Defence Research and Development Canada Reference Document

DRDC-RDDC-2020-D042

May 2020

CAN UNCLASSIFIED

CAN UNCLASSIFIED

Full thickness wound healing and safety of Reactive Skin Decontamination Lotion (RSDL) application in rabbits

Jessica Franken John Mikler DRDC – Suffield Research Centre

The body of this CAN UNCLASSIFIED document does not contain the required security banners according to DND security standards. However, it must be treated as CAN UNCLASSIFIED and protected appropriately based on the terms and conditions specified on the covering page.

CAN UNCLASSIFIED

Template in use: EO Publishing App for SR-RD-EC Eng 2018-12-19_v1 (new disclaimer).dotm © Her Majesty the Queen in Right of Canada (Department of National Defence), 2020

© Sa Majesté la Reine en droit du Canada (Ministère de la Défense nationale), 2020

CAN UNCLASSIFIED

IMPORTANT INFORMATIVE STATEMENTS

This document was reviewed for Controlled Goods by Defence Research and Development Canada (DRDC) using the Schedule to the Defence Production Act.

Disclaimer: This publication was prepared by Defence Research and Development Canada an agency of the Department of National Defence. The information contained in this publication has been derived and determined through best practice and adherence to the highest standards of responsible conduct of scientific research. This information is intended for the use of the Department of National Defence, the Canadian Armed Forces (“Canada”) and Public Safety partners and, as permitted, may be shared with academia, industry, Canada’s allies, and the public (“Third Parties”). Any use by, or any reliance on or decisions made based on this publication by Third Parties, are done at their own risk and responsibility. Canada does not assume any liability for any damages or losses which may arise from any use of, or reliance on, the publication.

In conducting the research described in this report, the investigators adhered to the “Guide to the Care and Use of Experimental Animals, Vol. I, 2nd Ed.” published by the Canadian Council on Animal Care.

Endorsement statement: This publication has been published by the Editorial Office of Defence Research and Development Canada, an agency of the Department of National Defence of Canada. Inquiries can be sent to: Publications.DRDC-RDDC@drdc-rddc.gc.ca.

DRDC-RDDC-2020-D042 i

Abstract

Reactive skin decontamination lotion (RSDL) was developed to remove chemical warfare agents (CWA),

including vesicants and organophosphorus nerve agents, from the epidermis. RSDL acts by breaking

down some agents and to decontaminate the surface more effectively than soapy water. Other products

have been developed but RSDL has proven to be superior in each case, except when open wounds are

present. Safety studies are lacking for how wound healing is effected if RSDL is used to decontaminate a

burn, is rinsed off, and then allowed to heal. This review set out to determine whether a proposed rabbit

chemical burn study is the most appropriate way to model wound healing.

ii DRDC-RDDC-2020-D042

Résumé

La lotion neutralisante pour la décontamination de la peau (LNDP) a été mise au point pour éliminer de

l’épiderme les agents de guerre chimique (CWA), notamment les agents vésicants et les agents

neurotoxiques organophosphorés. La LNDP agit en dégradant certains agents et est plus efficace que

l’eau savonneuse pour décontaminer les surfaces exposées. D’autres produits ont été mis au point, mais la

LNDP s’est révélée supérieure dans chaque cas, sauf en présence de plaies ouvertes. Il n’existe aucune

étude d’innocuité sur le processus de cicatrisation des plaies lorsqu’on utilise la LNDP pour décontaminer

une brûlure, puis que l’on rince la peau et qu’on la laisse cicatriser. Cet examen visait à déterminer si une

étude proposée sur les brûlures chimiques chez le lapin constitue le moyen le plus approprié de modéliser

la guérison des plaies.

DRDC-RDDC-2020-D042 iii

Table of Contents

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i

Résumé . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ii

Table of Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii

List of Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iv

1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

2 Statement of Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

3 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

List of Symbols/Abbreviations/Acronyms/Initialisms . . . . . . . . . . . . . . . . . . . 6

iv DRDC-RDDC-2020-D042

List of Tables

Table 1: Cytokine function and relation to wound healing phases. . . . . . . . . . . . . . 3

DRDC-RDDC-2020-D042 1

1 Background

Reactive Skin Decontamination Lotion (RSDL) was developed for the purpose of decontaminating

epidermal tissue exposed to chemical warfare agents (CWA), specifically vesicants and

organophosphorus nerve agents. Destruction and decontamination of CWA’s on the epidermis by RSDL

has been shown to be superior to other products developed for the same purpose. In order for Canada to

universalize use of RSDL by civilian first responders in emergency chemical exposures (including

accidental spills of a strong acid or base), further studies investigating safety of use in open wounds need

to be conducted. The proposed test system to investigate this question is rabbits and wound healing of full

thickness sulfuric acid burns that undergo decontamination with either RSDL or soapy water is to be

evaluated. A literature review was conducted to determine whether rabbits are the best choice and what

parameters ought to be measured and scored for the most accurate comparison of healing.

2 DRDC-RDDC-2020-D042

2 Statement of Results

Wound healing in mammals occurs in four overlapping phases: hemostasis, inflammation, proliferation,

and remodelling (Singer & Clark, 1999). The most appropriate non-human model for experimental wound

healing studies is unclear since swine, rabbits, guinea pigs, rats, and mice each present unique challenges.

Swine have the closest skin structure to humans with sparse body hair, but they are expensive and have

tough skin; in contrast rodents are hairy, loose skinned, and rely on wound contraction as an early step in

wound healing (Abdullahi, Amini-Nik, & Jeschke, 2014). Rabbits may be preferentially chosen for

wound healing studies since they are low cost, readily available, and they have a large enough skin

surface area to allow multiple in vivo assessments to be practical in a burn study (Kim, Mustoe, & Clark,

2015). Rabbits and humans express similar whole body metabolic responses when full thickness burn

wounds occur (Hu et al., 1998). Rabbit dermatology differs from humans through their conservation of a

panniculus carnosis (PC) around their torso, the PC is a layer of muscle directly underneath the dermis

which facilitates wound contraction early in the healing process (Naldaiz-Gastesi, Bahri, Lopez de

Munain, McCullagh, & Izeta, 2018). Healing is expected to be faster in rabbits than humans due to this

early wound contraction. Despite this difference rabbits still undergo the same main phases of wound

healing, including reepithelialisation during the proliferation and remodelling stages, allowing the same

inflammatory cells and growth factors to be evaluated throughout the healing process (Abdullahi et al.,

2014). Some studies have opted to excise the PC to better replicate human wound healing while others

have used the rabbit ear as it lacks PC entirely, although the disadvantage of this approach is decreased

vascularity compared to other body parts. For the purpose of evaluating wound healing after full thickness

sulfuric acid burns and decontamination measures this early wound contraction is not considered to

interfere with results because relative healing rates compared between the decontamination methods will

be the focus.

The most common methods among studies to evaluate wound healing are through observation, histology,

and tensile tissue strength (Hemmati et al., 2018). Various timepoints in studies have been chosen for

macroscopic observation of wound healing, but the suggested gold standard would be a daily evaluation

(Kim et al., 2015). Typically two histology stains are used for microscopic evaluation of the tissue,

hematoxyline-eosin stain for general visualization of reepithelialisation and inflammation while

Herovici’s stain (or Masson’s trichrome stain) is for observation of immature (collagen III) versus mature

collagen (I) in the tissue sample (Hemmati et al., 2018). Collagen content can also be tested using a kit

produced by Chondrex Incorporated, that measures tissue hydroxyproline, a by-product of the

biochemical pathway from collagen III to collagen I. Another common method to evaluate wound healing

is testing the tensile tissue strength using a tensiometer (Walters, Kauvar, Reeder, & Baer, 2007). More

recently, assessment using reverse transcriptase (RT)-PCR following RNA extraction is quantifying

certain cytokines linked to specific stages in the healing process. Some that may be beneficial to quantify

in the proposed study include platelet derived growth factor (PDGF), vascular endothelial growth factor

(VEGF), transforming growth factor β (TGF-β), and tumor necrosis factor α (TNFα), summarized in

Table 1 (Singer & Clark, 1999). Unfortunately there does not seem to be a useful endpoint that can be

determined while the rabbit is alive and healing, excluding daily observations, all histology and immune

or RT-PCR assays require tissue samples taken following euthanasia.

DRDC-RDDC-2020-D042 3

Table 1: Cytokine function and relation to wound healing phases.

Cytokine Function Healing stage

PDGF Chemotaxis (fibroblast and smooth muscle cells) Early inflammatory to early

proliferation

TGF-β Chemotaxis (fibroblast, smooth muscle, PDGF,

TNFα), collagen/collagenase expression

Early inflammatory to early

proliferation

TNFα Chemotaxis, fibroblast mitogen Proliferation

VEGF Neovascularization and angiogenesis Reepithelialization

During hemostasis, PDGF and TGF-β content in tissue will be rising and remain high throughout the

inflammatory period which is generally around 3 days (Singer & Clark, 1999). As the inflammatory

period ends and the proliferation period begins collagen formation has begun, TNFα will become

detectable. VEGF becomes prominent further into the proliferation phase. Complete reeplithelialization of

excisional wounds in most studies reviewed occurred by day 21. This research indicates that histology

and molecular biology would be most useful to detect possible differences in healing between

decontamination variables at days 3 and 21. RSDL has been shown to be safe to use on unbroken skin but

research is limited regarding RSDL’s effect on wound healing. As, (Walters et al., 2007) found RSDL

treated wounds did not differ from control wounds histologically but were weaker in the tensile strength

test and contained less collagen. There were many limitations in this study, particularly the method of

treating the wound with RSDL and neglecting to wash it off.

4 DRDC-RDDC-2020-D042

3 Conclusion

The decision to use rabbits as the test system for this decontamination and wound healing study is

appropriate. One cm2 acid burn wounds on rabbits would be expected to first undergo clotting and

achieve hemostasis, then inflammation and contraction of PC musculature starts healing the wound, lastly

reeipithelialization will occur. This process will likely take around three weeks. Histology must be

conducted to evaluate the healing stage, an additional stain specific for type III vs type I collagen may

want to be considered. Tissue tensile strength evaluation is extremely important to compare with

(Walters’ et al., 2007) results. Further analysis using RT-PCR of healing factors PDGF, TGF-β, TNFα,

and VEGF may be beneficial if part of the wound tissue can be used for RNA extraction.

RSDL is currently carried by CAF and several other armies to serve as the primary decontaminant for

exposures to CWAs since it is the most effective. Threat to civilian populations is possible and first

responders would benefit from carrying RSDL as their primary decontaminant. Currently

decontamination by first responders involves soapy water, which if the patient is suffering strong

acid/base burns does not affect wound healing. Scientific evidence that RSDL does not hinder wound

healing would be imperative to the decision to add RSDL to the kit used by civilian first responders.

DRDC-RDDC-2020-D042 5

References

Abdullahi, A., Amini-Nik, S., and Jeschke, M. G. (2014). Animal models in burn research. Cell Mol Life

Sci, 71(17), pp. 3241–3255. doi:10.1007/s00018-014-1612-5.

Hemmati, A. A., Larki-Harchegani, A., Shabib, S., Jalali, A., Rezaei, A., and Housmand, G. (2018).

Wound healing property of milk in full thickness wound model of rabbit. Int J Surg, 54(Pt A),

pp. 133–140. doi:10.1016/j.ijsu.2018.04.030.

Hu, R. H., Yu, Y. M., Costa, D., Young, V. R., Ryan, C. M., Burke, J. F., and Tompkins, R. G. (1998). A

rabbit model for metabolic studies after burn injury. J Surg Res, 75(2), pp. 153–160.

doi:10.1006/jsre.1998.5274.

Kim, D. J., Mustoe, T., and Clark, R. A. (2015). Cutaneous wound healing in aging small mammals: a

systematic review. Wound Repair Regen, 23(3), pp. 318–339. doi:10.1111/wrr.12290.

Naldaiz-Gastesi, N., Bahri, O. A., Lopez de Munain, A., McCullagh, K. J. A., and Izeta, A. (2018). The

panniculus carnosus muscle: an evolutionary enigma at the intersection of distinct research fields. J Anat.

doi:10.1111/joa.12840.

Singer, A. J., and Clark, R. A. (1999). Cutaneous wound healing. N Engl J Med, 341(10), pp. 738–746.

doi:10.1056/NEJM199909023411006.

Walters, T. J., Kauvar, D. S., Reeder, J., and Baer, D. G. (2007). Effect of reactive skin decontamination

lotion on skin wound healing in laboratory rats. Mil Med, 172(3), pp. 318–321.

6 DRDC-RDDC-2020-D042

List of Symbols/Abbreviations/Acronyms/Initialisms

CWA Chemical Warfare Agents

DND Department of National Defence

DRDC Defence Research and Development Canada

PC Panniculus Carnosis

PCR Polymerase Chain Reaction

PDGF Platelet Derived Growth Factor

R&D Research & Development

RSDL Reactive Skin Decontamination Lotion

RT Reverse Transcriptase

TGF-β Transforming Growth Factor β

TNFα Tumor Necrosis Factor α

VEGF Vascular Endothelial Growth Factor

DOCUMENT CONTROL DATA *Security markings for the title, authors, abstract and keywords must be entered when the document is sensitive

1. ORIGINATOR (Name and address of the organization preparing the document. A DRDC Centre sponsoring a contractor's report, or tasking agency, is entered in Section 8.)

DRDC – Suffield Research Centre Defence Research and Development Canada P.O. Box 4000, Station Main Medicine Hat, Alberta T1A 8K6 Canada

2a. SECURITY MARKING (Overall security marking of the document including special supplemental markings if applicable.)

CAN UNCLASSIFIED

2b. CONTROLLED GOODS

NON-CONTROLLED GOODS DMC A

3. TITLE (The document title and sub-title as indicated on the title page.)

Full thickness wound healing and safety of Reactive Skin Decontamination Lotion (RSDL) application in rabbits

4. AUTHORS (Last name, followed by initials – ranks, titles, etc., not to be used)

Franken, J.; Mikler, J.

5. DATE OF PUBLICATION (Month and year of publication of document.)

May 2020

6a. NO. OF PAGES

(Total pages, including Annexes, excluding DCD, covering and verso pages.)

10

6b. NO. OF REFS

(Total references cited.)

7

7. DOCUMENT CATEGORY (e.g., Scientific Report, Contract Report, Scientific Letter.)

Reference Document

8. SPONSORING CENTRE (The name and address of the department project office or laboratory sponsoring the research and development.)

DRDC – Suffield Research Centre Defence Research and Development Canada P.O. Box 4000, Station Main Medicine Hat, Alberta T1A 8K6 Canada

9a. PROJECT OR GRANT NO. (If appropriate, the applicable research and development project or grant number under which the document was written. Please specify whether project or grant.)

06da

9b. CONTRACT NO. (If appropriate, the applicable number under which the document was written.)

10a. DRDC PUBLICATION NUMBER (The official document number by which the document is identified by the originating activity. This number must be unique to this document.)

DRDC-RDDC-2020-D042

10b. OTHER DOCUMENT NO(s). (Any other numbers which may be assigned this document either by the originator or by the sponsor.)

11a. FUTURE DISTRIBUTION WITHIN CANADA (Approval for further dissemination of the document. Security classification must also be considered.)

Public release

11b. FUTURE DISTRIBUTION OUTSIDE CANADA (Approval for further dissemination of the document. Security classification must also be considered.)

12. KEYWORDS, DESCRIPTORS or IDENTIFIERS (Use semi-colon as a delimiter.)

RSDL; CWA

13. ABSTRACT (When available in the document, the French version of the abstract must be included here.)

Reactive skin decontamination lotion (RSDL) was developed to remove chemical warfare agents (CWA), including vesicants and organophosphorus nerve agents, from the epidermis. RSDL acts by breaking down some agents and to decontaminate the surface more effectively than soapy water. Other products have been developed but RSDL has proven to be superior in each case, except when open wounds are present. Safety studies are lacking for how wound healing is effected if RSDL is used to decontaminate a burn, is rinsed off, and then allowed to heal. This review set out to determine whether a proposed rabbit chemical burn study is the most appropriate way to model wound healing.

La lotion neutralisante pour la décontamination de la peau (LNDP) a été mise au point pour éliminer de l’épiderme les agents de guerre chimique (CWA), notamment les agents vésicants et les agents neurotoxiques organophosphorés. La LNDP agit en dégradant certains agents et est plus efficace que l’eau savonneuse pour décontaminer les surfaces exposées. D’autres produits ont été mis au point, mais la LNDP s’est révélée supérieure dans chaque cas, sauf en présence de plaies ouvertes. Il n’existe aucune étude d’innocuité sur le processus de cicatrisation des plaies lorsqu’on utilise la LNDP pour décontaminer une brûlure, puis que l’on rince la peau et qu’on la laisse cicatriser. Cet examen visait à déterminer si une étude proposée sur les brûlures chimiques chez le lapin constitue le moyen le plus approprié de modéliser la guérison des plaies.