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STUDY SPONSOR: 

41204330.doc/JS 

ACUTE ORAL TOXICITY IN THE RAT 

- FIXED DOSE METHOD 

PROJECT NUMBER: 41204330 

AUTHOR: 

TEST FACILITY: 

A Pooles 

Harlan Laboratories Ltd 

Shardlow Business Park 

Shard low 

Derbyshire 

DE72 2GD 

UK 

Telephone: +44 (0) 1332 792896 

Facsimile: +44 (0) 1332 799018 
PROJECT NUMBER: 41204330 PAGE2 

QUALITY ASSURANCE 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 

encompass the major phases of this study type at least once every three 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 of the data generated and of the procedures followed. 

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 appropriate to the type and schedule of this study were as follows: 

14 August 2012 Study Plan Compliance Audit 

03 July 2012 Test Item Preparation 

05 July 2012 Animal Preparation 

05 July 2012 Dosing 

05 July 2012 Assessment of Response 

11 July 2012 Necropsy 

§ 26 October 2012 Draft Report Audit 

§ Date of QA Signature Final Report Audit 

§ Evaluation specific to this study 

~-.h.~~~---·················· 1 4 NOV 2012 DATE: ............................................... . 

For the Quality Assurance Unit* 

*Authorised QA Signatures: 

Senior Audit Staff: J G Riley BSc (Hons) MRQA, J M Crowther MIScT MRQA, 

G Wren ONC MRQA, S Bevan BSc (Hons) MRQA, L Blaney MRQA 
PROJECT 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 of Directives 2004/9/EC and 2004/1 0/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 of being applied 

to the test system; it is assumed that the formulation was stable for this duration. This 

exception is considered not to affect the purpose or integrity of the study. 

This report fully and accurately reflects the procedures used and data generated . 

................... A.: .. 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 by scanning the paper original, 

and are considered of equivalent integrity and authenticity to versions produced by optical photocopy. However, in all cases the 

hand-signed paper original, held in secure archives, is the definitive document. 
PROJECT NUMBER: 41204330 PAGE4 

CONTENTS 

QUALITY ASSURANCE 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 of Data 10 

4. ARCHIVES 10 

5. RESULTS 11 

5.1 Dose Level- 3334 mg/kg (equivalent to 2000 mg active ingredient/kg 

bodyweight) 11 

5.2 Dose Level- 500 mg/kg (equivalent to 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 Flow Chart for the Sighting Test 19 

Appendix 2 Flow Chart for the Main Test 20 

Appendix 3 Statement of GLP Compliance in Accordance with Directive 2004/9/EC 21 
PROJECT NUMBER: 41204330 

ACUTE ORAL TOXICITY IN THE RAT 

- FIXED DOSE METHOD 

SUMMARY 

PAGES 

Introduction. The study was performed to assess the acute oral toxicity of the test item 

in the Wistar strain 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 bis Acute Toxicity (Oral) of Commission 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, 

due to the occurrence of clinical signs of toxicity that exceeded the severity limit set forth 

in the UK Home Office Project Licence. There were no deaths at a dose level of 

500 mg/kg (equivalent to 300 mg active ingredient/kg bodyweight). 

Clinical Observations. Signs of systemic 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 of the 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 of animals treated at a dose level of 500 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 of 500 - 3334 mg/kg bodyweight 

(equivalent to 300-2000 mg active ingredient/kg bodyweight) (Globally Harmonised 

Classification System- Category 4). 
PROJECT NUMBER: 41204330 

ACUTE ORAL TOXICITY IN THE RAT 

- FIXED DOSE METHOD 

1. INTRODUCTION 

PAGE? 

The study was performed to assess the acute oral toxicity of the test item in the Wistar 

strain 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 bis Acute Toxicity (Oral) of Commission Regulation (EC) No. 440/2008 

The test system was chosen because the rat has been shown to be a suitable model for 

this type of study and is recommended in the test method. The results of the study are 

believed to be of value in predicting the likely toxicity of the test item to man. 

The study was performed between 04 September 2012 and 11 October 2012. 

2. TEST ITEM AND EXPERIMENTAL PREPARATION 

2.1 Description, Identification and Storage Conditions 

Sponsor's identification 

Description 

Batch number 

Purity 

Date received 

Expiry date 

Storage conditions 

The integrity of supplied data relating to the identity, purity and stability of the test item is 

the responsibility of the Sponsor. 
PROJECT NUMBER: 41204330 PAGES 

2.2 Experimental Preparation 

For the purpose of the study the test item formulations were adjusted for the purity of the 

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 the test item was freshly prepared, as required, as a solution in distilled water. 

The test item was formulated within two hours of being applied to the test system. It is 

assumed that the formulation was stable for this 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 and Animal 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 of at 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 number written on a cage card. At the start of the 

study the animals were eight to twelve weeks of age. The bodyweight variation did not 

exceed ±20% of the bodyweight of the 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 of the 

study. 

The temperature and relative humidity were set to achieve limits of 19 to 25°C and 30 to 

70% respectively. Any occasional deviations from these targets were considered not to 
PROJECT NUMBER: 41204330 PAGE9 

have affected the purpose or integrity of the study. The rate of air exchange was 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 

not to contain any contaminant of a level that might have affected the purpose or integrity 

of the study. 

3.2 Procedure 

In the absence of data regarding the toxicity of the test item, 500 mg/kg was chosen as 

the starting dose. 

A single animal was treated as follows: 

Dose Level Concentration Dose Volume Number of 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 Number of 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 of animals was treated as follows: 

Dose Level Concentration Dose Volume Number of Rats 

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

500® 50 10 4 

A total of five animals were therefore treated at a dose level of 500 mg/kg in the study. 

® = Equivalent to 300 mg active ingredient/kg bodyweight 

E9 = Equivalent to 2000 mg active ingredient/kg bodyweight 
PROJECT 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 made twice daily. 

Individual bodyweights were recorded on Day 0 (the day of dosing) and on Days 7 and 

14 or at 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 of an external 

examination and opening of the abdominal and thoracic cavities. The appearance of any 

macroscopic abnormalities was recorded. No tissues were retained. 

3.3 Evaluation of Data 

The test item will be classified according to Annex 3 of the OECD Guidelines for Testing 

of Chemicals 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 of the number of animals that died during the 

study (or that were killed for humane reasons), and determination of the 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 of the next higher dose level could result in development of severe signs 

of toxicity and probable mortality. Effects on bodyweights and abnormalities noted at 

necropsy were also identified. 

Using the mortality data obtained, an estimate of the acute oral median lethal dose 

(LD5o) of the test 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, Shard low, UK archives for five years, after which 

instructions will be sought as to further retention or disposal. 
PROJECT NUMBER: 41204330 PAGE 11 

5. RESULTS 

5.1 Dose Level - 3334 mg/kg (equivalent to 2000 mg active ingredient/kg 

bodyweight) 

Individual clinical observations and mortality data are given in Table 1. 

5.1.1 Mortality 

The animal was killed for humane reasons, four hours after dosing, due to the 

occurrence of clinical signs of toxicity 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 of the extremities, 

emaciation and loss of righting reflex. 

5.1.3 Bodyweight 

The bodyweight on Day 0 and at death are given in Table 2. 

5.1.4 Necropsy 

Necropsy findings 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 (equivalent to 300 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 mortality data are given in Table 4. 
PROJECT NUMBER: 41204330 PAGE12 

5.2.1 Mortality 

There were no deaths. 

5.2.2 Clinical Observations 

No signs of systemic toxicity were 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 over the observation period. 

5.2.4 Necropsy 

Individual necropsy findings are given in Table 6. 

No abnormalities were noted at necropsy. 

6. 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 of 500- 3334 mg/kg bodyweight (equivalent to 

300-2000 mg active ingredient/kg bodyweight) (Globally Harmonised Classification 

System- Category 4). 
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 = Equivalent to 2000 mg active ingredient/kg bodyweight 

0 = No signs of systemic toxicity 

H = Hunched posture 

A= Ataxia 

L = Lethargy 

Pr = Prostration 

Rd = Decreased respiratory rate 

Rl = Laboured respiration 

Ho = Hypothermia 

E = Pallor of the extremities 

Em = Emaciation 

Rr = Loss of righting reflex 

X*= Animal killed for humane reasons due to the occurrence of clinical signs of toxicity that exceeded the severity limit set forth in the UK Home Office 

Pro·ect Licence 

PROJECT NUMBER: 41204330 PAGE13 
ACUTE ORAL TOXICITY IN THE RAT- FIXED DOSE METHOD 

Table 2 Individual Bodyweights and Bodyweight Changes - 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= Equivalent to 2000 mg active ingredient/kg bodyweight 

Animal dead 

PROJECT NUMBER: 41204330 

Bodyweight Gain (g) 

During Week 

1 2 

- -

PAGE14 
ACUTE ORAL TOXICITY IN THE RAT- FIXED DOSE METHOD 

Table 3 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 = Equivalent to 2000 mg active ingredient/kg bodyweight 

PROJECT NUMBER: 41204330 PAGE15 
ACUTE ORAL TOXICITY IN THE RAT- FIXED DOSE METHOD 

Table 4 Individual Clinical Observations and 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 

® = Equivalent to 300 mg active ingredient/kg bodyweight 

0 = No signs of systemic toxicity 

PROJECT NUMBER: 41204330 PAGE16 
ACUTE ORAL TOXICITY IN THE RAT- FIXED DOSE METHOD 

Table 5 Individual Bodyweights and Bodyweight Changes - 500 mg/kg® 

Dose Level Animal Number Bodyweight (g) at Day Bodyweight Gain (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 

® = Equivalent to 300 mg active ingredient/kg bodyweight 

PROJECT NUMBER: 41204330 PAGE17 
ACUTE ORAL TOXICITY IN THE RAT- FIXED DOSE METHOD 

Table 6 Individual Necropsy Findings - 500 mg/kg® 

Dose Level Animal Number Time of Death Macroscopic Observations mg/kg and Sex 

1-0 Female Killed Day 14 No abnormalities 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 

® = Equivalent to 300 mg active ingredienUkg bodyweight 

PROJECT NUMBER: 41204330 PAGE18 
Appendix 1 

ACUTE ORAL TOXICITY IN THE RAT- FIXED DOSE METHOD 

Flow Chart for the Sighting Test 

Evident toxicity 

No deaths or evident toxicity • 

* for outcome ~ at 5 mg/kg there is an optional supplementary 

procedure to confirm the GHS classification 

• outcome. differs from the OECD guideline which states 'No Toxicity'. This has been amended to clarify the dosing procedure intended in the guideline 

PROJECT NUMBER: 41204330 PAGE19 
Appendix 2 

ACUTE ORAL TOXICITY IN THE RAT- FIXED DOSE METHOD 

Flow Chart for the Main Test 

~2 deaths 

~1 with evident toxicity and/or :51 death 

No deaths or evident toxicity • 

Group size 

The 5 animals in each main study group will include any animal tested at that 

dose level in the sighting study 

* Animal welfare override 

If this dose level caused death in the sighting study, then no further animals will 

be tested. Go directly to outcome~ 

• outcome. differs from the OECD guideline which states 'No Toxicity'. This has been amended to clarify the dosing procedure intended in the guideline 

PROJECT NUMBER: 41204330 PAGE 20 
PROJECT NUMBER: 41204330 PAGE 21 

Appendix 3 Statement of GLP Compliance in Accordance with Directive 

2004/9/EC 

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