Final Report

Study Title : Acute Oral Toxicity

Study in the Female Rat

(Acute Toxic Class)

Author



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RESPONSIBLE PERSONNEL

Acute Oral Toxicity Study in the Female Rat (Acute Toxic Class)

The following staff were responsible for key elements of the study:

Quality Assurance C Clare

Formulations P Tout

Animal House Supervisor L Cooper

Animal Health and Welfare A Basford

Necropsy J Campey

Histology L Patterson
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ARCHIVE STATEMENT

Acute Oral Toxicity Study in the Female Rat (Acute Toxic Class)

All primary data, or authenticated copies thereof, specimens and the final report will

be retained in the archives for one year after issue of

the final report. At this time the Sponsor will be contacted to determine whether the

data should be returned, retained or destroyed on their behalf and notified of the

financial implications of each of these options.
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CONTENTS

STUDY DIRECTOR AUTHENTICATION AND GLP COMPLIANCE STATEMENT ......................2

QUALITY ASSURANCE STATEMENT...............................................................................................3

SCIENTIFIC REVIEWER’S STATEMENT...........................................................................................4

RESPONSIBLE PERSONNEL................................................................................................................5

ARCHIVE STATEMENT .......................................................................................................................6

CONTENTS.............................................................................................................................................7

1 SUMMARY ......................................................................................................................................9

2 INTRODUCTION...........................................................................................................................10

3 PROTOCOL ADHERENCE...........................................................................................................11

4 TEST ARTICLE .............................................................................................................................11

5 EXPERIMENTAL DESIGN........................................................................................................... 11

5.1 Regulatory test guidelines.................................................................................................11

5.2 Test article administration.................................................................................................11

5.3 Study design......................................................................................................................11

6 TEST ARTICLE FORMULATION ...............................................................................................12

6.1 Preparation........................................................................................................................12

7 TEST SYSTEM ..............................................................................................................................12

7.1 Justification for selection of the test system......................................................................12

7.2 Species, strain and supplier...............................................................................................12

7.3 Acclimatisation period and allocation to study.................................................................13

7.4 Caging...............................................................................................................................13

7.5 Diet, water and bedding ....................................................................................................13

7.6 Environment......................................................................................................................13

7.7 Identification of the test system ........................................................................................13

8 ANIMAL HEALTH AND WELFARE...........................................................................................14

8.1 Environmental enrichment................................................................................................14

9 EXPERIMENTAL OBSERVATIONS...........................................................................................14

9.1 Clinical signs.....................................................................................................................14

9.2 Body weights ....................................................................................................................14

10 TERMINAL PROCEDURES .........................................................................................................14

10.1 Preservation of tissues for histopathology ........................................................................14

11 DATA EVALUATION...................................................................................................................15

11.1 Estimation of acute median lethal dose.............................................................................15

12 RESULTS .......................................................................................................................................16

12.1 Mortality ...........................................................................................................................16

12.2 Clinical signs.....................................................................................................................16

12.3 Body weights ....................................................................................................................16
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12.4 Necropsy findings .............................................................................................................16

13 CONCLUSION...............................................................................................................................17

14 REFERENCE..................................................................................................................................18

TABLES.................................................................................................................................................19

Table 1 Mortality data.....................................................................................................................20

Table 2 Clinical signs following treatment .....................................................................................21

Table 3 Individual body weights and weekly increments ...............................................................22

Table 4 Necropsy findings ..............................................................................................................23

APPENDICES........................................................................................................................................24

Appendix 1 Flow Diagram for Study Starting dose 300 or 2000 mg/kg.........................................25

Appendix 2 Certificate of Analysis .................................................................................................26
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1 SUMMARY

This study was conducted to assess the acute toxicity of the test article,

following a single oral administration to

small groups of rats. The study design provides information for hazard assessment

and classification and enables a chemical to be assigned to toxicity classes but

severely restricts animal usage.

Groups of three female fasted rats were given the test article as a single dose on Day 1

by oral gavage at dose levels of 300 or 2000 mg/kg. The test article was dispersed in

corn oil and administered at a dose volume of 10 mL/kg. Surviving animals were

killed on Day 15 and subsequently underwent a full necropsy.

There were two deaths following a single oral dose of 

among rats dosed at 2000 mg/kg. The deaths occurred at approximately 1⁄2 and 1 hour

post-dose. No changes in body weight were recorded. Signs of reaction to treatment in

decedents included exophthalmus, ataxia, lethargy, twitching and tachypnoea.

Macroscopic examination of these animals revealed no abnormalities.

Among surviving rats, signs of reaction to treatment included piloerection,

tachypnoea, exophthalmus, ataxia, twitching, bradypnoea, hyperpnoea, tremors,

lethargy, palpebral closure, hunched posture and unkempt appearance. Recovery, as

judged by external appearance and behaviour, was complete by Day 8.

All surviving rats achieved body weight gains during the first and second weeks of the

study.

Necropsy on Day 15 revealed two rats with uterine distension, which was

accompanied by a vaginal nodule in one animal, and one rat with pale kidneys

accompanied by renal pelvic dilatation.

The acute median lethal oral dose of to rats was found to

be between 500 and 1000 mg/kg.

The test article was classified as Category 4 according to the Globally Harmonised

System for the classification of chemicals which cause acute toxicity.

The test material was classified as HARMFUL and the symbol “Xn” and risk phrase

R 22 “HARMFUL IF SWALLOWED” are required according to EU labelling

regulations Commission Directive 2001/59/EC relating to the classification,

packaging and labelling of dangerous preparations.
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2 INTRODUCTION

The objective of the study was to assess the acute toxicity of the test article following

a single oral administration to small groups of rats. The study design provides

information for hazard assessment and classification and enables a chemical to be

assigned to toxicity classes but severely restricts animal usage.

Determination of the acute oral toxicity of a particular chemical product is among the

first investigations carried out to assess the potential hazard it presents to

manufacturer and consumer populations. It provides information on the likely

consequences to man of short-term exposure to chemical products by ingestion.

Study dates

Protocol signed by Study Director: 18 April 2005

Animals on site: 26 April 2005

First treatment: 4 May 2005

Experimental completion date: 26 May 2005

The study completion date is the date that the final report is signed by the Study

Director.
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3 PROTOCOL ADHERENCE

The study was conducted in accordance with the agreed definitive protocol. There

were no deviations from the protocol.

4 TEST ARTICLE

The test article, an orange liquid, was identified as 

 and was received at as follows:

Test article Batch number Quantity supplied (mL) Date of receipt at

8488/035 100 18 April 2005

The purity and an expiry date for the test article were not stated by the Sponsor.

When not in use the test article was stored in a sealed container, at room temperature

in the dark.

A certificate of analysis for the test article was provided by the Sponsor and is

presented in the Appendices.

The vehicle for the test article was corn oil, supplied by Sigma-Aldrich Co Ltd, Poole,

UK. The vehicle was chosen because corn oil gave the most satisfactory results in

pre-study formulation trials conducted by the .

5 EXPERIMENTAL DESIGN

5.1 Regulatory test guidelines

The study was conducted in compliance with Method B1 tris of Commission

Directive 2004/73/EC and OECD Guidelines for Testing of Chemicals, Method 423

(adopted 17 December 2001).

5.2 Test article administration

Doses were administered orally, by gavage, using plastic syringes and rubber

catheters. Each rat was dosed once on Day 1 by passing the tip of a catheter along the

oesophagus and instilling the test article into the gastric lumen.

5.3 Study design

Groups of fasted animals were treated as follows:

Dose Level (mg/kg) Concentration

(mg/mL)

Dose Volume (mL/kg) Number of Animals

300 30 10 3

2000 200 10 3

300 30 10 3
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Treatment of animals was sequential. Sufficient time was allowed between each group

to confirm the survival of the previously dosed animals.

The Study Director selected the starting dose level that was considered to be most

likely to cause the deaths of one or two animals.

Once the treatment-related mortality caused by the first dosing regimen was

established, the regimen appropriate for a second group of three rats was read from

the flow diagram presented in Appendix 1. Successive groups of rats were treated

according to the mortality affecting the previous treated group until the flow diagram

indicated that the Globally Harmonised Classification System (GHS) could be applied

to the test article.1

At the discretion of the Study Director, following review of the clinical

observations/mortality data obtained, the sequence detailed in the flow chart was

amended in an attempt to reduce the number of animals used without affecting the

purpose or integrity of the study. The rationale for deviation from the flow diagram is

documented in the study record.

Dose levels were expressed gravimetrically and in terms of test article received

(without regard to purity or active content).

Individual doses (mL) were calculated using the fasted body weights of the rats on the

morning of dosing (Day 1) and the dose volume of 10 mL/kg.

6 TEST ARTICLE FORMULATION

6.1 Preparation

The test article was dispersed in corn oil. The formulated concentrations were

calculated from the selected dose level and the dose volume of 10 mL/kg. All

formulations were used within two hours of preparation.

Dose containers were repeatedly inverted prior to administration to ensure

homogeneity.

7 TEST SYSTEM

7.1 Justification for selection of the test system

The rat has been selected because it is the rodent species preferred by various

regulatory authorities. Background data are available. Females are used because, in

literature surveys of conventional LD50 tests where a difference between the sexes has

been seen, females have been shown to be slightly more sensitive than males.

7.2 Species, strain and supplier

Female (nulliparous, non-pregnant) rats of the Hsd.BrlHan:WIST strain were obtained

from Harlan UK Ltd, Bicester. All animals were given a clinical inspection for ill

health on arrival and a sample was weighed.
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7.3 Acclimatisation period and allocation to study

Rats were arbitrarily selected from the delivery boxes and allocated to the appropriate

number of cages using a sequence that reduced selective bias. The condition of the

animals was assessed daily throughout the acclimatisation period of 8 to 16 days. A

second inspection was performed prior to study commencement to ensure the animals

were suitable for the study. Overtly healthy animals were arbitrarily allocated to the

study groups at least one day prior to dosing.

At the start of the study the rats were approximately 8 to 11 weeks of age. The weight

variation did not exceed ±20% of the mean weight.

7.4 Caging

The animals were housed in groups of up to five during the acclimatisation period in

cages that conform to the 'Code of Practice for the Housing and Care of Animals Used

in Scientific Procedures' (Home Office, London, 1989). From the day prior to dosing

(Day –1), the rats were housed in groups of three in similar cages.

7.5 Diet, water and bedding

SQC(E) Rat and Mouse Maintenance Diet No 1, from Special Diets Services Ltd,

Witham, was freely available to the animals at all times other than the period of

fasting from the evening of the day prior to dosing (Day -1) until approximately

3 hours after dosing. Each batch of diet had been analysed for specific constituents

and contaminants by the manufacturer.

Mains water was provided, ad libitum, via cage-mounted water bottles. The water is

had been analysed periodically for specific contaminants.

Certificates of analysis for contaminant levels in each batch of diet or in the water

supply are consigned to central files at this laboratory.

Bedding was provided on a weekly basis to each cage by use of clean Aspen wood

chips (Datesand Ltd, Manchester). The bedding had been analysed for specific

contaminants and the results retained on file at 

No contaminants were present in diet, water or bedding at levels which might

interfere with achieving the objective of the study.

7.6 Environment

The animal rooms were designed to permit at least 15 air changes per hour and to

maintain temperatures of 19 to 25°C. Although not actively controlled the relative

humidity of the animal room was expected to remain within the range 40 to 70% RH.

Recordings of maximum and minimum temperature and humidity were made twice

daily during the week and once a day on Saturdays, Sundays and public holidays. The

rooms were illuminated by fluorescent strip-lights for twelve hours daily.

7.7 Identification of the test system

A number written on the tail in indelible ink on the day before dosing individually

identified the rats. A colour coded card on each cage gave information including study

number, group number, animal numbers and sex.
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8 ANIMAL HEALTH AND WELFARE

All procedures carried out on live animals as part of this study were subject to the

provisions of United Kingdom National Law, in particular the Animals (Scientific

Procedures) Act 1986.

8.1 Environmental enrichment

A number of methods are used at to enrich the environment of the animals as

detailed in standard operating procedures.

9 EXPERIMENTAL OBSERVATIONS

9.1 Clinical signs

Treated rats were observed closely for clinical signs of reaction to treatment. Clinical

signs in all rats were recorded at approximately 15 and 30 minutes post-dose, hourly

between 1 and 4 hours post-dose (inclusive), twice daily on Days 2, 3 and 4 and once

daily from the fifth to last day of the observation period. In addition, rats treated at

300 mg/kg were observed for clinical signs immediately post-dose and rats treated at

2000 mg/kg were observed at 6 hours post-dose. These additional observations were

made at the discretion of the observer. Individual records of clinical signs and times

of death were maintained for each treated rat.

9.2 Body weights

Rats were weighed on Day -1 (day before dosing) and on Days 1, 4, 8 and 15.

Decedent carcass weights were also recorded prior to necropsy.

10 TERMINAL PROCEDURES

Rats surviving treatment were killed by intraperitoneal injection of sodium

pentobarbitone on Day 15. After exsanguination a full macroscopic necropsy was

performed and all lesions recorded.

The necropsy procedure included inspection of external surfaces and orifices, all

viscera and tissue within the abdominal, thoracic and cranial cavities, free-hand

sectioning of the liver and kidneys and examination of representative sections of

mucosal surfaces of the stomach, small and large intestines.

Animals dying during the observation period were subject to a similar necropsy with

the least possible delay. Carcasses were stored in a refrigerator until necropsy

commenced.

10.1 Preservation of tissues for histopathology

Representative samples of macroscopic changes found at necropsy were retained in

10% neutral buffered formalin pending instructions from the Sponsor. However, the

Sponsor confirmed that microscopic examination was not required.
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11 DATA EVALUATION

11.1 Estimation of acute median lethal dose

The test article was given a GHS1

 category according to the details in Appendix 1.

The Acute Toxic Class study results are expressed in terms of one of five GHS

categories: ≤5 mg/kg; >5 to 50 mg/kg; >50 to 300 mg/kg; >300 to 2000 mg/kg; >2000

to 5000 mg/kg.
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12 RESULTS

12.1 Mortality

(Table 1)

Two animals died following oral administration of the test article at 2000 mg/kg. The

rats were found dead at approximately 1⁄2 and 1 hour post-dosing, respectively.

12.2 Clinical signs

(Table 2)

Three of the six rats dosed at 300 mg/kg displayed piloerection between 2 and 3 hours

post-dose and one of these also displayed tachypnoea at 2, 3 and 4 hours post-dose.

Recovery, as judged by external appearance and behaviour, was complete by Day 2.

The remaining rats dosed at 300 mg/kg showed no signs of reaction to treatment.

All rats dosed at 2000 mg/kg displayed exophthalmus and ataxia, and one was also

lethargic, at 1⁄4 hour post-dose. Two of these rats showed twitching and tachypnoea.

Other signs of reaction to treatment, which were seen in the surviving rat, were

bradypnoea, hyperpnoea, tremors, lethargy, palpebral closure, hunched posture and

unkempt appearance. Recovery, as judged by external appearance and behaviour, was

complete by Day 8.

12.3 Body weights

(Table 3)

All surviving rats gained weight during the first and second weeks of the observation

period.

12.4 Necropsy findings

(Table 4)

The isolated macroscopic changes that were apparent on Day 15 showed no trend

consistent with a treatment related effect on any target organ. Such changes included

two instances of slight bilateral uterine distension, one of which was accompanied by

a vaginal nodule, and one instance of moderately pale kidneys accompanied by

moderate renal pelvic dilatation. These macroscopic changes were limited to animals

dosed at 300 mg/kg. There were no macroscopic changes apparent for rats dosed at

2000 mg/kg.
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13 CONCLUSION

The acute median lethal oral dose of to rats was found to

be between 500 and 1000 mg/kg.

The test article was classified as Category 4 according to the Globally Harmonised

System for the classification of chemicals which cause acute toxicity.

The test material was classified as HARMFUL and the symbol “Xn” and risk phrase

R 22 “HARMFUL IF SWALLOWED” are required according to EU labelling

regulations Commission Directive 2001/59/EC relating to the classification,

packaging and labelling of dangerous preparations.
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14 REFERENCE

1. OECD (1998). Harmonised Integrated Hazard Classification for Human

Health and Environmental Effects of Chemical Substances as endorsed by the

28th Joint Meeting of the Chemicals Committee and the Working Party on

Chemicals in November 1998, Part 2, p 11.
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TABLES
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Table 1

Mortality data

Dose level

(mg/kg)

Mortality ratio Times of death

after dosing

300

2000

300

0/3

2/3

0/3

1⁄2 and 1 hour
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Table 2

Clinical signs following treatment

Dose level: 300 mg/kg

Sign noted after dosing on Day 1

(hours):

Sign noted on Day: Animal

number Clinical sign

Imm 1⁄4 1⁄2 1 2 3 4 2 3 4 5 6 7 8 9 to 15

Piloerection + + 223F Tachypnoea + + +

224F Piloerection +

225F Piloerection + +

Key:

Imm Immediately post-dose

+ Sign present (if no entry is made the sign is absent)

Dose level: 2000 mg/kg

Sign noted after dosing on Day 1

(hours):

Sign noted on Day: Animal

number Clinical sign

1⁄41⁄21 2 3 4 6 2 3 4 5 6 7 8 9 to 15

Exophthalmus +

Lethargy +

Ataxia + 226F

Found dead +

Exophthalmus ++++++

Ataxia + + + + + +

Tachypnoea +

Twitching + + + +

Bradypnoea +

Hyperpnoea +

Tremor +

Lethargy +

Palpebral closure +

Hunched posture +

227F

Unkempt + + + + + +

Exophthalmus + +

Ataxia + +

Tachypnoea +

Twitching +

228F

Found dead +

Key:

+ Sign present (if no entry is made the sign is absent)

Dose level: 300 mg/kg

Clinical sign Animal number

229F 230F 231F

Observations !!!

Key:

! No clinical signs seen
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Table 3

Individual body weights and weekly increments

Dose level Body weight (g) at: Increment (g)

(mg/kg)

Animal

number

and sex

Day -1 Day 1 Day 4 Day 8 Day 15 Day 1

to 8

Day 8

to 15

Decedent body

weights

223F 144 134 157 170 184 36 14

300 224F 137 131 150 160 173 29 13

225F 149 140 159 163 184 23 21

226F 168 153 (153)

2000 227F 166 151 162 179 194 28 15

228F 152 145 (145)

229F 176 160 179 188 199 28 11

300 230F 182 167 190 199 208 32 9

231F 160 144 164 167 174 23 7

Decedent weight at death is given in parentheses
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Table 4

Necropsy findings

Dose level: 300 mg/kg

Animal number Time and

manner of

death (Day)

Necropsy comments

223F 15T No macroscopic changes

224F 15T Uterus: distension, bilateral- slight #

225F 15T Uterus: distension, bilateral- slight #

Vagina: beige nodule – 3 mm diameter #

Dose level: 2000 mg/kg

Animal number Time and

manner of

death (Day)

Necropsy comments

226F 1D No macroscopic changes

227F 15T No macroscopic changes

228F 1D No macroscopic changes

Dose level: 300 mg/kg

Animal number Time and

manner of

death (Day)

Necropsy comments

229F 15T Kidneys: pale, bilateral - moderate

pelvic dilatation, unilateral – moderate #

230F 15T No macroscopic changes

231F 15T No macroscopic changes

D Animal died during course of study

T Animal killed by intraperitoneal injection of an overdose of sodium pentobarbitone at completion of

observation period

# Sample preserved in neutral buffered formalin
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APPENDICES
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Appendix 1

Flow Diagram for Study

Starting dose 300 or 2000 mg/kg

Starting dose of 300 mg/kg

2-3 deaths

0-1 deaths 2-3 deaths

0-1 death 3

(at 50)

Step 1

2-3 deaths

0-1 deaths 2-3 deaths Other

0-1 death 3

(at 300)

Step 1

2-3 deaths

0-1 deaths 2-3 deaths Other

0-1 death

3

2-3 deaths

2

0-1 deaths 2-3 deaths

0-1 death 1

0 deaths 0

both groups

Starting dose of 2000 mg/kg

2-3 deaths

0-1 deaths 2-3 deaths

0-1 death 3

(at 50)

Step 1

2-3 deaths

0-1 deaths 2-3 deaths Other

0-1 death 3

(at 300)

Step 1

2-3 deaths

0-1 deaths 2-3 deaths Other

0-1 death 3

(at 2000)

Step 1

2-3 deaths 2

(at 2000)

0-1 deaths 2-3 deaths Step 1

Other

0-1 death

1

0 deaths 0

both groups

300

500

2500

2500

5

50

200

50

5

>300-2000

2000

3

>50-300

5 or 5000

Unclassified Unclassified

5

>2000-5000

3 animals 3 animals 1000

@ 2000 mg/kg @ 2000 mg/kg

START

@ 300 mg/kg

4

3 animals

@ 300 mg/kg 3 animals

@ 50 mg/kg

3 animals 30

@ 50 mg/kg 3 animals

2

>5-50

25

@ 5 mg/kg

3 animals >0-5

@ 5 mg/kg 3 animals

category mg/kg BW

1

Classify LD50 

GHS cut-off

3

>50-300

2

>5-50

category mg/kg BW

Classify LD50 

5 or 5000

Unclassified Unclassified

5

>2000-5000

@ 2000 mg/kg 3 animals

@ 2000 mg/kg 2000

4

3 animals >300-2000 1000

300

500

3 animals 3 animals

@ 300 mg/kg @ 300 mg/kg

START 200

@ 50 mg/kg

3 animals 30

@ 50 mg/kg 3 animals

25

@ 5 mg/kg

3 animals >0-5

@ 5 mg/kg 3 animals

1

GHS cut-off
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Appendix 2

Certificate of Analysis

Certificate of Analysis

Infineum Product: 

Units Method Result

TBN mgKOH/g ASTM D2896 127.57

TAN mgKOH/g ASTM D664 129.28

CHN % mass SOP/EA/NW/77 C 72% H 12.1% N 2.7%

Viscosity (100% @ 20oC) cSt ASTM D445 110

IR IN HOUSE - BELOW