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### REPORT

: Acute Oral Toxicity to the Rat (Acute Toxic Class

Method)

HLS s tudy number: CEF0003 Version ID: Audited Draft 2 Report Issue date: 24 July 2013Huntingdon Life Sciences CEF0003

2 

## Sponsor and Te st Facility Details Sponsor

Test facility Huntingdon Life Sciences Huntingdon Research Centre Wo olley Road Alconbury Huntingdon Cambridgeshi re PE28 4HS UKHuntingdon Life Sciences CEF0003

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## Ta ble of Contents Sponsor and Test Facility Details
.......................................................................................... 2 Compliance with Good Laboratory Practice
.......................................................................... 5 Quality Assurance Statement
................................................................................................ 6 Contributing Scientist
............................................................................................................ 7 Summary
.............................................................................................................................. 8 1. Introduction
................................................................................................................ 10 1.1 Objective
......................................................................................................... 10 1.2 Regulatory compliance
................................................................................... 10 1.3 Test system
.................................................................................................... 10 1.4 Route of administration
................................................................................... 10 1.5 Treatment groups and doses
.......................................................................... 11 1.6 Control animals
............................................................................................... 11 2. Experimental Procedure
............................................................................................. 12 2.1 Study schedule
............................................................................................... 12 2.1.1 Time sched ule
..................................................................................... 12 2.2 Test substance and formulation
...................................................................... 12 2.2.1 Test substance
.................................................................................... 12 2.2.2 Vehicle
................................................................................................ 13 2.2.3 Formulation
......................................................................................... 13 2.3 Animal management
....................................................................................... 13 2.3.1 Animal supply, acclimatisation and allocation
...................................... 13 2.3.2 Animal housing, diet and water supply
................................................ 14 2.3.3 Administration
..................................................................................... 15 2.4 Serial observations
......................................................................................... 15 2.4.1 Mortality
.............................................................................................. 15 2.4.2 Clinical observations
........................................................................... 15 2.4.3 Bodyweight
......................................................................................... 15 2.5 Necropsy and macrop athology
....................................................................... 15 2.5.1 Method of kill
....................................................................................... 15 2.5.2 Macroscopic pathology
........................................................................ 15 2.6 Computer systems
.......................................................................................... 16 2.7 Quality Assurance and archiving procedure s .................................................. 16 2.7.1 Quality Assurance
............................................................................... 16 2.7.2 Archives
.............................................................................................. 16 2.8 Deviations from protocol
................................................................................. 16 3. Results
....................................................................................................................... 17 3.1 Mortal ity
.......................................................................................................... 17 3.2 Clinical signs
................................................................................................... 17 3.3 Bodyweight
..................................................................................................... 17 3.4 Macroscopic pathology
................................................................................... 18 4. Conclusion
................................................................................................................. 19 5. References
................................................................................................................. 20Huntingdon Life Sciences CEF0003

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## List of T ables Table 1 Mortality data
............................................................................................... 21 Table 2 Clinical signs
................................................................................................ 22 

## List of Appendices Appendix 1 Individual and group mean bodyweights (g)
................................................. 23 Appendix 2 Individual bodyweight changes (g)
............................................................... 24 Appendix 3 Macroscopic findings
................................................................................... 25 Appendix 4 Te st procedure
............................................................................................. 26 

## List of An nexes Annex 1 GLP Compliance Statement
......................................................................... 27Huntingdon Life Sciences CEF0003

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## Compliance with Good Laboratory Practice : Acute Oral Toxicity to the Rat (Acute Toxic Class Method) The study described in this report was conducted in co mp liance with the fo llo wing Good Laboratory

Practice standards and, wi th the exceptio n noted below, I consider the data generated to be valid: The UK Good Laboratory Practice Regulat io ns (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 Commissio n Di

rective 2004/10/EC of 11 February 2004 (Official Journal No. L 50/44). These principles of Good Laboratory Practice are accepted by

the regulatory

authori ti es of the United States of America and Japan on the basis of intergovernmental agreements. Chemical analysis of samples of the fo rm ulated test substance for determinat io n of stabilit y, ho mo geneit y and concentration was not undertaken for this study.

Clare J Chesher BSc (Hons) MSc Study Di rector Hunt ingdon Life Sciences DateHuntingdon Life Sciences CEF0003

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## Quality Assurance Statement Acute Oral Toxicity to the Rat (Acute Toxic Class Method) The fo llo wing inspect io ns and audits have been carried out in relation to this study: Study Phase Date of Inspection Date of Reporting to Study Director and Management Protoc ol Audit 28 Mar 2013 28 Mar 2013 Report Audit 06 Jun 2013
- 07 Jun 2013 07 Jun 2013 Process based inspections: Ator about the time this study was in progress inspect io ns of procedures emplo yed on this t ype of study were carried out. These were condu cted and reported to appropriate Company Management as indicated below: Process Based Inspections Date(s) of Inspection Date of Reporting to Management Study management and conduct 08 Apr 2013
- 09 Apr 2013 10 Apr 2013 Acute study terminal procedures 17 Apr 2013 17 Apr 2013 Dose administration and post dose Observations 25 Apr 2013 25 Apr 2013 Formulation procedures 02 May 2013 02 May 2013 Bodyweights 23 May 2013,28 May 2013 28 May 2013 Clinical signs examination 23 May 2013 24 May 2013 Clinical signs examinat io n 23 May 2013 24 May 2013
In addit io n, an inspect io n of the facilit y where thi s study was conducted was carried out on an annual basis. These inspect io ns were promptly reported to Company Management. Dilys Smit h MRQA Lead Audito r Department of Qualit y Assurance Hunt ingdon Life Sciences DateHuntingdon Life Sciences CEF0003

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## Contributing Scientist

: A cute Oral Toxicity to the Rat (Acute Toxic Class Method) 

### Study management Clare J Chesher BSc (Hons) MSc Study Di rectorHuntingdon Life Sciences CEF0003

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## Summary The study was performed to assess the acute oral toxicit y of an addit ive in laser sensit ive co lo ur change inks , to the rat. Two groups of three fasted female rats received a single oral gavage dose of the test substance, formulated in 1% met hylcel lulose , at a dose level of 300 mg/kg body weight. As resul ts at this dose level indicated the acute (median) lethal oral dose of the test substance to be greater than 300 mg/kg bodyweight, in co mp liance wit h the study guidelines a further two groups of thr ee fasted females were similarly dosed at 2000 mg/kg bodyweight to complete the study
. 

### Results One female dosed at 2000 mg/kg died on Day 1. Clinical signs prior to death comprised fast breathing, flat posture, tremors, convulsio n, partially or f ully clos ed eyelids, i

rregular breathing, underact ive behaviour, hindlimbs splayed, increased body tone, shallow breathing, salivat io n and reduced body tem perature. These signs were seen fro m approximately 20 minutes after dosing. A loss in body weight was noted f

or the decedent. Macroscopic examinat io n of the animal revealed congest io n (characteri sed by darkened ti ssues/organs) of the subcutaneous tissue, liver and small and large intestines, pallor of the lungs and white liquid fluid contents in the stomach. One female dosed at 2000 mg/kg died on Day 2. Clinical signs prior to death comprised fast breathing, hunched posture, tremors, pilo erect io n, partially clos ed eyelids, i

rregular breathing, underact ive behaviour, reduced body tone, flat posture, salivat io n and reduced body tem perature. These signs were seen fro m approximately 20 minutes after dosing. A lo ss in body

weight was noted for the decedent. Macroscopic examinat io n of the animal revealed congestion (characterised by darkened t is sues/organs) of the subcutaneous tissue, lungs and small and large intest ines, a small caecum and dark red liquid fluid contents in the small intest ines and yellow liquid fluid content in the large intestines. Clinical signs of reaction to treatment in surviving animals co mpr is ed fast breathing, tremors, piloerect io n, hunched posture and unsteady gait seen in all females dosed at 2000 mg/kg. Underact ive behaviour, irregular breathing, and reduced body tem perature were seen in three females dosed at 2000 mg/kg. Partially clo sed eyelids, reduced body tone, flat posture and thin build were seen in two females dosed at 20 0 0 mg/kg and lachrymat io n, brown staining on the head, convulsio n and increased body tone were observed for individual animals dosed at 2000 mg/kg. These signs we re fi rst noted approximately

20 minutes after dosing. Recovery of surviving animals, as judged by external appearance and behaviour, was com plete by Day 10. No clinical signs were seen in any animal dosed at 300 m

g/kg. A lo w body

weight gain was noted for two females receiving 300 mg/kg and two females receiving 2000 mg/kg on Day 15 and a body

weight loss was recorded for one female receiving 300 mg/kg on Day 15. All other surviving anima ls were considered to have achieved sat is factory

body weight gains thr oughout the study
. Macroscopic examinat io n at study terminat io n on Day

15 revealed a small stomach for one female treated at 2000 mg/kg. No abnormalit ies were revealed in any other animal at the macroscopic examinat io n at this time.Huntingdon Life Sciences CEF0003

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### Conclusion The acute median lethal oral dose (LD 50 ) to rats of was demo nstrated to be between 2000 and 5000 mg/kg bodyweight.Huntingdon Life Sciences CEF0003

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## 1. Introduction 1.1 Objective The obj ect ive o f thi s study was to assess the toxic potential o f

, fo llo wing a single oral dose in the rat. 

### 1.2 Regulatory compliance The study was designed to meet the requirements of the fo llo wing guidelines: EEC Methods for the determination o f toxici ty and other healt h effects
. Commissio n Regulat io n No. 440/2008, Par t B, Method B.1 tris. Acute Oral Toxicit y: Acute Toxic Class Method. 30 May 2008. OECD Guideline for Testing of Chemicals No.423 ‘Acute Oral Toxicit y - Acute Toxic Class Method’ Adopted 17 December 2001. EPA Health Effects Test Guidelines OPPTS 870.1100 Acute Oral Toxicit y EPA 712-C-02- 190. 2002. Japanese Ministry

of Agriculture, Forestry

and Fisheries, Test Data for Registration of Agricultural Chemicals, Acute oral toxicit y (2 -1- 1), 12 No u san No 8147, Agricultural Production Bureau, November 24, 2000. The acute toxic class method is a stepwise procedure that uses three animals of a single sex per step. Depending on the mortalit y and/or the moribund status of the animals, on average two to four steps may be necessary to allow a j udgement on the acute to xicit y of the test substance. The acute toxic class method is based on bio metric evaluat io ns and has been subjected to international validat io n ( Schlede et al. 1995 )
. The study was conducted in accordance wit h the requirem ents of current, internat io nally recogni sed Good Laboratory Practice Standards, and the applicable sect io ns of the United Kingdo m Animals (Scient ific Procedures) Act 1986. 

### 1.3 Test system The rat was chosen as it has been shown to be a suitable model for this ty pe of study and is the species recommended by the test guidelines. As there were no data to indicate males were likely to be more sensit ive to the test substance, the testing was performed in females only
. 

### 1.4 Route of administration The rats were dosed by oral gavage as the test substance may be ingested accidentally
.Huntingdon Life Sciences CEF0003

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### 1.5 Treatment groups and doses The dose level s fo r the study were chosen in co mp liance wit h the study guidelines. As no previous toxico lo gical informat io n was available the init ial dose was 300 m g/kg. Dates dosed Times of

dosing Dose (mg/kg) Concentration (mg/mL) Dose volume

(mL/kg) No. of rats

Female 09 April 2013 11.26
- 11.28 300 30 10 3 11 April 2013 11.07
- 11.08 300 30 10 3 16 April 2013 10.22
- 10.24 2000 200 10 3 19 April 2013 10.22
- 1 0.24 2000 200 10 3 

### 1.6 Control animals No control animals were included in this study
.Huntingdon Life Sciences CEF0003

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## 2. Experimental Procedure 2.1 Study schedule 2.1.1 Time schedule Study init iat io n: (Protocol signed by

Study Di rector) 27 March 2013 Experimental start date: ( Ini tial animal al lo cat io n) 28 March 2013 Ini ti al d ay of treatment: 09 April 2013 Experimental co mp let io n date: (D ate of last necropsy

) 03 May 2013 Study com plet io n: When Study Di rector signs final

report 

### 2.2 Test substance and formulation 2.2.1 Test substance Informat io n supplie d by the Sponsor regarding the test substance is contained in the test substance data sheet, which is retained in study records. The fo llo wing informat io n is given in summary : Ident ification: Chemical name: I nt ended use: Appearance: White crystalline powder Storage condit io ns: Room tem perature in the dark Supplier: Sponsor Batch or L ot number: 12342 Expi ry date: W ill be documented in the raw data and in the final report Puri ty W ill be documented in the raw data and in the final report Sam ple receipt: 0 4 March 2013Huntingdon Life Sciences CEF0003

13 The Sponsor was responsible for the characterisat ion of the test substance and the documentation of

the methods of synt hesis, fabrication or d erivat io n and stabilit y. 2.2.2 Vehicle The vehicle used on this study was 1% met hylcellulose . A vehicle trial performed with in purified water showed that it formed a white suspension that separated out quickly and was not suitable for dosing. A vehicle trial performed in 1% methylcellulo se resul ted in a white suspensio n that was suitable for dosing. 2.2.3 Formulation The test substance was formulated at concentration s of 30 and 200 mg/ mL in the vehicle and administered at a volume of 10 mL/kg body weight. The test substance formulat io ns were prepared on the day of dosing. The absorption of the test substance was not determined. Determinat io n of the ho mo geneit y, stabili ty and puri ty of the test substance or test substance fo rm ulat io ns were not undertaken as part of thi s study
. Detailed records of test substance usage were maintained. The amount of test substance necessary to prepare the formulat io ns and the amount actually used were determined on each occasion. The difference between these amounts was checked b ef ore the form

ulat io ns were di spensed. 

### 2.3 Animal management 2.3.1 Animal supply, acclimatisation and allocation H eal thy nulliparous and non-pregnant female CD Crl:CD (SD ) albino rats were obtained fro m Charles River (UK) Ltd. The animals were allocated without cons cious bias to cages within the treatment groups. They were housed in groups of up to th ree rats , i n solid bottomed polycarbonate cages with a stainless steel mesh lid. Each cage contained a quant it y of autoclaved wood flake bedding. Cages, food hoppers, water bottles and bedding were changed at appropriate intervals. Each animal was assigned an alpha -numeric code and ident ified uniquely wit hin the study by tail marking. Each cage label was co lo ur- co ded and was ident ified uniquely wit h the study number, dose level

and animal mark. The animals were allowed to acclimat ise to the condit io ns described below for atleast 12 days before treatment. For those animals selected for this study, thei r body weights were in the range 230 to 270 g and they were approxima tely ei ght to nine weeks of age prior to dosing (Day

1).Huntingdon Life Sciences CEF0003

14 2.3.2 Animal housing, diet and water supply Animals were housed inside a barriered rodent facilit y (Building F21 , Room 044/045) . The facilit y was designed and operated to minimise the entry of external b iolo gical and chemical agents and to minimise the transference of such agents between rooms. The animal room was kept at positive pressure with respect to the outside by its own supply of f iltered fresh air, which was passed to atmosphere and not re
- circul ated. The tem perature and relative humidit y control s were set to maintain the range of 19 to 23  C and 40 to 70% respectively. Any minor deviat io ns fro m these ranges would not have had an adverse effect on the animals and would not affect the integrit y or validit y of the study . Artificial light ing was controlled to give a cy cle of 12 hours continuous light and 12 hours continuous dark per 24 hours. Environmental parameters are archived with the departmental raw data. Periodic checks were made on the numb er of ai r changes in the animal rooms. Temperature and humidit y wer e moni

tored daily. Alarms were activated if there was any failure of the vent ilat io n system, or temperature limits were exceeded. A stand
- by electrici ty supply was available to be automat ically brought into operation should the public supply fail. The cages were so lid bottomed polycarbonate cages with a stainless steel mesh lid. Each cage contained a quant it y of autoclaved softwood bark
- free fibre bedding. Cages, food hoppers, water bott les and bedding were changed at appropriate intervals. The animals were allowed free access to a standard rodent diet (Rat and Mouse No. 1 Maintenance Diet), except for overnight prior to and approximately four hours after dosing. This diet contained no a dded antibiot ic or other chemotherapeutic or prophylactic agent. Potable water taken fro m the public supply was f reely available via polycarbonate bottles fi tted wi th sipper tubes. Each cage of animals was provided with an Aspen chew block for environmenta l enrichment. Chew blocks were provided throughout the study and were replaced when necessary . Each cage of animals was provided wit h a plast ic shelter for environmental enrichment, which was replaced at the same time as the cages. Each batch of diet was analysed routinely by the supplier for various nutrit io nal com ponents and chemical and microbio lo gical contaminants. Supplier’s analy ti cal certificates were scrutinised and approved before any b atch of di et was released for use. The qualit y of the water supply is governed by regulat io ns published by the Department f

or Envi ronment, Food and Rural Affairs. Certificates of analysis were received routinely fro m the water supplier. Certificates of analysis were received routinely f rom the supplier of the ch ew blocks. Since the resul ts of these various analyses did not provide evidence of contaminat io n that might have prejudiced the study, they are not presented. No other specific contaminants that were likely to have been present in the diet or water were analysed, as none that may have interfered with or prejudiced the outcome of the study was known.Huntingdon Life Sciences CEF0003

15 2.3.3 Administration The appropri ate dose volume o f the test substance was administered to each rat by oral gavage using a plast ic syringe and plast ic catheter
. A record of the weight of each formulat io n dispensed and the amount remaining after dosing was made. The balance of these two weights was compared with the predicted usage as a check that the doses had been administered correctly
. Form ulat io ns were stirre d before and throughout the dosing procedure. 

### 2.4 Serial observations 2.4.1 Mortality Cages of rats were checked at least twice daily for any mortalit ies. 2.4.2 Clinical observations Animals were observed soon after dosing and at frequent int ervals for the remainder of Da y 1. On subsequent days, surviving anima ls were observed once in the morning and again at the end of the experimental day (wi th the exception

of Day 15
- mo rning only

). The nature and severit y, where appropriate, of the clinical signs and the time were r ecorded at each observat io n. All surviving animals were observed for 14 days after dosing. 2.4.3 Bodyweight The weight of each rat was recorded on Days 1 (prior to dosing), 8 and 15 or at death
. Individual weekly body

weight changes and group mean body weights we re calculated. 

### 2.5 Necropsy and macropathology 2.5.1 Method of kill All surviving animals were humanely killed on Day 15 by carbon dioxide asphyxiat io n. 2.5.2 Macroscopic pathology All animals were subject to a macroscopic examination which consisted of opening the crania l, thoracic and abdo minal cavit ies. The macroscopic appearance of all examined organs was recorded.Huntingdon Life Sciences CEF0003

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### 2.6 Computer systems The computer systems that were used on this study to acquire and quant ify data include: System name* System function Xybion Pristima Used for Pharmacy test substance management.
* All versio

n numbers of the systems are maintained by Huntingdon Life Sciences 

### 2.7 Quality Assurance and archiving procedures 2.7.1 Quality Assurance Details o f the Qualit y Assurance inspections and audits are presen ted on the Qualit y Assurance Statement. 2.7.2 Archives Fo llo wing com

plet io n of this study

all raw data were store d in the archives of Hunt ingdon Life Sciences. A copy of the final report will

b e retained indefinit ely and all Qualit y Assurance inspection record s for a period of 20 years. All other appropriate specimens and records will be re tained fo r a minimum period of one year from the date of issue of the final report. Atthe end of this retention period the Sponsor will be contacted and advice sought on future archiving requi rements. Under no circumstances will any item be discarded without the Sponsor’s knowledge. 

### 2.8 Deviations from protocol The pre treatment body

weights for animals R10, R11 and R12 were recorded on Day -2 rather than Day
- 1 in error. This d eviation f

rom the Protocol

has no effect on the integrit y or validit y of the study
. There were no other deviations fro m protocol
.Huntingdon Life Sciences CEF0003

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## 3. Results 3.1 Mortality Table 1 One female (R9 ) dosed at 2000 mg/kg died on Day 1 . Clinical signs prior to death comprised fast breathing, flat posture, tremors, convulsio n, partially or fully closed eyelids, irregular breathing, underact ive behaviour, hindlimbs splayed, increased body tone, shallow breathing, salivat io n and reduced body tem perature . These signs were seen fro m appr oximately 20 minutes af

ter dosing. A loss in body weight was noted for the decedent. Macroscopic examinat io n of the animal revealed congest io n (characteri sed by darkened ti ssues/organs) of the subcutaneous tissue, liver and small and large intestines, pal lo r of the l ungs and white liquid fluid contents in the stomach. One female (R10 ) dosed at 2000 mg/kg died on Day 2
. Clinical signs prior to death com prised fast breathing, hunched posture, tremors, pilo erect io n, partially clos ed eyelids, irregular breath ing, underactive behaviour , reduced body tone, flat posture, salivat io n and reduced body

tem perature. These signs were seen fro m approximately 20 minutes after dosing. A loss in body

weight was noted for the decedent. Macroscopic examinat io n of the anima l revealed congest io n (characterised by darkened t is sues/organs) of the subcutaneous ti ssue, l ungs and small and large int estines, a small caecum and dark red liqui d fluid contents in the small intest ines and yellow liquid fluid content in the large intest ines. 

### 3.2 Clinical signs Table 2 Clinical signs of reaction to treatment comprised fast breathing, tremors, piloerection, hunched posture and unsteady gai t seen in all females dosed at 2000 mg/kg. Underact ive behaviour, irregular breathing, and reduced body t em perature were seen in three females dosed at 2000 mg/kg. Partially closed eyelids, reduced body tone, flat posture and thin build were seen in two females dosed at 20 0 0 m g/kg and lachrymat io n, brown staining on the head, convulsio n and increased body to ne were observed for individual animals dosed at 2000 mg/kg. These signs we re first noted approximately 20 minutes af ter dosing. Recovery of surviving anima ls, as judged by external appearance and behaviour, was complete by Day 10 . No clinical signs wer e seen in any animal dosed at 300 mg/kg. 

### 3.3 Bodyweight Appendix 1 and Appendix 2 A lo w body

weight gain was noted for two females ( R1 and R3 ) receiving 300 mg/kg and two females (R7 and R8) receiving 2000 mg/kg on Day 15 and a body

weight loss was recorded fo r one female (R4) receiving 300 mg/kg on Day 15 . All other surviving animals were considered to have achieved sat is factory body weight gains throughout the study
.Huntingdon Life Sciences CEF0003

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### 3.4 Macroscopic pathology Appendix 3 Macroscopic examinat io n at study te rminat io n on Day

15 reveal ed a small stomach for one female ( R8 ) treated at 2000 mg/kg. No abnormalit ies were revealed in any other animal at the macroscopic examinat io n at thi s time.Huntingdon Life Sciences CEF0003

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## 4. Conclusion The acute median lethal oral dose (LD 50 ) to rats of was demo nstrated to be bet ween 2000 and 5000 mg/kg bodyweight.Huntingdon Life Sciences CEF0003

20 

## 5. References Schlede E , Mi schke U , Di ener W and Kayser D (1995). The International Validat io n Study of the Acute
- Toxic- Class Method (Oral). Arch Toxicol
. 69 , 659
- 670.Huntingdon Life Sciences CEF0003

21 Table 1 Mortality data Dose (mg/kg) No. of

deaths in groups of 3 Day*

1 2 3 to 15 <5m ~4.5h a b a b 300 0/3F
- - - - - - 300 0/3F
- - - - - - 2000 1/3F 0 1 0 0 0 0 2000 1/3F 0 0 1 0 0 0
* The day

/time indicated is the time that the animal was found dead F Female m Minute h Hour a First observation b Second observation

- Not applicableHuntingdon Life Sciences CEF0003

22 Table 2 Clinical signs Clinical signs No. of rats in groups of 3 showing signs Dose (mg/kg) 300 2000 Fast breathing 0 0 3 3 Tremors 0 0 3 3 Piloerection 0 0 3 3 Un deractive behaviour 0 0 3 2 Irregular breathing 0 0 3 2 Reduced body temperature 0 0 3 2 Hunched posture 0 0 2 3 Partially closed eyelids 0 0 2 3 Reduced body tone 0 0 3 1 Flat posture 0 0 3 1 Inc reased body tone 0 0 2 0 Un steady gait 0 0 2 2 Convulsion 0 0 2 0 Thin build 0 0 2 0 Salivation 0 0 1 1 Closed eyelids 0 0 1 0 Hindlimbs splayed 0 0 1 0 Shallow breathing 0 0 1 0 Br own staining head 0 0 1 0 Lachry mation 0 0 0 1Huntingdon Life Sciences CEF0003

23 Appendix 1 Individual and group mean bodyweights (g) Dose (mg/kg) Sex Animal Number Bodyweights (g) at Day

1* 8 15 Death

300 Female R1 256 292 294
- R2 264 316 333
- R3 252 291 294
- Mean 257 300 307
- Female R4 240 269 268
-

R5 253 289 299
- R6 258 301 312
- Mean 250 286 293
-

2000 Female R7 231 257 264
- R8 240 280 287
- R9 230
- - 221 Mean 234 269 276
- Female R10 257
- - 242

R11 270 322 354
- R12 252 285 303
- Mean 260 304 329
- * Prio

r to dosing

- Not applicableHuntingdon Life Sciences CEF0003

24 Appendix 2 Individual bodyweight changes (g) Dose (mg/kg) Sex Animal Number Bodyweight changes (g) at Days 1 - 8 8 - 15

300 Female R1 36 2 R2 52 17 R3 39 3 Female R4 29 (1)

R5 36 10 R6 43 11

2000 Female R7 26 7 R8 40 7 R9
- - Female R10
- -

R11 52 32 R12 33 18

- Not applicable ( ) Loss in bodyweightHuntingdon Life Sciences CEF0003

25 Appendix 3 Macroscopic findings Dose (mg/kg) 300 2000 Female Female Animal Number R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 R11 R12 Fate of animal K K K K K K K K D D K K Day of death 15 15 15 15 15 15 15 15 1 2 15 15 Tissues examined Findings Subcutaneous tissue         2 2   Br ain             Heart             Lungs         4 2   Liver         2    Spleen             Kidneys             Stomach        6 9    Duodenum             Small Intestines         2 2 ,9   Large Intestines         2 2 ,9   Caecum          6   Ur inary Bladder             K Killed at study termination D Sporadic death  No abnormalitie s detected 2 Congestion (darkened tissue/organs) 4 Pallor of tissue/organs 6 Small (atrophy) 9 Fluid contents ( wh ite, y ellow or dark red liquid)26Huntingdon Life SciencesCEF0003

Appendix 4 Test procedure

C. Test procedure with a starting dose of 300 mg/kg body weight

5mg/kg 3 animals 5mg/kg 3 animals

5mg/kg 3 animals 50mg/kg 3 animals

5 25 30 50 200 300 500 1000 2000 2500 5000  2-3 0-1 2-3

0-1 2-3 50mg/kg 3 animals 50mg/kg 3 animals 300mg/kg 3 animals 300mg/kg 3 animals 2000mg/kg 3 animals 2000mg/kg 3 animals

300mg/kg 3 animals 2000mg/kg 3 animals

Category 1 Category 2 Category 3 Category 4 Category 5 Category 5 or Unclassified > 0-5 > 5 - 50 > 50 - 300 > 300 - 2000 > 2000 - 5000 Start

0-1 2-3 0-1 2-3 0-1 2-3 0-1

2-3 0-1 2-3 0-1

LD50 cut - off mg/kg b.w. GHS

3(at 50) at 1 ststep other 3(at 300) at 1 st step 3 2 1 0 other 0 0

-  : unclassified - Testing at 5000 mg/kg b.w. - per step three animals of a single sex ( normally females ) are used - 0,1,2,3: Number of moribund or dead animals at each step - GHS: Globally Harmonized Classification System (mg/kg b.w.)Huntingdon Life Sciences CEF0 003

27 Annex 1 GLP Compliance StatementHuntingdon Life Sciences CEF0003

28 HUNTINGDON RESEARCH CENTRE GLP COMPLIANCE STATEMENT 2012