN G~l!&~** **~~$1~).**