# Final Report

Study Title Dermal Irritation Study in the Rabbit
Author A Robertson SponsorFinal Report

- 6 - ARCHIVE STATEMENT
Transcutaneous Electrical Resistance Assay
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.Final Report

- 8 - Appendix 1 Certificate of Analysis ............................................................................................. ....23Final Report

- 9 - 1 SUMMARY This study was conducted to assess the corrosivity to skin by measurement of transcutaneous electrical resistance (TER). It was designed to determine the skin corrosivity potential of the test article, , using ex vivo techniques. Corrosive substances produce an irreversible loss of normal stratum corneum integrity and function which can be measured as a reduction in the inherent TER below a corrosive threshold level (5 kohm). A dye binding assessment is used to identify chemicals that give false low readings for electrical resistance in the absence of visual damage. The results obtained from the electrical resistance measurements and dye binding assessments are indicators used in the prediction of severe cutaneous effects in vivo

. A measured volume of liquid test material (0.15 mL) was applied, without dilution, to the epidermal surface of pelt discs by injection into the central well of a polyterafluoroethylene (PTFE) tube. A set of three skin discs was exposed to for 24 hours at 20 to 23°C. Two additional sets of three skin discs were similarly prepared and dosed with negative and positive control chemicals. The control chemicals were purified water and 10 M hydrochloric acid for both the TER and dye binding measurements. gave a mean resistance value of 1.64 kohms. The negative control, purified water, gave a mean resistance value of 14.27 kohms and the positive control, 10 M hydrochloric acid, gave a mean resistance value of 0.63 kohms. The mean dye concentration recorded for the test article was 333.7 μg/disc. The mean dye concentration recorded for the negative control was 39.6 μg/disc. The mean dye concentration recorded for the positive control was 147.7 μg/disc. The test article was considered to have the potential to be corrosive to skin in vivo under the conditions of the test.Final Report

- 10 - 2 INTRODUCTION This study was conducted to assess the corrosivity to skin by measurement of transcutaneous electrical resistance (TER). It was designed to determine the skin corrosivity potential of the test article using ex vivo techniques. Corrosive substances produce an irreversible loss of normal stratum corneum integrity and function which can be measured as a reduction in the inherent TER below a corrosive threshold level (5 kohm). A dye binding assessment is used to identify chemicals that give false low readings for electrical resistance in the absence of visual damage. The results obtained from the electrical resistance measurements and dye binding assessments are indicators used in the prediction of severe cutaneous effects in vivo

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Study dates Protocol signed by Study Director: 18 April 2005 Animals on site: 26 April 2005 First treatment: 4 May 2005 Experimental completion date: 6 May 2005 The study completion date is the date that the final report is signed by the Study Director.Final Report

- 11 - 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, was received at as follows:

Test article Batch number Quantity supplied (mL) Date of receipt at Covance 100 18 April 2005

The purity and 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.

5 EXPERIMENTAL DESIGN 5.1 Regulatory test guidelines The study was conducted to meet the known requirements of the Commission Directive 2000/33/EC Method B40 Skin Corrosion. The study was also conducted to meet the known requirements of the OECD Guideline for Testing of Chemicals Method 430 (adopted 13 April 2004); this guideline was mistakenly omitted from the study protocol.
5.2 Preparation of antibiotic solution A standard antibiotic wash was prepared containing 800 μg/mL penicillin, 10 μg/mL chloramphenicol and 10 μg/mL amphotericin in lukewarm purified water.
5.3 Animal preparation All animals were given a clinical inspection for ill health on arrival. The condition of the animals was assessed daily throughout the acclimatisation period. The animals were aged 23 to 25 days old at the end of the acclimatisation period. Following acclimatisation, the animals were shaved to remove all hair from the full length of the dorsum and flanks using small animal electric clippers. Rats showing pre-existing damage or irritation were eliminated at this stage. The animals were then prepared by submerging the clipped area in antibiotic wash and carefully wiping the immersed skin with cotton wool. The rats were washed again using the sameFinal Report

- 13 - during these procedures were removed by gently tapping the PTFE tube. The stainless steel electrodes of the databridge assembly were placed on either side of the disc and the resistance measurement in kohms was recorded immediately. The first reading of the databridge was recorded (as resistance decreases with time). The electrodes were secured at the top by the Kelvin Clips and inserted into the PTFE tube (3 mm diameter electrode) or the receptor chamber (1 mm diameter electrode). The clip holding the inner (thick) electrode was rested on top of the PTFE tube to ensure a consistent length of electrode was submerged in the magnesium sulphate solution. The outer (thin) electrode was placed in the receptor chamber resting on the base of the chamber. The spring clip holding the PTFE tube was positioned such that the length of tube inside the holding vessel was consistent (70 mm), thereby reducing any error introduced by varying displacement of the magnesium sulphate solution.
5.7 Sulforhodamine B dye application and removal Certain non-corrosive materials such as neutral organics and surfactants have the ability to reduce the skin resistance below the threshold level of 5 kohms indicative of a corrosive material. The mean TER value of the discs treated with the test article was less than 5 kohms with no visual signs of damage. Therefore an assessment of sulforhodamine B dye penetration was carried out on the control and test article treated tissues in order to determine if the TER values obtained were the result of increased skin permeability or skin corrosion. After completion of the 24 hour post-dosing resistance measurements, the appropriate PTFE tubes were removed from the receptor chamber and the magnesium sulphate solution was washed away with a jet of tap water. The skin was carefully examined for obvious damage. As there was no obvious major damage the epidermal surface of each skin disc was subjected to direct application of 150 μL 10% w/v sulforhodamine B dye in purified water. Two hours later the skin discs were jet-washed with tap water for approximately 10 seconds to remove any excess or unbound dye. Each disc was then carefully removed from the PTFE tube and placed in a glass vial containing 8 mL of purified water. The vials were gently agitated for 5 minutes to remove any further excess/unbound dye. The rinsing procedure was repeated and then each skin disc was removed and placed into another scintillation vial containing 5 mL 30% w/v sodium dodecyl sulphate in purified water (SDS) and incubated overnight at 60°C [SDS was prepared on the previous day and maintained at 60°C]. After incubation each skin disc was removed from SDS and discarded. The remaining solution was centrifuged at approximately 175 G for 8 minutes at room temperature. A 1 mL aliquot of the supernatant was diluted with 4 mL SDS and mixed by gentle agitation of the sealed container. A sample of the diluted supernatant was transferred to a spectrophotometry cuvette and the optical density measured at 565 nM (the sample was maintained at 60°C until the spectrophotometer was available).

6 TEST SYSTEM 6.1 Justification for selection of the test system The species and strain used to provide tissues for this assay are defined in the EU test method.Final Report

- 14 - 6.2 Species, strain and supplier Male rats of the Hsd.BrlHan:WIST strain were obtained from Harlan UK Ltd, Bicester.
6.3 Specification The animals were ordered to be 20 to 22 days old on arrival to ensure they were in the telogen phase of hair growth throughout the acclimatisation period and at the time of skin disc preparation. All animals were given a clinical inspection for ill-health on arrival. The condition of the animals was assessed daily throughout the acclimatisation period.
6.4 Caging Animals were housed in groups of up to four in cages that conform to the ‘Code of practice for the housing and care of animals used in scientific procedures’ (Home Office, London, 1989).
6.5 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. Any minor deviations from the expected ranges were noted in the study data. The rooms were illuminated by fluorescent strip-lights for twelve hours daily.
6.6 Environmental enrichment During acclimatisation, a number of methods are used at enrich the environment of the animals as detailed in standard operating procedures. 6.7 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.Final Report

- 15 - 7 PRE-EXPERIMENTAL PROCEDURES 7.1 Acclimatisation and health procedures All animals were given a clinical inspection for ill health on arrival. They were acclimatised for three days and a second inspection was performed before pelt preparation to ensure their suitability for study.
7.2 Identification of the test system Each pelt was identified by a unique number. Each PTFE tube was identified by a number written in indelible ink. The numbers of the tubes assigned to the test or control skin discs were recorded in the study data. These identification numbers were also used on the scintillation vials as required.

8 DATA EVALUATION The mean TER results were accepted if the concurrent positive and negative control values fell within acceptable ranges for the method. Acceptable resistance ranges are given below:
Control Substance Resistance range (kohms) Positive 10 M Hydrochloric acid 0.5 – 1.0 Negative Purified water 10 – 25 The mean dye binding results were accepted on condition that concurrent values fell within the acceptable ranges for the method. Acceptable dye content ranges for the control substances are given below:
Control Substance Dye content range (μg/disc) Positive 10 M Hydrochloric acid 40 – 100 Negative Purified water 15 – 35
The test material is considered to be non-corrosive to the skin if the mean TER value obtained for the test material was greater than 5 kohms, or

• The mean TER value was less than or equal to 5 kohms and

• The skin disc was showing no obvious damage, and

• The mean disc dye content was well below the mean disc dye content for the 10 M hydrochloric acid obtained concurrently
The test material is considered to be corrosive to skin if the mean TER value was less than or equal to 5 kohms and the skin disc was obviously damaged, or

• The mean TER value was less than or equal to 5 kohms, and

• The skin disc was showing no damage, butFinal Report

- 16 - • The mean disc dye content was greater than or equal to the mean disc dye content of the 10 M hydrochloric acid obtained concurrently
8.1 Calculation of dye content per disc The concentration of the dye in solution was calculated using the optical density (absorbance) value as follows:
optical density x molecular weight Concentration of dye (g/L) = Molar extinction coefficient Since the dye was extracted in 5 mL of solvent and this solution was diluted 1:5 v/v, the dye content of the disc was calculated from:
concentration of dye x 5 x 5 x 10 6 Dye content ( μ g/disc) = 1000 Where: Optical density = value obtained Molar Extinction Coefficient for Sulforhodamine B dye = 8.7 x 10 4 Molecular Weight of Sulforhodamine B dye = 580 The sulforhodamine B dye content was determined for each skin disc separately. A mean dye content was then calculated for the three discs.Final Report

- 18 - TABLESFinal Report

- 19 - Table 1 Pelt viability measurements

Resistance readings (kohms) Rat number Trial Disc 1 Trial Disc 2 Trial Disc 3 Accept/Reject 169 41.05 5.46 22.29 Accept 170 34.20 44.70 NR Accept
NR the third disc was not required because the first two discs recorded resistance greater than 10 kohmsFinal Report

- 20 - Table 2 Resistance measurements

Test article Tube number Resistance measurements (kohms) Classification

1 1.43 2 2.27 3 1.21 Mean 1.64Final Report

- 22 - APPENDIXFinal Report

- 23 - Appendix 1 Certificate of Analysis Certificate of Analysis