final report test facility study no. 785047

FINAL REPORT

Test Facility Study No. 785047

The In Vitro Percutaneous Absorption of Benzalkonium Chloride from 2 Formulations Through Human Skin (Analysis by LC-MS/MS)

TEST FACILITY: Charles River Laboratories Edinburgh Ltd

Elphinstone Research Centre Tranent, East Lothian

EH33 2NE UK

SPONSOR: Lonza Inc

412 Mt. Kemble Avenue, Suite 200S Morristown, NJ 07960

USA

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DocuSign Envelope ID: 7B4F10-9Appendix 2

Appendix 2


TABLE OF CONTENTS

1 COMPLIANCE STATEMENT .................................................................. 7

2 QUALITY ASSURANCE STATEMENT ................................................. 8

3 RESPONSIBLE PERSONNEL .................................................................. 9

4 SUMMARY ................................................................................................ 10

5 INTRODUCTION ...................................................................................... 12

5.1 Study Location ............................................................................................ 12

5.2 Study Dates ................................................................................................. 12

5.3 Archive Location ........................................................................................ 12

5.4 Regulatory Citations and References ....................................................... 12

6 EXPERIMENTAL PROCEDURES ........................................................ 13

6.1 Materials ..................................................................................................... 13

6.1.1 Test Item ..................................................................................................... 13

6.1.2 Reference Item ............................................................................................ 13

6.1.3 Internal Standard ....................................................................................... 14

6.2 Other Materials .......................................................................................... 14

6.3 Human Skin Samples ................................................................................. 14

6.4 Preparation of Split-Thickness Skin Membranes ................................... 14

6.5 Static Diffusion Cell Apparatus ................................................................ 14

6.6 Receptor Fluid ............................................................................................ 15

6.7 Solubility of Benzalkonium Chloride in Receptor Fluid ........................ 15

6.8 Static Diffusion Cell Preparation .............................................................. 15

6.9 Barrier Integrity Assessment .................................................................... 15

6.10 Pre-dose Receptor Fluid Collection .......................................................... 16

6.11 Preparation of Formulations ..................................................................... 16

6.12 Application of Formulation to Human Skin ............................................ 16

6.13 Receptor Fluid Sampling ........................................................................... 16

6.14 Terminal Exposure Procedures (30 min Post Dose) ............................... 17



6.15 Terminal Post Exposure Procedures (102 h Post Dose) ......................... 17

6.16 Storage of Samples ..................................................................................... 17

6.17 Sample Analysis .......................................................................................... 17

6.18 Electronic Data Acquisitions and Systems ............................................... 17

6.19 Data Presentation ....................................................................................... 18

7 DEFINITIONS ........................................................................................... 18

8 PROTOCOL ADHERENCE .................................................................... 18

9 REFERENCES ........................................................................................... 18

10 RESULTS AND DISCUSSION ................................................................ 18

10.1 Benzalkonium Chloride in Antibacterial Foaming Handwash (0.131%, w/w) ............................................................................................. 18

10.2 Benzalkonium Chloride in Antibacterial Liquid Hand Soap (0.14%, w/w) ............................................................................................... 19

11 CONCLUSION ........................................................................................... 20

12 FIGURES .................................................................................................... 21

13 TABLES ...................................................................................................... 28

14 APPENDICES ............................................................................................ 38



LIST OF FIGURES

Figure 1 Individual Absorption Profiles (% Applied Dose) for Benzalkonium Chloride in Receptor Fluid at 102 h Post Dose Following Topical Application of Antibacterial Foaming Handwash (0.131%, w/w) to Human Split-Thickness Skin ............................................................................ 21

Figure 2 Individual Absorption Profiles (% Applied Dose) for Benzalkonium Chloride in Receptor Fluid at 102 h Post Dose Following Topical Application of Antibacterial Foaming Handwash (0.131%, w/w) to Human Split-Thickness Skin (Excluding Cell 7) ............................................. 22

Figure 3 Cumulative Absorption Profiles (ng/cm2) for Benzalkonium Chloride in Receptor Fluid at 102 h Post Dose Following Topical Application of Antibacterial Foaming Handwash (0.131%, w/w) to Human Split-Thickness Skin (Mean ± SD, n =11) ....................................................... 23

Figure 4 Flux Profile (ng/cm2/h) for Benzalkonium Chloride in Receptor Fluid Following Topical Application of Antibacterial Foaming Handwash (0.131%, w/w) to Human Split-Thickness Skin (Mean ± SD, n =11) .............. 24

Figure 5 Individual Absorption Profiles (% Applied Dose) for Benzalkonium Chloride in Receptor Fluid at 102 h Post Dose Following Topical Application of Antibacterial Liquid Hand Soap (0.14%, w/w) to Human Split-Thickness Skin ............................................................................ 25

Figure 6 Cumulative Absorption Profiles (ng/cm2) for Benzalkonium Chloride in Receptor Fluid at 102 h Post Dose Following Topical Application of Antibacterial Liquid Hand Soap (0.14%, w/w) to Human Split-Thickness Skin (Mean ± SD, n =12) ....................................................... 26

Figure 7 Flux Profile (ng/cm2/h) for Benzalkonium Chloride in Receptor Fluid Following Topical Application of Antibacterial Liquid Hand Soap (0.14%, w/w) to Human Split-Thickness Skin (Mean ± SD, n =12) ................ 27



LIST OF TABLES

Table 1 Distribution of Benzalkonium Chloride (% Applied Dose) at 102 h Post Dose Following Topical Application of Antibacterial Foaming Handwash (0.131%, w/w) to Human Split-Thickness Skin ............................. 28

Table 2 Cumulative Absorption of Benzalkonium Chloride (% Applied Dose) into Receptor Fluid Following Topical Application of Antibacterial Foaming Handwash (0.131%, w/w) to Human Split-Thickness Skin .............. 29

Table 3 Distribution of Benzalkonium Chloride (µg/cm2) at 102 h Post Dose Following Topical Application of Antibacterial Foaming Handwash (0.131%, w/w) to Human Split-Thickness Skin ............................................... 30

Table 4 Cumulative Absorption of Benzalkonium Chloride (ng/cm2) into Receptor Fluid Following Topical Application of Antibacterial Foaming Handwash (0.131%, w/w) to Human Split-Thickness Skin .............. 31

Table 5 Flux Profile (ng/cm2/h) of Benzalkonium Chloride into Receptor Fluid Following Topical Application of Antibacterial Foaming Handwash (0.131%, w/w) to Human Split-Thickness Skin ............................................... 32

Table 6 Distribution of Benzalkonium Chloride (% Applied Dose) at 102 h Post Dose Following Topical Application of Antibacterial Liquid Hand Soap (0.14%, w/w) to Human Split-Thickness Skin .............................. 33

Table 7 Cumulative Absorption of Benzalkonium Chloride (% Applied Dose) into Receptor Fluid Following Topical Application of Antibacterial Liquid Hand Soap (0.14%, w/w) to Human Split-Thickness Skin .................. 34

Table 8 Distribution of Benzalkonium Chloride (µg /cm2) at 102 h Post Dose Following Topical Application of Antibacterial Liquid Hand Soap (0.14%, w/w) to Human Split-Thickness Skin ................................................. 35

Table 9 Cumulative Absorption of Benzalkonium Chloride (ng/cm2) into Receptor Fluid Following Topical Application of Antibacterial Liquid Hand Soap (0.14%, w/w) to Human Split-Thickness Skin .............................. 36

Table 10 Flux Profile (ng/cm2/h) of Benzalkonium Chloride into Receptor Fluid Following Topical Application of Antibacterial Liquid Hand Soap (0.14%, w/w) to Human Split-Thickness Skin ................................................. 37



LIST OF APPENDICES

Appendix 1 GLP Certificate ................................................................................................. 38 Appendix 2 Certificate of Analysis and Chain of Custody of Benzalkonium

Chloride Formulations ...................................................................................... 39 Appendix 3 Certificate of Analysis of Deuterated Benzalkonium Chloride ........................ 41 Appendix 4 Certificate of Analysis of Nifedipine ................................................................ 42 Appendix 5 Human Skin Donor Details ............................................................................... 43 Appendix 6 Thickness of Full and Split-Thickness Skin Membranes ................................. 43 Appendix 7 Cross Reference of Cell Number with Donor Number and Electrical

Resistance ......................................................................................................... 44 Appendix 8 Epidermis Removal by Tape Stripping ............................................................ 45 Appendix 9 Analytical Procedure ........................................................................................ 46 Appendix 10 OECD Guidance Document No. 28 Glossary of Terms ................................... 78



1 COMPLIANCE STATEMENT

Study Title: The In Vitro Percutaneous Absorption of Benzalkonium Chloride from 2 Formulations Through Human Skin (Analysis by LC-MS/MS) I, the undersigned, hereby declare that this study was performed in accordance with the OECD Principles of Good Laboratory Practice as incorporated into the United Kingdom Statutory Instrument for GLP and as accepted by Regulatory Authorities throughout the European Union, United States of America (FDA and EPA) and Japan (MHLW, MAFF and METI), and other countries that are signatories to the OECD Mutual Acceptance of Data Agreement. The study was conducted according to the procedures herein described and this report represents a true and accurate record of the results obtained.

Frank Toner, BSc MRSB Study Director

DocuSign Envelope ID: 7B4F107D-500C-47F3-8AB9-94502F0E2932


2 QUALITY ASSURANCE STATEMENT

Study Title: The In Vitro Percutaneous Absorption of Benzalkonium Chloride from 2 Formulations Through Human Skin (Analysis by LC-MS/MS) The Charles River Quality Assurance Unit conducted a protocol review, protocol amendment review, process inspections and report audits on this study, as detailed below.

QA INSPECTION DATES

Date findings submitted to Date(s) of Audit Phase(s) Audited Study Director Management 18 Oct 2019 Terminal Procedures N/A 18 Oct 2019 22 Oct 2019 Dose Administration N/A 23 Oct 2019 30 Oct 2019 Barrier Integrity Assessment N/A 01 Nov 2019 13 Nov 2019 Split-thickness skin preparation N/A 13 Nov 2019 20 Nov 2019 Final Protocol 29 Nov 2019 29 Nov 2019 02 Dec 2019 Receipt of Skin N/A 02 Dec 2019 27 Dec 2019 Protocol Amendment 01 27 Dec 2019 27 Dec 2019 11 Mar 2020 – 12 Mar 2020 Report 20 Mar 2020 20 Mar 2020 16 Mar 2020 – 20 Mar 2020 17 Mar 2020 – 20 Mar 2020 Data Review – Bioanalysis &

Immunology 20 Mar 2020 20 Mar 2020

02 June 2020 Final Report 02 June 2020 02 June 2020 Process-based inspections relevant to this study are scheduled once every quarter. The outcome of each inspection is reported to Management and, where relevant, the Study Director. Facilities relevant to this study are included in Charles River’s annual facility inspection programme. The outcome of each inspection is reported to Management. This report is considered to describe accurately and completely the procedures used in the study and the results obtained.

Alison McNaughton, HNC RQAP-GLP MRQA Quality Assurance



3 RESPONSIBLE PERSONNEL

Study Director: Frank Toner, BSc MRSB Report Compilation: Leanne Page, BSc Principal Analyst: Rona McGregor, BSc Quality Assurance: Alison McNaughton, HNC RQAP-GLP MRQA

Jamie White, BSc Sponsor Representative: Mielke Burkhard, PhD Sponsor Monitor: Dr. Nicholas P. Skoulis, Ph.D



4 SUMMARY

The test item, Benzalkonium Chloride, is used in antibacterial soap formulations developed by the Sponsor. As part of the risk assessment, a study was required to assess the rate and extent of absorption of the test item following topical application of the formulations to human skin. The analytical method was validated in all relevant samples (i.e. human skin, receptor fluid, tape strips, skin wash, tissue swabs, pipette tips, donor chamber wash and receptor chamber wash) under Charles River Study No. 329863 (GLP compliant). Details of the tested formulations are provided below:

• Antibacterial Foaming Handwash (0.131%, w/w) • Antibacterial Liquid Hand Soap (0.14%, w/w)

Split-thickness human skin membranes were mounted into static diffusion cells in vitro. Receptor fluid was added to the receptor chamber (nominal volume 5 mL, nominal exposure area 0.64 cm2). The skin surface temperature was maintained at 32°C ± 1°C throughout the experiment. An electrical resistance barrier integrity assessment was performed and any skin sample exhibiting resistance lower than 10.9 kΩ was excluded from subsequent absorption measurements. The formulations were applied (ca 10 mg/cm2) to human split-thickness skin membranes from six different donors and the cells were left open to the atmosphere. The application of the test preparation was repeated at timepoints as summarised in the following table:

Dosing times Day 1 0, 1.5 h, 3.0 h, 4.5 h, 6 h Day 2 24 h, 25.5 h, 27 h, 28.5 h, 30 h Day 3 48 h, 49.5 h, 51 h, 52.5 h, 54 h

Percutaneous absorption was assessed by collecting receptor fluid periodically at timepoints summarised in the following table:

Receptor fluid sampling times Day 1 0, 1.5 h, 3.0 h, 4.5 h, 6 h, 7.5 h Day 2 24 h, 25.5 h, 27 h, 28.5 h, 30 h, 31.5 h Day 3 48 h, 49.5 h, 51 h, 52.5 h, 54 h, 55.5 h Day 4 72 h, 75 h, 78 h Day 5 96 h, 99 h, 102 h

After each application the formulations remained in contact with the skin for 30 min. Following which the skin surface was rinsed with deionised water and the surface dried with tissue swabs. At 102 h post initial dose, the skin was then removed from the static cells, the stratum corneum tape stripped and the skin divided into exposed and unexposed skin. The bulk receptor fluid was collected from the receptor chamber and retained for analysis. The receptor chambers were rinsed with methanol and the samples retained for analysis. All samples were analysed by LC-MS/MS using the validated analytical procedures.



A summary of the Benzalkonium Chloride absorption through human split-thickness skin is provided in the following table:

Test Item Benzalkonium Chloride Formulation Foaming Handwash Liquid Hand Soap Applied rate (mg/cm2) 10.06 9.95 Distribution % Applied Dose Total Dislodgeable Dose 96.17 92.85 Stratum Corneum 0.38 0.13 Unexposed Skin 0.01 0.01 Total Unabsorbed 96.56 92.99 Exposed Skin 0.35 0.10 Total Absorbed Dose 0.11 0.06 Dermal Delivery 0.46 0.16 Mass Balance 97.02 93.15 Distribution µg/cm2 Total Dislodgeable Dose 189.73 185.05 Stratum Corneum 0.76 0.26 Unexposed Skin 0.02 0.02 Total Unabsorbed 190.51 185.33 Exposed Skin 0.70 0.21 Total Absorbed Dose 0.21 0.11 Dermal Delivery 0.91 0.32 Mass Balance 191.42 185.65

Total Dislodgeable dose = total skin wash + total tissue swab + total pipette tip + donor chamber wash. Stratum corneum = tape strips 1 to 20. Total unabsorbed dose = total dislodgeable dose + stratum corneum + unexposed skin. Total absorbed dose = cumulative receptor fluid + receptor chamber wash. Dermal delivery = absorbed dose + exposed skin. Mass balance = unabsorbed dose + dermal delivery.

In conclusion, following topical application Benzalkonium Chloride in Antibacterial Foaming Handwash (0.131%, w/w) and Antibacterial Liquid Hand Soap (0.14%, w/w) to human skin in vitro, the total absorbed dose was 0.11% (0.21 µg/cm2) and 0.06% (0.11 µg/cm2), respectively. Dermal delivery was 0.46% (0.91 µg/cm2) and 0.16% (0.32 µg/cm2) of the applied dose, respectively. The mass balance was 97.02% (191 µg/cm2) and 93.15% (186 µg/cm2) of the applied dose.



5 INTRODUCTION

The test item, Benzalkonium Chloride, is used in antibacterial soap formulations developed by the Sponsor. As part of the risk assessment, a study was required to assess the rate and extent of absorption of the test item following topical application of the formulations to human skin. The analytical method was validated in all relevant samples (i.e. human skin, receptor fluid, tape strips, skin wash, tissue swabs, pipette tips, donor chamber wash and receptor chamber wash) under Charles River Study No. 329863 (GLP compliant). Details of the tested formulations are provided below:

• Antibacterial Foaming Handwash (0.131%, w/w) • Antibacterial Liquid Hand Soap (0.14%, w/w)

5.1 Study Location

The study was conducted at Charles River Laboratories Edinburgh Ltd, Elphinstone Research Centre, Tranent, East Lothian, EH33 2NE, UK. 5.2 Study Dates

Key dates in the conduct of the study were as follows: Study Initiation: 19th November 2019 Experimental Start Date: 26th November 2019 Experimental Completion Date: 23rd December 2019 Study Completion Date: See Compliance Statement page for date of Study

Director’s Signature 5.3 Archive Location

All raw data generated and recorded during this study will be stored in the Scientific Archive of Charles River Laboratories Edinburgh Ltd for two years after the issue of the final report. After the two-year period the Sponsor will be consulted regarding the disposal, transfer or continued storage of the raw data. Electronic data generated by the Test Facility were archived as noted above, except for the reporting files stored on Shared Document Management System (SDMS), which were archived at the Charles River Laboratories facility located in Wilmington, MA. A reserve sample of the formulations will be retained in the Scientific Archive of Charles River Laboratories Edinburgh Ltd for a period of at least two years or until the quality of the material no longer affords evaluation. The original signed copy of the final report will be stored indefinitely in the Scientific Archives of Charles River Laboratories Edinburgh Ltd. 5.4 Regulatory Citations and References

This study was performed in accordance with Good Laboratory Practice regulations. A copy of the GLP Certificate for Charles River is provided in Appendix 1. The study was performed in accordance with the following document:



• OECD Guideline for Testing of Chemicals, Guideline 428: Skin Absorption: In Vitro

Method (2004). • OECD Environmental Health and Safety Publications Series on Testing and

Assessment No. 28. Guidance Document for the Conduct of Skin Absorption Studies (2004).

• Scientific Committee on Consumer Safety. Notes of Guidance for Testing of Cosmetic Ingredients and their Safety Evaluation, 10th Revision, 24-25 October 2018.

• COLIPA (1997). Cosmetic Ingredients: Guidelines for Percutaneous Absorption/Penetration. The European Cosmetic, Toiletry and Perfumery Association.

6 EXPERIMENTAL PROCEDURES

6.1 Materials

6.1.1 Test Item

Identification: Antibacterial Foaming Handwash Supplier Henkel Batch (Lot) Number: 7424-44 Physical Appearance: Light blue liquid Purity: 100% Concentration 0.131%, w/w Storage Conditions: Ambient Expiration Date 11 October 2022

Identification: Antibacterial Liquid Hand Soap Supplier: Colgate Palmolive Batch (Lot) Number: PCSB19-364 Physical Appearance: Reddish Orange Liquid Purity: 100% Concentration: 0.14%, w/w Storage Conditions: Ambient Expiration Date: 12 September 2021

An archive sample of formulations has been retained at Charles River. A copy of the Certificate of Analysis and Chain of Custody for Antibacterial Foaming Handwash and Antibacterial Liquid Hand Soap, respectively, are presented in Appendix 2. 6.1.2 Reference Item

Identification: Deuterated Benzalkonium Chloride Supplier: EAG Laboratories Batch (Lot) Number: V1JAT100-19485 Physical Appearance: Off white solid Purity: 98.7% Storage Conditions: Ambient/protected from light Recertification Date: 11 March 2021



A copy of the Certificate of Analysis is presented in Appendix 3. 6.1.3 Internal Standard

Identification: Nifedipine Supplier: Sigma-Aldrich Batch (Lot) Number: MKBW8746V Physical Appearance: Yellow powder Purity: 99% Storage Conditions: Refrigerator set to maintain 4oC Recertification Date: Not available

A copy of the Certificate of Analysis is presented in Appendix 4. 6.2 Other Materials

Phosphate buffered saline, penicillin-streptomycin, sodium azide and bovine serum albumin were obtained from Sigma Aldrich Ltd. Sodium hydroxide solution was obtained from Merck. Methanol was obtained from Fisher Chemicals. All other materials were obtained by Charles River. 6.3 Human Skin Samples

Samples of full-thickness human skin (Abdomen) were obtained from female donors aged 34 to 50 years old. One skin sample was obtained from NHS Lothian on ice. On arrival, the subcutaneous fat and connective tissue was removed from the skin using a scalpel. The skin was washed under cold running tap water and dried with paper towels. The skin was then cut into pieces, wrapped in aluminium foil and/or vacuum packed and stored at -20ºC until used in the study. Five samples were obtained from Tissue Solutions. The samples arrived at Charles River deep frozen on dry ice and were stored in a freezer set to maintain a temperature of -20°C until used on the study. The age and sex of the donors and site from which the skin was taken were recorded centrally and in the study records. The details are shown in Appendix 5. 6.4 Preparation of Split-Thickness Skin Membranes

Human skin samples were removed from -20°C storage and allowed to thaw at ambient temperature. The thickness of the full-thickness skin membranes was measured using a micrometer. Split-thickness membranes were prepared by pinning the full-thickness skin, stratum corneum uppermost, onto a raised cork board and cutting with an electric dermatome (Zimmer®) at a setting equivalent to 200-400 µm depth. The thickness of the membranes was measured using a micrometer. Membranes were then wrapped in foil, placed into a self-sealing bag and stored in a freezer, set to maintain a temperature of -20°C, for a maximum period of two months. The thickness of the full-thickness and split-thickness human skin membranes is provided in Appendix 6. 6.5 Static Diffusion Cell Apparatus

A static diffusion cell system (PermeGear Inc) was used (see the following photograph). The glass static diffusion cells were placed in a manifold on a magnetic stirrer plate heated via a circulating water bath to maintain the skin surface temperature at 32°C ± 1°C. The actual cell



temperatures (ranging from 31.3°C to 32.6°C) were calibrated prior to mounting the skin membranes. The surface area of exposed skin within the cells was 0.64 cm2. The receptor chamber volume was nominally 5 mL, with each receptor chamber individually marked with the actual volume by the manufacturer. A Photograph of a Static Diffusion Cell and a Static Diffusion Cell in the Heated Manifold

6.6 Receptor Fluid

The receptor fluid chosen for use in this study was phosphate buffered saline containing bovine serum albumin (BSA, ca 5%, w/v), sodium azide (ca 0.01%, w/v), streptomycin (0.1 mg/mL) and penicillin (100 units/mL). The receptor fluid was degassed by sonication, the pH adjusted to pH 7.4 ± 0.1 (actual pH: 7.40) and stored in a fridge set to maintain a temperature of ca +4°C prior to use. 6.7 Solubility of Benzalkonium Chloride in Receptor Fluid

The solubility of Benzalkonium Chloride in the receptor fluid was determined under the method validation (Charles River Study No. 329863). 6.8 Static Diffusion Cell Preparation

The receptor chambers were placed in a manifold and connected to a circulating waterbath. Magnetic stirrer bars were placed in the receptor fluid chambers which were filled with receptor fluid (Section 6.6). Once ambient temperature was reached, sections of split-thickness skin (ca 1.5 x 1.5 cm) were cut and mounted in the diffusion cells between the donor and receptor chamber. The donor chamber was tightened into place with a clamp. Cells were visually checked to ensure no cells were leaking and no air bubbles were present in the receptor fluid chamber. 6.9 Barrier Integrity Assessment

Skin samples were allowed to equilibrate at 32°C ± 1°C for ca 5 min. Phosphate buffered saline (1 mL) was then added to the donor chamber and the skin samples were allowed to equilibrate for a further ca 30 min. The electrical resistance was then measured using a Tinsley Databridge (Model 6401) set at low voltage alternating current, 1000 Hz with a maximum voltage of 300 mV root-mean-squared (rms) in the parallel equivalent circuit mode. Any skin sample exhibiting a resistance less than 10.9 kΩ was excluded from subsequent



absorption measurements. A cross reference of skin cell number, donor number and electrical resistance (kΩ) is presented in Appendix 7. The phosphate buffered saline was removed from the skin surface; the skin was rinsed with water and dried with tissue paper. 6.10 Pre-dose Receptor Fluid Collection

Prior to dosing, a 300 µL sample of receptor fluid was removed from the receptor chamber collection arm. The receptor fluid volume was then maintained by the addition of fresh receptor fluid up to the calibration line on the receptor chamber collection arm. Following sample collection, the receptor chamber collection arm was sealed with Parafilm® to prevent evaporation of receptor fluid. 6.11 Preparation of Formulations

The formulations, Antibacterial Foaming Hand Wash and Antibacterial Liquid Hand Soap, were supplied ready-formulated at a Benzalkonium Chloride concentration of 0.131% (w/w) and 0.14% (w/w), respectively. 6.12 Application of Formulation to Human Skin

Each formulation was applied to 12 human split-thickness skin membranes at a rate of ca 10 mg/cm2 (ca 6.4 mg) at time 0 h (initial dose). A positive displacement pipette was used to apply the formulation evenly over the stratum corneum surface of the skin. Representative weighed aliquots were removed at the time of dosing to ensure the correct mass of each of the formulations was applied. One of the representative aliquots taken at each dosing occasion and a random aliquot per day, were analysed (6 aliquots per day). The test preparation was then applied at 1.5, 3, 4.5, 6, 24, 25.5, 27, 28.5, 30, 48, 49.5, 51, 52.5 and 54 h post initial dose, with further representative weighed aliquots taken. Samples were analysed as described below in Section 6.17. The results of the analysis are shown below.

Test Preparation Target Test Item Concentration

(mg/g)

Test Item Concentration Mean (mg/g) CV (%)

Antibacterial Foaming Hand Wash 1.31 1.308 3.92 Antibacterial Liquid Hand Soap 1.40 1.335 4.29

6.13 Receptor Fluid Sampling

Absorption was assessed by collecting receptor fluid samples (300 μL) at 1.5, 3, 4.5, 6, 7.5, 24, 25.5, 27, 28.5, 30, 31.5, 48, 49.5, 51, 52.5, 54, 55.5, 72, 75, 78, 96, 99 and 102 h post initial dose. Following collection, the receptor fluid samples were either retained at ambient temperature until transferred for analysis then stored in a freezer set to maintain a temperature of -20°C or stored immediately in a freezer set to maintain a temperature of -20°C. The receptor fluid volume in the receptor fluid chamber was maintained by replenishment of fresh receptor fluid (300 μL) after each receptor fluid removal, with the exception at 102 h. The development of air bubbles beneath the skin discs was avoided and bubbles removed where applicable. The receptor fluid samples were analysed by LC-MS/MS.



6.14 Terminal Exposure Procedures (30 min Post Dose)

Each exposure period was terminated at 30 mins after each dosing (Section 6.12). The skin surface was rinsed with ultrapure (deionized) water (10 mL). The water was aspirated with a pipette tip. The skin surface was dried with two tissue swabs. Skin wash, pipette tips and tissue swab samples were retained and analysed as described in Section 6.17. 6.15 Terminal Post Exposure Procedures (102 h Post Dose)

At 102 h post dose, the donor chambers were transferred to a pot containing methanol (ca 15 mL). Donor wash pots were left to extract the test item for >30 min prior to sonication for ca 10 min. The skin was removed from each cell and placed on a piece of tissue to remove any remaining receptor fluid from the underside of the skin. This tissue was placed into the receptor chamber wash vial for that cell. The stratum corneum was removed with 20 successive tape strips (D-Squame tape discs). The skin sample was rotated 90° after each tape strip. Rotation was stopped if the epidermis/dermis junction became fragile or if epidermis was removed. The tape strips were pooled into groups; SC1-2, SC3-5, SC6-10, SC11-15 and SC16-20. Blank tape strips samples were added to SC1-2 and SC3-5 to ensure all samples contained five tape strips. Where a piece of epidermis was removed, this was recorded and is presented in Appendix 8. Where all of the epidermis was removed tape stripping was stopped. The skin under the cell flange (unexposed skin) was cut away from the exposed skin. Exposed and unexposed samples were retained in separate vials for analysis. Bulk receptor fluid (ca 5 mL) was removed from each receptor chamber and retained in a vial. The receptor chambers were rinsed with methanol (20 mL). The solvent was pooled as a single sample into the receptor wash vial. All terminal exposure samples were analysed by LC-MS/MS except for the bulk receptor fluid which was retained against future interest. 6.16 Storage of Samples

All samples were stored in a freezer set to maintain a temperature of -20°C prior to analysis. Following analysis, bulk samples were stored in a freezer set to maintain a temperature of -20°C. 6.17 Sample Analysis

Receptor fluid, tissue swab, pipette tip, skin wash, skin, tape strip and mock dose samples were analysed for Benzalkonium Chloride content using the established LC-MS/MS methods (AP.329863A.01 and AP.329863B.01. Appendix 9). 6.18 Electronic Data Acquisitions and Systems

LC-MS/MS data acquisition and integration was performed using Analyst (Version 1.6.2). The LIMS system used was Watson Version 7.4.2.



The data was transferred into Microsoft® Office Excel® 2013 for calculation of result tables and graphs. 6.19 Data Presentation

Data presented in results, tables, figures and appendices are computer generated and rounded appropriately for inclusion in the report. Consequently, calculation of values from data presented will, in some instances, yield minor variations. 7 DEFINITIONS

The definitions used are taken directly from the OECD Guidance Document No. 28. For the definitions see Appendix 10. 8 PROTOCOL ADHERENCE

The study was performed in accordance with the protocol for Charles River Study No. 785047 and Protocol Amendment 1 with the following deviations: Protocol section 10 states the concentration of the test item in the formulations will be determined prior to the experimental phase. This was performed at the time of dosing, as the results were suitable this does not impact on the study integrity. Protocol section 15.7 states the skin will be washed for 30 s with deionised water. Washing the skin samples for 30 s was not positively recorded, however, the process of applying 20 x 0.5 mL aliquots cannot be conducted in less than 30 s. The washing procedure was shown to be robust based on the results obtained. Protocol section 16. For a single analytical batch, the high QC samples were outside the acceptance criteria, however batch has been accepted with data up to the mid QC level as all parameters up to this level were acceptable in accordance with the protocol. Any samples above this level (40 ng/mL) were successfully repeated in another analytical batch. Therefore, this deviation has no impact upon the integrity of the study. Protocol Amendment 3 section 16 states a -80°C storage however the actual storage was -20°C as detailed in the analytical procedure. This typographical error in the protocol amendment 3 has no impact upon the integrity of the study as the analytical procedure was correctly followed. 9 REFERENCES

Page, L (2019). Comparison of Receptor Fluids: An In Vitro Percutaneous Absorption of Radiolabelled Testosterone Through Human Skin. Charles River Study No. 995724, Report No.40762. 10 RESULTS AND DISCUSSION

10.1 Benzalkonium Chloride in Antibacterial Foaming Handwash (0.131%, w/w)

A total of 12 samples of human split-thickness skin membranes obtained from 6 different donors were dosed topically with Benzalkonium Chloride in Antibacterial Foaming Handwash. Overall, the absorption profiles looked similar for all samples, with absorption of



Benzalkonium Chloride generally increasing to 102 h post dose. Absorption calculated throughout the course of the experiment was above the limit of reliable measurement for all samples from 48 h post dose onwards. The individual absorption profiles are provided in Figure 1 and Figure 2 (excluding cell 7). Cell 7 was excluded from the mean and standard deviation calculations as this sample was observed to become visibly damaged during the absorption assessment. This was further confirmed by the profile which shows a rapid increase in absorption from 54 h post dose. Cell 7 was an outlier for absorbed dose (mean + two standard deviations). The mass balance for all individual samples was within 100 ± 15%. The following results are provided as mean values (n = 11). The distribution of Benzalkonium Chloride at 102 h post dose is provided in Table 1. The mean mass balance was 97.02% of the applied dose at 102 h post dose. The majority of the applied dose was removed by washing the skin (15 times) 30 min post application of the formulation (94.94%, 0.72% and 0.33% was recovered in the skin wash, tissue swab and pipette tips, respectively). A further 0.18% was recovered in the donor chamber wash. Therefore, the total dislodgeable dose was 96.17% of the applied dose. The material recovered in the donor wash was almost certainly material that was dislodged from the skin during the washing procedure. The mean total unabsorbed dose was 96.56% of the applied dose. This consisted of the dislodgeable dose, unexposed skin (0.01%) and the radioactivity associated with the stratum corneum (0.38%). The total absorbed dose (0.11%) was the sum of the receptor fluid (0.09%) and receptor chamber wash (0.02%). The exposed skin contained 0.35% of the applied dose. Dermal delivery (0.46%) was the sum of the absorbed dose and the exposed skin. The absorption profile is provided in Table 2. The distribution of Benzalkonium Chloride by mass, at 102 h post dose, is shown in Table 3. The mass balance, total dislodgeable dose, total unabsorbed dose, total absorbed dose and dermal delivery were 191, 190, 191, 0.21 and 0.91 µg/cm2, respectively. The absorption profile, by mass, is provided in Table 4 and Figure 3. The flux profiles are provided in Table 5 and graphically in Figure 4. 10.2 Benzalkonium Chloride in Antibacterial Liquid Hand Soap (0.14%, w/w)

A total of 12 samples of human split-thickness skin membranes obtained from 6 different donors were dosed topically with Benzalkonium Chloride in Antibacterial Liquid Hand Soap. Overall, the absorption profiles looked similar for all samples, with absorption of Benzalkonium Chloride increasing to 102 h post dose. Absorption calculated throughout the course of the experiment was below the limit of reliable measurement for most samples. The individual absorption profiles are provided in Figure 5. The mass balance for all individual samples was within 100 ± 15%. The following results are provided as mean values (n = 12). The distribution of Benzalkonium Chloride at 102 h post dose is provided in Table 6. The mean mass balance was 93.15% of the applied dose at 102 h post dose. The majority of the applied dose was removed by washing the skin (15 times) 30 min post application of the formulation (91.92%, 0.55% and 0.30% was recovered in the skin wash, tissue swab and pipette tips, respectively). A further 0.08% was recovered in the donor chamber wash. Therefore, the total dislodgeable dose was 92.85% of the applied dose. The material recovered in the donor wash was almost certainly material that was dislodged from the skin during the washing procedure. The mean total unabsorbed dose was 92.99% of the applied dose. This consisted of the dislodgeable dose, unexposed skin (0.01%) and the radioactivity associated with the stratum corneum (0.13%). The total absorbed dose (0.06%) was the sum



of the receptor fluid (0.04%) and receptor chamber wash (0.02%). The exposed skin contained 0.10% of the applied dose. Dermal delivery (0.16%) was the sum of the absorbed dose and the exposed skin. The absorption profile is provided in Table 7. The distribution of Benzalkonium Chloride by mass, at 102 h post dose, is shown in Table 8. The mass balance, total dislodgeable dose, total unabsorbed dose, total absorbed dose and dermal delivery were 186, 185, 185, 0.11 and 0.32 µg/cm2, respectively. The absorption profile, by mass, is provided in Table 9 and Figure 6. The flux profiles are provided in Table 10 and graphically in Figure 7. 11 CONCLUSION

In conclusion, following topical application Benzalkonium Chloride in Antibacterial Foaming Handwash (0.131%, w/w) and Antibacterial Liquid Hand Soap (0.14%, w/w) to human skin in vitro, the total absorbed dose was 0.11% (0.21 µg/cm2) and 0.06% (0.11 µg/cm2), respectively. Dermal delivery was 0.46% (0.91 µg/cm2) and 0.16% (0.32 µg/cm2) of the applied dose, respectively. The mass balance was 97.02% (191 µg/cm2) and 93.15% (186 µg/cm2) of the applied dose.



12 FIGURES

Figure 1 Individual Absorption Profiles (% Applied Dose) for Benzalkonium Chloride in Receptor Fluid at 102 h Post Dose Following Topical Application of Antibacterial Foaming Handwash (0.131%, w/w) to Human Split-Thickness Skin

0.0

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Figure 2 Individual Absorption Profiles (% Applied Dose) for Benzalkonium Chloride in Receptor Fluid at 102 h Post Dose Following Topical Application of Antibacterial Foaming Handwash (0.131%, w/w) to Human Split-Thickness Skin (Excluding Cell 7)



Figure 3 Cumulative Absorption Profiles (ng/cm2) for Benzalkonium Chloride in Receptor Fluid at 102 h Post Dose Following Topical Application of Antibacterial Foaming Handwash (0.131%, w/w) to Human Split-Thickness Skin (Mean ± SD, n =11)



Figure 4 Flux Profile (ng/cm2/h) for Benzalkonium Chloride in Receptor Fluid Following Topical Application of Antibacterial Foaming Handwash (0.131%, w/w) to Human Split-Thickness Skin (Mean ± SD, n =11)



Figure 5 Individual Absorption Profiles (% Applied Dose) for Benzalkonium Chloride in Receptor Fluid at 102 h Post Dose Following Topical Application of Antibacterial Liquid Hand Soap (0.14%, w/w) to Human Split-Thickness Skin

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Figure 6 Cumulative Absorption Profiles (ng/cm2) for Benzalkonium Chloride in Receptor Fluid at 102 h Post Dose Following Topical Application of Antibacterial Liquid Hand Soap (0.14%, w/w) to Human Split-Thickness Skin (Mean ± SD, n =12)

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Figure 7 Flux Profile (ng/cm2/h) for Benzalkonium Chloride in Receptor Fluid Following Topical Application of Antibacterial Liquid Hand Soap (0.14%, w/w) to Human Split-Thickness Skin (Mean ± SD, n =12)

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13 TABLES

Table 1 Distribution of Benzalkonium Chloride (% Applied Dose) at 102 h Post Dose Following Topical Application of Antibacterial Foaming Handwash (0.131%, w/w) to Human Split-Thickness Skin

Cell No. and Donor No.

Mean SD Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 Cell 7 Cell 8 Cell 9 Cell 10 Cell 11 Cell 12

1141 1138 1144 1132 1149 1083 1141 1138 1144 1132 1149 1083

Skin Wash (total in 15 washes) 95.97 95.68 93.12 93.55 94.07 94.45 96.77 102.79 91.48 94.50 96.63 92.08 94.94 3.04

Tissue Swabs (total in 15 washes) 0.53 0.68 0.75 0.83 0.50 0.60 0.52 0.72 0.88 0.93 0.50 0.95 0.72 0.17

Pipette Tip (total in 15 washes) 0.30 0.29 0.34 0.32 0.30 0.25 0.25 0.49 0.29 0.38 0.33 0.35 0.33 0.06

Donor Wash 0.03 0.13 0.32 0.07 0.20 0.23 0.07 0.14 0.16 0.16 0.11 0.45 0.18 0.12

Total Dislodgeable Dose 102 h 96.84 96.78 94.53 94.77 95.07 95.53 97.62 104.14 92.82 95.97 97.57 93.83 96.17 2.99

Stratum Corneum 1-2 0.15 0.07 0.17 0.11 0.06 0.04 0.25 0.07 0.13 0.07 0.06 0.05 0.09 0.04

Stratum Corneum 3-5 0.11 0.12 0.11 0.09 0.07 0.04 0.10 0.07 0.12 0.06 0.06 0.06 0.08 0.03

Stratum Corneum 6-10 0.10 0.11 0.16 0.16 0.04 0.06 0.09 0.06 0.19 0.10 0.06 0.06 0.10 0.05

Stratum Corneum 11-15 0.05 0.04 0.01 0.08 0.04 0.05 0.07 0.04 0.13 0.09 0.04 0.05 0.06 0.03

Stratum Corneum 16-20 0.01 0.03 0.12 0.08 0.01 0.03 0.10 0.07 0.04 0.17 0.02 0.05 0.06 0.05

Stratum Corneum 0.42 0.38 0.56 0.52 0.22 0.21 0.61 0.30 0.61 0.49 0.24 0.28 0.38 0.14

Unexposed Skin 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.03 0.01 0.01

Total Unabsorbed 97.26 97.16 95.10 95.29 95.30 95.75 98.24 104.45 93.43 96.47 97.82 94.14 96.56 2.94

Exposed Skin 0.06 0.28 0.24 0.28 0.27 0.27 0.34 0.59 0.25 0.22 0.13 1.30 0.35 0.34

Receptor Wash 0.02 0.02 0.02 0.02 0.02 0.02 0.05 0.02 0.02 0.02 0.02 0.03 0.02 0.00

Receptor Fluid 0.03 0.12 0.08 0.06 0.11 0.13 0.95 0.14 0.08 0.04 0.03 0.17 0.09 0.05

Total Absorbed 0.05 0.14 0.10 0.08 0.13 0.15 1.01 0.16 0.09 0.05 0.05 0.20 0.11 0.05

Dermal Delivery 0.10 0.42 0.33 0.36 0.40 0.42 1.35 0.75 0.35 0.27 0.18 1.50 0.46 0.38

Mass Balance 97.37 97.59 95.44 95.65 95.70 96.17 99.59 105.19 93.78 96.74 98.00 95.64 97.02 2.96 Cell 7 excluded from mean + SD as cell noted to be damaged



Table 2 Cumulative Absorption of Benzalkonium Chloride (% Applied Dose) into Receptor Fluid Following Topical Application of Antibacterial Foaming Handwash (0.131%, w/w) to Human Split-Thickness Skin


Mean SD Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 Cell 7 Cell 8 Cell 9 Cell 10 Cell 11 Cell 12 Time (h) 1141 1138 1144 1132 1149 1083 1141 1138 1144 1132 1149 1083 0 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.5 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.000 3 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.000 4.5 0.004 0.031 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.007 0.008 6 0.005 0.006 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.000 7.5 0.005 0.007 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.000 24 0.005 0.011 0.005 0.005 0.016 0.008 0.005 0.006 0.005 0.005 0.005 0.007 0.007 0.003 25.5 0.005 0.013 0.005 0.005 0.016 0.008 0.005 0.008 0.005 0.005 0.005 0.008 0.008 0.004 27 0.006 0.014 0.006 0.006 0.014 0.009 0.006 0.010 0.006 0.006 0.006 0.008 0.008 0.003 28.5 0.006 0.015 0.008 0.006 0.022 0.009 0.006 0.010 0.006 0.006 0.006 0.010 0.009 0.005 30 0.006 0.016 0.007 0.006 0.021 0.012 0.007 0.011 0.007 0.006 0.006 0.012 0.010 0.005 31.5 0.013 0.017 0.008 0.006 0.025 0.012 0.008 0.012 0.008 0.006 0.006 0.013 0.012 0.006 48 0.010 0.034 0.021 0.010 0.047 0.033 0.015 0.025 0.024 0.008 0.009 0.035 0.023 0.013 49.5 0.011 0.037 0.024 0.011 0.049 0.036 0.017 0.026 0.025 0.009 0.010 0.037 0.025 0.014 51 0.010 0.036 0.022 0.011 0.046 0.034 0.015 0.026 0.025 0.009 0.009 0.034 0.024 0.013 52.5 0.010 0.036 0.023 0.011 0.051 0.035 0.018 0.029 0.026 0.010 0.010 0.038 0.025 0.014 54 0.012 0.039 0.025 0.013 0.048 0.044 0.050 0.033 0.031 0.012 0.011 0.049 0.029 0.015 55.5 0.013 0.043 0.030 0.015 0.056 0.046 0.128 0.036 0.034 0.012 0.012 0.054 0.032 0.017 72 0.019 0.078 0.052 0.030 0.087 0.092 0.721 0.069 0.053 0.022 0.021 0.093 0.056 0.030 75 0.021 0.084 0.053 0.033 0.089 0.095 0.802 0.081 0.058 0.022 0.022 0.162 0.066 0.043 78 0.020 0.084 0.054 0.034 0.100 0.089 0.752 0.084 0.063 0.023 0.021 0.106 0.062 0.033 96 0.030 0.120 0.077 0.056 0.115 0.123 0.910 0.120 0.073 0.033 0.030 0.152 0.084 0.044 99 0.029 0.121 0.081 0.056 0.114 0.121 0.976 0.123 0.072 0.036 0.029 0.159 0.086 0.045 102 0.033 0.124 0.081 0.059 0.113 0.130 0.952 0.139 0.077 0.037 0.030 0.170 0.090 0.048

Cell 7 excluded from mean + SD as cell noted to be damaged Data calculated from less than the low limit of quantification (<1 ng/mL)



Table 3 Distribution of Benzalkonium Chloride (µg/cm2) at 102 h Post Dose Following Topical Application of Antibacterial Foaming Handwash (0.131%, w/w) to Human Split-Thickness Skin



1141 1138 1144 1132 1149 1083 1141 1138 1144 1132 1149 1083




Donor Wash 0.06 0.25 0.63 0.13 0.40 0.46 0.15 0.27 0.32 0.32 0.22 0.90 0.36 0.24


Stratum Corneum 1-2 0.29 0.15 0.33 0.22 0.12 0.07 0.50 0.14 0.26 0.13 0.12 0.10 0.17 0.09

Stratum Corneum 3-5 0.21 0.24 0.22 0.17 0.13 0.07 0.20 0.13 0.23 0.13 0.12 0.12 0.16 0.06

Stratum Corneum 6-10 0.20 0.23 0.31 0.32 0.08 0.12 0.17 0.12 0.37 0.19 0.11 0.12 0.20 0.10

Stratum Corneum 11-15 0.10 0.08 0.02 0.16 0.08 0.10 0.14 0.08 0.26 0.18 0.09 0.11 0.11 0.06

Stratum Corneum 16-20 0.03 0.06 0.23 0.16 0.03 0.06 0.20 0.13 0.08 0.34 0.04 0.10 0.11 0.10

Stratum Corneum 0.83 0.75 1.10 1.03 0.44 0.42 1.21 0.59 1.20 0.96 0.48 0.55 0.76 0.28

Unexposed Skin 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.06 0.02 0.01


Exposed Skin 0.11 0.56 0.47 0.56 0.53 0.54 0.67 1.16 0.50 0.43 0.26 2.56 0.70 0.67

Receptor Wash 0.03 0.03 0.03 0.03 0.03 0.03 0.11 0.04 0.03 0.03 0.03 0.06 0.03 0.01

Receptor Fluid 0.06 0.24 0.16 0.12 0.22 0.26 1.88 0.27 0.15 0.07 0.06 0.34 0.18 0.09

Total Absorbed 0.10 0.28 0.19 0.15 0.25 0.29 1.99 0.31 0.18 0.10 0.09 0.40 0.21 0.10

Dermal Delivery 0.20 0.83 0.66 0.71 0.78 0.83 2.65 1.47 0.68 0.53 0.35 2.96 0.91 0.75

Mass Balance 192.09 192.53 188.28 188.71 188.80 189.73 196.47 207.53 185.02 190.85 193.35 188.69 191.42 5.84 Cell 7 excluded from mean + SD as cell noted to be damaged



Table 4 Cumulative Absorption of Benzalkonium Chloride (ng/cm2) into Receptor Fluid Following Topical Application of Antibacterial Foaming Handwash (0.131%, w/w) to Human Split-Thickness Skin


Mean SD LLOQ Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 Cell 7 Cell 8 Cell 9 Cell 10 Cell 11 Cell 12 Time (h) 1141 1138 1144 1132 1149 1083 1141 1138 1144 1132 1149 1083 0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.5 7.81 7.81 7.81 7.81 7.81 7.81 7.81 7.81 7.81 7.81 7.81 7.81 7.81 0.00 7.81 3 8.28 8.28 8.28 8.28 8.28 8.28 8.28 8.28 8.28 8.28 8.28 8.28 8.28 0.00 8.28 4.5 8.75 61.41 8.75 8.75 8.75 8.75 8.75 8.75 8.75 8.75 8.75 8.75 13.54 15.88 8.75 6 9.22 12.38 9.22 9.22 9.22 9.22 9.22 9.22 9.22 9.22 9.22 9.22 9.51 0.95 9.22 7.5 9.69 12.85 9.69 9.69 9.69 9.69 9.69 9.69 9.69 9.69 9.69 9.69 9.97 0.95 9.69 24 10.16 21.05 10.16 10.16 31.64 15.70 10.16 11.25 10.16 10.16 10.16 14.06 14.06 6.78 10.16 25.5 10.63 25.26 10.63 10.63 32.15 15.80 10.63 15.61 10.63 10.63 10.63 16.56 15.38 7.18 10.63 27 11.09 27.72 11.09 11.09 28.28 17.42 12.27 19.42 11.09 11.09 11.09 16.59 16.00 6.68 11.09 28.5 11.56 28.78 15.39 11.56 42.60 18.08 12.73 18.96 12.19 11.56 11.56 19.23 18.32 9.64 11.56 30 12.03 31.85 13.51 12.03 41.53 23.80 13.81 22.25 14.10 12.03 12.03 24.25 19.95 9.86 12.03 31.5 26.41 33.40 15.46 12.50 48.62 22.86 16.02 23.57 16.64 12.50 12.50 25.61 22.73 11.00 12.50 48 19.27 66.21 40.85 20.23 92.35 65.01 29.65 48.58 46.80 15.78 17.50 68.78 45.58 25.64 12.97 49.5 21.01 72.70 46.80 21.92 97.08 71.50 33.59 50.34 49.90 17.67 19.80 72.05 49.16 26.96 13.44 51 20.22 70.88 42.50 21.39 91.27 67.16 29.88 51.05 49.84 18.07 17.51 67.48 47.03 25.46 13.91 52.5 20.66 71.25 45.38 22.02 99.98 69.53 35.37 57.51 51.81 20.17 19.10 75.24 50.24 27.45 14.38 54 23.12 77.62 50.04 25.60 94.44 85.92 98.15 65.08 60.70 23.37 22.22 96.47 56.78 29.70 14.84 55.5 25.44 83.96 58.92 28.64 110.41 90.28 253.35 72.01 66.40 24.37 23.85 105.81 62.74 33.15 15.31 72 37.62 154.40 103.13 59.15 172.50 181.09 1421.46 135.75 104.46 43.50 41.93 184.03 110.69 58.33 15.78 75 41.25 166.45 104.28 65.56 176.56 187.88 1583.02 159.68 114.17 44.35 43.55 320.56 129.48 84.73 16.25 78 39.93 166.28 105.91 66.48 197.58 175.79 1483.65 165.12 123.37 45.36 42.33 208.48 121.51 64.96 16.72 96 58.35 236.73 151.25 109.70 226.42 242.90 1794.90 236.04 143.74 65.93 58.58 299.60 166.29 86.23 17.19 99 58.09 238.33 159.10 110.40 224.45 239.35 1924.90 242.01 141.57 70.74 57.82 314.40 168.75 88.26 17.66 102 64.15 244.05 159.20 117.07 223.35 256.79 1877.55 274.21 152.17 72.45 59.19 336.28 178.08 94.81 18.13

Cell 7 excluded from mean + SD as cell noted to be damaged Data calculated from less than the low limit of quantification (<1 ng/mL)



Table 5 Flux Profile (ng/cm2/h) of Benzalkonium Chloride into Receptor Fluid Following Topical Application of Antibacterial Foaming Handwash (0.131%, w/w) to Human Split-Thickness Skin



Cell 7 excluded from mean + SD as cell noted to be damaged Where flux was calculated to be negative, this is shown as 0.00



Table 6 Distribution of Benzalkonium Chloride (% Applied Dose) at 102 h Post Dose Following Topical Application of Antibacterial Liquid Hand Soap (0.14%, w/w) to Human Split-Thickness Skin



1141 1138 1144 1132 1149 1083 1141 1138 1144 1132 1149 1083




Donor Wash 0.05 0.03 0.16 0.08 0.05 0.03 0.07 0.05 0.16 0.04 0.03 0.26 0.08 0.07


Stratum Corneum 1-2 0.02 0.08 0.05 0.03 0.02 0.01 0.04 0.02 0.04 0.02 0.02 0.03 0.03 0.02

Stratum Corneum 3-5 0.04 0.08 0.06 0.01 0.01 0.04 0.01 0.02 0.03 0.02 0.02 0.02 0.03 0.02

Stratum Corneum 6-10 0.05 0.05 0.03 0.03 0.01 0.01 0.05 0.03 0.03 0.03 0.02 0.02 0.03 0.01

Stratum Corneum 11-15 0.02 0.02 0.03 0.03 0.01 0.01 0.05 0.02 0.03 0.04 0.02 0.01 0.02 0.01

Stratum Corneum 16-20 0.01 0.01 0.02 0.03 0.01 0.01 0.01 0.01 0.05 0.02 0.02 0.01 0.02 0.01

Stratum Corneum 0.14 0.23 0.19 0.13 0.06 0.07 0.15 0.11 0.18 0.13 0.09 0.09 0.13 0.05

Unexposed Skin 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.00


Exposed Skin 0.01 0.04 0.36 0.16 0.03 0.11 0.03 0.14 0.08 0.07 0.05 0.18 0.10 0.10

Receptor Wash 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.00

Receptor Fluid 0.01 0.01 0.06 0.02 0.01 0.09 0.03 0.04 0.02 0.04 0.01 0.12 0.04 0.03

Total Absorbed 0.03 0.03 0.08 0.04 0.02 0.10 0.05 0.05 0.04 0.06 0.03 0.13 0.06 0.03

Dermal Delivery 0.04 0.07 0.44 0.20 0.05 0.21 0.08 0.19 0.12 0.13 0.07 0.31 0.16 0.12

Mass Balance 92.50 90.19 91.22 93.05 94.42 87.52 94.62 93.91 99.68 95.94 92.42 92.37 93.15 3.02



Table 7 Cumulative Absorption of Benzalkonium Chloride (% Applied Dose) into Receptor Fluid Following Topical Application of Antibacterial Liquid Hand Soap (0.14%, w/w) to Human Split-Thickness Skin



Data calculated from less than the low limit of quantification (<1 ng/mL)



Table 8 Distribution of Benzalkonium Chloride (µg /cm2) at 102 h Post Dose Following Topical Application of Antibacterial Liquid Hand Soap (0.14%, w/w) to Human Split-Thickness Skin



1141 1138 1144 1132 1149 1083 1141 1138 1144 1132 1149 1083




Donor Wash 0.10 0.07 0.32 0.15 0.11 0.06 0.14 0.09 0.32 0.09 0.05 0.52 0.17 0.14


Stratum Corneum 1-2 0.04 0.15 0.10 0.06 0.03 0.02 0.08 0.05 0.09 0.05 0.04 0.06 0.06 0.04

Stratum Corneum 3-5 0.09 0.15 0.11 0.02 0.03 0.08 0.02 0.04 0.06 0.04 0.04 0.04 0.06 0.04

Stratum Corneum 6-10 0.10 0.10 0.06 0.06 0.03 0.02 0.10 0.07 0.07 0.07 0.04 0.05 0.06 0.03

Stratum Corneum 11-15 0.04 0.03 0.06 0.07 0.02 0.02 0.10 0.04 0.05 0.08 0.04 0.03 0.05 0.02

Stratum Corneum 16-20 0.02 0.02 0.04 0.06 0.02 0.02 0.02 0.02 0.10 0.03 0.04 0.02 0.03 0.02

Stratum Corneum 0.28 0.45 0.38 0.26 0.12 0.14 0.31 0.22 0.36 0.26 0.18 0.19 0.26 0.10

Unexposed Skin 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.00


Exposed Skin 0.03 0.09 0.72 0.31 0.06 0.22 0.06 0.27 0.17 0.14 0.09 0.35 0.21 0.19

Receptor Wash 0.03 0.03 0.04 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.00

Receptor Fluid 0.03 0.03 0.12 0.05 0.02 0.18 0.06 0.08 0.04 0.09 0.02 0.23 0.08 0.07

Total Absorbed 0.06 0.06 0.16 0.08 0.05 0.21 0.09 0.11 0.08 0.12 0.05 0.26 0.11 0.07

Dermal Delivery 0.09 0.15 0.88 0.39 0.11 0.43 0.15 0.38 0.24 0.26 0.15 0.61 0.32 0.24

Mass Balance 184.35 179.74 181.80 185.45 188.16 174.42 188.56 187.15 198.66 191.20 184.19 184.08 185.65 6.02



Table 9 Cumulative Absorption of Benzalkonium Chloride (ng/cm2) into Receptor Fluid Following Topical Application of Antibacterial Liquid Hand Soap (0.14%, w/w) to Human Split-Thickness Skin


Mean SD LLOQ Cell 13 Cell 14 Cell 15 Cell 16 Cell 17 Cell 18 Cell 19 Cell 20 Cell 21 Cell 22 Cell 23 Cell 24 Time (h) 1141 1138 1144 1132 1149 1083 1141 1138 1144 1132 1149 1083 0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.5 7.97 7.81 7.81 7.81 7.81 7.81 7.81 7.81 7.81 7.81 7.81 7.81 7.83 0.05 7.81 3 8.44 8.28 8.28 8.28 8.28 8.28 8.28 8.28 8.28 8.28 8.28 8.28 8.29 0.05 8.28 4.5 8.91 8.75 8.75 8.75 8.75 8.75 8.75 8.75 8.75 8.75 8.75 8.75 8.76 0.05 8.75 6 9.38 9.22 9.22 9.22 9.22 9.22 9.22 9.22 9.22 9.22 9.22 9.22 9.23 0.05 9.22 7.5 9.84 9.69 9.69 9.69 9.69 9.69 9.69 9.69 9.69 9.69 9.69 9.69 9.70 0.05 9.69 24 10.31 10.16 10.16 10.16 10.16 10.16 10.16 10.16 10.16 10.16 10.16 10.16 10.17 0.05 10.16 25.5 10.78 10.63 10.63 10.63 10.63 10.63 10.63 10.63 10.63 10.63 10.63 10.63 10.64 0.05 10.63 27 11.25 11.09 11.09 11.09 11.09 11.09 11.09 11.09 11.09 11.09 11.09 11.09 11.11 0.05 11.09 28.5 11.72 11.56 11.56 11.56 11.56 11.56 11.56 11.56 11.56 11.56 11.56 11.56 11.58 0.05 11.56 30 12.19 12.03 12.03 12.03 12.03 12.03 12.03 12.03 12.03 12.03 12.03 12.03 12.04 0.05 12.03 31.5 12.66 12.50 12.50 12.50 12.50 12.50 12.50 12.50 12.50 12.50 12.50 12.50 12.51 0.05 12.50 48 13.13 12.97 12.97 12.97 12.97 33.20 12.97 18.05 12.97 23.28 12.97 26.48 17.08 6.89 12.97 49.5 13.59 13.44 14.06 13.44 13.44 36.21 13.44 18.59 13.44 24.21 13.44 29.56 18.07 7.77 13.44 51 14.06 13.91 14.80 13.91 13.91 35.87 13.91 19.35 13.91 24.43 13.91 33.37 18.78 8.08 13.91 52.5 14.53 14.38 15.64 14.38 14.38 36.18 14.38 21.35 14.38 26.63 14.38 33.73 19.53 8.16 14.38 54 15.00 14.84 18.99 14.84 14.84 44.76 14.84 24.22 14.84 28.20 17.42 40.12 21.91 10.55 14.84 55.5 15.47 15.31 24.38 15.31 15.31 50.65 15.94 25.10 15.31 31.82 18.44 44.14 23.93 12.24 15.31 72 20.08 19.22 59.43 24.92 15.78 123.80 32.93 45.89 23.91 67.00 19.55 131.66 48.68 40.49 15.78 75 20.32 19.89 70.44 27.81 16.25 124.80 36.22 54.13 28.77 66.80 19.60 116.81 50.15 37.85 16.25 78 20.92 19.79 72.54 28.24 16.72 129.38 35.64 50.15 30.43 70.52 18.75 169.41 55.21 48.68 16.72 96 25.90 24.95 119.64 42.06 17.19 161.98 54.22 70.71 42.27 87.33 21.02 221.69 74.08 63.96 17.19 99 28.43 24.60 120.22 45.48 17.66 177.44 57.34 74.56 39.52 91.60 21.20 226.60 77.05 66.80 17.66 102 26.83 25.58 122.81 48.98 18.13 175.39 60.58 76.18 44.07 85.73 22.53 229.56 78.03 66.64 18.13

Data calculated from less than the lower limit of quantification (<1 ng/mL)



Table 10 Flux Profile (ng/cm2/h) of Benzalkonium Chloride into Receptor Fluid Following Topical Application of Antibacterial Liquid Hand Soap (0.14%, w/w) to Human Split-Thickness Skin



Where flux was calculated to be negative, this is shown as 0.00



14 APPENDICES

Appendix 1 GLP Certificate



Appendix 2 Certificate of Analysis and Chain of Custody of Benzalkonium Chloride Formulations



Appendix 2 (Continued)



Appendix 3 Certificate of Analysis of Deuterated Benzalkonium Chloride



Appendix 4 Certificate of Analysis of Nifedipine



Appendix 5 Human Skin Donor Details

Charles River Donor No. Sex/Age Site Supplier 1083 F/50Y Abdomen NHS Lothian 1132 F/34Y Abdomen Tissue Solutions 1138 F42Y Abdomen Tissue Solutions 1141 F47Y Abdomen Tissue Solutions 1144 F/38Y Abdomen Tissue Solutions 1149 F/42Y Abdomen Tissue Solutions

Appendix 6 Thickness of Full and Split-Thickness Skin Membranes

Charles River Donor No.. Membrane Thickness (µm) Full Thickness Skin Split-Thickness Skin

1083 900 400 1132 1000 380 1138 900-1000 400 1141 1440 390 1144 1100 400 1149 1920 400



Appendix 7 Cross Reference of Cell Number with Donor Number and Electrical Resistance

Cell Number Charles River Donor No. Electrical Resistance (kΩ) 1 1141 20.26 2 1138 13.16 3 1144 14.59 4 1132 14.64 5 1149 12.57 6 1083 14.85 7 1141 18.57 8 1138 11.43 9 1144 17.13 10 1132 13.91 11 1149 16.15 12 1083 12.45 13 1141 23.92 14 1138 12.08 15 1144 19.21 16 1132 15.94 17 1149 17.22 18 1083 11.00 19 1141 17.74 20 1138 11.26 21 1144 20.24 22 1132 14.47 23 1149 18.51 24 1083 11.20

After the initial set up of the split-thickness skin membranes in the diffusion cell system, electrical resistance measurements were taken. Where the resistance was < 10.9 kΩ (i.e. the acceptance criterion was not met), the skin was replaced with a new piece and the electrical resistance re-measured. Details for all failed skin samples, which were subsequently replaced, are provided in the table below. Details for the final measurements for all samples which passed are provided in the table above.

Cell Number Charles River Donor No. Electrical Resistance (kΩ) Reject/ Not Used 14 1138 10.36 Rejected 14 (1st repeat) 1138 7.913 Rejected 18 1083 10.62 Rejected 18 (1st repeat) 1083 10.40 Rejected 20 1138 10.48 Rejected 22 1132 4.466 Rejected 24 1083 6.193 Rejected 24 (1st repeat) 1083 9.956 Rejected



Appendix 8 Epidermis Removal by Tape Stripping The stratum corneum was removed with twenty successive tape strips. Where epidermis was removed on a tape strip this is reported in the table below, with approximate fraction removed given in parenthesis.

Cell Number Tape Strip Number (Fraction of Epidermis Removed) 1 16-20 (1/2) 7 1-2 (trace), 11-15 (1/2), 16-20 (remaining) 10 16-20 (3/4) 13 16-20 (1/4) *15 1-2 (trace), 3-5 (trace), 6-10 (trace), 11-15 (trace), 16-20 (trace) 19 11-15 (1/2), 16-20 (remaining) *21 16-20 (trace)

*Epidermis damaged prior to tape stripping.



Appendix 9 Analytical Procedure



Appendix 10 OECD Guidance Document No. 28 Glossary of Terms


final report test facility study no. 785047

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