# Final Report

Study Title : Acute Oral Toxicity Study in the Female Rat (Acute Toxic Class) AuthorFinal Report

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### RESPONSIBLE PERSONNEL Acute Oral Toxicity Study in the Female Rat (Acute Toxic Class) The following staff were responsible for key elements of the study: Quality Assurance C Clare Formulations P Tout Animal House Supervisor L Cooper Animal Health and Welfare A Basford Necropsy J Campey Histology L PattersonFinal Report

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### ARCHIVE STATEMENT Acute Oral Toxicity Study in the Female Rat (Acute Toxic Class) All primary data, or authenticated copies thereof, specimens and the final report will be retained in the archives for one year after issue of the final report. At this time the Sponsor will be contacted to determine whether the data should be returned, retained or destroyed on their behalf and notified of the financial implications of each of these options.Final Report

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### CONTENTS STUDY DIRECTOR AUTHENTICATION AND GLP COMPLIANCE STATEMENT ......................2 QUALITY ASSURANCE STATEMENT...............................................................................................3
SCIENTIFIC REVIEWER’S STATEMENT...........................................................................................4
RESPONSIBLE PERSONNEL.......................................................................................................... ......5
ARCHIVE STATEMENT .............................................................................................................. .........6
CONTENTS....................................................................................................................... ......................7
1 SUMMARY ....................................................................................................................... ...............9
2 INTRODUCTION.................................................................................................................. .........10
3 PROTOCOL ADHERENCE...........................................................................................................1 1
4 TEST ARTICLE .................................................................................................................. ...........11
5 EXPERIMENTAL DESIGN........................................................................................................... 11 5.1 Regulatory test guidelines.................................................................................................1 1 5.2 Test article administration................................................................................................. 11 5.3 Study design................................................................................................................ ......11 6 TEST ARTICLE FORMULATION ...............................................................................................12 6.1 Preparation................................................................................................................. .......12 7 TEST SYSTEM ................................................................................................................... ...........12 7.1 Justification for selection of the test system......................................................................12 7.2 Species, strain and supplier...............................................................................................1 2 7.3 Acclimatisation period and allocation to study.................................................................13 7.4 Caging...................................................................................................................... .........13 7.5 Diet, water and bedding ....................................................................................................1 3 7.6 Environment................................................................................................................. .....13 7.7 Identification of the test system ........................................................................................13 8 ANIMAL HEALTH AND WELFARE...........................................................................................14 8.1 Environmental enrichment................................................................................................14 9 EXPERIMENTAL OBSERVATIONS...........................................................................................14 9.1 Clinical signs.............................................................................................................. .......14 9.2 Body weights ................................................................................................................ ....14 10 TERMINAL PROCEDURES .........................................................................................................1 4 10.1 Preservation of tissues for histopathology ........................................................................14 11 DATA EVALUATION.............................................................................................................. .....15 11.1 Estimation of acute median lethal dose.............................................................................15 12 RESULTS ...................................................................................................................... .................16 12.1 Mortality .................................................................................................................. .........16 12.2 Clinical signs............................................................................................................. ........16 12.3 Body weights ............................................................................................................... .....16Final Report

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14 REFERENCE.................................................................................................................... ..............18
TABLES......................................................................................................................... ........................19 Table 1 Mortality data......................................................................................................... ............20 Table 2 Clinical signs following treatment ..................................................................................... 21 Table 3 Individual body weights and weekly increments ...............................................................22 Table 4 Necropsy findings ...................................................................................................... ........23 APPENDICES..................................................................................................................... ...................24 Appendix 1 Flow Diagram for Study Starting dose 300 or 2000 mg/kg.........................................25 Appendix 2 Certificate of Analysis ............................................................................................. ....26Final Report

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### 1 SUMMARY This study was conducted to assess the acute toxicity of the test article, following a single oral administration to small groups of rats. The study design provides information for hazard assessment and classification and enables a chemical to be assigned to toxicity classes but severely restricts animal usage. Groups of three female fasted rats were given the test article as a single dose on Day 1 by oral gavage at dose levels of 300 or 2000 mg/kg. The test article was dispersed in corn oil and administered at a dose volume of 10 mL/kg. Surviving animals were killed on Day 15 and subsequently underwent a full necropsy. There were two deaths following a single oral dose of among rats dosed at 2000 mg/kg. The deaths occurred at approximately 1⁄2 and 1 hour post-dose. No changes in body weight were recorded. Signs of reaction to treatment in decedents included exophthalmus, ataxia, lethargy, twitching and tachypnoea. Macroscopic examination of these animals revealed no abnormalities. Among surviving rats, signs of reaction to treatment included piloerection, tachypnoea, exophthalmus, ataxia, twitching, bradypnoea, hyperpnoea, tremors, lethargy, palpebral closure, hunched posture and unkempt appearance. Recovery, as judged by external appearance and behaviour, was complete by Day 8. All surviving rats achieved body weight gains during the first and second weeks of the study. Necropsy on Day 15 revealed two rats with uterine distension, which was accompanied by a vaginal nodule in one animal, and one rat with pale kidneys accompanied by renal pelvic dilatation. The acute median lethal oral dose of to rats was found to be between 500 and 1000 mg/kg. The test article was classified as Category 4 according to the Globally Harmonised System for the classification of chemicals which cause acute toxicity. The test material was classified as HARMFUL and the symbol “Xn” and risk phrase R 22 “HARMFUL IF SWALLOWED” are required according to EU labelling regulations Commission Directive 2001/59/EC relating to the classification, packaging and labelling of dangerous preparations.Final Report

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### 2 INTRODUCTION The objective of the study was to assess the acute toxicity of the test article following a single oral administration to small groups of rats. The study design provides information for hazard assessment and classification and enables a chemical to be assigned to toxicity classes but severely restricts animal usage. Determination of the acute oral toxicity of a particular chemical product is among the first investigations carried out to assess the potential hazard it presents to manufacturer and consumer populations. It provides information on the likely consequences to man of short-term exposure to chemical products by ingestion.
Study dates Protocol signed by Study Director: 18 April 2005 Animals on site: 26 April 2005 First treatment: 4 May 2005 Experimental completion date: 26 May 2005 The study completion date is the date that the final report is signed by the Study Director.Final Report

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### 3 PROTOCOL ADHERENCE The study was conducted in accordance with the agreed definitive protocol. There were no deviations from the protocol.

4 TEST ARTICLE The test article, an orange liquid, was identified as and was received at as follows:
Test article Batch number Quantity supplied (mL) Date of receipt at
8488/035 100 18 April 2005 

### The purity and an expiry date for the test article were not stated by the Sponsor. When not in use the test article was stored in a sealed container, at room temperature in the dark. A certificate of analysis for the test article was provided by the Sponsor and is presented in the Appendices. The vehicle for the test article was corn oil, supplied by Sigma-Aldrich Co Ltd, Poole, UK. The vehicle was chosen because corn oil gave the most satisfactory results in pre-study formulation trials conducted by the .

5 EXPERIMENTAL DESIGN 5.1 Regulatory test guidelines The study was conducted in compliance with Method B1 tris of Commission Directive 2004/73/EC and OECD Guidelines for Testing of Chemicals, Method 423 (adopted 17 December 2001). 5.2 Test article administration Doses were administered orally, by gavage, using plastic syringes and rubber catheters. Each rat was dosed once on Day 1 by passing the tip of a catheter along the oesophagus and instilling the test article into the gastric lumen. 5.3 Study design Groups of fasted animals were treated as follows: Dose Level (mg/kg) Concentration (mg/mL) Dose Volume (mL/kg) Number of Animals
300 30 10 3 2000 200 10 3 300 30 10 3Final Report

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### Treatment of animals was sequential. Sufficient time was allowed between each group to confirm the survival of the previously dosed animals. The Study Director selected the starting dose level that was considered to be most likely to cause the deaths of one or two animals. Once the treatment-related mortality caused by the first dosing regimen was established, the regimen appropriate for a second group of three rats was read from the flow diagram presented in Appendix 1. Successive groups of rats were treated according to the mortality affecting the previous treated group until the flow diagram indicated that the Globally Harmonised Classification System (GHS) could be applied to the test article. 1 

### At the discretion of the Study Director, following review of the clinical observations/mortality data obtained, the sequence detailed in the flow chart was amended in an attempt to reduce the number of animals used without affecting the purpose or integrity of the study. The rationale for deviation from the flow diagram is documented in the study record. Dose levels were expressed gravimetrically and in terms of test article received (without regard to purity or active content). Individual doses (mL) were calculated using the fasted body weights of the rats on the morning of dosing (Day 1) and the dose volume of 10 mL/kg.

6 TEST ARTICLE FORMULATION 6.1 Preparation The test article was dispersed in corn oil. The formulated concentrations were calculated from the selected dose level and the dose volume of 10 mL/kg. All formulations were used within two hours of preparation. Dose containers were repeatedly inverted prior to administration to ensure homogeneity.

7 TEST SYSTEM 7.1 Justification for selection of the test system The rat has been selected because it is the rodent species preferred by various regulatory authorities. Background data are available. Females are used because, in literature surveys of conventional LD 50 

### tests where a difference between the sexes has been seen, females have been shown to be slightly more sensitive than males. 7.2 Species, strain and supplier Female (nulliparous, non-pregnant) rats of the Hsd.BrlHan:WIST strain were obtained from Harlan UK Ltd, Bicester. All animals were given a clinical inspection for ill health on arrival and a sample was weighed.Final Report

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### 7.3 Acclimatisation period and allocation to study Rats were arbitrarily selected from the delivery boxes and allocated to the appropriate number of cages using a sequence that reduced selective bias. The condition of the animals was assessed daily throughout the acclimatisation period of 8 to 16 days. A second inspection was performed prior to study commencement to ensure the animals were suitable for the study. Overtly healthy animals were arbitrarily allocated to the study groups at least one day prior to dosing. At the start of the study the rats were approximately 8 to 11 weeks of age. The weight variation did not exceed ±20% of the mean weight. 7.4 Caging The animals were housed in groups of up to five during the acclimatisation period in cages that conform to the 'Code of Practice for the Housing and Care of Animals Used in Scientific Procedures' (Home Office, London, 1989). From the day prior to dosing (Day –1), the rats were housed in groups of three in similar cages. 7.5 Diet, water and bedding SQC(E) Rat and Mouse Maintenance Diet No 1, from Special Diets Services Ltd, Witham, was freely available to the animals at all times other than the period of fasting from the evening of the day prior to dosing (Day -1) until approximately 3 hours after dosing. Each batch of diet had been analysed for specific constituents and contaminants by the manufacturer. Mains water was provided, ad libitum , via cage-mounted water bottles. The water is had been analysed periodically for specific contaminants. Certificates of analysis for contaminant levels in each batch of diet or in the water supply are consigned to central files at this laboratory. Bedding was provided on a weekly basis to each cage by use of clean Aspen wood chips (Datesand Ltd, Manchester). The bedding had been analysed for specific contaminants and the results retained on file at No contaminants were present in diet, water or bedding at levels which might interfere with achieving the objective of the study. 7.6 Environment The animal rooms were designed to permit at least 15 air changes per hour and to maintain temperatures of 19 to 25 ° C. Although not actively controlled the relative humidity of the animal room was expected to remain within the range 40 to 70% RH. Recordings of maximum and minimum temperature and humidity were made twice daily during the week and once a day on Saturdays, Sundays and public holidays. The rooms were illuminated by fluorescent strip-lights for twelve hours daily. 7.7 Identification of the test system A number written on the tail in indelible ink on the day before dosing individually identified the rats. A colour coded card on each cage gave information including study number, group number, animal numbers and sex.Final Report

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### 8 ANIMAL HEALTH AND WELFARE All procedures carried out on live animals as part of this study were subject to the provisions of United Kingdom National Law, in particular the Animals (Scientific Procedures) Act 1986. 8.1 Environmental enrichment A number of methods are used at to enrich the environment of the animals as detailed in standard operating procedures.

9 EXPERIMENTAL OBSERVATIONS 9.1 Clinical signs Treated rats were observed closely for clinical signs of reaction to treatment. Clinical signs in all rats were recorded at approximately 15 and 30 minutes post-dose, hourly between 1 and 4 hours post-dose (inclusive), twice daily on Days 2, 3 and 4 and once daily from the fifth to last day of the observation period. In addition, rats treated at 300 mg/kg were observed for clinical signs immediately post-dose and rats treated at 2000 mg/kg were observed at 6 hours post-dose. These additional observations were made at the discretion of the observer. Individual records of clinical signs and times of death were maintained for each treated rat. 9.2 Body weights Rats were weighed on Day -1 (day before dosing) and on Days 1, 4, 8 and 15. Decedent carcass weights were also recorded prior to necropsy.

10 TERMINAL PROCEDURES Rats surviving treatment were killed by intraperitoneal injection of sodium pentobarbitone on Day 15. After exsanguination a full macroscopic necropsy was performed and all lesions recorded. The necropsy procedure included inspection of external surfaces and orifices, all viscera and tissue within the abdominal, thoracic and cranial cavities, free-hand sectioning of the liver and kidneys and examination of representative sections of mucosal surfaces of the stomach, small and large intestines. Animals dying during the observation period were subject to a similar necropsy with the least possible delay. Carcasses were stored in a refrigerator until necropsy commenced. 10.1 Preservation of tissues for histopathology Representative samples of macroscopic changes found at necropsy were retained in 10% neutral buffered formalin pending instructions from the Sponsor. However, the Sponsor confirmed that microscopic examination was not required.Final Report

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### 11 DATA EVALUATION 11.1 Estimation of acute median lethal dose The test article was given a GHS 1 

### category according to the details in Appendix 1. The Acute Toxic Class study results are expressed in terms of one of five GHS categories: ≤ 5 mg/kg; >5 to 50 mg/kg; >50 to 300 mg/kg; >300 to 2000 mg/kg; >2000 to 5000 mg/kg.Final Report

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### 12 RESULTS 12.1 Mortality (Table 1) Two animals died following oral administration of the test article at 2000 mg/kg. The rats were found dead at approximately 1⁄2 and 1 hour post-dosing, respectively. 12.2 Clinical signs (Table 2) Three of the six rats dosed at 300 mg/kg displayed piloerection between 2 and 3 hours post-dose and one of these also displayed tachypnoea at 2, 3 and 4 hours post-dose. Recovery, as judged by external appearance and behaviour, was complete by Day 2. The remaining rats dosed at 300 mg/kg showed no signs of reaction to treatment. All rats dosed at 2000 mg/kg displayed exophthalmus and ataxia, and one was also lethargic, at 1⁄4 hour post-dose. Two of these rats showed twitching and tachypnoea. Other signs of reaction to treatment, which were seen in the surviving rat, were bradypnoea, hyperpnoea, tremors, lethargy, palpebral closure, hunched posture and unkempt appearance. Recovery, as judged by external appearance and behaviour, was complete by Day 8. 12.3 Body weights (Table 3) All surviving rats gained weight during the first and second weeks of the observation period. 12.4 Necropsy findings (Table 4) The isolated macroscopic changes that were apparent on Day 15 showed no trend consistent with a treatment related effect on any target organ. Such changes included two instances of slight bilateral uterine distension, one of which was accompanied by a vaginal nodule, and one instance of moderately pale kidneys accompanied by moderate renal pelvic dilatation. These macroscopic changes were limited to animals dosed at 300 mg/kg. There were no macroscopic changes apparent for rats dosed at 2000 mg/kg.Final Report

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### 13 CONCLUSION The acute median lethal oral dose of to rats was found to be between 500 and 1000 mg/kg. The test article was classified as Category 4 according to the Globally Harmonised System for the classification of chemicals which cause acute toxicity. The test material was classified as HARMFUL and the symbol “Xn” and risk phrase R 22 “HARMFUL IF SWALLOWED” are required according to EU labelling regulations Commission Directive 2001/59/EC relating to the classification, packaging and labelling of dangerous preparations.Final Report

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### 14 REFERENCE 1. OECD (1998). Harmonised Integrated Hazard Classification for Human Health and Environmental Effects of Chemical Substances as endorsed by the 28 th 

### Joint Meeting of the Chemicals Committee and the Working Party on Chemicals in November 1998, Part 2, p 11.Final Report

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### TABLESFinal Report

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### Table 1 Mortality data
Dose level

(mg/kg) Mortality ratio Times of death after dosing 300 2000 300 0/3 2/3 0/3 1⁄2 and 1 hourFinal Report

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### Table 2 Clinical signs following treatment Dose level: 300 mg/kg Sign noted after dosing on Day 1 (hours): Sign noted on Day: Animal number Clinical sign Imm 1⁄4 1⁄2 1 2 3 4 2 3 4 5 6 7 8 9 to 15 Piloerection + + 223F Tachypnoea + + + 224F Piloerection + 225F Piloerection + +
Key: Imm Immediately post-dose
+ Sign present (if no entry is made the sign is absent)


### Dose level: 2000 mg/kg Sign noted after dosing on Day 1 (hours): Sign noted on Day: Animal number Clinical sign 1⁄41⁄21 2 3 4 6 2 3 4 5 6 7 8 9 to 15 Exophthalmus +

Lethargy +

Ataxia + 226F Found dead + Exophthalmus ++++++ Ataxia + + + + + + Tachypnoea + Twitching + + + + Bradypnoea + Hyperpnoea +

Tremor + Lethargy + Palpebral closure + Hunched posture + 227F

Unkempt + + + + + + Exophthalmus + + Ataxia + + Tachypnoea + Twitching + 228F
Found dead +
Key:
+ Sign present (if no entry is made the sign is absent)


### Dose level: 300 mg/kg Animal number Clinical sign 229F 230F 231F Observations !!!
Key: ! No clinical signs seenFinal Report

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### Table 3 Individual body weights and weekly increments Body weight (g) at: Increment (g) Dose level

(mg/kg) Animal number and sex Day -1 Day 1 Day 4 Day 8 Day 15 Day 1 to 8 Day 8 to 15 Decedent body weights
223F 144 134 157 170 184 36 14 224F 137 131 150 160 173 29 13 300 225F 149 140 159 163 184 23 21 226F 168 153 (153) 227F 166 151 162 179 194 28 15 2000 228F 152 145 (145) 229F 176 160 179 188 199 28 11 230F 182 167 190 199 208 32 9 300 231F 160 144 164 167 174 23 7
Decedent weight at death is given in parenthesesFinal Report

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### Table 4 Necropsy findings
Dose level: 300 mg/kg

Animal number Time and manner of death (Day) Necropsy comments
223F 15T No macroscopic changes 224F 15T Uterus: distension, bilateral- slight # 225F 15T Uterus: distension, bilateral- slight # Vagina: beige nodule – 3 mm diameter #



### Dose level: 2000 mg/kg

Animal number Time and manner of death (Day) Necropsy comments
226F 1D No macroscopic changes 227F 15T No macroscopic changes 228F 1D No macroscopic changes



### Dose level: 300 mg/kg

Animal number Time and manner of death (Day) Necropsy comments
229F 15T Kidneys: pale, bilateral - moderate pelvic dilatation, unilateral – moderate # 230F 15T No macroscopic changes 231F 15T No macroscopic changes

D Animal died during course of study T Animal killed by intraperitoneal injection of an overdose of sodium pentobarbitone at completion of observation period # Sample preserved in neutral buffered formalinFinal Report

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### APPENDICESFinal Report

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### Appendix 1 Flow Diagram for Study Starting dose 300 or 2000 mg/kg

Starting dose of 300 mg/kg

2-3 deaths
0-1 deaths 2-3 deaths
0-1 death 3 (at 50) Step 1 2-3 deaths
0-1 deaths 2-3 deaths Other
0-1 death 3 (at 300) Step 1 2-3 deaths
0-1 deaths 2-3 deaths Other
0-1 death
3 2-3 deaths 2 0-1 deaths 2-3 deaths
0-1 death 1

0 deaths 0 both groups

Starting dose of 2000 mg/kg

2-3 deaths
0-1 deaths 2-3 deaths
0-1 death 3 (at 50) Step 1 2-3 deaths
0-1 deaths 2-3 deaths Other
0-1 death 3 (at 300) Step 1 2-3 deaths
0-1 deaths 2-3 deaths Other
0-1 death 3 (at 2000) Step 1 2-3 deaths 2 (at 2000) 0-1 deaths 2-3 deaths Step 1 Other 0-1 death 1
0 deaths 0 both groups 300

500

2500 2500

5

50

200 50 5

>300-2000

2000 3 >50-300

5 or 5000 Unclassified Unclassified 5 >2000-5000 3 animals 3 animals 1000 @ 2000 m g /k g @ 2000 m g /k g

START @ 300 m g /k g 4 3 animals @ 300 m g /k g 3 animals @ 50 m g /k g 3 animals 30 @ 50 m g /k g 3 animals 2 >5-50
25 @ 5 m g /k g 3 animals >0-5 @ 5 m g /k g 3 animals cate g or y m g /k g BW 1 Classif y LD50

GHS cut-off

3 >50-300

2 >5-50 cate g or y m g /k g BW Classif y LD50

5 or 5000 Unclassified Unclassified 5 >2000-5000 @ 2000 m g /k g 3 animals @ 2000 m g /k g 2000 4 3 animals >300-2000 1000 300
500 3 animals 3 animals @ 300 m g /k g @ 300 m g /k g

START 200 @ 50 m g /k g 3 animals 30 @ 50 m g /k g 3 animals 25 @ 5 m g /k g 3 animals >0-5 @ 5 m g /k g 3 animals 1 GHS cut-offFinal Report

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### Appendix 2 Certificate of Analysis Certificate of Analysis
Infineum Product:

Units Method Result TBN mgKOH/g ASTM D2896 127.57 TAN mgKOH/g ASTM D664 129.28 CHN % mass SOP/EA/NW/77 C 72% H 12.1% N 2.7% Viscosity (100% @ 20oC) cSt ASTM D445 110 IR IN HOUSE - BELOW