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Attia.org.au

Compilation and Review of
Published and Unpublished
Tea Tree Oil Literature
A report for the Rural Industries Research
and Development Corporation
by CF Carson, KA Hammer, TV Riley RIRDC Publication No 05/151 RIRDC Project No UWA-75A
2005 Rural Industries Research and Development Corporation.
All rights reserved.
ISBN 1 74151 214 X
ISSN 1440-6845
Compilation and Review of Published and Unpublished Tea Tree Oil Literature
Publication No. 05/151
Project No. UWA-75A

The views expressed and the conclusions reached in this publication are those of the author and not
necessarily those of persons consulted. RIRDC shall not be responsible in any way whatsoever to any person
who relies in whole or in part on the contents of this report.
This publication is copyright. However, RIRDC encourages wide dissemination of its research, providing the
Corporation is clearly acknowledged. For any other enquiries concerning reproduction, contact the
Publications Manager on phone 02 6272 3186.
Researcher Contact Details
Microbiology (M502) School of Biomedical and Chemical Sciences
The University of Western Australia
35 Stirling Hwy
Crawley WA 6009
Australia
Phone: (08) 9346 3690
Fax: (08) 9346 2912
Email: triley@cyllene.uwa.edu.au
In submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form.
RIRDC Contact Details
Rural Industries Research and Development Corporation
Level 1, AMA House
42 Macquarie Street
BARTON ACT 2600
PO Box 4776
KINGSTON ACT 2604

Phone: 02 6272 4539
Fax: 02 6272 5877
Email: rirdc@rirdc.gov.au.
Website: http://www.rirdc.gov.au

Published in September 2005
Printed on environmentally friendly paper by Union Offset Foreword

The aim of this project was to assess, compile and review the available published and unpublished
data on tea tree oil. This information is presented in the form of a Database of Tea Tree Oil
Literature, a review of the literature for publication in a scientific journal and a Material Safety Data
Sheet appropriate for use by all suppliers of tea tree oil.
There are now many scientific publications describing many aspects of tea tree oil, ranging from tree
breeding, harvesting and oil distillation to its effectiveness in reducing inflammation, treating
dandruff and cold sores. However, much of this scientific literature is not readily accessible for
industry stakeholders. Similarly, no significant compilation and review of the data has occurred
recently, meaning that areas of research that have been overlooked are not obvious. Finally, the
compilation of available data into a standard tea tree oil Material Safety Data Sheet is useful to all
industry stakeholders in the tea tree oil industry.
This report includes a review of the available tea tree oil literature (including eco-toxicity), a tea tree
oil literature database, and a Material Safety Data Sheet created for tea tree oil.
This project was funded from industry revenue which is matched by funds provided by the Australian
Government. This report, a new addition to RIRDC's diverse range of over 1500 research
publications, forms part of our Tea tree oil R&D Program, which aims to support the continued
development of a profitable tea tree oil industry.
Most of our publications are available for viewing, downloading or purchasing online through our
website:

• purchases at www.rirdc.gov.au/eshop
Peter O'Brien
Managing Director Rural Industries Research and Development Corporation Acknowledgments

We would like to acknowledge the many publishers and authors who gave their permission to
reproduce tea tree oil publications in the literature database. We are also grateful to The University of
Western Australia and the Western Australian Centre for Pathology and Medical Research for
institutional support.
Abbreviations

Concentration per litre of aqueous solution lethal to 50% of test organisms Dose per kilogram of body weight that is lethal to 50% of test organisms Concentration per litre of aqueous solution lethal to 0% of test organisms Concentration per litre of aqueous solution lethal to 100% of test organisms Material safety data sheet portable document file parts per million Contents

Foreword .
Abbreviations.
Executive Summary .
Chapter 1. Introduction .
Chapter 2. Objectives.
Chapter 3. Review of literature.
Chapter 4. Review of tea tree oil ecotoxicity data .
4.1 Acute toxicity of tea tree oil to aquatic organisms . 4.2 Acute toxicity of tea tree oil to terrestrial insects. 4.3 Other acute toxicity data. 4.4 Conclusions . Chapter 5. Literature Database .
5.1 Methods. 5.2 Results . Chapter 6. Material safety data sheet.
Chapter 7. Recommendations for further studies .
References .
Appendix 1 – Literature database .
Appendix 2 – Responses of publishers to permission requests.
Appendix 3 – Material Safety Data Sheet .
Executive Summary

The body of literature about tea tree (Melaleuca alternifolia) oil has grown dramatically over the last 10 years as a number of Australian and international researchers investigate tea tree oil. Collectively, their results represent a significant advance in our knowledge of tea tree oil and its properties. The inclusion of a literature review in this report is therefore a timely contribution to our understanding of tea tree oil. The tea tree oil literature was reviewed focusing on the more recent literature but incorporating older publications for historical perspective. This review article will be published in a peer-review scientific journal and will be a significant benefit to the tea tree oil industry as it strives to establish tea tree oil as a bona fide alternative therapeutic agent. The ecotoxicity of tea tree oil was reviewed. However, since very little data are available describing the ecotoxicity of tea tree oil, information from secondary related sources was sought. Data pertaining to the ecotoxicity of other essential oils and essential oil components against various aquatic and terrestrial vertebrates and invertebrates were compiled and reviewed. While these data may not substitute for tea tree oil data, they can guide and inform future ecotoxicity studies. This area of tea tree oil research has not received adequate attention. The tea tree oil literature was compiled into a database designed to be accessible via the internet, potentially through the ATTIA web site. More than 500 tea tree oil publications were found, including research articles, reviews, conference abstracts or presentations, books and theses. Over 100 requests were made to publishers for copyright permissions to reproduce articles in full. In many cases this was granted or permission was granted to reproduce abstracts only. The availability of such a collection to industry stakeholders is unique and should facilitate greater appreciation of the work that has been done to date and the areas that require more attention. A Material Safety Data Sheet (MSDS) was compiled from scientific publications, industry documents and various regulatory codes. This represents the most up to date and comprehensive MSDS available for tea tree oil. It is designed for universal application throughout the tea tree oil industry and will be of significant benefit to tea tree oil exporters. It may also enhance the standing of ATTIA to stakeholders, purchasers and regulatory agencies. The collection and evaluation of all of this literature, and the construction of the MSDS made it possible to identify areas of research that have been overlooked or neglected, and to make recommendations regarding future tea tree oil studies. The two areas that require significant further work fall into two broad categories; (1) Safety and toxicity and (2) Clinical efficacy. Expansion of research in these two areas is an absolute requirement for further regulatory approvals for tea tree oil. In conclusion, the contents of this report represent a unique collection and digest of the existing tea tree oil literature. This report should help industry stakeholders have a better understanding of the state of tea tree oil research and development. Chapter 1. Introduction

Compared to the early 1990's, there is now an extraordinary amount of literature available describing
the properties of tea tree oil. In particular, ATTIA and industry participants have compiled an
extensive collection of tea tree oil literature. However, the usefulness of these data collections could
be significantly enhanced, as described below.
The existing scientific literature are somewhat incomplete and are not readily accessible to most
industry stakeholders. A comprehensive collection of tea tree oil literature in the form of an
electronic database would therefore be extremely valuable. Furthermore, the availability of the
database to industry stakeholders would be a valuable resource for the industry.
As a natural extension of this, a collation and review of the safety, toxicity and eco-toxicology data is
required. This could reduce or eliminate future duplication of effort, and highlight areas of research
that have been overlooked. Since these areas of oversight are likely to be numerous, a review is
unlikely to reduce research expenditures for the industry but will at least allow the industry to focus
their limited resources on areas of priority.

Preparation of generic Material Safety Data Sheet (MSDS) for TTO suitable for multiple importing
destinations will be of significant benefit to tea tree oil exporters. It could also enhance the standing
of ATTIA to stakeholders, purchasers and regulatory agencies. The preparation of the MSDS may
also highlight areas of research or inquiry that require further work.
In conclusion, despite the amount of literature available on tea tree oil, data have not been compiled
into a readily accessible format for industry participants. The benefits of a review and compilation of
tea tree oil literature to industry are numerous, and the preparation of an up-to-date, generic MSDS
may simplify matters for both Australian exporters and overseas importers.
Chapter 2. Objectives
The objectives of this work, in order of priority, are: 1. A review published in a peer-reviewed journal and for RIRDC of the following for TTO: safety
and toxicity, clinical efficacy 2. An assessment and/or review of the eco-toxicology of TTO based on direct and indirect data. If
sufficient data are available a review will be published in a peer-reviewed journal and RIRDC report. 3. Conversion of tea tree oil literature into an electronic resource for researchers, industry
participants and stakeholders 4. Production of a Material Safety Data Sheet (MSDS) suitable for multiple importing
5. Written recommendation for future TTO studies
Chapter 3. Review of literature
Tea tree oil
Many complementary and alternative medicines have enjoyed increased popularity in recent decades. Efforts to justify their use have seen their putative biological properties come under increasing scrutiny in vitro and, in some cases, in vivo. One such product is tea tree oil (TTO), the volatile essential oil derived mainly from the Australian native plant Melaleuca alternifolia. Employed largely for its antimicrobial properties, TTO is incorporated as the active ingredient in many topical formulations used to treat cutaneous infections. It is widely available over-the-counter in Australia, Europe and North America and is marketed as a remedy for various ailments. Composition and chemistry
TTO is composed of terpene hydrocarbons, mainly monoterpenes, sesquiterpenes and their associated alcohols. Terpenes are volatile, aromatic hydrocarbons and may be considered as polymers of isoprene which has the formula C5H8 (Sharp, 1983). Early reports on the composition of TTO described 12 (Guenther, 1968), 21 (Laakso, 1965 cited in Altman, 1988) and 48 (Swords & Hunter, 1978) components. The seminal paper by Brophy and colleagues (1989) examined over 800 TTO samples by gas chromatography and gas chromatography mass spectrometry and reported approximately 100 components and their range of concentrations. Given the scope for variation, it is fortunate that the composition of oil sold as TTO is regulated by an international standard for "Oil of Melaleuca –terpinen-4-ol type" which sets maxima and/or minima for 14 components of the oil (International Organisation for Standardisation, 1996) (see Table 3.1). Notably the standard does not dictate the species of Melaleuca from which the TTO must be sourced. Instead, it sets out physical and chemical criteria for the desired chemotype. There are several varieties, or chemotypes, of M. alternifolia and each produces oil with a distinct chemical composition. Six chemotypes have been described as follows: terpinen-4-ol chemotype (1), terpinolene chemotype (2), and four 1,8-cineole chemotypes (3-6) (Homer et al., 2000). The terpinen-4-ol chemotype typically contains levels of terpinen-4-ol of between 30-40% (Homer et al., 2000) and is the chemotype used in commercial TTO production. This is the chemotype that will be discussed below. The components specified by the standard were selected for a variety of reasons including biological activity and provenance verification. For example, terpinen-4-ol is a major component of TTO and has long been considered the main antimicrobial component of the oil. Consequently, to optimise antimicrobial activity, a lower limit of 30% and no upper limit were set for terpinen-4-ol content. An upper limit of 15% and no lower limit were set for 1,8-cineole, although the rationale for this may not have been entirely sound; for many years cineole was erroneously considered to be a skin and mucous membrane irritant fuelling efforts to minimise its level in TTO. Recent work soundly refutes this notion but since cineole levels are usually inversely proportional to terpinen-4-ol levels, minimising cineole content in order to maximise terpinen-4-ol content remains an important consideration. Despite the scope for batch to batch variation in TTO, no obvious differences in its bioactivity have been noted so far. The suggestion that oil from a particular M. alternifolia clone possesses enhanced cidal activity has been made (May et al., 2000) but the evidence is not compelling. TTO has a relative density of 0.885-0.906 (International Organisation for Standardisation, 1996), is only sparingly soluble in water and is miscible with non-polar solvents. Some of the chemical and physical properties of TTO components are shown in Table 3.2. The composition of TTO may change considerably during storage with ρ-cymene levels increasing and α- and γ-terpinene levels declining (Brophy et al., 1989). Light, heat, exposure to air and moisture all affect oil stability and TTO should be stored in dark, cool, dry conditions preferably in a
vessel that contains little air.
Provenance and nomenclature
The provenance of TTO is not always clear from its common name or those of its sources. It is
known by a number of synonyms including "melaleuca oil" and "ti tree oil", "ti tree" being a Maori
and Samoan common name for plants in the genus Cordyline (Weiss, 1997). Even the term
"melaleuca oil" is potentially ambiguous since several chemically distinct oils are distilled from other
Melaleuca species such as cajuput oil (also cajeput or cajaput) from M. cajuputi and niaouli oil from
M. quinquenervia (often misidentified as M. viridiflora) (Lassak and McCarthy, 1983; Southwell &
Lowe, 1999). However, the term has been adopted by the Australian Therapeutic Goods
Administration as the official name for TTO. The use of common plant names further confounds the
issue. In Australia, "tea trees" are also known as "paperbark trees" and collectively these terms may
refer to species in the Melaleuca or Leptospermum genera of which there are several hundred. For
instance, common names for M. cajuputi include "swamp tea tree" and "paperbark tea tree" while
those for M. quinquenervia include "broad-leaved tea tree" and "broad-leaved paperbark" (Lassak &
McCarthy, 1983). Many Leptospermum species are cultivated as ornamental plants and are often
mistakenly identified as the source of TTO. In addition, the essential oil kanuka and manuka derived
from the New Zealand plants Kunzea ericoides and Leptospermum scoparium, respectively, are
referred to as New Zealand TTOs (Christoph et al., 2000) although they are very different in
composition from Australian TTO (Perry et al., 1997). In this review article, the term TTO will refer
only to the oil of M. alternifolia.
As explained above, the international standard for TTO does not specify which Melaleuca species
must be used to produce oil. Rather it sets out the requirements for an oil chemotype. Oils that meet
the requirements of the standard have been distilled from Melaleuca species other than M.
alternifolia
including M. dissitiflora, M. linariifolia and M. uncinata (Murtagh, 1999). However, in
practice commercial TTO is produced from M. alternifolia (Maiden and Betche) Cheel. The
Melaleuca genus belongs to the Myrtaceae family and contains approximately 230 species almost all
of which are native to Australia (Craven, 1999). When left to grow naturally, M. alternifolia grows to
a tree reaching heights of approximately 5-8 metres (Colton & Murtagh, 1999). Trees older than
three years flower typically in October and November (Lassak & McCarthy, 1983, Baker, 1999) and
flowers are produced in loose, white to creamy coloured terminal spikes, which can give trees a
"fluffy" appearance (Weiss, 1997).
Commercial production
The commercial TTO industry was born after its medicinal properties were first reported by Penfold in the 1920s as part of a larger survey into Australian essential oils with economic potential. During that nascent stage, TTO was produced from natural bush stands of plants, ostensibly M. alternifolia, that produced oil with the appropriate chemotype. The native habitat of M. alternifolia is low-lying, swampy, sub-tropical, coastal ground around the Clarence and Richmond Rivers in north-eastern New South Wales and southern Queenland (Swords & Hunter, 1978) and, unlike several other Melaleuca species, it does not occur naturally outside Australia. The plant material was hand-cut and often distilled on the spot in make-shift, mobile, wood-fired bush stills. The industry continued in this fashion for several decades. Legend has it that the oil was considered so important for its medicinal uses that Australian soldiers were supplied oil as part of their military kits during World War II and that bush-cutters were exempt from national service (Carson & Riley, 1993). However, no evidence to corroborate these accounts could be found (A.-M Conde & M. Pollard, Australian War Memorial, Canberra, Australia, personal communication). Production ebbed after World War II as demand for the oil declined presumably due to the development of effective antibiotics and the waning image of natural products. Interest in the oil was rekindled in the 1970s as part of the general renaissance in natural products. Commercial plantations were established in the 1970s and 1980s allowing the industry to mechanise and produce large quantities of a consistent product (Brophy et al., 1989; Johns et al., 1992). Today there are plantations in Western Australia, Queensland and New South Wales although the majority is in New South Wales around the Lismore region. Typically, plantations are established from seedlings sowed and raised in greenhouses prior to planting out in the field at high density. The time to first harvest varies from 1-3 years depending on the climate and rate of plant growth. Harvesting is by a coppicing process in which the whole plant is cut off close to ground level and chipped into smaller fragments prior to oil extraction. Oil extraction TTO is produced by steam distillation of the leaves and terminal branches of M. alternifolia. Once
condensed, the clear to pale yellow oil is separated from the aqueous distillate. The yield of oil is
typically 1-2% of the wet weight of the plant material. Alternative extraction methods have been
considered including those using microwave technology but none has been utilised on a commercial
scale.
Antimicrobial activity
Of all the properties claimed for TTO, it is those regarding antimicrobial activity that have received
the most attention. The earliest reported use of the M. alternifolia plant that presumably exploited
this property is the traditional use by the Bundjalung Aborigines of northern New South Wales.
Crushed leaves of "tea trees" were inhaled to treat coughs and colds, or were sprinkled on wounds
after which a poultice was applied (Shemesh & Mayo, 1991). In addition, tea tree leaves were soaked
to make an infusion to treat sore throats or skin ailments (Low, 1990; Shemesh & Mayo, 1991). The
oral history of Australian Aborigines also tells of healing lakes which were lagoons into which M.
alternifolia
leaves had fallen and decayed over time (Altman, 1988). Use of the oil itself, as opposed
to the unextracted plant material, did not become common practice until Penfold published the first
reports of its antimicrobial activity in a series of papers in the 1920s and 1930s. In evaluating the
antimicrobial activity of M. alternifolia oil and other oils, he made comparisons with the disinfectant
carbolic acid or phenol, the gold standard of the day, in a test known as the Rideal-Walker (RW)
coefficient. TTO's activity was compared directly with that of phenol and rated at 11 times as active
(Penfold & Grant, 1925). The RW coefficient of several of the components of TTO were also
reported including cineole (3.5) and cymene (8) (Penfold & Grant, 1923), linalool (13) (Penfold &
Grant, 1924), terpinen-4-ol (13.5) and terpineol (16) (Penfold & Grant, 1925). As a result, TTO was
promoted as a therapeutic agent (Anon., 1930; Anon., 1933a; Anon., 1933b). It must be mentioned
that in terms of the evidence they provide for the medicinal properties of TTO, these and many other
early publications (Humphery, 1930; MacDonald, 1930; Halford, 1936; Penfold & Morrison, 1946;
Feinblatt, 1960; Peña, 1962) are of limited value since by today's standards the data they provide are
mostly anecdotal.
In contrast, contemporary data clearly show that the broad-spectrum antimicrobial activity of TTO
includes antibacterial, antifungal, antiviral and anti-protozoal activity. Not all the activity has been
characterised well in vitro and in the few cases where in vivo work has been done, data are promising
but thus far inadequate. In vitro, methodological issues have plagued evaluation of the oil's
antimicrobial activity since the lipophilic oil does not lend itself to standard aqueous test systems.
Despite this, considerable work has been done, particularly on the antibacterial activity of the oil.
Antibacterial activity
Evaluation of the antimicrobial activity of TTO has been impeded by its physical properties; TTO is only sparingly soluble in water and this limits its miscibility in test media. The solubility of several components of TTO is shown in Table 3.2. Different strategies have been used to counteract this problem, the addition of surfactants to broth and agar test media being used most widely (Atkinson & Brice, 1955; Beylier, 1979; Carson et al., 1995a,b; Griffin, Markham & Leach, 2000; Banes-Marshall et al., 2001). Dispersion of TTO in liquid media usually results in a turbid suspension that makes determination of endpoints in susceptibility tests difficult. Occasionally dyes have been used as visual indicators of the MIC with mixed success (Chand et al., 1994; Carson et al., 1995a,b Mann & Markham, 1998). TTO has been tested in vitro against a wide variety of bacteria. Only a few reports of the antibacterial activity of TTO appear in the literature from 1940 to the 1980s. The earliest of these was published by Atkinson and Brice (1955), who assessed plants of the Myrtaceae family for antibacterial activity by both agar and broth dilution assays. Antibacterial titres (% v/v) as determined by agar and broth dilution assays were 0.63 and 0.31, respectively, for Staphylococcus aureus, 1.25 and 0.24 for Salmonella typhi and 0.31 and 0.10 for Mycobacterium phlei (Atkinson & Brice, 1955). Similarly, Low et al. (1974) described the antibacterial activity of a number of essential oils from the Myrtaceae family. They used the agar dilution method of Atkinson and Brice and found MICs (% v/v) of 0.062 for S. aureus and 0.031 for Salm. typhi. They also used an assay where test organisms were exposed to each neat essential oil for 10 minutes only, after which viable organisms were recovered. With M. alternifolia oil, S. aureus could not be recovered whereas viable Pseudomonas aeruginosa were recovered (Low et al., 1974). In the study by Beylier (1979), more than 100 oils were initially examined for antimicrobial activity, and 10 of these (including M. alternifolia oil) were selected for further investigation. The MIC (% v/v) ranges were 0.25 - 0.5 for S. aureus, 0.125 - 0.25 for Escherichia coli and 4 for P. aeruginosa (Beylier, 1979). MICs for Candida albicans and Aspergillus niger were also determined in this study and these will be discussed below. Walsh and Longstaff (1987) used both broth and agar dilution methods to assess ‘Melasol', a product containing 40% TTO, 13% isopropyl alcohol and 47% water, for activity against oral pathogens. MICs (% v/v) of Melasol were 0.08 for S. aureus, 0.16 for Streptococcus faecalis, 0.16 for P. aeruginosa and 0.08 for E. coli, by the agar method (Walsh & Longstaff, 1987). These MICs are low compared to those obtained in the previous studies, especially considering that Melasol contains only 40% TTO, however, the alcohol in the solution may have accounted for this activity. A range of oral microorganisms, such as Actinomyces viscosus, Bacteroides gingivalis, Eikenella corrodens and Strep. mutans, was tested also and MICs ranged from 0.02 - 0.08% (Walsh & Longstaff, 1987). From the early 1990s onwards, many reports detailing the antimicrobial activity of TTO have appeared in the scientific literature. Although there was still a degree of discrepancy between the methods used in the different publications, often the MIC values reported were relatively similar. A summary of some of the published in vitro susceptibility data for bacteria is shown in Table 3.3. The majority of MICs and MBCs are in the range of 0.06% - 1.0%, however, MICs of more than 2% have been reported for some commensal skin staphylococci and micrococci, Enterococcus faecalis and P. aeruginosa (Hammer et al., 1996; Banes-Marshall et al., 2001). The activity of TTO against antibiotic-resistant bacteria has attracted considerable attention with methicillin-resistant S. aureus (MRSA) receiving the most attention thus far. Since the potential to use TTO against MRSA was first hypothesised (Walsh & Longstaff, 1987), several groups have evaluated the activity of TTO against MRSA beginning with Carson et al. (1995a) who examined 64 MRSA from Australia and the United Kingdom, including 33 mupirocin-resistant isolates. The MIC and MBC of the Australian isolates were 0.25% and 0.5%, respectively, while those for the UK isolates were 0.312% and 0.625%, respectively. Subsequent reports on the susceptibility of MRSA to TTO have given similar results (Nelson, 1997; Chan & Loudon,1998; Elsom & Hide, 1999; May et al., 2000; Hada et al., 2001). Resistance to TTO Decreased susceptibility to TTO has been reported for a number of bacteria including P. aeruginosa (Hammer et al., 1996; Griffin et al., 2000, Banes-Marshall et al., 2001). The mechanism by which P. aeruginosa tolerates higher concentrations of TTO has begun to be explored and appears to be associated with the outer membrane (Mann, Cox & Markham, 2000; Griffin, Wyllie & Markham, 2001). Resistance to TTO per se has not been reported despite medicinal use of the oil in Australia since the 1920s. However, the question of whether or not true resistance to TTO can be induced in vitro or may occur spontaneously in vivo remains unanswered. It is possible that the multi-component nature of TTO may reduce the potential for resistance to occur spontaneously since multiple simultaneous mutations may be required to overcome all the antimicrobial components of TTO. These are important issues if TTO is to be used more widely, particularly against antibiotic-resistant organisms. There has been one report of induced resistance to TTO in S. aureus (Nelson, 2000) where stepwise exposure of five MRSA isolates to increasing TTO concentrations yielded three isolates whose MIC had risen to 1% and one isolate each whose MIC had increased to 2% and 16% TTO. All isolates had initial MICs of 0.25%. There has also been one report suggesting that E. coli which harbour mutations in the multiple antibiotic resistance (mar) operon, so-called Mar mutants, may exhibit decreased susceptibility to TTO. However, the decrease in susceptibility seen in this work by time-kill and broth dilution methods was marginal and cannot be considered strong evidence of this phenomenon (Gustafson et al., 2001), although it remains feasible and more data should be sought. Mechanism of antibacterial action The mechanism of action of TTO has now been partly elucidated. Prior to the availability of data, assumptions about its mechanism of action were made on the basis of its hydrocarbon structure and attendant lipophilicity. Hydrocarbons partition preferentially into biological membranes and disrupt their vital functions (Sikkema, deBont & Poolman, 1995) and TTO and its components were presumed to behave in this manner. This premise is further supported by data showing that TTO permeabilises model liposomal systems (Cox et al., 2000). In previous work with hydrocarbons not found in TTO (Jackson & deMoss, 1965; Uribe et al., 1990) and with terpenes found at low concentrations in TTO (Andrews, Parks & Spence, 1980; Uribe, Ramirez & Peña, 1985), lysis and the loss of membrane integrity and function manifested by the leakage of ions and the inhibition of respiration were demonstrated. Treatment of S. aureus with TTO precipitates the leakage of potassium ions (Cox et al., 2000; Hada et al., 2003) and 260 nm-absorbing materials (Carson, Mee & Riley, 2002) and inhibits respiration (Cox et al., 2000). TTO also sensitizes previously tolerant cells to sodium chloride (Carson, Mee & Riley, 2002) and produces morphological changes apparent under electron microscopy (Reichling et al., 2002). However, no significant lysis of whole cells was observed by electron microscopy (Reichling et al., 2002) or spectrophotometrically (Carson, Mee & Riley, 2002), no cytoplasmic membrane damage as evidenced by lactate dehydrogenase release could be detected (Reichling et al., 2002) and only modest uptake of propidium iodide was observed (Cox et al., 2001b) after treatment with TTO. In E. coli, detrimental effects on potassium homeostasis (Cox et al., 1998), glucose-dependent respiration (Cox et al., 1998), morphology (Gustafson et al., 1998) and ability to exclude propidium iodide have been observed. A modest loss of 280 nm-absorbing material has also been reported (Cox et al., 2001b). In contrast to the absence of whole cell lysis seen in S. aureus treated with TTO, lysis occurs in E. coli treated with TTO (Gustafson et al., 1998) and this effect is exacerbated by co-treatment with EDTA (C Carson, unpublished data). All of these effects confirm that TTO compromises the structural and functional integrity of bacterial membranes. When the effects on S. aureus of terpinen-4-ol and α-terpineol, two of the main antibacterial components of TTO, and 1,8-cineole were examined, none was found to induce autolysis and all were found to cause the leakage of 260 nm-absorbing material and render cells susceptible to sodium chloride (Carson, Mee & Riley, 2002). Interestingly, the greatest effects were seen with 1,8-cineole, a component often considered to be marginally antimicrobial. This raises the possibility that while cineole may not be one of the primary antimicrobial components of TTO, it may permeabilise bacterial membranes and facilitate the entry of other more active components. Little work on the effects of TTO components on cell morphology has been reported. Electron microscopy of terpinen- 4-ol treated S. aureus cells (Carson, Mee & Riley, 2002) revealed lesions similar to those seen after TTO treatment (Reichling et al., 2002), including mesosomes. The loss of viability, inhibition of glucose-dependent respiration and induction of lysis seen after TTO treatment all occur to a greater degree with organisms in the exponential rather than stationary phase of growth (Cox et al., 1997; Gustafson et al., 1998). The increased vulnerability of actively growing cells was also apparent in the greater degree of morphological changes seen in these cells by electron microscopy (Cox et al., 1997). The differences in susceptibility seen with bacteria in different phases of growth suggest that additional targets may be involved. In vivo antibacterial activity Despite the increasing amount of in vitro data for bacteria, few in vivo (or clinical) investigations have been performed. Clinical studies investigating the effects of TTO treatment on acne, dental plaque formation and the elimination of MRSA colonisation have been published. In an investigation of acne treatment, Bassett et al. (1990) compared the efficacy of 5% TTO and 5% benzoyl peroxide for therapy, with 58 and 61 evaluable patients in each treatment group, respectively (Bassett et al., 1990). Patients were assessed at commencement, and at 1, 2, and 3 months. Parameters assessed were the numbers of inflamed and non-inflamed lesions, and a grade was given for oiliness, erythema, scaling, pruritis and dryness. The major findings of the study were that both treatments reduced the numbers of inflamed lesions, although benzoyl peroxide performed significantly better than TTO. The benzoyl peroxide group also showed significantly less oiliness than the tea tree group, however the tea tree group showed significantly less scaling, pruritis and dryness. Erythema did not differ between groups. Interestingly, significantly fewer overall side effects were reported by the TTO group (27 of 61 patients) than the benzoyl peroxide group (50 of 63 patients). A study comparing the effects of mouthwashes containing either approximately 0.34% TTO, 0.1% chlorhexidine or placebo on plaque formation and vitality was performed using eight volunteers (Arweiler et al., 2000). On day zero, volunteers had their teeth professionally cleaned, and for the next four days they rinsed twice daily with one of the treatments and did not clean their teeth in any other manner. Teeth were clinically evaluated on days 1, 2, 3 and 4. Each mouthwash was evaluated in this manner, with a wash-out period of 10 days between the end of one treatment and the beginning of the next. The plaque index and plaque vitality from the TTO mouthwash treatment did not differ from placebo mouthwash on any day, whereas the chlorhexidine mouthwash differed significantly on all days. Thus the TTO treatment was considered ineffective at reducing plaque regrowth or the vitality of plaque organisms (Arweiler et al., 2000). In contrast, a small study evaluating the effect of a 0.2% TTO mouthwash on oral flora suggested that TTO could reduce the number of mutans streptococci, and the total number of oral bacteria and that residual activity maintained these reduced levels for two subsequent weeks (Groppo et al., 2002). A pilot study conducted by Caelli et al. (2000) examined the effectiveness of a 4% TTO nasal ointment and a 5% TTO body wash for the eradication of MRSA carriage, as compared to conventional treatment of mupirocin nasal ointment and Triclosan body wash. Of the 15 patients receiving conventional treatment, two were cleared and eight were chronic carriers at the end of therapy, compared to the tea tree group where five were cleared and three were chronic carriers. In addition, five patients from the conventional treatment group and seven from the TTO group did not complete therapy. Due to the low patient numbers, these differences were not statistically significant, although they indicate that TTO therapy may be effective in decolonising MRSA carriers. In addition to these clinical studies, there is a single case report of a woman who treated herself successfully with a 5 day course of TTO pessaries after having been clinically diagnosed with bacterial vaginosis (Blackwell, 1991b). Of the three studies described above, two are limited by low numbers of patients and all have some ambiguous or equivocal outcomes, indicating that much
remains unknown about optimising TTO efficacy in vivo.
Antifungal activity
Published studies investigating the antifungal activity of TTO have focussed on assessing either the in vitro activity of the oil against medically relevant fungi, or the use of TTO to treat human fungal infections. These studies will be discussed here. The development of protocols for evaluating the susceptibility of fungi to antifungal agents has lagged behind similar methods that have been developed for bacteria and only recently have standard methods been published for evaluating the in vitro activity of antifungal agents (Rex et al., 2001). Prior to the publication of these standard methods, researchers used a variety of different assays to assess in vitro activity, which means that data from these studies is often difficult to compare. Another limitation of some of these published studies is that very often only one isolate of a given species is tested in any particular investigation, meaning that generalisations about susceptibility are limited. A range of yeasts from the genera Candida, Malassezia and Trichosporon are susceptible in vitro to concentrations of TTO of less than 1.0%. Since Candida yeasts (in particular C. albicans) are commonly chosen as test organisms, a moderate amount of susceptibility data are available for these organisms. Individual MICs and MIC90s that have been reported for C. albicans, by either the broth or agar dilution assay include (%) 0.04 (Beylier, 1979), 0.2 (Griffin, Markham & Leach, 2000), 0.25 (Vazquez et al., 2000), 0.3 (Christoph et al., 2000) and 0.44 (Nenoff et al., 1996). Several other Candida species, such as C. parapsilosis, C. glabrata, C. tropicalis, C. kefyr and C. krusei, have been tested against TTO in vitro and MICs ranged from 0.25 to 0.5% and minimum fungicidal concentrations (MFCs) ranged from 0.5 to 1.0% (Vazquez et al., 2000; Banes-Marshall et al., 2001; D'Auria et al., 2001). Malassezia yeasts also appear to be susceptible to TTO with MICs in the range of 0.06 – 0.44% (Nenoff et al., 1996; Griffin & Markham, 2000). TTO has activity against single isolates of T. cutaneatum, Schizosaccharomyces pombe and Debaromyces hansenii with MICs of 0.22% (Nenoff et al., 1996), 0.5% and 0.5%, respectively (D'Auria et al., 2001). Two studies have used the disc diffusion method to investigate the activity of TTO against dermatophytes. In both studies, zones of inhibition were seen adjacent to discs containing either 10 or 20 μl of neat TTO, using isolates of Epidermophyton floccosum, M. audouinii, M. canis, T. mentagrophytes, T. rubrum and T. tonsurans (Ånséhn, 1990; Concha et al., 1998). The exception was one strain of E. floccosum which showed no zone of inhibition (Concha et al., 1998). Several studies have investigated the activity of TTO against dermatophytes in more depth and have shown MICs of 0.7% for E. floccosum (Christoph et al., 2000), 0.11 – 0.5% for M. canis (Nenoff et al., 1996; D'Auria et al., 2001), 0.25% for M. gypseum (D'Auria et al., 2001) 0.12 – 0.75% for T. mentagrophytes (Bassett et al., 1990; Nenoff et al., 1996; Griffin & Markham, 2000; D'Auria et al., 2001) and 0.12 – 1.0% for T. rubrum (Bassett et al., 1990; Nenoff et al., 1996; Griffin & Markham, 2000; D'Auria et al., 2001). MFCs of TTO have been determined as follows; 0.25 – 0.5% for M. canis and T. mentagrophytes, 0.5% for M. gypseum and 0.25 – 1.0% for T. rubrum (D'Auria et al., 2001). Similar to studies performed with the dermatophytes, several methods have been used to investigate the activity of TTO against other filamentous fungi. With a few exceptions, these fungi are susceptible. All isolates of Aspergillus niger, Rhizopus oligosporus and Penicillium spp. showed zones of inhibition to either 20 μl or 35 μl oil on a paper disc (Concha et al., 1998; Chao et al., 2000). MICs for the filamentous fungi, mostly obtained by the agar dilution method, were in the range of 0.2 – 1.0% for isolates of A. flavus, A. niger, Penicillium spp., Rhizopus spp. and Scopulariopsis spp. (Beylier, 1979; Bassett et al., 1990; Southwell, 1993; Rushton et al., 1997; Christoph et al., 2000; Griffin & Markham, 2000). However, isolates of A. fumigatus and A. nidulans were not inhibited at 2% TTO in another study (Vazquez et al., 2000). Fungicidal data for these organisms have not been published. In vivo antifungal activity A small number of trials has been published investigating the efficacy of TTO for fungal infections. The earliest of these was by Walker (1972), who published a series of his observations of patients treated with a TTO solution for a range of foot problems, including tinea pedis and onychomycosis. More recently, two comparative trials investigating onychomycosis (Buck et al., 1994; Syed et al., 1999), two investigating tinea pedis (Tong et al., 1992; Satchell et al., 2002b) and one investigating dandruff (Satchell et al., 2002a) have been published. In the first of the onychomycosis trials (Buck et al., 1994), patients were treated twice daily with either neat TTO or 1% clotrimazole solution for a total of 6 months of treatment. After this time, of 64 patients treated with TTO, 18% were culture negative with a total of 60% of participants having full or partial resolution. This compared to the clotrimazole treatment group (n = 53) of whom 11% were culture negative and 61% had full or partial resolution. Overall, there were no statistically significant differences between the two treatment groups. The second onychomycosis trial (Syed et al., 1999) compared two creams, one containing 5% TTO alone and the other containing 5% TTO and 2% butenafine, both applied 3 times daily for 8 weeks. At completion of treatment the overall cure rate in patients treated with 5% TTO was 0%, compared to 80% for patients treated with both butenafine and TTO. The observation that TTO may be useful adjunct therapy for onychomycosis has also been made by Klimmek et al. (2002). In the first trial investigating tinea pedis, patients were treated with 10% TTO in sorbolene, 1% tolnaftate or placebo, applied twice daily for 4 weeks (Tong et al., 1992). At completion of treatment, patients treated with TTO had mycological cure and clinical improvement rates of 30% and 65%, respectively. This compares to mycological cure rates of 21% in patients receiving placebo and 85% in patients receiving tolnaftate. Similarly, clinical improvement was seen in 41% of patients receiving placebo and 68% of patients receiving tolnaftate. In the second trial, the efficacy of 25% and 50% TTO solutions in ethanol and polyethylene glycol solutions was evaluated by comparison to treatment with placebo (vehicle) (Satchell et al., 2002b). Patients applied their randomly assigned treatment twice daily for four weeks and were assessed after 2 and 4 weeks of treatment. Marked clinical responses were seen in 72% and 68% of the 25% and 50% TTO treatment groups, respectively, compared to 39% in the placebo group. Similarly, there were mycological cures in 55% and 64% of the 25% and 50% TTO treatment groups, respectively, compared to 31% in the placebo group. Dermatitis occurred in one patient in the 25% TTO group and three patients in the 50% TTO group resulting in the recommendation that 25% TTO be considered an alternative treatment for tinea since it was associated with fewer adverse reactions and was as effective as 50% TTO. These studies highlight the importance of considering the formulation of the TTO product when conducting in vivo work, since it is likely that the sorbolene vehicle used in the first trial significantly compromised the antifungal activity of the oil. The efficacy of TTO in the treatment of mild to moderate dandruff was evaluated in a large, randomised, single-blind, placebo-controlled trial in which patients used their allocated treatment daily for 4 weeks (Satchell et al., 2002a). In this study, the 5% TTO group (n = 63) showed statistically significant improvements in the investigator-assessed whole scalp lesion score, total area of involvement score and total severity score, as well as in the patient-assessed itchiness and greasiness scores compared to the placebo group (n = 62). There were no serious adverse events in either treatment group and only three patients in the TTO group reported events compared to eight in the placebo group. The data from this trial show that 5% TTO is well-tolerated and appears effective in the treatment of mild to moderate dandruff. Lastly, a case series of patients using TTO mouthwash for oropharyngeal candidiasis has been published (Jandourek et al., 1998). The 13 patients included in the series were HIV positive patients who had already failed treatment with a 14 day course of oral fluconazole. Patients were treated with 15ml of tea tree solution four times a day for up to 28 days. After treatment of the 12 evaluable
patients, two were cured, six were improved, four were unchanged and one patient had deteriorated.
Overall, eight patients had a clinical response, and seven had a mycological response. In summarising
the outcomes of these trials it seems apparent that treatment with TTO does not elicit a high rate of
infection cure. This is most likely due to many factors such as length and frequency of treatment and
the formulation of the trial product. In addition, it is believed that onychomycosis is unresponsive to
topical treatment therefore a high rate of cure should not be expected (Weitzman & Summerbell,
1995).
Antiviral activity
The few studies that have investigated the antiviral properties of TTO support the anecdotal notion
that TTO has antiviral properties. The antiviral activity of TTO was first shown using tobacco mosaic
virus and tobacco plants with agricultural applications in mind (Bishop, 1995). A field trial was
conducted in which Nicotiniana glutinosa plants were sprayed with 100, 250 or 500 ppm TTO or
control solutions, and all plants were then experimentally infected with tobacco mosaic virus. After
10 days, there were significantly fewer lesions per square centimetre of leaf of plants treated with
TTO as compared to controls (Bishop, 1995).
Schnitzler et al. (2001) investigated the activity of tea tree and eucalyptus oils against herpes simplex
virus (HSV). Briefly, the activity of TTO was determined by incubating virus with varying
concentrations of TTO, and then using these treated viruses to infect cell monolayers. After 4 days,
the numbers of plaques formed by virus treated with TTO, or untreated control virus, were
determined and compared. The concentration of TTO inhibiting 50% of plaque formation, as
compared to controls, was 0.0009% for HSV1 and 0.0008% for HSV2. These studies also showed
that at the higher concentration of 0.003%, TTO reduced HSV1 titres by 98.2% and HSV2 titres by
93.0%. Also, by applying TTO at different stages in the virus replicative cycle, TTO was shown to
have the greatest effect on free virus (prior to infecting cells) although when TTO was applied during
the adsorption period a reduction in plaque formation was seen also.
Some activity against bacteriophages, or viruses that infect bacteria, has also been reported with
exposure to 50% TTO at 4°C for 24 h reducing the number of plaques formed on a bacterial lawn
(Chao et al., 2000).
Further evidence for antiviral activity comes from a pilot study investigating the treatment of
recurrent herpes labialis (cold sores) with a 6% TTO gel or a placebo gel without TTO (Carson et al.,
2001). Comparison of each patient group (both containing nine evaluable patients) at the end of the
study showed that re-epithelialisation after treatment occurred after 9 days for the tea tree group and
after 12.5 days for the placebo group. Other measures such as duration of virus positivity by culture
or polymerase chain reaction, viral titres and time to crust formation were not significantly different,
possibly due to small patient numbers. Interestingly, when TTO was evaluated for its protective
efficacy in an in vivo mouse model of genital HSV type 2 infection it did not perform well (Bourne
et al., 1999). In contrast, 1,8-cineole, a component of TTO, performed well protecting 7 of 16
animals from disease.
The results of these studies indicate that TTO may act against viruses in several ways. In addition to
lethal effects directly on virus particles, TTO can also affect the way virus adsorbs to tissue culture
cells and can cause a reduced rate of infection in tobacco plants.
Anti-protozoal activity
TTO also has anti-protozoal activity although the data on this are limited to two publications. TTO caused a 50% reduction in growth (as compared to controls) of the protozoa Leishmania major and Trypanosoma brucei at concentrations of 403 μg/ml and 0.5 μg/ml, respectively (Mikus et al., 2000). Further investigation showed that terpinen-4-ol contributed significantly to this activity. In a different study, TTO at 300 μg/ml killed all cells of Trichomonas vaginalis (Viollon et al., 1996). This
combined with anecdotal in vivo evidence that Trichomonas vaginalis infections may be successfully
treated with TTO (Peña, 1962) suggest that further work is warranted.
Antimicrobial components of TTO
Considerable attention has been paid to which components of TTO are responsible for the
antimicrobial activity, mainly the antibacterial and antifungal activities. Early indications from RW
coefficients were that much of the activity could be attributed to terpinen-4-ol and α-terpineol
(Penfold & Grant, 1925). Data available today confirm that these two components contribute
substantially to the oil's activity (Carson & Riley, 1995; Raman, Weir & Bloomfield, 1995; Hammer
et al., 2003). However, of the components tested it seems that most possess at least some degree of
antimicrobial activity (Carson & Riley, 1995; Raman, Weir & Bloomfield, 1995; Hammer et al.,
2003) and while some may be considered less active, none can be considered inactive.
The possibility that components in TTO may exert synergistic or antagonistic effects on the overall
antimicrobial activity has been explored in vitro (Cox et al., 2001a) as has the potential for
interactions with other essential oils, such as lavender (Cassella et al., 2002), and other essential oil
components such as β-triketones from manuka oil (Christoph, Kaulfers & Stahl-Biskup, 2001;
Christoph, Stahl-Biskup & Kaulfers, 2001). Given the numerous components of TTO, the scope for
such effects is enormous and much more work is required to examine this question.
Anti-inflammatory activity
Anti-inflammatory activity has also been attributed to TTO but for many years only anecdotal
evidence was available. In vitro work over the last decade has demonstrated that terpinen-4-ol can
inhibit the production of several inflammatory mediators (such as interleukins) by human peripheral
blood monocytes (Hart et al., 2000). This suggests a mechanism by which TTO may reduce the
normal inflammatory response. Terpinen-4-ol also suppresses superoxide production by agonist-
stimulated monocytes, but not neutrophils (Brand et al., 2001). In vivo, topically applied TTO has
been shown to modulate the oedema associated with the efferent phase of a contact hypersensitivity
response (Brand et al., 2002a). This activity was attributed primarily to terpinen-4-ol and α-terpineol.
Similarly, topical TTO reduced histamine-induced skin oedema of the type that is often associated
with immediate type allergic hypersensitivities (Brand et al., 2002b). This activity also appeared to
be due mainly to terpinen-4-ol.
Agricultural applications
The broad-spectrum antimicrobial activity of TTO lends itself to uses other than human and animal medicine. There has also been interest in exploiting its properties for disease protection in crops and produce. The greatest interest seems to have been in the antifungal properties of the oil and there are data from in vitro antifungal assays, greenhouse studies and field trials against many important agricultural pathogens. The plethora of methods used in this work preclude direct comparisons. However, of the in vitro data, Bishop & Thornton (1997) showed that the following organisms could be inhibited by exposure to TTO in a disc diffusion method or to TTO vapour: Alternaria brassicicola, Alternaria solani, Botrytis cinerea, Fusarium solani, Myocentrospora acerina, Pythium ultimum, Rhizoctonia solani, Rhizopus sexualis, Rhizopus stolonifer, Sclerotinia sclerotiorum, Sclerotium cepivorum and Serpula lacrymans. Further work on B. cinerea using a bioassay with Brassica oleracea var. capitata (Dutch White cabbage) demonstrated that a concentration of 3.2% TTO compared favourably with three commercial fungicides (Bishop & Reagan, 1998). Vapourised TTO also inhibited the germination of B. cinerea spores at 12.5% but not 6.25% TTO (Wilson et al., 1997). Caolo-Tanski et al. (2002a,b) showed that TTO can inhibit a similar range of fungal pathogens in vitro, and in some cases in greenhouse studies and field trials. The fungi tested were Alternaria alternata, A. solani, Cercospora beticola, Cochliobolus sativum, Fusarium graminearum, Phytophthora infestans, Pythium paroecandrum, Rhizoctonia solani and S. sclerotiorum. Greenhouse studies also confirmed the potential for 1% TTO to be used for the control of powdery mildew of
cucurbits caused by Sphaerotheca fuliginea (Olsen et al., 1988). The same concentration of oil
proved effective in greenhouse trials in controlling false smut of palms caused by Graphiola
phoenicis
(Polizzi & Agosteo, 1995). Although the microbial pathogens were not identified, TTO
vaporised at a concentration of 100 μl l-1 reduced post-harvest decay in raspberries (Rubus idaeus L.)
(Wang, 2003). Washington and colleagues (1999) showed in field trials on strawberries that TTO
could control leather rot caused by Phytophthora cactorum and anthracnose (or blackspot) caused by
Colletotrichum acutatum. Earlier work by this group had shown that a commercially available TTO
crop treatment could reduce numbers of the predatory two spotted mites, Typhlodromus occidentalis
(Washington et al., 1991).
In one of the few investigations to examine bacterial disease in plants, TTO applied to soil infested
with Ralstonia solanacearum failed to reduce the subsequent incidence of wilt in tomatoes
(Pradhanang et al., 2002). Similarly, TTO did not successfully control Xanthomonas campestris pv.
campestris when applied to Brassica oleracea var. capitata (Dillard et al., 2000). Antiviral activity
has also attracted some attention and spray solutions containing TTO significantly decreased lesion
numbers in tobacco plants when applied prior to inoculation with tobacco mosaic virus (Bishop
1995).
Insecticidal activity
Anecdotally many essential oils have been mooted as insecticidal agents. Few have been investigated
scientifically. TTO has been evaluated in vitro against Pediculus humanus capitis (head lice) (Veal,
1996; Downs et al., 2000) and a shampoo containing several plant extracts including TTO performed
well in a pilot study of lice treatment (McCage et al., 2002). Scabies sarcopti (scabies) has also been
tested in vitro and found to be susceptible to the oil (Walton, Myerscough & Currie, 2000). In
contrast, in one in vitro test, a natural mosquito repellent product containing TTO provided almost no
protection against Aedes aegypti (Chou et al., 1997).
The activity of TTO against house dust mites has also been shown in two studies. In the first, the
activity of several essential oils (including TTO) against house dust mites was compared to that of
benzyl benzoate, a standard treatment. Oils of citronella and tea tree were as effective as 0.5% benzyl
benzoate and TTO at a concentration of 0.8% killed 79% of mites after a 10 min exposure time
(McDonald & Tovey, 1993). In the second study, TTO was the most effective at killing the house
dust mite Dermatophagoides pteronyssinus, when compared to lavender and lemon essential oils
(Priestley et al., 1998). TTO at a concentration of 10% caused 100% immobility after 30 min and
100% mortality after 2 h.
Other properties
It is likely that TTO possesses other properties common to the terpene family of chemicals. Many of the components in TTO have been shown to improve the percutaneous penetration of topically applied drugs. Most notable of these are cineole (Obata et al., 1991; Yamane et al., 1995; Gao & Singh, 1997;1998), and terpinen-4-ol and α-terpineol (Magnusson et al., 1997; Godwin & Michniak, 1999). By inference, it seems likely that TTO can also enhance the transdermal penetration of other compounds although it has not been reported to date. If TTO can penetrate the outer layers of the dermis this may help implementation of its antimicrobial properties; rather than inhibiting or killing microorganisms in the uppermost skin layers only, it may penetrate deeper eliminating underlying organisms and preventing relapses. Anecdotally, TTO is also credited with anti-pruritic activity. No scientific data exist for this property although it may be linked to its anti-inflammatory activity since the pathophysiology of itch is thought to be related to the inflammatory response (Hägermark & Wahlgren, 1992). Claims of analgesic properties have been made for TTO (Markham, 1999) but there are almost no
data to support them and it is impossible to confirm or refute these claims. The exception is one paper
describing reductions in the degree of pain and the total use of analgesics post-operatively after twice
daily inhalation of tea tree and peppermint oils (Takahashi et al., 2002).
Other applications
Numerous other applications have been suggested for TTO. One for which several products have
been developed but for which little research has been done is aerosolised TTO. There are anecdotal
reports of aerosolised TTO reducing hospital acquired infections (Bowden, 2001) but no scientific
data. Some preliminary in vitro work has been done with several researchers showing that vaporised
TTO can inhibit bacteria including Mycobacterium avium ATCC 4676 (Maruzzella & Sicurella,
1960) and the respiratory pathogens E. coli, Haemophilus influenzae, Streptococcus pyogenes and
Strep. pneumoniae (Inouye, Takizawa & Yamaguchi, 2001), and fungi (Inouye, Uchida &
Yamaguchi, 2001). Related work on agricultural applications of TTO has made similar observations
(Bishop & Thornton, 1997; Wilson et al., 1997).
TTO products designed for application to burns have also been developed but scientific data on their
suitability for use are lacking and their use has been questioned (Faoagali, George & Leditschke,
1997; Price, 1998). Some preliminary work has been done (Smith, 1995; Faoagali, George &
Leditschke, 1997; Jandera et al., 2000; Osti & Osti, 2002) but substantial additional research is
required before informed conclusions about their role can possibly be made.
There has been some interest in using TTO in veterinary medicine and it has been suggested for the
treatment of chronic dermatitis in dogs (Fitzi et al., 2002).
Consideration has been given to using M. alternifolia in constructed wetlands for sewage treatment
(Bolton & Greenway, 1997; 1999a,b). In addition to water treatment, this type of scheme offers the
benefits of native habitat rehabilitation and TTO production.
TTO has been assessed as an alternative solvent for the gutta-percha solvents used in dentistry
(Kaplowitz, 1990; Kaplowitz, 1991; Görduysus et al., 1997).
Product formulation issues
TTO's physical characteristics present certain difficulties for the formulation of products. Its
lipophilicity leads to miscibility problems in aqueous based products while its volatility means that
packaging must provide a suitable barrier to losses through volatilisation. Consideration must also be
given to the properties of the finished product. Early suggestions that TTO's antimicrobial activity
may be compromised by organic matter came from disc diffusion studies in which the addition of
blood to agar medium decreased zone sizes (Ånséhn, 1990). This observation contrasts sharply with
the old claim that the activity of TTO may be enhanced in the presence of organic matter such as
blood and pus. Data from Hammer and colleagues comprehensively refuted this idea (Hammer,
Carson & Riley, 1999) and also showed that product excipients may compromise activity.
Some work on the characteristics and behaviour of TTO within formulations has been conducted.
Caboi et al. (2002) examined the potential of a monoolein/water system as a carrier for TTO and
terpinen-4-ol. However, if stable, biologically-active formulations of TTO are going to be developed,
much remains to be done.
Safety and toxicity
Most TTO is used topically. Anecdotal evidence from almost 80 years of use suggests that topical use is safe, and that adverse events are minor and occasional. The toxicity of TTO can be considered in three major areas; toxicity from ingestion, from topical application and eco-toxicity. Topical or dermal toxicity can be further divided into allergic and irritant types of reaction. Oral toxicity Tea tree is categorised as a Schedule 6 poison in Australia. According to the Drugs, Poisons and Controlled Substances Act 1981, substances classed within this category have "a moderate potential for causing harm, the extent of which can be reduced through the use of distinctive packaging with strong warnings and safety directions on the label". To this end, neat TTO is labelled that it must be kept out of the reach of children, is packaged with a childproof cap and is labelled ‘not to be taken internally'. TTO can be toxic if ingested, as evidenced by studies with animals and from cases of human poisoning. An established laboratory method for measuring the toxicity of a substance is to determine the LD50, which is the ingested dose that is lethal to 50% of a test population. This is expressed as units of toxic substance per kilogram of body weight. The LD50 for TTO in a rat model is 1.9 – 2.6 ml/kg (Russell, 1999). Although values determined in animal models are not necessarily directly related to human toxicity, the animal model data indicate that TTO is orally toxic and therefore not suitable for internal use. Several incidences of oral poisoning in humans have been reported in the literature. Such occurrences tend to be more dramatic in children because of their low body weight compared to an adult. One such case report involved a 23 month old child who drank approximately 10 ml of TTO. After a nap of approximately 30 min, the child was unsteady on his feet and appeared as if ‘drunk'. The child was taken to a hospital and treated with activated charcoal and sorbitol via a naso-gastric tube, and approximately 5 h later he appeared to be asymptomatic. All other signs (such as respiratory rate, oxygen saturation, pupil reactivity, electrolytes and blood glucose) were normal throughout (Jacobs & Hornfeldt, 1994). The authors attribute the clinical symptoms to a central nervous system depression caused by the ingested TTO. A case of poisoning in an adult occurred when a patient drank approximately half a tea cup of TTO corresponding to a dose of approximately 0.5-1.0 ml/kg body weight (Seawright, 1993). The patient was comatose for 12 h, and semi-conscious and hallucinatory for the following 36 h. Symptoms of abdominal pain and diarrhoea continued for approximately 6 weeks after this. In another incident, a 60 year old man who swallowed one and a half teaspoonfuls of TTO as a preventative for a cold presented with a red rash which covered his feet, knees, upper body and arms including his palms and elbows (Elliott, 1993). His hands, feet and face were also swollen. The rash and other symptoms gradually disappeared and approximately one week later he had more or less recovered. Apart from these reports, there are no data on the systemic toxicity of TTO in humans. However, the ingestion of TTO should not be recommended. Despite this, deliberate ingestion is occasionally suggested (Belaiche 1985; Blackwell, 1991a) or reported. Dermal toxicity Systemic effects from topical TTO application in humans or other animals appear to be very rare, judging by published reports. The topical application of significant quantities of eucalyptus oil (containing approximately 80% 1,8-cineole) to a 6 year old girl caused systemic effects, including slurred speech, drowsiness, vomiting, ataxia and unconsciousness, although the girl recovered fully within approximately 6 h (Darben et al., 1998). Severe systemic effects following dermal application of TTO to cats have been reported (Bischoff & Guale, 1998). Three cats with shaved but intact skin had approximately 120 ml of neat TTO applied to them topically as a flea repellant. Within 5 h all three cats were experiencing symptoms such as hypothermia, uncoordination, dehydration and trembling, and one was comatose (Bischoff & Guale, 1998). All cats were treated by a veterinarian and two recovered after 24 and 48 h, respectively, but the third cat was found dead 3 days after admission. Irritant reactions Irritant reactions are an inflammatory type of response caused when an irritating substance comes into contact with a body surface, usually the skin. Importantly, these reactions are often concentration dependent, but are not dependent on previous exposure to the irritating agent. Irritant reactions may usually be avoided through the use of lower concentrations of the irritant and this bolsters the case for discouraging the use of neat oil and promoting the use of well-formulated products. The irritant capacity of TTO has been evaluated in both animal models and human trials, however, only the human data will be discussed below. The irritant capacity of TTO has been investigated using an occlusive patch test method with Finn chambers (Southwell et al., 1997). TTO was prepared in white soft paraffin at a concentration of 25% and this mixture was applied in patch tests on the backs or upper arms of volunteers. After 24 h, patches were removed and the skin was checked for any reactions. A new chamber was then applied to the same area, and checked again 24 h later. This was repeated at subsequent 24 h intervals for a total of 21 days. None of the 25 participants produced an irritant reaction from these tests. However, three of the original 28 participants showed distinct allergic reactions and were withdrawn from the trial. The TTO component 1,8-cineole, which has a reputation as a skin irritant, was also tested at concentrations up to and including 28% and did not produce any irritant reactions in the 25 (non-allergic) participants (Southwell et al., 1997). Another study similarly found that of 20 patients patch-tested with 1% TTO, none had irritant reactions (Knight & Hausen, 1994). This study also showed that TTO was a ‘weak sensitiser' after attempts were made to experimentally sensitise guinea pigs to TTO. Subsequent experiments have confirmed that newly distilled TTO has a relatively low sensitising capacity whereas TTO that had been exposed to light, oxygen, warmth and moisture, and was considered ‘degraded', was a moderate to strong sensitiser (Hausen et al., 1999). Contact allergy Contact allergy is defined as a cutaneous reaction caused by direct contact with an allergen to which the patient has become sensitised (Hensyl, 1990). Once an allergic reaction to TTO has occurred it is likely that all subsequent exposures to TTO, no matter what concentration, will elicit further allergic reactions. A series of seven such patients were described in a report by Knight & Hausen (1994). All patients reacted to 1% TTO when tested by patch testing using Finn chambers. In addition, these patients also reacted to one or more of the components d-limonene, α-terpinene, aromadendrene, terpinen-4-ol and α-phellandrene at 1, 5 or 10%. In the study by Southwell discussed above, the three participants having allergic type reactions to 25% TTO were tested against TTO components and reacted mostly to the sesquiterpenoid fractions but not the pure monoterpenes (Southwell et al., 1997). These studies indicate that contact allergy to TTO can occur, although the rate of occurrence is still not known. Toxicity against cell lines in vitro The testing of human or animal cells in vitro is seen as a modern alternative to animal testing to determine toxicity. Several studies have investigated the toxic effects of TTO and/or components on (human) cell lines in vitro. The amounts of TTO that reduced the growth of cells by 50% as compared to controls (IC50) after 24 h, ranged from 20 to 2700 μg/ml for HeLa, K562, CTVR-1, Molt-4 and Hep G2 cells (Hayes et al., 1997). IC50 values determined in other studies were 43.0 μg/ml for human HL-60 cells (Mikus et al., 2000), 0.006% for RC-37 cells (Schnitzler et al., 2001), 575 μg/ml for human fibroblasts and about 450 μg/ml for human epithelial cells (Söderberg et al., 1996). In addition, TTO produced toxic effects against human monocytes at concentrations of ≥0.004% (Hart et al., 2000) or ≥0.016% (Brand et al., 2001) and at ≥0.016% against human neutrophils (Brand et al., 2001). Eco-toxicity Literature on the ecotoxicity of TTO has been summarised and reviewed in Chapter 4. Table 3.1. Composition of M. alternifolia (tea tree) oil
Component Composition
ISO 4730 Range1 Typical 1 International Organisation for Standardisation 2 Brophy et al., 1989 3 no upper or lower limit set Table 3.2. Properties of TTO components
Type of compound monocyclic terpene alcohol C10H180 1491 3.26 monocyclic terpene monocyclic terpene monocyclic terpene alcohol C10H180 907 2.84 monocyclic terpene monocyclic terpene dicyclic terpene monocyclic terpene alcohol C10H180 1827 3.28 aromadendrene sesquiterpene sesquiterpene C15H24 (+)-limonene monocyclic sabinene dicyclic monoterpene C10H16 globulol sesquiterpene 1 Griffin et al., 1999b 2 Griffin et al., 1999a Table 3.3 Susceptibility data for bacteria tested against M. alternifolia oil (% v/v)
Bacterial species
Acinetobacter baumannii Actinomyces viscosus Actinomyces spp. Bacillus cereus Bacteroides spp. 0.061, 0.51 0.06-0.121 Corynebacterium sp. 0.2-0.32, 2.08 2.08 Enterococcus faecalis 0.5-0.752 Enterococcus faecalis (vancomycin R) 0.5-14, >810 0.5-14, >810 Escherichia coli 0.253, 7, 0.0811 0.253, 7 Fusobacterium nucleatum Klebsiella pneumoniae 0.258, 0.32 0.258 Lactobacillus spp. 1.014, 2.01 2.01, 14 Micrococcus luteus 0.06-0.58 0.25-6.08 Peptostreptococcus anaerobius 0.26, 0.251 0.03-0.121 Porphyromonas endodentalis 0.025-0.114 0.025-0.114 Porphyromonas gingivalis Prevotella spp. 0.031, 0.251 0.031 Prevotella intermedia 0.003-0.114 0.003-0.114 Propionibacterium acnes 0.052, 0.31-0.635 0.513 Proteus vulgaris 0.0811, 0.32, 2.010 4.010 Pseudomonas aeruginosa 1->2.02 ,1-810, 3.08 2->810, 3.08 Staphylococcus aureus 0.63-1.255, 0.57, 10 1.010, 2.07 Staphylococcus aureus (methicillin R) 0.0411, 0.254, 9 0.54, 0.59 Staphylococcus epidermidis 0.63-1.255, 1.08 4.08 Staphylococcus hominis Streptococcus pyogenes Veillonella spp. 0.016-1.014 0.03-1.014 1 Hammer et al., 1999a; 2 Griffin et al., 2000; 3 Gustafson et al., 1998; 4 Nelson, 1997; 5 Raman et al., 1995; 6 Shapiro et al., 1994; 7 Carson et al., 1995b; 8 Hammer et al., 1996; 9 Carson et al., 1995a; 10 Banes-Marshall et al., 2001; 11 Mann & Markham, 1998; 12 Carson et al., 1996; 13 Carson & Riley, 1994; 14 Hammer et al., 2003 Chapter 4. Review of tea tree oil
ecotoxicity data

Ecotoxicology can be loosely defined as the effects of pollutants on natural ecosystems. Although
data from acute toxicity testing of single animal or insect species may be regarded as overly
simplistic, they are often the starting point for assessing ecotoxicity.
Data describing the ecotoxicity of tea tree oil are very limited. The toxicity of tea tree oil against fish,
amphibians, insects, worms or other aquatic and terrestrial species, or ecosystems, has not been
assessed to any great extent.
4.1 Acute toxicity of tea tree oil to aquatic organisms
Two publications have assessed the potential for tea tree oil to be used as an antifungal agent in fish aquaculture (Campbell et al., 2001; Marking et al., 1994). Whilst both studies tested the efficacy of tea tree oil against aquatic fungi, Marking et al. (1994) also assessed the toxicity of tea tree oil to rainbow trout eggs. They found that tea tree oil was non-toxic to rainbow trout eggs at a concentration of 1500 ppm. Ecotoxicity data for two essential oils and some essential oil components are shown in Table 4.1. In addition, clove oil (containing 90% eugenol) has been evaluated as an anaesthetic for fish. It has been shown to anaesthetise fish at concentrations of 6 - 200 mg/l (Afifi et al., 2001; Sladky et al., 2001) but data are not available describing lethal concentrations. The values shown in Table 4.1 show that thyme oil and eugenol are for the most part categorised as slightly toxic, having LC50 values of between 10 and 100 mg/l (Kamrin, 1997). Lovage oil and it's component ocimene are categorised as practically non-toxic (with LC50 values of > 100 mg/l) whereas cumene is categorised as moderately toxic (with LC50 values of 1 – 10 mg/l). Ecotoxicity data for several components of tea tree oil are shown in Table 4.2. Using the toxicity categories described above, and the limited data for tea tree oil components, limonene and cymene are classified as slightly toxic, a-terpineol is moderately toxic, α- pinene appears to be practically non-toxic and data for β-pinene are equivocal. Notably absent are any data for the tea tree oil components terpinen-4-ol or γ-terpinene, the two components present in the highest proportions in tea tree oil. Whilst ecotoxicity data for essential oils other than tea tree oil, or essential oil components, can only be used as a guide, they suggest that tea tree oil may fall into the ‘slightly toxic' category, with LC50 values of between 10 – 100 μg/l. Table 4.1 Acute toxicity data for thyme oil, eugenol, lovage oil, ocimene and cumene
Volatile oil

Aquatic species
Reference
Rainbow trout1 LC50 = 16.1 mg/la Bull Env Contam Toxicol 1998; 60: 923-930 LC50 = 21.1 mg/la Bull Env Contam Toxicol 1998; 60: 923-930 salmon2 LC50 = 67.6 mg/la Bull Env Contam Toxicol 1998; 60: 923-930 trout1 LC50 = 61.5 mg/la Bull Env Contam Toxicol 1998; 60: 923-930 trout1 LC50 = 9 mg/l c Aqua Res 1998; 29: 89-101 LC50 = 125 mg/l b J Aquatic Animal Health 2000; 12: 224-229 LC50 = 63 mg/l b J Aquatic Animal Health 2000; 12: 224-229 LC50 = 250 mg/l b J Aquatic Animal Health 2000; 12: 224-229 Brine shrimp3 LD50 = 228 ppm Int J Aromather 2001; 11: 145-151 Brine shrimp3 LD50 = 697 ppm Int J Aromather 2001; 11: 145-151 LC50 = 8.1 mg/l a Ecotoxicol Env Saf 1995; 31: 287-289 trout1 LC50 = 6.4 mg/l a Ecotoxicol Env Saf 1995; 31: 287-289 flea4 LC50 = 4.8 mg/l a Ecotoxicol Env Saf 1995; 31: 287-289 1Onchorhynchus mykiss 2Onchorhynchus kisutch 3Artemia salina 4Daphnia magna 5Onchorhynchus masou 6Carassius aurantus a24 h exposure time b60 min exposure time cestimated over 8 – 96 h Table 4.2 Acute toxicity of components of tea tree oil to aquatic species
Component Aquatic

Data Reference
(life stage)
Water flea 4 LC50 = 68 mg/l a Bull Env Contam Toxicol 1980; 24: 684-691 Int J Aromather 2001; 11: 145-151 3 LD50 = 491 ppm Int J Aromather 2001; 11: 145-151 Rainbow trout (fry) 1 LC50 = 1.2 mg/l d J Great Lakes Res 1995; 21: 373-383 Z. Wasser-Abwasser Forsch 1978; 11(5): Int J Aromather 2001; 11: 145-151 Toxic dose range: Water Res. 1976; 10: 303-306 10 – 100 mg/l e LC50 = 6.8 mg/l a Bull Env Contam Toxicol 1998; 60: 923-930 LC50 = 6.7 mg/l a Bull Env Contam Toxicol 1998; 60: 923-930 LC50 = 9.4 mg/l a Bull Env Contam Toxicol 1980; 24: 684-691 Sheepshead minnow Bull Env Contam Toxicol 1981; 27: 596-604 1Onchorhynchus mykiss; 2Onchorhynchus kisutch; 3Artemia salina; 4Daphnia magna; 5Onchorhynchus masou; 6Carassius aurantus; 7Leuciscus idius melanotus a24 h exposure time; b60 min exposure time; cestimated over 8 – 96 h; d60 day exposure time; e96 exposure time 4.2 Acute toxicity of tea tree oil to terrestrial insects
The acute toxicity of essential oils and components has most commonly been evaluated in the context
of using essential oils as crop fumigants and protectants. Data describing the toxic effects of tea tree
oil on insects are limited. However, the LD50 of tea tree oil against the rice weevil Sitophilus oryzae
(L.) has been determined as >150 μl/l of air (Lee et al., 2001). In addition, varroa mites, which are
parasitic to honey bees, have been shown to be susceptible to tea tree oil. After 6 h treatment, 59.4%
of mites exposed to tea tree oil in air had died, compared to only 20% of control mites (Sammataro et
al
., 1998). The toxicity of several essential oils to insects is shown in Table 4.3. Whilst these data are
a useful indication of which concentrations of essential oil are toxic, it remains to be determined
whether tea tree oil has similar toxicity.
Table 4.3 Selected acute toxicity data for essential oils and terrestrial insects
Oil or Component

Insect species
Data Reference
(life stage)
Rice weevil 3 LD50 = > 150 μl/l of air Crop Prot 2001; 20: 317-320 Rice weevil 3 LD50 = 54 μl/l of air Crop Prot 2001; 20: 317-320 Oregano (Oreganum LD50 = 5.6 μl/fly J Agric Food Chem 1998; 46: 1111- vulgare subsp. hirtum) Fruit fly 2 LD50 = 2.09 J Agric Food Chem 1997; 45: 2690- (Mentha pulegium) Rice weevil 3 LD50 = 30.5 μl/l of air Crop Prot 2001; 20: 317-320 LD50 = 3.3 μl/fly J Agric Food Chem 1998; 46: 1111- (Satureja thymbra) Spearmint Fruit fly 2 LD50 = 1.12 J Agric Food Chem 1997; 45: 2690- (Mentha spicata) Rice weevil 3 LD50 = > 150 μl/l of air Crop Prot 2001; 20: 317-320 Rice weevil 3 LD50 = 63.9 μl/l of air Crop Prot 2001; 20: 317-320 Spodoptera litura LD50 = 43.7 μg/larva J Agric Food Chem 2001; 49: 715- LD50 = 6.78 μl/fly J Agric Food Chem 1998; 46: 1111- a24 h exposure time; 1Musca domestica; 2Drosophila melanogaster; 3Sitophilus oryzae In addition to toxicity tests with whole oils, several studies have determined the toxicity of essential oil components. These data may give a general indication of the likely toxicity of tea tree oil to insects. Data are shown in Table 4.4. Table 4.4 Selected acute toxicity data for essential oil components and terrestrial
insects
Component

Insect species
Reference
Spodoptera litura LD50 = 1.6 μg/larva J Agric Food Chem 2001; 49: 715-720 LD50 = 42.7 μg/larva J Agric Food Chem 1998; 46: 1111-1115 1,8-Cineole Rice LD50 = 23.5 μl/l of air Crop Prot 2001; 20: 317-320 Rice weevil 3 LD50 = 25.0 μl/l of air Crop Prot 2001; 20: 317-320 Spodoptera litura LD50 = 273.7 μg/larva J Agric Food Chem 2001; 49: 715-720 LD50 = 61.5 μl/l of air Crop Prot 2001; 20: 317-320 LD50 = 700 μg/insecta J Pest Sci 1988; 13: 287-290 LD50 = 90 μg/insecta J Pest Sci 1988; 13: 287-290 LD50 = 50.4 μg/flya J Agric Food Chem 2002; 50: 4576-4580 LD50 = 39.2 μl/l of air Crop Prot 2001; 20: 317-320 LD50 = 111.5 μg/flya J Agric Food Chem 2002; 50: 4576-4580 LD50 = 117.2 μg/flya J Agric Food Chem 2002; 50: 4576-4580 LD50 = 71.2 μl/l of air Crop Prot 2001; 20: 317-320 Terpinen-4-ol Rice LD50 = 25.6 μl/l of air Crop Prot 2001; 20: 317-320 Spodoptera litura LD50 = 130.4 μg/larva J Agric Food Chem 2001; 49: 715-720 Rice weevil 3 LD50 = 69.1 μl/l of air Crop Prot 2001; 20: 317-320 LD50 = 175.7 μg/flya J Agric Food Chem 2002; 50: 4576-4580 Spodoptera litura LD50 = 141.3 μg/larva J Agric Food Chem 2001; 49: 715-720 Spodoptera litura LD50 = 25.4 μg/larva J Agric Food Chem 2001; 49: 715-720 LD50 = 2.6 μg/larva J Agric Food Chem 1998; 46: 1111-1115 a24 h exposure time; †component not found in tea tree oil 1Musca domestica; 2Drosophila melanogaster; 3Sitophilus oryzae; 4Blatella germanica
In addition, another study showed that the treatment of insects for 14 h with several compounds at a
concentration 0.05 μg/l of air resulted in varying mortalities. Treatment with linalool resulted in
100% mortality in house flies, German cockroaches and saw-toothed grain beetles, 10% mortality in
red flour beetles and 0% in rice weevils. Terpineol resulted in 100% mortality in saw-toothed grain
beetles, no mortality in German cockroaches, red flour beetles or rice weevils and 20% in house flies.
Treatment with cineole resulted in 100% mortality in all insects. Similarly, treatment with limonene
resulted in 100% mortality for all insects except red flour beetles, which had a mortality rate of 60%
(Lee et al., 2003).
In addition to acute toxicity data, several studies have indicated that essential oils can have other
effects, such as inhibition of larval growth (Hummerbrunner & Isman, 2001), inhibition of
reproduction (Regnault-Roger & Hamraoui, 1995) and deterrence of feeding (Hummerbrunner &
Isman, 2001).
The above studies with both whole oils and components indicate that tea tree oil is likely to be toxic
to insects. However, it has been stated that the acute toxicity of monoterpenes to insects is relatively
low, compared to conventional insecticides (Lee et al., 1997). Whether this is also true for tea tree oil
remains unknown.
4.3 Other acute toxicity data
One study has shown that d-limonene is toxic to the earthworm Eisenia fetida (Savigny) (Karr et al., 1990). The LD50 by topical application was 60 ppm, and when earthworms were exposed to 12.6 ppm it took 4.9 h for 50% of the organisms to die. Chronic exposure to limonene also resulted in weight
loss.
4.4 Conclusions
Although very limited data are available regarding the ecotoxicity of tea tree oil, it can be extrapolated from data for other essential oils and components that tea tree oil should be considered slightly to moderately toxic, as a conservative estimate of overall ecotoxicity. Chapter 5. Literature Database

The existing scientific literature on TTO are not readily accessible to most industry stakeholders. A
comprehensive collection of TTO literature in the form of an electronic database available through
the ATTIA web site would be a valuable resource for the industry.
5.1 Methods
The following electronic databases were searched for publications that include data on TTO, TTO components, Melaleuca alternifolia and other Melaleuca species of relevance to the TTO industry: Agricola (1979 to current), Biological Abstracts (1995 to current), CAB Abstracts (1973 to current), Current Contents (1993 week 27 to current), EMBASE (1988 to current) and Medline (1966 to current). Articles up to and including those indexed on these databases by June 2003 were included. Information on popular press books was primarily found from general internet searches, including searches of websites specifically dealing with books. Additional articles were sourced from the extensive literature collections of C. Carson and K. Hammer. Publications containing any of the above keywords were manually scrutinised to identify additional papers. Publications containing substantial reference to tea tree oil, in the form of one or more paragraphs, were included in the database. The journal articles cited in the database came from approximately 200 different publications, from approximately 100 different publishers. Wherever possible, the contact details for each publisher were obtained. A request was sent to each publisher requesting permission to reproduce the relevant article(s) in full in the tea tree oil database. The majority of publishers were contacted by May 31, 2003. Articles that were written entirely in a language other than english were not contacted. In addition, journals for which no contact details could be found, despite extensive searching, were not contacted. Where permission was granted to include the full text of articles, they were either obtained in the form of PDF files from journal websites or authors, or each page of the article was scanned to create an image and a PDF file was constructed. 5.2 Results
Citations
More than 500 tea tree oil publications were found, including research articles, reviews, conference
abstracts or presentations, books and theses. Several citations were omitted from the database,
usually because they had been made redundant by subsequent publication, were never officially
published, or because of insufficient quality.
Obtaining permission for reproduction of material
Results of permission requests to publishers are shown in Appendix 2. Due to the modest budget for
the project the decision was made to not pay fees if payment was required in order to reproduce
articles. Permission to reproduce the full text of articles was requested on the basis that the articles
would be available through the ATTIA web site, to ATTIA members only and that the database
would be password protected.
Database construction and delivery
The database was structured as an introductory page, a list of citations (Appendix 1), with links to either the abstract or full text or both, and a list of abstracts. The list of abstracts is not included in an appendix because copyright permission to reproduce them in this report was not granted. It is intended that the database will be available through the ATTIA web site (http://www.teatree.org.au/). ATTIA bears responsibility for making the database available through its web site, for ensuring that it is available only to members and that it is password protected. It should be noted that permission has been granted for the use of the articles and abstracts within this database for ATTIA members for personal educational purposes only. Permission has not been granted for ATTIA members to reproduce or distribute this information. To do so would be an infringement of copyright. Chapter 6. Material safety data sheet
A material safety data sheet (MSDS) was created for tea tree oil (Appendix 3), following the guidelines set out in the National Code of Practice for the Preparation of Material Safety Data Sheets (National Occupational Safety and Health Commission, 2003). Industry personnel, scientific literature and the following data sources were consulted to obtain correct, up to date information for tea tree oil: 1. International Standards Organisation (1996) Oil of Melaleuca, terpinen-4-ol type (tea tree oil). International Standard ISO 4730:1996(E), International Standards Organisation, Geneva 2. Sweet DV. (Editor) (1997) Registry of toxic effects of chemical substances (RTECS), comprehensive guide. U.S. Department of Health and Human Services, Cincinnati, Ohio. 3. Standard for the uniform scheduling of drugs and poisons (2002) Commonwealth Department of Health Published Canberra: Australian Government Publishing Service. March 2002, 16. 4. Australian code for the transport of dangerous goods by road and rail (ADG code) (1999). Commonwealth Department of Transport and Regional Services, Canberra, 6th Edition. 5. Approved criteria for classifying hazardous substances (NOHSC: 1008(1994)) National Occupational Health and Safety Commission. Australian Govt. Pub. Service, Canberra, 1994. 6. The Australian inventory of chemical substances (1992). Department of the Arts, Sport, the Environment and Territories, Commonwealth Environment Protection Agency, AGPS Press, Canberra. 7. National Occupational Health and Safety Commission (2003) National Code of Practice for the Preparation of Material Safety Data Sheets [NOHSC:2011 (2003)]. Australian Government Publishing Service, Canberra, April 2003. Chapter 7. Recommendations for further

The recommendations for further studies listed below represent the opinions of the authors of this
report. It is quite plausible that other experts, such as toxicologists, dermatologists or medical doctors
may arrive at a different set of conclusions regarding what further tea tree oil research needs to be
conducted. As such, this list must not be regarded as exhaustive, definitive or in order of priority.
The recommendations as to further research that should be conducted into tea tree oil can be
subdivided into two broad categories, shown below.
1) Safety and toxicity
a. Toxicity studies, using an animal model • Safety of inhaled tea tree oil • Absorption through broken skin and/or wounds • Reproductive toxicity, including mutagenicity and teratology studies • Chronic toxicity b. Toxicity studies, using human volunteers • Absorption through skin, looking for oil or metabolites in blood and urine • Repeat application studies • Mucous membrane irritation studies c. Ecotoxicity studies • Aquatic and terrestrial insects • Fish species • Plant species
2) Clinical efficacy (pilot studies and full clinical trials)
• Wounds (diabetic ulcers, chronic wounds) • Head lice • Impetigo • Vaginal candidiasis • Mouthrinse for gingivitis • Pre and post-operative wound infections In addition, the work investigating the anti-inflammatory properties of tea tree oil must be continued and expanded. Corroboration of existing clinical data for infections or conditions such as tinea, dandruff, acne, MRSA carriage, onychomycosis and oral candidiasis is imperative. There are several other potential clinical applications for tea tree oil that first require significant preliminary in vitro work. • Tea tree oil has great potential as an intra-vaginal microbicide • The activity of tea tree oil against microbial biofilms needs to be determined, with a view to using it to impregnate indwelling medical devices such as catheters. • The efficacy of aerosolised tea tree oil against bacteria and microbial biofilms requires attention, as inhaled tea tree oil may be a potential therapy for lung infections such as cystic fibrosis
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Appendix 1 – Literature database

The following is a list of published documents that relate to tea tree oil. This is the most exhaustive
and comprehensive bibliography of tea tree oil literature published to date. The abstracts and full text
of many of the documents are available on the database. The remainder may be available through
document delivery services such as Infotrieve, Ingenta, Loansome Doc on Public Medline or Science
Direct. Visit these web sites for further information.
Adamson S. (1997) Infection Control. Hospital & Healthcare 28(6): 24. [News]
Ahmad AW, Mansor P, Abdul Malek, Y & Jaafar H. (1998) Distillation of tea-tree (Melaleuca
alternifolia
) oil. I. Establishment of basic parameters and standard conditions for a test distiller and
evaluation of two prototype distillers. Journal of Tropical Agriculture & Food Science 26: 175-187.
[Research article] Reproduced with permission.
Ahmad AW, Mansor P & Tunku Kassim TAR. (1998) Distillation of tea-tree (Melaleuca
alternifolia
) oil. II. Comparison of different fuels for steam generation using a prototype distiller.
Journal of Tropical Agriculture & Food Science 26: 189-194. [Research article] Reproduced with permission.
Ahmed S & Tsang M. (2001) A study of the anti-bacterial activity of a number of constituents of
‘tea tree' oil against E. coli and the effect of varying the constituent ratio. British Pharmaceutical
Conference Abstract Book
. p. 113 [Conference abstract]
Allen P. (2001) Tea tree oil: the science behind the antimicrobial hype. Lancet. 358: 1245.
[News] No abstract.
Altman PM. (1988) Australian tea tree oil. The Australian Journal of Pharmacy 69: 276-278.
[Review] Reproduced with permission.
Altman PM. (1988) Potential use of essential oils in cosmetics. Cosmetics, Aerosols and Toiletries
in Australia
2: 13-14,22. [Review] No abstract.
Altman PM. (1989) Australian tea tree oil - a natural antiseptic. Australian Journal of Biotechnology
3: 247-248. [Review] Reproduced with permission.
Altman PM. (1991) Australian tea tree oil - an update. Cosmetics, Aerosols and Toiletries in
Australia
5: 27-29. [Review]
Anderson JN & Fennessy PA. (2000) Can tea tree (Melaleuca alternifolia) oil prevent MRSA?
Medical Journal of Australia. 173: 489.[Letter] Copyright 2000, The Medical
Journal of Australia. Reproduced with permission.

Anon (1930) A retrospect. Medical Journal of Australia i: 85-89. [Letter]
Copyright 1930, The Medical Journal of Australia. Reproduced with permission.
Anon (1936) Revised medical and dental data of ti-trol (antiseptic oil) and melasol (antiseptic
solution). Australian Essential Oils Ltd., Sydney, NSW. [Booklet]
Anon (1933) An Australian antiseptic oil. British Medical Journal i: 966. [Letter]
Anon (1933) Ti-trol oil. British Medical Journal ii: 927. [Letter]
Anon (1990) Tea-tree oil and acne. Lancet Dec 8: 1438. [Letter]
Anon (1995) Special takeout feature: Tea tree oil. CAJ 111: 19-21.
Anon (1997) Tea tree oil – skin reactions. Bulletin from Swedish Adverse Drug Reactions Advisory
Committee (SADRAC) 66: 4. [Letter]
Anon (1997) Tea tree oil – universal agent? Soap, Perfumery and Cosmetics May 1997: 50-52
[News]
Anon (1997) Lessons I've learned. Complementary Therapies in Medicine 5: 241-242. [News]
Anon (2002) Tea tree oil, latest alternative medicine. Journal of the National Medical Association
92(8): A14 [News] Reproduced with permission
Anon (2002) Aussie tea tree oil wins international recognition – export boost to follow. News release
from Standards Australia. [News]
Ånséhn S. (1990) The effect of tea tree oil on human pathogenic bacteria and fungi in a laboratory
study [Swedish]. Swedish Journal of Biological Medicine 2: 5-8. [Research article]
Apted JH. (1991) Contact dermatitis associated with the use of tea-tree oil. Australasian Journal of
Dermatology
. 32: 177. [Letter] Full text (PDF) Reproduced with permission from Blackwell
Publishers

Arweiler NB, Donos N, Netuschil L, Reich E & Sculean A. (2000) Clinical and antibacterial effect
of tea tree oil - a pilot study. Clinical Oral Investigations 4: 70-73. [Research article]
Atkinson N & Brice HE. (1955) Antibacterial substances produced by flowering plants. Australian
Journal of Experimental Biology
and Medicine 33: 547-554. [Research article] No abstract.
Austin N. (1987) Tea-tree oil may become megabuck earner. The Bulletin 109 (November 10): 38.
[News]
Bailey PCE, Watkins SC, Morris KL, Boon PI. (2003) Do Melaleuca ericifolia Sm. leaves
suppress organic matter decay in freshwater wetlands? Archiv fur Hydrobiologie 156: 225-240.
[Research article] Baker GR, Lowe RF & Southwell IA. (2000) Comparison of oil recovered from tea tree leaf by
ethanol extraction and steam distillation. Journal of Agricultural & Food Chemistry 48: 4041-4043.
[Research article] Banes-Marshall L, Cawley P & Phillips Carol A. (2001) In vitro activity of Melaleuca alternifolia
(tea tree) oil against bacterial and Candida spp. isolates from clinical specimens. British Journal of
Biomedical Science
58: 139-145. [Research article]
Bang B & Agner T. (2000) Picture of the month. Allergic contact dermatitis. [Swedish] Ugeskrift
for Laeger
162: 3867. [Case report] No abstract.
Bassett IB, Pannowitz DL & Barnetson RStC. (1990) A comparative study of tea-tree oil versus
benzoylperoxide in the treatment of acne. Medical Journal of Australia. 153: 455-458. [Research
article] edical Journal of Australia. Reproduced
with permission.

Baumann LS. (2002) Cosmeceutical critique - tea tree oil. eSkin and Allergy News 33: 14. [News]
Becker D. (2001) Sensitizations to tea tree oil and propolis are increasing. Tw Dermatologie. 5: 30.
[Letter]
Beckman B & Ippen H. (1998) Tea tree oil [German]. Dermatosen 46: 120-124. [Research article]
Bedi MK & Shenefelt PD. (2002) Herbal therapy in dermatology. Archives of Dermatology 138:
232-242. [Review]
Beer C. (1986) Tea tree oil loves skin. Nature and Health 7: 16-17. [News]
Beer C. (1987) Australian tea tree oil. Nature and Health 8: 3-7. [News]
Belaiche P. (1985) Germicidal properties of the essential oil of Melaleuca alternifolia (Cheel) for the
idiopathic, chronic urinary tract infections caused by coliforms [French]. Phytotherapy 15: 9-11.
[Research article] French abstract only.
Belaiche P. (1985) Treatment of cutaneous infections with the essential oil of Melaleuca alternifolia
Cheel [French]. Phytotherapy 15: 15-17. [Research article] French abstract only.
Belaiche P. (1985) Treatment of vaginal infections of Candida albicans with the essential oil of
Melaleuca alternifolia (Cheel) [French]. Phytotherapy 15: 13-14. [Research article] French abstract
only.
Betts TJ, Moir CM & Tassone AI. (1991) Use of a liquid crystal stationary phase at temperatures
below its melting point for the gas chromatographic study of some volatile oil constituents. Journal
of Chromatography A
. 547: 335-344. [Research article]
Beylier MF. (1979) Bacteriostatic activity of some Australian essential oils. Perfumer and Flavourist
4: 23-25. [Research article] No abstract.
Bhushan M & Beck MH. (1997) Allergic contact dermatitis from tea tree oil in a wart paint.
Contact Dermatitis 36: 117-118. [Case report] Full text (PDF) Reproduced with permission from
Blackwell Publishers

Bischoff K & Guale F. (1998) Australian tea tree (Melaleuca alternifolia) oil poisoning in three
purebred cats. Journal of Veterinary Diagnostic Investigation 10: 208-210. [Research article] Reproduced with permission.
Bishop CD. (1995) Antiviral activity of the essential oil of Melaleuca alternifolia (Maiden &
Betche) Cheel (tea tree) against tobacco mosaic virus. Journal of Essential Oil Research 7: 641-644.
[Research article]
Bishop CD & Reagan J. (1998) Control of the storage pathogen Botrytis cinerea on Dutch White
cabbage (Brassica oleracea var. capitata) by the essential oil of Melaleuca alternifolia. Journal of
Essential Oil Research
10: 57-60. [Research article]
Bishop CD & Thornton IB. (1997) Evaluation of the antifungal activity of the essential oils of
Monarda citriodora var. citriodora and Melaleuca alternifolia on post-harvest pathogens. Journal of
Essential Oil Research
9: 77-82. [Research article]
Blackwell AL. (1991) Tea tree oil and anaerobic (bacterial) vaginosis. Lancet. 337: 300. [Letter]
Blackwell R. (1991) An insight into aromatic oils: lavender and tea tree. British Journal of
Phytotherapy
2: 26-30.
Blamey C. (2001) Case history of infected eczema treated with essential oils. Grand Rounds 5: 11-
14. [Case report]
Bolton KGE & Greenway M. (1997) A feasibility study of Melaleuca trees for use in constructed
wetlands in subtropical Australia. Water Science & Technology 35: 247-254. [Research article]Bolton KGE & Greenway M. (1999) Nutrient sinks in a constructed Melaleuca wetland receiving
secondary treated effluent. Water Science & Technology 40: 341-347. [Research article]Bolton KGE & Greenway M. (1999) Pollutant removal capability of a constructed Melaleuca
wetland receiving primary settled sewage. Water Science & Technology 39: 199-206. [Research
article]
Boon PI & Johnstone L. (1997) Organic matter decay in coastal wetlands: an inhibitory role for
essential oil from Melaleuca alternifolia leaves? Archiv Fur Hydrobiologie 138: 433-449. [Research
article] Bouic F. (2000) Use of tea tree oil in the treatment of head lice. British Medical Journal 2000:
[Letter - electronic publication only]Bourne KZ, Bourne N, Reising SF & Stanberry LR. (1999) Plant products as topical microbicide
candidates: assessment of in vitro and in vivo activity against herpes simplex virus type 2. Antiviral
Research
42: 219-226. [Research article]
Bowden L. (2001) The effectiveness of tea tree oil in reducing hospital acquired infections in care of
the elderly ward. Infection Control Nurses Association Annual Infection Control Conference. 24-28
September 2001. Blackpool, UK p. 23. [Conference abstract]
Brand C, Ferrante A, Prager RH, Riley TV, Carson CF, Finlay-Jones JJ & Hart PH. (2001)
The water-soluble components of the essential oil of Melaleuca alternifolia (tea tree oil) suppress the
production of superoxide by human monocytes, but not neutrophils, activated in vitro. Inflammation
Research
50: 213-219. [Research article]
Brand C, Grimbaldeston MA, Gamble JR, Drew J, Finlay-Jones JJ & Hart PH. (2002) Tea tree
oil reduces the swelling associated with the efferent phase of a contact hypersensitivity response.
Inflammation Research 51: 236-244. [Research article] Brand C, Townley SL, Finlay-Jones JJ & Hart PH. (2002) Tea tree oil reduces histamine-induced
oedema in murine ears. Inflammation Research 51: 283-289. [Research article]
Brenan JA, Dennerstein GJ, Sfameni SF, Drinkwater P, Marin G & Scurry JP. (1996)
Evaluation of patch testing in patients with chronic vulvar symptoms. Australasian Journal of
Dermatology
37: 40-43. [Research article] Full text (PDF) Reproduced with permission
from Blackwell Publishers

Brophy JJ, Davies NW, Southwell IA, Stiff IA & Williams LR. (1989) Gas chromatographic
quality control for oil of Melaleuca terpinen-4-ol type (Australian tea tree). Journal of Agricultural &
Food Chemistry
37: 1330-1335. [Research article] Bruynzeel DP. (1999) Contact dermatitis due to tea tree oil. Tropical Medicine & International
Health
4: 630. [Letter] Reproduced with permission from Blackwell Publishers
Buchbauer G. (1997) About tea tree oil [German]. Eurocosmetics i: 21-24. [News]
Buchness MR. (1998) Alternative medicine and dermatology. Seminars in Cutaneous Medicine &
Surgery
17: 284-290. [Review] Buck DS, Nidorf DM & Addino JG. (1994) Comparison of two topical preparations for the
treatment of onychomycosis: Melaleuca alternifolia (tea tree) oil and clotrimazole. Journal of Family
Practice
38: 601-605. [Research article]
Budhiraja SS, Cullum ME, Sioutis SS, Evangelista L & Habanova ST. (1999) Biological activity
of Melaleuca alternifolia (tea tree) oil component, terpinen-4-ol, in human myelocytic cell line HL-
60. Journal of Manipulative & Physiological Therapeutics 22: 447-453. [Research article]
Bunnell T. (2000) Tea tree oil antiseptic cream: a new treatment for ringworm and sarcoptic mange
in the hedgehog (Erinaceus europaeus). Journal of the American Holistic Veterinary Association 19:
29-31 [Research article] No abstract.
Burdzenia O. (2002) The use of volatile oils in sports [Polish]. Medycyna sportowa 18: 33-38
[Review]
Burfield T. & Sheppard-Hanger S. (2000) Super clone "88" Melaleuca alternifolia – what is its
value? First International Phyto-Aromatic Conference, Nice France, March 24-26, 2000. [Conference
abstract]
Butcher PA. (1995) Genetic diversity in Melaleuca alternifolia: implications for breeding to
improve production of Australian tea tree oil. Australian National University [PhD Thesis]
Butcher PA, Bell JC & Moran GF. (1992) Patterns of genetic diversity and nature of the breeding
system in Melaleuca alternifolia (Myrtaceae). Australian Journal of Botany 40: 365-375. [Research
article] Butcher PA, Byrne M & Moran GF. (1995) Variation within and among the chloroplast genomes
of Melaleuca alternifolia and M. linariifolia (Myrtaceae). Plant Systematics & Evolution 194: 69-81.
[Research article] Butcher PA, Doran JC & Slee MU. (1994) Intraspecific variation in leaf oils of Melaleuca
alternifolia
(Myrtaceae). Biochemical Systematics & Ecology 22: 419-430. [Research article]Butcher PA, Matheson AC & Slee MU. (1996) Potential for genetic improvement of oil production
in Melaleuca alternifolia and M. linariifolia. New Forests 11: 31-51. [Research article]Byrne W. (1996) Tea tree oil for thrush. Australian Pharmacist 15: 104,109. [News]
Reprinted with permission.
Byrnes NB. (1986) A revision of Melaleuca Myrtaceae in northern and eastern Australia 3.
Austrobaileya 2: 254-273. [Research article] Caboi F, Murgia S, Monduzzi M & Lazzari P. (2002) NMR investigation on Melaleuca
alternifolia
essential oil dispersed in the monoolein aqueous system: phase behaviour and dynamics.
Langmuir 18: 7916-7922. [Research article]
Caelli M, Riley TV, Heller R & Carson CF. (1998) Tea tree oil - an alternative topical
decolonisation agent for adult inpatients with methicillin-resistant Staphylococcus aureus (MRSA) –
a pilot study. Journal of Hospital Infection 40(Suppl A): P.9.2.20. [Conference abstract]
Caelli M, Porteous J, Carson CF, Heller R & Riley TV. (2000) Tea tree oil as an alternative
topical decolonization agent for methicillin-resistant Staphylococcus aureus. Journal of Hospital
Infection
46: 236-237. [Research note] Campbell RE, Lilley JH, Taukhid, Panyawachira V & Kanchanakhan S. (2001) In vitro
screening of novel treatments for Aphanomyces invadans. Aquaculture Research 32: 223-233.
[Research article] Reproduced with permission from Blackwell Publishers
Campbell AJ & Maddox CDA. (1997) Controlling insect pests in tea tree using pyrgo beetle as the
basis. RIRDC project DAN-91A, RIRDC publication R97/062, 64pp. [Government publication]
Caolo-Tanski JM, Hanson LE, Hill AL & Hill JP. (2002) Use of Melaleuca alternifolia oil for
plant disease control. Presented at the American Phytopathological Society's annual meeting in
Milwaukee, Wisconsin, July 27-31, 2002 [Conference abstract]
Caolo-Tanski JM, Hanson LE, Hill AL & Hill JP. (2002) The potential use of Australian tea tree
oil (Melaleuca alternifolia) as a method of control for several plant pathogens. Proceedings of the 6th
Annual Rocky Mountain Plant Biotechnology and Molecular Biology Symposium at Colorado State
University, Ft.Collins April 17, 2002, pg. 22. [Conference abstract]
Carr A. (1998) Therapeutic properties of New Zealand and Australian tea trees (Leptospermum and
Melaleuca). New Zealand Pharmacy 18: 29-31. [Review] No abstract.
Carson CF. (1998) Tea tree oil – applications and implications. Cosmetics & Toiletries Manufacture
Worldwide
1998: 17-21. [News]
Carson CF. (1999) Antimicrobial activity of the essential oil of Melaleuca alternifolia (tea tree oil)
The University of Western Australia. [PhD Thesis]
Carson CF. (1999) Tea tree essential oil – fact and fiction. Aromatherapy Today 10: 6-10. [News]
Reproduced with permission
Carson CF, Ashton L, Dry L, Smith DW & Riley TV. (2001) Melaleuca alternifolia (tea tree) oil
gel (6%) for the treatment of recurrent herpes labialis. Journal of Antimicrobial Chemotherapy 48:
450-451. [Letter] No abstract.
Carson CF, Cookson BD, Farrelly HD & Riley TV. (1995) Susceptibility of methicillin-resistant
Staphylococcus aureus to the essential oil of Melaleuca alternifolia. Journal of Antimicrobial
Chemotherapy
35: 421-424. [Research article]Carson CF, Hammer KA & Riley TV. (1995) Broth micro-dilution method for determining the
susceptibility of Escherichia coli and Staphylococcus aureus to the essential oil of Melaleuca
alternifolia
(tea tree oil). Microbios 82: 181-185. [Research article] Carson CF, Hammer KA & Riley TV. (1997) In-vitro activity of the essential oil of Melaleuca
alternifolia
against Streptococcus spp. Journal of Antimicrobial Chemotherapy 37: 1177-1178.
[Letter] No abstract.
Carson CF, Hammer KA & Riley TV. (1997) Use of the essential oil of Melaleuca alternifolia (tea
tree oil) in cutaneous fungal infections. Journal of British Podiatric Medicine 52: iv-v. [Letter] No
abstract.

Carson CF, Hammer KA & Riley TV. (1998) Antimicrobial activity of Melaleuca alternifolia (tea
tree) oil against wound organisms. In Program and Abstracts of the 4th International Conference of
the Hospital Infection Society, 13-17 September 1998, Edinburgh. Abstr. P.9.2.16. [Conference
abstract]
Carson CF, Hammer KA & Riley TV. (1998) A brief review of antifungal activity of the essential
oil of Melaleuca alternifolia (tea tree oil). Mikologia Lekarska 5: 205-207. [Review]
Carson CF, Mee BJ & Riley TV. (2002) Mechanism of action of Melaleuca alternifolia (tea tree)
oil on Staphylococcus aureus determined by time-kill, lysis, leakage, and salt tolerance assays and
electron microscopy. Antimicrobial Agents & Chemotherapy 46: 1914-20. [Research article]
Carson CF & Riley TV. (1993) Antimicrobial activity of the essential oil of Melaleuca alternifolia.
Letters in Applied Microbiology 16: 49-55. [Review] Abstract Full text (PDF) Reproduced with
permission from Blackwell Publishers

Carson CF & Riley TV. (1994) The antimicrobial activity of tea tree oil. Medical Journal of
Australia
160: 236. [Letter] Copyright 1994, The Medical Journal of Australia.
Reproduced with permission.

Carson CF & Riley TV. (1994) Susceptibility of Propionibacterium acnes to the essential oil of
Melaleuca alternifolia. Letters in Applied Microbiology. 19: 24-25. [Research article] Full
text (PDF)
Reproduced with permission from Blackwell Publishers
Carson CF & Riley TV. (1995) Antimicrobial activity of the major components of the essential oil
of Melaleuca alternifolia. Journal of Applied Bacteriology 78: 264-269. [Research article]
Full text (PDF)
Reproduced with permission from Blackwell Publishers
Carson CF & Riley TV. (1995) Toxicity of the essential oil of Melaleuca alternifolia or tea tree oil.
Journal of Toxicology - Clinical Toxicology
33: 193-194. [Letter] No abstract.
Carson CF & Riley TV. (1996) Working with and against tea tree oil - issues of synergy and
antagonism. In Program and Abstracts of the 19th International Federation of the Societies of
Cosmetic Chemists Congress. Sydney, Australia, Oct 22 - 25 1996. [Conference abstract]
Carson CF & Riley TV. (1997) Investigations into the mechanism of action of Melaleuca
alternifolia
(tea tree) oil. In Program and Abstracts of the Australian Society for Microbiology
Annual Scientific Meeting and Exhibition. Australian Society for Microbiology, Adelaide. Abstr.
P02.38, p. A109. [Conference abstract]
Carson CF & Riley TV. (1998) Antimicrobial activity of tea tree oil. RIRDC Project No UWA-
24A, RIRDC Publication No 98/070, 63pp. [Government publication]
Carson CF & Riley TV. (2001) Safety, efficacy and provenance of tea tree (Melaleuca alternifolia)
oil. Contact Dermatitis 45: 65-67.[Review] Reproduced with permission
from Blackwell Publishers

Carson CF & Riley TV. (2003) Non-antibiotic therapies for infectious diseases. Communicable
Diseases Intelligence
27(Suppl): S143-146. [Journal Article]
Carson CF, Riley TV & Cookson BD. (1998) Efficacy and safety of tea tree oil as a topical
antimicrobial agent. Journal of Hospital Infection 40: 175-178. [Review] No abstract.
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Hammer KA, Carson, CF & Riley TV. (1996) The in-vitro susceptibility of Malassezia furfur to
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Hammer KA, Carson CF & Riley TV. (1998) In-vitro activity of essential oils, in particular
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Hammer KA, Carson CF & Riley TV. (1999) Antimicrobial activity of essential oils and other
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Hammer KA, Carson CF & Riley TV. (1999) In vitro susceptibility of yeasts to Melaleuca
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Hammer KA, Carson CF & Riley TV. (2000) Activity of Melaleuca alternifolia (tea tree) oil
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Appendix 2 – Responses of publishers to

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Prime National Publishing Corporation Urban and Fischer Control Publications Contact: H Hugel Contact: Dr Shu-Kun Lin Contact: J Kreeger Institue of Forest Tekno Scienze sr, Italy National Herbalists Association Luciano de Fiore California Agriculture Diagnostic Investig and Human Toxicology Full Journal Title
American Journal of Clinical Dermatology Adverse Drug Reactions & Toxicological Reviews Am J Alzheimers Dis Other De Medical Journal of Australia Journal of Tropical Agriculture & Food Science Alternative and Com Int. Journal of Alternative & Com American Bee Journal International Dental Journal Journal of Veterinary The Australian Journal of Hospital Pharm Australian Forestry Manufacturing Chemist Australian Farm Journal Australian Journal of Medical Herba Annali italiani di Dermatologia allergologi California Agriculture Australasian Journal of Expe Journal of the National Medical Association Response of publisher to permission request Full text granted
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aceutical Journal and Essential oil records Full Journal Title Journal of the Science of Food and Agriculture Veterinary Clinical and Experim Australasian Journal of Dermatology Contact Derm Derm Immunology Journal of Applied Microbiology Journal of the European Academ Letters in Applied Microbiology My Oral Microbiology Tropical Medicine & International Health Molecular Ecology British Journal of Derm Aquaculture Res Australian Journal of Biotechnology The Foot Allergo Journal Medy Nursing times Ganzheitliche Tierm Inflammation research Mikologia lekarska Perfumery Acta Botanica Sinica Grand Rounds HRC Journal of High Resolution Chromatography Canadian Pharm British Journal of Phy Soap Perfumery Schweizer Archiv fur Tierheilkunde Microbiology Journal of British Podiatric Medicine Advances in Food Science Journal of the American Holistic Veteri Cosmetics and Toiletries Manufacture Worl Phy Antiviral Research BioMed Central Surgery
Response of publisher to permission request No response No No No response No response No response No No response No No response No response No response No No response No No No response No response No response No response No No response No response No response No No No response No response No response No response No response No response No response No response No response No response No response No response No response No response
Experimental Biology of Tropical Medicine & Hy Side Publications, Inc a Iberoamericana de Micologia Publisher Elsevier
Fed. American Soc. of Georg Thieme Verlag Manor Enterprises P/L Marcel Dekker, Inc. Soc. Chiropodists & P Step Publishing Ltd, UK of Tropical Medicine & Hy Control Elsevier zin und Klassische Naturheil – Clinical Toxico Therapies in Nursing & Midwifery Full Journal Title ESkin and Allergy European Journal of Pharmaco Lancet Elsevier Obstetrics and Gy Thermochimia Acta Industrial Crops & Products Biochemical Sy Plant Science Fitoterapia Elsevier Journal of Microbiological Methods Journal of Chromatography Journal of Hospital Infection Burns Elsevier Clinics in Dermatology American Journal of Infection Toxicology Clinics in Podiatric Medicine a Complementary The Foot Journal of Allergy Journal of the American Food and Chemical Toxicology Seminars in Cutaneous Medicine Journal of Manipulative and Phy Complementary International Journal of Aromat FASEB Journal Aktuelle Dermatologie Planta Medica Water Science and Technology Forschende Komplementarmedi AIDS Lippincott Journal of Endodontics Cosmetics Aerosols and Toiletries in Aust Journal of Toxicology Asthm Revista Iberoamericana de Micologia Transactions of the Roy Podiatry British Journal of Biom
right belongs to each author) right belongs to each author) Response of publisher to permission request No response No response No response No response No response Not contacted Not contacted Not contacted Not contacted Not contacted Not contacted Not contacted Not contacted Not contacted Not contacted
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Appendix 3 – Material Safety Data Sheet

NOTE:

1) Do not photocopy this MSDS.
2) The document in its original format can be obtained from ATTIA.
Classified as hazardous according to the criteria of NOHSC Australia
1. IDENTIFICATION
Name:

Other names:
melaleuca oil, Melaleuca alternifolia oil, T36-C7, teebaumol Recommended use:
Topical antibacterial agent, antiseptic, anti-inflammatory agent SUSDP name:
Melaleuca oil (tea-tree oil) Supplier name:
(Manufacturer to complete) Street address:
(Manufacturer to complete) (Manufacturer to complete) Telephone:
(Manufacturer to complete) Emergency contact:
(Manufacturer to complete) 2. HAZARDS IDENTIFICATION
Hazard classification:

Classified as Hazardous according to the criteria of NOHSC Australia. Classified as Dangerous Goods for the purpose of transport by road or rail. Risk phrases:
R22 Harmful if swallowed R36/37/38 Irritating to eyes, respiratory system and skin Safety phrases:
S26 In case of contact with eyes, rinse immediately with plenty of water and contact a doctor or Poisons Information Centre (13 11 26, Australia wide). S36 Wear suitable protective clothing HAG phrases:
(9) Form: liquid (62) Avoid personal/skin contact (83) Fire fighting: foam (18) Combustible (85) Fire fighting: dry agent (51) Does not mix with water RTECS number
3. COMPOSITION
Chemical identity:

Melaleuca oil (tea-tree oil) Common names:
melaleuca oil, Melaleuca alternifolia oil, T36-C7, teebaumol ,Tea tree (melaleuca alternifolia) oil 68647-73-4, 85085-48-9, 8022-72-8 4. FIRST AID MEASURES
Poison Information Centres can provide additional assistance on 13 11 26 (Australia wide).
Eye: Irrigate with copious amounts of water. Seek immediate medical attention.
Inhalation: If over-exposure occurs leave exposure area immediately. If other than minor symptoms are displayed seek
immediate medical attention.
Skin: Gently flush affected areas with water. Remove contaminated clothing and wash thoroughly before re-use. Seek
medical attention if irritation develops.
Ingestion: If swallowed do NOT induce vomiting. Give a glass of water. Seek immediate medical attention.
Facilities: Eye wash facilities and safety shower are recommended.
5. FIRE FIGHTING MEASURES
Suitable extinguishing media: Dry agent, carbon dioxide, foam or water fog. Do not use full water jet.
Hazards from combustion products: May evolve toxic gases (hydrocarbons, carbon oxides) if burning.
Precautions and special protective equipment: Evacuate area and contact emergency services. Remain upwind and
notify those downwind of hazard. Wear full protective equipment including Self Contained Breathing Apparatus when
combating fire. Use waterfog to cool intact containers and nearby storage areas.
Hazchem code: 3[Y]
Product name: Tea tree oil 6. ACCIDENTAL RELEASE MEASURES
Spillage: In case of spillage (bulk), wear splash-proof goggles, PVC/rubber gloves, coveralls and rubber boots (see
section 8). Keep people away, evacuate area.
Containment and clean up: Absorb spill with sand or similar, collect and place in sealable containers using non-
sparking tools and transport outdoors for disposal. Ventilate area and wash spill site after material pick-up is complete.
Prevent spill from entering drains or waterways. Caution: slippery when spilt.
7. HANDLING AND STORAGE
Handling: Measures should be taken to prevent materials from being splashed into the eyes or on the skin. Wear eye-
shields and protective clothing. Smoking should not be permitted in work areas. Provide adequate ventilation.
Storage: Store in a cool, dry, well-ventilated area, away from oxidising agents (eg hypochlorites), acids (eg sulfuric
acid), heat and light sources, and foodstuffs. Ensure containers are adequately labelled, protected from physical
damage and sealed when not in use. Keep only in original container. Check regularly for leaks or spills. Large storage
areas should have appropriate ventilation systems. This material is a Scheduled Poison (S6) and must be stored,
maintained and used in accordance with the relevant regulations.
8. EXPOSURE CONTROLS/PERSONAL PROTECTION
National exposure standards: No exposure standard allocated
Biological limits: No biological limit allocated
Engineering controls: Ensure adequate ventilation. In poorly ventilated areas, mechanical explosion-proof extraction
ventilation is recommended. Keep containers closed when not in use.
PPE: Wear coveralls, splash-proof goggles and PVC or rubber gloves. Where an inhalation risk exists, wear a Type A
(organic vapour) Respirator. In a laboratory situation, wear a laboratory coat.
9. PHYSICAL AND CHEMICAL PROPERTIES
Appearance:
Colourless to pale yellow liquid
Odour: Characteristic, myristic
Solubility: Insoluble in water, 1 part miscible with 2 parts ethanol (85% v/v) at 20°C.
Vapour pressure:
Vapour density:
Boiling point/range:
116° – 265°C Freezing point:
Specific density:
0.885 - 0.906 at 20°C. Flash point:
57° - 60°C (closed cup) Fire point:
72°C (Cleveland open cup (IP 36)) Upper flammable limit in air:
Lower flammable limit in air:
Ignition temperature:
Specific heat value:
Percent volatile:
Refractive index:
1,475 0 – 1,482 0 at 20°C. Optical rotation:
Between +5° and +15° at 20°C. 10. STABILITY AND REACTIVITY
Chemical stability: Stable
Conditions to avoid: Heat, light, open flames and other sources of ignition
Incompatible materials: Strong oxidising or reducing agents. Protect from air.
Hazardous decomposition products: Carbon monoxide and carbon dioxide (from combustion).
Hazardous reactions: Hazardous polymerisation will not occur.
Product name: Tea tree oil 11. TOXICOLOGICAL INFORMATION
ACUTE EFFECTS
Eye contact: Severe irritant
Skin contact: Irritant. May cause erythema, irritation or oedema. Repeated or prolonged skin contact may lead to
allergic contact dermatitis.
Inhalation: Potential irritant. Over-exposure at high levels may result in mucous membrane irritation of the nose and
throat with coughing.
Ingestion: May be harmful if swallowed. Swallowing can result in allergic dermatitis, hallucinations, ataxia, diarrhoea,
central nervous system depression, sleep or coma.
Acute toxicity*:
Ear TD (guinea pig): 100% (instilled for 30 min) Toxic effects: D40 (change in acuity)11 Dermal LD50 (rabbit): >5 g/kg1 Dermal LDLo (rabbit): 5 g/kg1 Dermal TD (cat): 5-7 mL/kg2 Toxic effects: F19 (ataxia); P72 (changes in leucocyte count) Dermal TD (dog): 0.143 – 0.164 g/kg3 Toxic effects: F07 (somnolence), F19 (ataxia), partial paralysis Dermal TD (human adult): > 25% (in white soft paraffin, applied for 21 d)4 Oral LD50 (rat): 1.9 g/kg (1.4 – 2.7 g/kg)1 Oral LD50 (rat): 1.9 – 2.6 g/kg13 Oral TD (rat): 1.5 g/kg5 Toxic effects: F07 (somnolence) F18 (muscle weakness), F19 (ataxia), partial paralysis Oral TD (human adult): 21 μL/kg (after repeated low dose exposure)6 Toxic effects: P20 (changes in cell count (unspecified)); R01 (dermatitis, allergic); R03 (dermatitis, other))4 Oral TD (human adult): 0.5-1.0 mL/kg7 Toxic effects: F08 (hallucinations, distorted perceptions); F24 (coma); K12 (hypermotility, diarrhoea) Oral TD (human child): 0.5 mL/kg8 Toxic effects: F04 (sleep); F19 (ataxia) Oral TD (human child): 0.5 mL/kg9 Toxic effects: F08 (hallucinations, distorted perceptions); F19 (ataxia)5 Oral TD (human child): 0.6 mL/kg (approx.)10 Toxic effects: F07 (somnolence), F19 (ataxia), F24 (coma) Chronic toxicity:
No information available Sensitisation potential:
Low (modified FCA method, guinea pig model)12 Not mutagenic as determined by the AMES test
* see Toxic Effects Code from the Registry of Toxic Effects of Chemical Substances (RTECS)
12. ECOLOGICAL INFORMATION
Not acutely toxic to fish (LC50 > 100 mg/l OECD 206) Readily biodegradeable (OECD301F) Mobility:
No information available Product name: Tea tree oil 13. DISPOSAL CONSIDERATIONS
Disposal methods:

Dispose of small amounts at an approved landfill site. For larger amounts contact a licensed professional waste disposal service. Prevent contamination of drains or waterways. 14. TRANSPORT INFORMATION
UN number:

UN proper shipping name:
TERPENE HYDROCARBONS, N.O.S. Un Packing group:
ADG proper shipping name:
Not listed in ADG code Class and subsidiary risk(s):
Class 3. No subsidiary risks listed. Hazchem:
Special precautions for user: Classified as dangerous goods for the purpose of transport by road or rail. Class 3
Flammable Liquid. Do not transport with chemicals of class ; 1 (Explosives), 2.1/2.3 (Flammable/Toxic gases), 4.2
(Spontaneously combustibles), 5.1 (Oxidising agents), 5.2 (Organic peroxides), 6 (Toxics), 7 (Radioactives) and
foodstuffs.
15. REGULATORY INFORMATION
Poison Schedule:
This material is listed on the Australian Inventory of Chemical substances This material is listed on the European Inventory of Existing Commercial Substances 16. OTHER INFORMATION
This document was last modified on: 18th July 2003
ABBREVIATIONS
ADG
(Australian Dangerous Goods); AICS (Australian Inventory of Chemical Substances); CAS (Chemical Abstract
Service); EINECS (European Inventory of Existing Commercial Substances); EPG (Emergency Procedure Guide); FCA
(Freund's Complete Adjuvant); HAG (Hazmat Action Guide); LD50 (Dose lethal for 50% of the test population); LDLo
(Lowest Published Lethal Dose); N.O.S. (Not Otherwise Specified); NOHSC (National Occupational Health and Safety
Commission); PPE (Personal Protective Equipment); RTECS (Registry of Toxic Effects of Chemical Substances);
SUSDP (Standard for the Uniform Scheduling of Drugs and Poisons); TD (Toxic Dose); TDLo (Lowest Published Toxic
Dose); UN (United Nations)
REFERENCES
(1) Ford RA. Food Chem Toxicol 1988; 26: 407; (2) Bischoff K & Guale F. J Vet Diagn Invest 1998; 10: 208-210; (3)
Kaluzienski M. J Toxicol Clin Toxicol 2000; 38: 518-519; (4) Southwell IA et al. J Essent Oil Res 1997; 9: 47-52; (5) Kim
D. et al. American Chemical Society National Meeting 2002. 223: 114-MEDI Part 2; (6) Elliot C. Med J Aust 1993; 159:
830-831; (7) Seawright A. Med J Aust 1993; 159: 831; (8) Del Beccaro MA. Vet Human Toxicol 1995; 37: 557-558; (9)
Jacobs MR & Hornfeldt CS. J Toxicol – Clin Toxicol 1994; 32: 461-464; (10) Morris MC et al. Pediatric Emergency Care
2003; 19: 169-171; (11) Zhang SY & Robertson D. Audiol Neuro-Otol 1999; 5: 64-68; (12) Hausen BM et al. Am J
Contact Dermatitis
1999; 10: 68-77; (13) Bolt AG. Report for the Australian Tea Tree Oil Industries Association, 1989
DATA SOURCES
International Standards Organisation (1996) Oil of Melaleuca, terpinen-4-ol type (tea tree oil). International Standard ISO 4730:1996(E), International Standards Organisation, Geneva. Sweet DV. (Editor) (1997) Registry of toxic effects of chemical substances (RTECS), comprehensive guide. U.S. Department of Health and Human Services, Cincinnati, Ohio.
Disclaimer: This Material Safety Data Sheet was prepared according to the National Code of Practice for the
Preparation of Material Safety Data Sheets [NOHSC:2011(2003)]. The above information is believed to be correct but
does not claim to be all inclusive and shall be used only as a guide.
- END OF REPORT -
Product name: Tea tree oil

Source: http://www.attia.org.au/mce_doc.php?id=7

Ps201500508_pap_ps 1.3

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