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Working for a better future

HFCPE: Acute Oral Toxicity to the Rat 

(Acute Toxic Class Method) 

HLS study number: VHJ0007 

Version ID: Final 

Issue date: 10 March 2010 
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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 

 UK 
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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 ................................................................................................................... 20
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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 ........................................................................ 27


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Contributing Scientist 

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

Study management 

Rachel Clarke BSc (Hons) 

Study Director 
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Summary 

The study was performed to assess the acute oral toxicity of HFCPE (used in electronic 

applications), to the rat. 

A group of three fasted female rats received a single 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, elevated gait, brown staining on the head, nose and 

forepaws, tremors, reduced body tone and thin build. Bodyweight losses were recorded for 

both decedents. Macroscopic examination of the animals revealed pallor of the kidneys and 

congestion (characterised by darkened tissues/organs) of the subcutaneous 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 the 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, piloerection, loose faeces, brown staining on the head, 

nose and forepaws, hunched posture, irregular breathing and elevated gait. These signs were 

first observed from approximately 2 hours after dosing and recovery, as judged by external 

appearance and behaviour, was complete by Day 9. 

Clinical signs of reaction to treatment in females dosed at 50 mg/kg comprised loose faeces, 

piloerection and wet urinogenital area. These signs were first observed from approximately 

2 hours after dosing and recovery, as judged by external appearance 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 all other animals throughout the 

study. 

The macroscopic examination at study termination on Day 15 revealed a small (atrophic) 

stomach for one female treated at 50 mg/kg. 

No abnormalities were revealed in the surviving animal treated at 300 mg/kg or in any other 

animal treated at 50 mg/kg at the macroscopic examination. 
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Conclusion 

The acute median lethal oral dose (LD50) to rats of HFCPE was demonstrated 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). 
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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 requirements of the following guidelines: 

EEC Methods for the determination of toxicity. 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 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. 

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 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 mortality and/or the 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 Practice Standards, and the applicable 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 test substance may be ingested accidentally. 
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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 information was available the initial 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. 
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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 the 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: Electronic 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 characterisation of the test substance and the 

documentation of the methods of synthesis, fabrication or derivation and stability. 
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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 concentration 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 substance was not determined. 

Determination of the homogeneity, stability and purity of the test substance or test substance 

formulations were not undertaken as part of this study. 

Detailed records of test substance usage were maintained. The amount of test substance 

necessary to prepare the formulations 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 from 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 bottles and bedding were changed at appropriate 

intervals. 

Each animal was assigned an alpha-numeric code and identified uniquely within the study by 

tail marking. Each cage label was colour-coded and was identified uniquely with the study 

number, dose level and animal mark. 

The animals were allowed to acclimatise to the conditions described below for a minimum of 

5 days before treatment. For those animals selected for this study, their bodyweights were in 

the range 192 to 235 g and they were approximately eight to twelve weeks 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 the entry of external biological 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 filtered fresh air, which was passed to atmosphere and not re-circulated. The temperature 
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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 and would not affect the integrity 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 parameters are archived with the departmental 

raw data. 

Periodic checks were made on the number of air 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 public supply fail. 

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 

(see deviations from protocol section). This diet contained no added antibiotic or other 

chemotherapeutic or prophylactic agent. 

Potable water taken from the public supply was 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 enrichment. 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 any 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 were 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 results of these various analyses did not provide evidence of 

contamination 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. 

2.3.3 Administration 

The appropriate dose volume of the test substance 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. 
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2.4 Serial observations 

2.4.1 Mortality 

Cages of rats were checked at least 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 experimental day (with the exception of Day 15 - 

morning only). The nature and severity, where appropriate, of the clinical signs and the time 

were recorded at each observation. 

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. 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 changes and group mean 

bodyweights were calculated. 

2.5 Necropsy and macropathology 

2.5.1 Method of kill 

All surviving animals were humanely killed 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. 
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2.6 Quality Assurance and archiving procedures 

2.6.1 Quality Assurance 

Details of the Quality Assurance inspections and audits are presented on the 

Quality Assurance Statement. 

2.6.2 Archives 

Following completion of this study all raw data were stored in the archives of 

Huntingdon Life Sciences. 

A copy of the final report and all Quality Assurance inspection records 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 year retention period the 

Sponsor will be contacted and advice sought on the above requirements. Under no 

circumstances will any item be discarded 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 affected the integrity or validity of the study. 
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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. Clinical signs observed in both 

animals prior to death comprised urine staining of the perigenital area, piloerection, loose 

faeces, hunched posture, irregular breathing, hindlimbs splayed, elevated gait and brown 

staining on the head, nose and forepaws. In addition, one animal also had tremors, reduced 

body tone and thin build. Bodyweight losses were recorded for both decedents. Macroscopic 

examination of the animals revealed pallor of the kidneys and congestion (characterised by 

darkened tissues/organs) of the subcutaneous 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 the duodenum and small and large intestines, in addition, in 

one animal the stomach was also congested. 

3.2 Clinical signs 

Table 2

Clinical signs of reaction to treatment in the surviving female (no. B1) dosed at 300 mg/kg 

comprised urine staining of the perigenital area, piloerection, loose faeces, brown staining on 

the head, nose and forepaws, hunched posture, irregular breathing, and elevated gait. These 

signs were first observed from approximately 2 hours after dosing and recovery, as judged by 

external appearance and behaviour, was complete by Day 9. 

Clinical signs of reaction to treatment in females dosed at 50 mg/kg comprised loose faeces 

observed in four females (nos. B4, B5, B7 and B9), piloerection observed in three females 

(nos. B4, B5 and B6) and wet urinogenital area observed in two females (nos. B5 and B6). 

These signs were first observed from approximately 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 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 (nos. B6 and B7, respectively). 

Satisfactory bodyweight gains were recorded for all other animals throughout the study. 
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3.4 Macroscopic pathology 

Appendix 3

The macroscopic examination at study termination on Day 15 revealed a small (atrophic) 

stomach for one female (no. B4) treated at 50 mg/kg. 

No abnormalities were revealed in the surviving animal treated at 300 mg/kg or in any other 

animal treated at 50 mg/kg at the macroscopic examination. 
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4. Conclusion 

The acute median lethal oral dose (LD50) to rats of HFCPE was demonstrated 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). 
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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 toxicity, 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. 

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 Bureau, November 24, 2000. 

OECD (2000a) Guidance document on Acute Oral 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 animals 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. 
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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 applicable 
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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 0 

 
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Appendix 1 Individual and group 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/kg 
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Appendix 2 Individual bodyweight 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 bodyweight 
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Appendix 3 Macroscopic 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) 
26 

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 

2-3 0-1

2-3

2-3 0-1

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 C

 > 0-5 > 5 - 50 > 50 - 300 > 300 - 2000 

Start

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

2-3 0-1 2-3 

LD50 cut -offmg/kg b.w.GHS

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

- ∞ : 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.)
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Annex 1 GLP Compliance Statements 
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HUNTINGDON RESEARCH CENTRE GLP COMPLIANCE STATEMENT 2008 
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HUNTINGDON RESEARCH CENTRE GLP COMPLIANCE STATEMENT 2009