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Working for a better future

REPORT

: Acute Oral Toxicity to the Rat (Acute Toxic Class 

Method)

HLS study number: CEF0003

Version ID: Audited Draft 2 Report

Issue date: 24 July 2013
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Sponsor and Test Facility Details

Sponsor

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 ................................................ 14

2.3.3 Administration ..................................................................................... 15

2.4 Serial observations ......................................................................................... 15

2.4.1 Mortality .............................................................................................. 15

2.4.2 Clinical observations ........................................................................... 15

2.4.3 Bodyweight ......................................................................................... 15

2.5 Necropsy and macropathology ....................................................................... 15

2.5.1 Method of kill....................................................................................... 15

2.5.2 Macroscopic pathology........................................................................ 15

2.6 Computer systems.......................................................................................... 16

2.7 Quality Assurance and archiving procedures .................................................. 16

2.7.1 Quality Assurance ............................................................................... 16

2.7.2 Archives .............................................................................................. 16

2.8 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 Statement.........................................................................27
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Compliance with Good Laboratory Practice

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

The study described in this report was conducted in compliance with the following Good 

Laboratory Practice standards and, with the exception noted below, I consider the data 

generated to be valid:

The UK Good Laboratory Practice Regulations (Statutory Instrument 1999 

No. 3106, as amended by Statutory Instrument 2004 No. 994).

OECD Principles of Good Laboratory Practice (as revised in 1997), 

ENV/MC/CHEM(98)17.

EC Commission Directive 2004/10/EC of 11 February 2004 (Official Journal 

No. L 50/44).

These principles of Good Laboratory Practice are accepted by the regulatory authorities of 

the United States of America and Japan on the basis of intergovernmental agreements.

Chemical analysis of samples of the formulated test substance for determination of stability,

homogeneity and concentration was not undertaken for this study.

Clare J Chesher BSc (Hons) MSc

Study Director

Huntingdon Life Sciences

Date
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Quality Assurance Statement

 Acute Oral Toxicity to the Rat (Acute Toxic Class Method)

The following inspections and audits have been carried out in relation to this study:

Study Phase Date of Inspection Date of Reporting 

to Study Director 

and Management

Protocol Audit 28 Mar 2013 28 Mar 2013

Report Audit 06 Jun 2013-07 Jun 2013 07 Jun 2013

Process based inspections: At or about the time this study was in progress inspections of 

procedures employed on this type of study were carried out. These were conducted and 

reported to appropriate Company Management as indicated below:

Process Based Inspections Date(s) of Inspection Date of Reporting to 

Management

Study management and conduct 08 Apr 2013-09 Apr 2013 10 Apr 2013

Acute study terminal procedures 17 Apr 2013 17 Apr 2013

Dose administration and post dose 

Observations

25 Apr 2013 25 Apr 2013

Formulation procedures 02 May 2013 02 May 2013

Bodyweights 23 May 2013,28 May 2013 28 May 2013

Clinical signs examination 23 May 2013 24 May 2013

Clinical signs examination 23 May 2013 24 May 2013

In addition, an inspection of the facility where this study was conducted was carried out on an 

annual basis. These inspections were promptly reported to Company Management.

Dilys Smith MRQA 

Lead Auditor

Department of Quality Assurance

Huntingdon Life Sciences

Date
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Contributing Scientist

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

Study management

Clare J Chesher BSc (Hons) MSc

Study Director
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Summary

The study was performed to assess the acute oral toxicity of an additive in laser 

sensitive colour change inks, to the rat.

Two groups of three fasted female rats received a single oral gavage dose of the test 

substance, formulated in 1% methylcellulose, at a dose level of 300 mg/kg bodyweight. As 

results at this dose level indicated the acute (median) lethal oral dose of the test substance to 

be greater than 300 mg/kg bodyweight, in compliance with the study guidelines a further two 

groups of three fasted females were similarly dosed at 2000 mg/kg bodyweight to complete 

the study.

Results

One female dosed at 2000 mg/kg died on Day 1. Clinical signs prior to death comprised fast 

breathing, flat posture, tremors, convulsion, partially or fully closed eyelids, irregular 

breathing, underactive behaviour, hindlimbs splayed, increased body tone, shallow breathing, 

salivation and reduced body temperature. These signs were seen from approximately 20 

minutes after dosing. A loss in bodyweight was noted for the decedent. Macroscopic 

examination of the animal revealed congestion (characterised by darkened tissues/organs) of 

the subcutaneous tissue, liver and small and large intestines, pallor of the lungs and white 

liquid fluid contents in the stomach.

One female dosed at 2000 mg/kg died on Day 2. Clinical signs prior to death comprised fast 

breathing, hunched posture, tremors, piloerection, partially closed eyelids, irregular 

breathing, underactive behaviour, reduced body tone, flat posture, salivation and reduced 

body temperature. These signs were seen from approximately 20 minutes after dosing. A 

loss in bodyweight was noted for the decedent. Macroscopic examination of the animal 

revealed congestion (characterised by darkened tissues/organs) of the subcutaneous tissue, 

lungs and small and large intestines, a small caecum and dark red liquid fluid contents in the 

small intestines and yellow liquid fluid content in the large intestines.

Clinical signs of reaction to treatment in surviving animals comprised fast breathing, tremors, 

piloerection, hunched posture and unsteady gait seen in all females dosed at 2000 mg/kg. 

Underactive behaviour, irregular breathing, and reduced body temperature were seen in three 

females dosed at 2000 mg/kg. Partially closed eyelids, reduced body tone, flat posture and 

thin build were seen in two females dosed at 2000 mg/kg and lachrymation, brown staining 

on the head, convulsion and increased body tone were observed for individual animals dosed 

at 2000 mg/kg. These signs were first noted approximately 20 minutes after dosing. 

Recovery of surviving animals, as judged by external appearance and behaviour, was 

complete by Day 10. No clinical signs were seen in any animal dosed at 300 mg/kg.

A low bodyweight gain was noted for two females receiving 300 mg/kg and two females 

receiving 2000 mg/kg on Day 15 and a bodyweight loss was recorded for one female 

receiving 300 mg/kg on Day 15. All other surviving animals were considered to have 

achieved satisfactory bodyweight gains throughout the study.

Macroscopic examination at study termination on Day 15 revealed a small stomach for one 

female treated at 2000 mg/kg. No abnormalities were revealed in any other animal at the 

macroscopic examination at this time.
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Conclusion

The acute median lethal oral dose (LD50) to rats of was demonstrated to be 

between 2000 and 5000 mg/kg bodyweight.
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1. Introduction

1.1 Objective

The objective of this study was to assess the toxic potential of 

, 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 and other health effects. 

Commission Regulation No. 440/2008, Part B, Method B.1 tris. Acute Oral 

Toxicity: Acute Toxic Class Method. 30 May 2008.

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 Nousan 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 was 300 mg/kg.

Dates dosed Times of 

dosing

Dose (mg/kg) Concentration 

(mg/mL)

Dose volume 

(mL/kg)

No. of rats

Female

09 April 2013 11.26-11.28 300 30 10 3

11 April 2013 11.07-11.08 300 30 10 3

16 April 2013 10.22-10.24 2000 200 10 3

19 April 2013 10.22-10.24 2000 200 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)

27 March 2013

Experimental start date:

(Initial animal allocation)

28 March 2013

Initial day of treatment: 09 April 2013

Experimental completion date:

(Date of last necropsy)

03 May 2013

Study completion: When Study Director signs final report

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:

Chemical name:

Intended use:

Appearance: White crystalline powder

Storage conditions: Room temperature in the dark

Supplier: Sponsor

Batch or Lot number: 12342

Expiry date: Will be documented in the raw data and in 

the final report

Purity Will be documented in the raw data and in 

the final report

Sample receipt: 04 March 2013
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The Sponsor was responsible for the characterisation of the test substance and the 

documentation of the methods of synthesis, fabrication or derivation and stability.

2.2.2 Vehicle

The vehicle used on this study was 1% methylcellulose.

A vehicle trial performed with in purified water showed that it formed a white 

suspension that separated out quickly and was not suitable for dosing. A vehicle trial

performed in 1% methylcellulose resulted in a white suspension that was suitable for dosing.

2.2.3 Formulation

The test substance was formulated at concentrations of 30 and 200 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

Healthy nulliparous and non-pregnant female CD Crl:CD (SD) albino rats were 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 up to three rats, 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 at least 

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

in the range 230 to 270 g and they were approximately eight to nine weeks of age prior to 

dosing (Day 1).
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2.3.2 Animal housing, diet and water supply

Animals were housed inside a barriered rodent facility (Building F21, Room 044/045). 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 

and relative humidity controls were set to 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 cages were solid bottomed polycarbonate cages with a stainless steel mesh lid. Each 

cage contained a quantity of autoclaved softwood bark-free fibre bedding. Cages, food 

hoppers, water bottles and bedding were changed at appropriate intervals.

The animals were allowed free access to a standard rodent diet (Rat and Mouse No. 1 

Maintenance Diet), except for overnight prior to and approximately four hours after dosing. 

This diet contained no 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.

Each cage of animals was provided with an Aspen chew block for environmental enrichment. 

Chew blocks were provided throughout the study and were replaced when necessary. Each 

cage of animals was provided with a plastic shelter for environmental enrichment, which was 

replaced at the same time as the cages.

Each batch of diet was analysed routinely by the supplier for various 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 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.
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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.

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 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. 

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 Computer systems

The computer systems that were used on this study to acquire and quantify data include: 

System name* System function

Xybion Pristima Used for Pharmacy test substance management.

* All version numbers of the systems are maintained by Huntingdon Life Sciences

2.7 Quality Assurance and archiving procedures

2.7.1 Quality Assurance

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

Quality Assurance Statement.

2.7.2 Archives

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

Huntingdon Life Sciences. 

A copy of the final report will be retained indefinitely and all Quality Assurance inspection 

records for a period of 20 years. All other appropriate specimens and records will be retained 

for a minimum period of one year from the date of issue of the final report. At the end of this 

retention period the Sponsor will be contacted and advice sought on future archiving 

requirements. Under no circumstances will any item be discarded without the Sponsor’s 

knowledge.

2.8 Deviations from protocol

The pre treatment bodyweights for animals R10, R11 and R12 were recorded on Day -2 

rather than Day -1 in error.

This deviation from the Protocol has no effect on the integrity or validity of the study.

There were no other deviations from protocol.
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3. Results

3.1 Mortality

Table 1

One female (R9) dosed at 2000 mg/kg died on Day 1. Clinical signs prior to death comprised

fast breathing, flat posture, tremors, convulsion, partially or fully closed eyelids, irregular 

breathing, underactive behaviour, hindlimbs splayed, increased body tone, shallow breathing, 

salivation and reduced body temperature. These signs were seen from approximately 20 

minutes after dosing. A loss in bodyweight was noted for the decedent. Macroscopic 

examination of the animal revealed congestion (characterised by darkened tissues/organs) of 

the subcutaneous tissue, liver and small and large intestines, pallor of the lungs and white 

liquid fluid contents in the stomach.

One female (R10) dosed at 2000 mg/kg died on Day 2. Clinical signs prior to death 

comprised fast breathing, hunched posture, tremors, piloerection, partially closed eyelids, 

irregular breathing, underactive behaviour, reduced body tone, flat posture, salivation and 

reduced body temperature. These signs were seen from approximately 20 minutes after 

dosing. A loss in bodyweight was noted for the decedent. Macroscopic examination of the 

animal revealed congestion (characterised by darkened tissues/organs) of the subcutaneous 

tissue, lungs and small and large intestines, a small caecum and dark red liquid fluid contents 

in the small intestines and yellow liquid fluid content in the large intestines.

3.2 Clinical signs

Table 2

Clinical signs of reaction to treatment comprised fast breathing, tremors, piloerection, 

hunched posture and unsteady gait seen in all females dosed at 2000 mg/kg. Underactive 

behaviour, irregular breathing, and reduced body temperature were seen in three females 

dosed at 2000 mg/kg. Partially closed eyelids, reduced body tone, flat posture and thin build 

were seen in two females dosed at 2000 mg/kg and lachrymation, brown staining on the head,

convulsion and increased body tone were observed for individual animals dosed at 

2000 mg/kg. These signs were first noted approximately 20 minutes after dosing. Recovery 

of surviving animals, as judged by external appearance and behaviour, was complete by 

Day 10. No clinical signs were seen in any animal dosed at 300 mg/kg.

3.3 Bodyweight

Appendix 1 and Appendix 2

A low bodyweight gain was noted for two females (R1 and R3) receiving 300 mg/kg and two 

females (R7 and R8) receiving 2000 mg/kg on Day 15 and a bodyweight loss was recorded 

for one female (R4) receiving 300 mg/kg on Day 15. All other surviving animals were 

considered to have achieved satisfactory bodyweight gains throughout the study.
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3.4 Macroscopic pathology

Appendix 3

Macroscopic examination at study termination on Day 15 revealed a small stomach for one 

female (R8) treated at 2000 mg/kg. No abnormalities were revealed in any other animal at 

the macroscopic examination at this time.
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4. Conclusion

The acute median lethal oral dose (LD50) to rats of was demonstrated to be 

between 2000 and 5000 mg/kg bodyweight.
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5. References

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.
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Table 1 Mortality data

Dose 

(mg/kg)

No. of 

deaths in 

groups of 3

Day*

1 2 3 to 15

<5m ~4.5h a b a b

300 0/3F - - - - - -

300 0/3F - - - - - -

2000 1/3F 0 1 0 0 0 0

2000 1/3F 0 0 1 0 0 0

* The day/time indicated is the time that the animal was found dead

F Female

m Minute

h Hour

a First observation

b Second observation

- Not applicable
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Table 2 Clinical signs

Clinical signs

No. of rats in groups of 3 showing signs

Dose (mg/kg)

300 2000

Fast breathing 0 0 3 3

Tremors 0 0 3 3

Piloerection 0 0 3 3

Underactive behaviour 0 0 3 2

Irregular breathing 0 0 3 2

Reduced body temperature 0 0 3 2

Hunched posture 0 0 2 3

Partially closed eyelids 0 0 2 3

Reduced body tone 0 0 3 1

Flat posture 0 0 3 1

Increased body tone 0 0 2 0

Unsteady gait 0 0 2 2

Convulsion 0 0 2 0

Thin build 0 0 2 0

Salivation 0 0 1 1

Closed eyelids 0 0 1 0

Hindlimbs splayed 0 0 1 0

Shallow breathing 0 0 1 0

Brown staining head 0 0 1 0

Lachrymation 0 0 0 1
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Appendix 1 Individual and group mean bodyweights (g)

Dose (mg/kg) Sex Animal 

Number

Bodyweights (g) at Day

1* 8 15 Death

300

Female

R1 256 292 294 -

R2 264 316 333 -

R3 252 291 294 -

Mean 257 300 307 -

Female

R4 240 269 268 -

R5 253 289 299 -

R6 258 301 312 -

Mean 250 286 293 -

2000

Female

R7 231 257 264 -

R8 240 280 287 -

R9 230 - - 221

Mean 234 269 276 -

Female

R10 257 - - 242

R11 270 322 354 -

R12 252 285 303 -

Mean 260 304 329 -

* Prior to dosing

- Not applicable
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Appendix 2 Individual bodyweight changes (g)

Dose (mg/kg) Sex Animal Number Bodyweight changes (g) at Days

1-8 8-15

300

Female

R1 36 2

R2 52 17

R3 39 3

Female

R4 29 (1)

R5 36 10

R6 43 11

2000

Female

R7 26 7

R8 40 7

R9 - -

Female

R10 - -

R11 52 32

R12 33 18

- Not applicable

( ) Loss in bodyweight
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Appendix 3 Macroscopic findings

Dose (mg/kg)

300 2000

Female Female

Animal Number R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 R11 R12

Fate of animal K K K K K K K K D D K K

Day of death 15 15 15 15 15 15 15 15 1 2 15 15

Tissues examined Findings

Subcutaneous tissue         2 2  

Brain            

Heart            

Lungs         4 2  

Liver         2   

Spleen            

Kidneys            

Stomach        6 9   

Duodenum            

Small Intestines         2 2,9  

Large Intestines         2 2,9  

Caecum          6  

Urinary Bladder            

K Killed at study termination

D Sporadic death

 No abnormalities detected

2 Congestion (darkened tissue/organs)

4 Pallor of tissue/organs

6 Small (atrophy)

9 Fluid contents (white, yellow or dark red liquid)
26

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Appendix 4 Test procedure

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

5mg/kg

3 animals

5mg/kg

3 animals

5mg/kg

3 animals

50mg/kg

3 animals

 5 25 30 50 200 300 500 1000 2000 2500 5000 

2-3 0-1

2-3

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 Category 5 Category 5 or

 > 0-5 > 5 - 50 > 50 - 300 > 300 - 2000 > Unclassified 2000 - 5000

Start

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

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

LD50 cut -off

mg/kg b.w.

GHS

3(at 50)

at 1st step

3(at 300) other

at 1st step

other 3 2 1 0

0

0

-  : unclassified

 - Testing at 5000 mg/kg b.w.

- per step three animals of a single sex (normally females ) are used

- 0,1,2,3: Number of moribund or dead animals at each step

- GHS: Globally Harmonized Classification System (mg/kg b.w.)
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Annex 1 GLP Compliance Statement
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HUNTINGDON RESEARCH CENTRE GLP COMPLIANCE STATEMENT 2012