Final Report

Study Title Acute Dermal Toxicity

Study in the Rat

Author



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RESPONSIBLE PERSONNEL

Acute Dermal Toxicity Study in the Rat

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

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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......................................................................................................................12

5.3.1 Preliminary test...................................................................................................12

5.3.2 Main study - limit test.........................................................................................12

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.................................................................12

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

9.3 Dermal reactions ...............................................................................................................14

10 TERMINAL PROCEDURES .........................................................................................................15

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

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

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

12.1 Mortality ...........................................................................................................................16
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12.2 Clinical signs.....................................................................................................................16

12.3 Dermal reactions ...............................................................................................................16

12.4 Body weights ....................................................................................................................16

12.5 Necropsy findings .............................................................................................................16

13 CONCLUSION...............................................................................................................................16

TABLES.................................................................................................................................................17

Table 1 Mortality data.....................................................................................................................18

Table 2 Clinical signs following treatment .....................................................................................18

Table 3 Dermal reactions ................................................................................................................19

Table 4 Individual body weights and weekly increments ...............................................................20

Table 5 Necropsy findings ..............................................................................................................21

APPENDIX ............................................................................................................................................22

Appendix 1 Certificate of Analysis .................................................................................................23
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1 SUMMARY

 following a single (24 hour) semi-occluded

topical application to the rat.

In the preliminary test the test article was applied as an undiluted liquid to the clipped

dorsum of a group comprising one male rat and one female rat at a dose level of

2000 mg/kg.

In the main study the test article was applied as an undiluted liquid to the clipped

dorsum of a group of five male and five female rats (which included the two

preliminary test rats) on Day 1. Each rat received a single topical application at a dose

level of 2000 mg/kg. The treated areas of dorsum were covered by a semi-occlusive

dressing for 24 hours. All animals were killed on Day 15 and subsequently underwent

a full necropsy.

No animal died and there were no clinical signs of reaction to treatment. Dermal

reactions were limited to atonia and desquamation. No dermal reactions were

observed in any of the males. One female also showed no dermal reactions.

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

No macroscopic changes were observed at necropsy.

The acute median lethal dermal dose of to rats was found

to exceed 2000 mg/kg.

Based on the results of this study, the General Classification and Labelling

Requirements for Dangerous Substances and Preparations, as stated in Commission

 in respect of its acute dermal toxicity.
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2 INTRODUCTION

The objective of the study was to determine the acute dermal toxicity of the test

article following a single (24 hour) semi-occluded topical application to the rat.

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

the first investigations carried out to assess the hazard it presents to manufacturer and

consumer populations. It provides information on likely consequences to man of

short-term exposure to chemical products following dermal contamination and a

common basis for classification and labelling.

Study dates

Protocol signed by Study Director: 18 April 2005

Animals on site: 10 May 2005

First treatment:

Preliminary Study 19 May 2005

Main Study 25 May 2005

Experimental completion date: 8 June 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 major deviations from the protocol. Minor deviations, which did not affect

the integrity or outcome of the study, are presented in the body of the report.

4 TEST ARTICLE

The test article, an orange liquid was identified as 
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The weight of the dose container with contents was determined before and after

dosing. The weight of the test formulation removed was compared with the weight of

the sum of the individual dose volumes and any discrepancies (outside the range -5%

to +10% of nominal) accounted for.

5.3 Study design

5.3.1 Preliminary test

The Study Director selected a dosing regimen that was intended either to demonstrate

that the maximum dose level was unlikely to be lethal in the main study, or to

determine a broad range of dose levels that included the acute median lethal dose. The

maximum dose level did not exceed 2000 mg/kg, the "limit test" dose level specified

in the test guidelines. A pair of animals (one male and one female) received a single

dermal dose of test article at 2000 mg/kg.

5.3.2 Main study - limit test

Since no compound-related mortality occurred in the preliminary test, one group of

five male and five female rats was subjected to a single dermal application of the test

article at 2000 mg/kg. This group consisted of the two animals used in the preliminary

test plus a further four male and four female rats to give the required group size of

five males and five females.

6 TEST ARTICLE FORMULATION

6.1 Preparation

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

(without regard to purity or active content). The test article was administered as

supplied by the Sponsor. The intervals between dispensing and completion of

administration of the test article were two hours 32 minutes (preliminary test) and two

hours 55 minutes (main study). The protocol specified that administration was to have

been within two hours of preparation. This deviation is not considered to have

affected the integrity or outcome of the study.

7 TEST SYSTEM

7.1 Justification for selection of the test system

The rat was selected because it is a rodent species recommended by various

regulatory authorities. The rat has been found to be the most tractable species for the

dosing procedures used in this type of study at this laboratory. The strain of rat was

selected as it is widely used for this type of study and a literature base is available for

comparative purposes.

7.2 Species, strain and supplier

Male and female (nulliparous, non-pregnant) rats of the HsdBrlHan: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.

7.3 Acclimatisation period and allocation to study

The condition of the animals was assessed daily throughout the acclimatisation period

of 9 to 15 days. A clinical inspection was performed prior to study commencement to
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ensure the animals were suitable for the test procedures. Overtly healthy animals were

arbitrarily allocated to the study at least one day before treatment commenced.

Animals were in a body weight range of 226 to 259 g (males) and 161 to 179 g

(females) on Day 1. Based on information from the supplier the rats were

approximately 8 to 10 weeks old on Day 1.

7.4 Caging

Up to five rats of the same sex were accommodated in cages that conform with 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) until after

completion of the Day 3 observations each rat was individually housed in a similar

cage. Animals allocated to the main study were returned to group housing after

completion of the Day 3 observations.

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. 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 had

been periodically analysed for specific contaminants.

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

supply were 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 o 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 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. Rats on the main study were also

observed immediately post-dose. Individual records of clinical signs 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.

9.3 Dermal reactions

The condition of the dermal test site was recorded following removal of the dressing

on Day 2 and once daily thereafter for the duration of the study period. Erythema and

oedema were scored as follows:

Erythema & eschar Grade

No erythema 0

Very slight erythema 1

Well defined erythema 2

Moderate erythema 3

Severe erythema (beet redness) or eschar formation preventing

grading of erythema 4

Oedema Grade

No oedema 0

Very slight oedema 1

Slight oedema

(edges of area well-defined by definite raising)

2

Moderate oedema 3

Severe oedema (raised more than 1 mm and

extending beyond the dermal test site)

4
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Other dermal changes were recorded in detail and are presented in Table 3. A glossary

of terminology and scoring scales is incorporated into the study record.

10 TERMINAL PROCEDURES

Rats 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, the

dermal test site, all viscera and tissue within the abdominal, thoracic and cranial

cavities, free-hand sectioning of the dermal test site, liver and kidneys.

11 DATA EVALUATION

11.1 Estimation of acute median lethal dose

The acute median lethal dose was determined using the mortality data.
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12 RESULTS

12.1 Mortality

(Table 1)

There were no deaths following a single dermal application of the test article at a dose

level of 2000 mg/kg.

12.2 Clinical signs

(Table 2)

No clinical signs of reaction to treatment were observed in any animal.

12.3 Dermal reactions

(Table 3)

Dermal reactions were limited to atonia, which was observed in one female on Day 2,

and desquamation, which was observed in up to four females between Day 3 and the

end of the observation period. One female and all of the males remained free of any

signs of dermal reaction throughout the study.

12.4 Body weights

(Table 4)

All rats gained weight during the first and second weeks of the study.

12.5 Necropsy findings

(Table 5)

No macroscopic changes were observed for animals killed on Day 15.

13 CONCLUSION

A single (24 hour) semi occluded topical application of the test article at a dose level

of 2000 mg/kg caused no deaths in a group of ten rats. No adverse clinical signs were

observed during the observation period. The acute median lethal dermal dose level

(LD50) was found to exceed 2000 mg/kg.

Based on the results of this study, the General Classification and Labelling

Requirements for Dangerous Substances and Preparations, as stated in Commission

Directive 2001/59/EC, indicate that no symbol and risk phrase are required for the test

article in respect of its acute dermal toxicity.
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TABLES


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Table 4

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

173M 229 237 234 256 285 19 29

174M 223 226 222 235 256 9 21

175M 249 259 257 271 292 12 21

176M 240 241 244 255 274 14 19

177M 238 245 238 250 267 5 17

241F 162 169 168 181 195 12 14

242F 171 169 172 183 191 14 8

243F 156 161 159 166 176 5 10

244F 176 179 173 189 199 10 10

2000

245F 166 170 166 177 187 7 10


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Appendix 1

Certificate of Analysis

Certificate of Analysis