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## HFCPE: Acute Oral Toxicity to the Rat (Acute Toxic Class Method)

HLS study number: VHJ0007

Version ID: Final

Issue date: 10 March 2010Huntingdon Life Sciences VHJ0007

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## Sponsor and Test Facility Details

Sponsor ZEON Corporation Shin Marunouchi Center Building 1-6-2 Marunouchi Chiyoda-ku Tokyo 100-8246 JAPAN

Test facility Huntingdon Life Sciences Huntingdon Research Centre Woolley Road Alconbury Huntingdon Cambridgeshire PE28 4HS UKHuntingdon Life Sciences VHJ0007

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## Table 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 schedule ...................................................................................... 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 ................................................. 13 2.3.3 Administration ....................................................................................... 14 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 macropathology ......................................................................... 15 2.5.1 Method of kill ........................................................................................ 15 2.5.2 Macroscopic pathology ......................................................................... 15 2.6 Quality Assurance and archiving procedures ................................................... 16 2.6.1 Quality Assurance ................................................................................ 16 2.6.2 Archives ................................................................................................ 16 2.7 Deviations from protocol ................................................................................... 16

3. Results .......................................................................................................................

.. 17

3.1 Mortality ............................................................................................................ 17 3.2 Clinical signs .................................................................................................... 17 3.3 Bodyweight ....................................................................................................... 17 3.4 Macroscopic pathology ..................................................................................... 18

4. Conclusion ................................................................................................................... 19

5. References ................................................................................................................... 20Huntingdon Life Sciences VHJ0007

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## List of Tables

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 Test procedure .............................................................................................. 26



## List of Annexes

Annex 1 GLP Compliance Statements ........................................................................ 27Huntingdon Life Sciences VHJ0007

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## Contributing Scientist

HFCPE: Acute oral toxicity to th e rat (acute toxic class method)


## Study management

Rachel Clarke BSc (Hons) Study DirectorHuntingdon Life Sciences VHJ0007

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## Summary

The study was performed to assess the acute or al toxicity of HFCPE (used in electronic applications), to the rat.

A group of three fasted female rats received a si ngle oral gavage dose of the test substance, formulated in corn oil, at a dose level of 300 mg/kg bodyweight. As results at this dose level indicated the acute median lethal dose to be below 300 mg/kg bodyweight, in compliance with the study guidelines, a further two groups of three fasted females were similarly dosed at 50 mg/kg bodyweight to complete the study.



## Results

One female dosed at 300 mg/kg was found dead on Day 6 and one female dosed at 300 mg/kg was killed for welfare reasons on Day 7. Clinical signs observed prior to death comprised urine staining of the perigenital area, piloerection, loose faeces, hunched posture, irregular breathing, hindlimbs splayed, elevat ed gait, brown staining on the head, nose and forepaws, tremors, reduced body tone and thin bu ild. Bodyweight losses were recorded for both decedents. Macroscopic examination of th e animals revealed pallor of the kidneys and congestion (characterised by darken ed tissues/organs) of the subc utaneous tissue, heart, liver and lungs, the lungs were also small (atrophic) in one animal. In addition, the caecum and spleen were small (atrophy), a pale spleen was also noted in one animal, and a small stomach (atrophy) was recorded for one animal. Both animals had congestion (characterised by blood vessels injected) and red fluid contents contained within th e duodenum and small and large intestines, in addition, in one animal the stomach was congested.

Clinical signs of reaction to treatment in the surviving female dosed at 300 mg/kg comprised urine staining of the perigenital area, piloer ection, loose faeces, br own staining on the head, nose and forepaws, hunched posture, irregular breat hing and elevated gait. These signs were first observed from approximately 2 hours afte r dosing and recovery, as judged by external appearance and behaviour, was complete by Day 9.

Clinical signs of reaction to treatment in fe males dosed at 50 mg/kg comprised loose faeces, piloerection and wet urinogenita l area. These signs were fi rst observed from approximately 2 hours after dosing and recove ry, as judged by external a ppearance and behaviour, was complete by Day 2.

The surviving female dosed at 300 mg/kg had a bodyweight loss between Day 1 and 5, satisfactory bodyweight gains were recorded for this animal thereafter. Bodyweight stasis or a low bodyweight gain was recorded between Day 8 and 15 for two animals receiving 50 mg/kg. Satisfactory bodyweight gains were recorded for al l other animals throughout the study.

The macroscopic examination at study termin ation on Day 15 revealed a small (atrophic) stomach for one female treated at 50 mg/kg.

No abnormalities were revealed in the survivi ng animal treated at 300 mg/kg or in any other animal treated at 50 mg/kg at the macroscopic examination.Huntingdon Life Sciences VHJ0007

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## Conclusion

The acute median lethal oral dose (LD50) to ra ts of HFCPE was demons trated to be between 50 and 300 mg/kg bodyweight.

HFCPE is included in Category 3, according to the Globally Harmonised System (GHS), ( UNITED NATIONS, 2005 ).Huntingdon Life Sciences VHJ0007

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## 1. Introduction

1.1 Objective

The objective of this study was to assess the toxic potential of HFCPE, used in electronic applications, following a single oral dose in the rat. 

## 1.2 Regulatory compliance

The study was designed to meet the re quirements of the following guidelines:

EEC Methods for the determination of t oxicity. Annex to Commission Directive 2004/73/EC (Official Journal No. L152, 30.4.2004) , Part B, Method B.1 tris. Acute Oral Toxicity: Acute Toxic Class Method.

OECD Guideline for Testing of Chemical s No.423 ‘Acute Oral Toxicity - Acute Toxic Class Method’ Adopted 17 December 2001.

EPA Health Effects Test Guidelines OPPTS 870.1100 Acute Oral Toxicity EPA 712-C-02-190. 2002.

Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, Acute oral toxicity (2-1-1), 12 Nohsan No 8147, Agricultural Production Bu reau, November 24, 2000.

The acute toxic class method is a stepwise proce dure that uses three animals of a single sex per step. Depending on the mortality and/or th e moribund status of the animals, on average two to four steps may be necessary to allow a judgement on the acute toxicity of the test substance. The acute toxic class method is based on biometric evaluations and has been subjected to international validation ( Schlede et al. 1995 )

. The study was conducted in accordance with the requirements of current, internationally recognised Good Laboratory Practi ce Standards, and the appli cable sections of the United Kingdom Animals (Scientific Procedures) Act 1986.


## 1.3 Test system

The rat was chosen as it has been shown to be a suitable model for this type of study and is the species recommended by the test guidelines. As there were no data to indicate males were likely to be more sensitive 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 te st substance may be i ngested accidentally.Huntingdon Life Sciences VHJ0007

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## 1.5 Treatment groups and doses

The dose levels for the study were chosen in compliance with the study guidelines. As no previous toxicological inform ation was available the initi al dose level was 300 mg/kg.

Dates dosed Dose (mg/kg) Concentration (mg/mL) Dose volume (mL/kg) No. of rats Female

20 October 2009 300 30 10 3 27 October 2009 50 5 10 3 3 November 2009 50 5 10 3



## 1.6 Control animals

No control animals were included in this study.Huntingdon Life Sciences VHJ0007

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## 2. Experimental Procedure

2.1 Study schedule

2.1.1 Time schedule

Study initiation: (Protocol signed by Study Director) 08 October 2009

Experimental start date: (initial animal allocation) 15 October 2009

Date of initial treatment: 20 October 2009

Experimental completion date: (date of last necropsy) 17 November 2009

Study completion: 10 March 2010



## 2.2 Test substance and formulation

2.2.1 Test substance

Information supplied by the Sponsor regarding th e test substance is contained in the test substance data sheet, which is retained in study records.

The following information is given in summary:

Identification: HFCPE

Chemical name: 1,3,3,4,4,5,5-Heptafluorocyclopentene

Intended use: Electr onic applications

Appearance: Colourless liquid

Storage conditions: Room temperature

Supplier: Sponsor

Lot number: 20090601

Expiry date: End of June 2010

Purity/composition: 99.3%

Date of receipt: 22 June 2009

The Sponsor was responsible for the charac terisation of the test substance and the documentation of the methods of synthesis, fabrication or derivation and stability.Huntingdon Life Sciences VHJ0007

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### 2.2.2 Vehicle

The vehicle used was corn oil.

2.2.3 Formulation

The test substance was formulated at a concen tration of 30 or 5 mg/mL in the vehicle and administered at a volume of 10 mL/kg bodyweight.

The test substance formulations were prepared on the day of dosing.

The absorption of the test s ubstance was not determined.

Determination of the homogeneity, stability and pur ity of the test substance or test substance formulations were not undert aken as part of this study.

Detailed records of test substance usage were maintained. The amount of test substance necessary to prepare the formul ations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed. 

## 2.3 Animal management

2.3.1 Animal supply, acclimatisation and allocation

Animals chosen for this study were selected fr om a stock supply of healthy nulliparous and non-pregnant female CD (Crl:CD ‘SD’) rats obtained from Charles River (UK) Ltd.

The animals were allocated without conscious bias to cages within the treatment groups. They were housed in groups of three rats of the same sex, in solid bottomed polycarbonate cages with a stainless steel mesh lid. Each cage contained a quantity of autoclaved wood flake bedding. Cages, food hoppers, water bottle s and bedding were changed at appropriate intervals.

Each animal was assigned an alpha-numeric c ode and identified unique ly within the study by tail marking. Each cage label was colour-code d and was identified uniquely with the study number, dose level and animal mark.

The animals were allowed to acclimatise to th e conditions described be low for a minimum of 5 days before treatment. For those animals se lected for this study, their bodyweights were in the range 192 to 235 g and they were approximate ly eight to twelve w eeks of age prior to dosing (Day 1).
2.3.2 Animal housing, diet and water supply

Animals were housed inside a barriered rodent facility (Building F21, Room 7ab). The facility was designed and operated to minimise th e entry of external biological and chemical agents and to minimise the transfer ence of such agents between rooms.

The animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to atmosphere and not re-circulated. The temperatureHuntingdon Life Sciences VHJ0007

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### and relative humidity controls were set to generally maintain the range of 19 to 23 ° C and 40 to 70% respectively. Any minor deviations from these ranges would not have had an adverse effect on the animals a nd would not affect the integrit y or validity of the study. Artificial lighting was controlled to give a cycle of 12 hours continuous light and 12 hours continuous dark per 24 hours. Environmental para meters are archived with the departmental raw data.

Periodic checks were made on the number of ai r changes in the animal rooms. Temperature and humidity were monitored daily.

Alarms were activated if there was any failure of the ventilation system, or temperature limits were exceeded. A stand-by electricity supply was available to be automatically brought into operation should the pub lic supply fail.

The animals were allowed free access to a standard rodent diet (Rat and Mouse No. 1 Maintenance Diet), except for overnight prio r to and approximately four hours after dosing (see deviations from protocol section). This diet contained no a dded antibiotic or other chemotherapeutic or prophylactic agent.

Potable water taken from the public supply wa s freely available via polycarbonate bottles fitted with sipper tubes.

During the acclimatisation period, each cage of animals was provided with a soft white untreated chew block for environmental enrich ment. The wood blocks were removed from the cage of animals for the same period as the food on the day prior to dosing.

Each batch of diet was analysed routinely by the supplier for various nutritional components and chemical and microbiological contaminants . Supplier’s analytical certificates were scrutinised and approved before a ny batch of diet was released for use. The quality of the water supply is governed by regulations published by the Department for Environment, Food and Rural Affairs. Certificates of analysis we re received routinely from the water supplier. Certificates of analysis were received routinely from the supplier of the wood flake bedding and chew blocks. Since the re sults of these various analyses did not provide evidence of contamination that might have prejudiced the study, they ar e 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.

2.3.3 Administration

The appropriate dose volume of the test subs tance was administered to each rat by oral gavage using a plastic syringe and plastic catheter.

A record of the weight of each formulation 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.

Formulations were stirred before and throughout the dosing procedure.Huntingdon Life Sciences VHJ0007

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## 2.4 Serial observations

2.4.1 Mortality

Cages of rats were checked at leas t twice daily for any mortalities.

2.4.2 Clinical observations

Animals were observed soon after dosing and at frequent intervals for the remainder of Day 1. On subsequent days, surviving animals were observed, as a minimum, once in the morning and again at the end of the experi mental day (with the exception of Day 15 - morning only). The nature and severity, where appropriate, of the clin ical signs and the time were recorded at each observation.

All surviving animals were obser ved for 14 days after dosing.
2.4.3 Bodyweight

The weight of each rat was recorded on Days 1 (p rior to dosing), 8 and 15 or at death. An additional weight of each rat dosed at 300 mg/ kg was recorded on Day 5 due to the severity of clinical signs observed. Individual weekly bodyweight ch anges and group mean bodyweights were calculated.


## 2.5 Necropsy and macropathology

2.5.1 Method of kill

All surviving animals were humanely k illed on Day 15 by carbon dioxide asphyxiation.
2.5.2 Macroscopic pathology

All animals were subject to a macroscopic examination which consisted of opening the cranial, thoracic and abdominal cavities. The macroscopic appearance of all examined organs was recorded.Huntingdon Life Sciences VHJ0007

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## 2.6 Quality Assurance a nd archiving procedures

2.6.1 Quality Assurance

Details of the Quality Assurance inspec tions and audits are presented on the Quality Assurance Statement.

2.6.2 Archives

Following completion of this study all raw da ta were stored in the archives of Huntingdon Life Sciences.

A copy of the final report and all Quality A ssurance inspection reco rds will be retained indefinitely. All other appropriate records will be retained for a minimum period of one year from the date of issue of the final report. At the end of the one y ear retention period the Sponsor will be contacted and advice s ought on the above requirements. Under no circumstances will any item be discar ded without the Sponsor’s knowledge. 

## 2.7 Deviations from protocol

The following deviation from protocol occurred:

The protocol stated that animals were to be re-fed approximately 4 hours after the first dose administration, however, on 3 November 2009, animal numbers B7, B8 and B9 were fed approximately 5 hours after dose administration.

This deviation was considered to have not a ffected the integrity or validity of the study.Huntingdon Life Sciences VHJ0007

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## 3. Results

3.1 Mortality

Table 1 and Appendix 1

, Appendix 2 and Appendix 3

One female (no. B3) dosed at 300 mg/kg was found dead on Day 6 and one female (no. B2) dosed at 300 mg/kg was killed for welfare reasons on Day 7. C linical signs observed in both animals prior to death comprise d urine staining of the perige nital area, piloerection, loose faeces, hunched posture, irregular breathing, hi ndlimbs splayed, elevated gait and brown staining on the head, nose and forepaws. In a ddition, one animal also had tremors, reduced body tone and thin build. Bodyw eight losses were recorded for both decedents. Macroscopic examination of the animals rev ealed pallor of the kidneys an d congestion (characterised by darkened tissues/organs) of the subcutaneous tissue, heart, liv er and lungs, the lungs were also small (atrophic) in one animal. In addition, the caecum and spleen were small (atrophy), a pale spleen was also noted in one animal, and a small stomach (atr ophy) was recorded for one animal. Both animals had congestion (cha racterised by blood vesse ls injected) and red fluid contents contained within the duodenum and small and larg e intestines, in addition, in one animal the stomach was also congested. 

## 3.2 Clinical signs

Table 2

Clinical signs of reaction to treatment in th e surviving female (no. B1) dosed at 300 mg/kg comprised urine staining of the perigenital ar ea, piloerection, loose faeces, brown staining on the head, nose and forepaws, hunched posture, ir regular breathing, and elevated gait. These signs were first observed from approximately 2 hours after dosing and recovery, as judged by external appearance and beha viour, was complete by Day 9.

Clinical signs of reaction to treatment in fe males dosed at 50 mg/kg comprised loose faeces observed in four females (nos. B4, B5, B7 a nd B9), piloerection obser ved in three females (nos. B4, B5 and B6) and wet urinogenital area ob served in two females (nos. B5 and B6). These signs were first observed from approxi mately 2 hours after dosing and recovery, as judged by external appearance and behaviour, was complete by Day 2. 

## 3.3 Bodyweight

Appendix 1 and Appendix 2

The surviving female (no. B1) dosed at 300 mg /kg had a bodyweight loss between Day 1 and 5, satisfactory bodyweight gains were reco rded for this animal thereafter.

Bodyweight stasis or a low bodyweight gain was recorded between Day 8 and 15 for two animals receiving 50 mg/kg (nos. B6 and B7, respectively).

Satisfactory bodyweight gains were recorded for all other animals throughout the study.Huntingdon Life Sciences VHJ0007

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## 3.4 Macroscopic pathology

Appendix 3

The macroscopic examination at study termin ation on Day 15 revealed a small (atrophic) stomach for one female (no. B4) treated at 50 mg/kg.

No abnormalities were revealed in the survivi ng animal treated at 300 mg/kg or in any other animal treated at 50 mg/kg at the macroscopic examination.Huntingdon Life Sciences VHJ0007

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## 4. Conclusion

The acute median lethal oral dose (LD50) to ra ts of HFCPE was demons trated to be between 50 and 300 mg/kg bodyweight.

HFCPE is included in Category 3, according to the Globally Harmonised System (GHS), ( UNITED NATIONS, 2005 ).Huntingdon Life Sciences VHJ0007

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## 5. References

OECD Guideline for Testing of Chemicals No. 423 ‘Acute Oral Toxicity - Acute Toxic Class Method’. Adopted 17 December 2001.

EPA Health Effects Test Guidelines OPPTS. 870.1100 Acute Oral Toxicity. EPA 712-C-02-190, 2002.

SCHLEDE, E., MISCHKE, U., DIENER, W., and KAYSER, D. (1995). The International Validation Study of the Acute- Toxic-Class Method (Oral). Arch Toxicol

. 69 , 659-670.

EEC Methods for the determination of toxic ity, Annex to Commission Directive 2004/73/EC (Official Journal No. L152, 30.4.2004), Part B, Met hod B.1 tris. Acute Oral Toxicity - Acute Toxic Class Method.

Japanese Ministry of Agriculture, Forestry a nd Fisheries, Test Data for Registration of Agricultural Chemicals, Acut e oral toxicity (2-1-1), 12 Nohsan No. 8147, Agricultural Production Bureau, November 24, 2000.

OECD (2000a) Guidance document on Acute Or al Toxicity. Environmental Health and Safety Monograph Series on Testing and Assessment No. 24.

OECD (2000b) Guidance document on the Recognition, Assessment and use of clinical signs as Humane Endpoints for Experimental animal s used in Safety Evaluation Environmental Health and Safety Monograph series on Testing and Assessment No. 19.

UNITED NATIONS (2005). Part 3 (Health Hazards) of the Globally Harmonised System of Classification and Labelling of Chemicals, ISSN: 92-1-116927-5.Huntingdon Life Sciences VHJ0007

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### Table 1 Mortality data

Dose (mg/kg) No. of deaths in groups of 3 Day* 6 7 a b a b

300 2/3F 1 0 1 0 50 0/3F - - - - 50 0/3F - - - -

* The day/time indicated is the time that the animal was found dead or sacrificed on humane grounds. F Female a am observation b pm observation - Not applicableHuntingdon Life Sciences VHJ0007

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### Table 2 Clinical signs

Clinical signs No. of rats in groups of 3 showing signs Dose (mg/kg) 300 50 50

Piloerection 3 3 0 Loose faeces 3 2 2 Urine staining (perigenital area) 3 0 0 Hunched 3 0 0 Brown staining on head/nose and front paws 3 0 0 Irregular breathing 3 0 0 Elevated gait 3 0 0 Hindlimbs splayed 2 0 0 Unsteady gait 2 0 0 Reduced body tone 1 0 0 Thin build 1 0 0 Underactivity 1 0 0 Tremors 1 0 0 Wet urinogenital region 0 2 0Huntingdon Life Sciences VHJ0007

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### Appendix 1 Individual and gr oup mean bodyweights (g)

Dose (mg/kg) Sex Animal Number Bodyweights (g) at Day 1* 5† 8 15 Death

300 Female B1 207 195 221 234 - B2 199 171 - - 147 B3 225 206 - - 195 Mean 210 191 - - -

50 Female B4 215 - 231 245 - B5 235 - 268 279 - B6 219 - 244 244 - Mean 223 - 248 256 -

Female B7 199 - 228 231 - B8 202 - 232 251 - B9 192 - 221 231 - Mean 198 - 227 238 -

* Prior to dosing - Not applicable † Additional weight recorded for rats dosed at 300 mg/kgHuntingdon Life Sciences VHJ0007

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### Appendix 2 Individual bod yweight changes (g)

Dose (mg/kg) Sex Animal Number Bodyweight changes (g) at Days 1-5 1-8 8-15

300 Female B1 (12) 14 13 B2 (28) - - B3 (19) - -

50 Female B4 - 16 14 B5 - 33 11 B6 - 25 0

Female B7 - 29 3 B8 - 30 19 B9 - 29 10

- Not applicable ( ) Loss in bodyweightHuntingdon Life Sciences VHJ0007

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### Appendix 3 Macro scopic findings

Dose (mg/kg)

300 50 Female Female

Animal Number B1 B2 B3 B4 B5 B6 B7 B8 B9 Fate of animal K S D K K K K K K Day of death 15 7 6 15 15 15 15 15 15

Tissues examined Findings

Subcutaneous tissue 9 2B 2B 9 9 9 9 9 9 Brain 9 9 9 9 9 9 9 9 9 Heart 9 2B 2B 9 9 9 9 9 9 Lungs 9 2B 2B, 6B 9 9 9 9 9 9 Liver 9 2B 2B 9 9 9 9 9 9 Spleen 9 6B 4B, 6B 9 9 9 9 9 9 Kidneys 9 4B 4B 9 9 9 9 9 9 Stomach 9 6B 3B 6B 9 9 9 9 9 Duodenum 9 3B, 9(i)B 3B, 9(i)B 9 9 9 9 9 9 Small Intestines 9 3B, 9(i)B 3B, 9(i)B 9 9 9 9 9 9 Large Intestines 9 3B, 9(i)B 3B, 9(i)B 9 9 9 9 9 9 Caecum 9 6B 6B 9 9 9 9 9 9 Urinary Bladder 9 9 9 9 9 9 9 9 9

K Killed at study termination D Sporadic death S Sacrificed due to the severity of the clinical signs seen 9 No abnormalities detected 2B Congestion (darkened tissues/organs) 3B Congestion (blood vessels injected) 4B Pallor of tissues/organs 6B Small (atrophy) 9(i)B Fluid contents (red)Huntingdon Life Sciences VHJ0007

26Appendix 4 Test procedureC. Test procedure with a startingdose of 300 mg/kgbodyweight5mg/kg3 animals5mg/kg3 animals5mg/kg3 animals50mg/kg3 animals5 25 30 50 200 300 500 1000 2000 2500 5000∞2-30-12-30-12-350mg/kg3 animals50mg/kg3 animals300mg/kg3 animals300mg/kg3 animals2000mg/kg3 animals2000mg/kg3 animals300mg/kg3 animals2000mg/kg3 animalsCategory 1Category 2 Category 3 Category 4 Category 5Category 5 orUnclassified>0-5>5 - 50>50 - 300>300 - 2000>2000 - 5000Start0-12-30-12-30-12-30-12-30-12-30-1LD50 cut -offmg/kg b.w.GHS3(at 50)at 1ststepother3(at 300)at 1ststep3210other00-∞: 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 VHJ0007

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### Annex 1 GLP Compliance StatementsHuntingdon Life Sciences VHJ0007

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### HUNTINGDON RESEARCH CENTRE GLP COMPLIANCE STATEMENT 2008Huntingdon Life Sciences VHJ0007

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### HUNTINGDON RESEARCH CENTRE GLP COMPLIANCE STATEMENT 2009