In their review paper Carson et al. (1996) stated that the antiviral activity of TTO was first shown
using tobacco mosaic virus and tobacco plants.
In field trials TTO (spray concentration 0, 100, 250 or 500 ppm) was sprayed on plants that were then
experimentally infected with tobacco mosaic virus. After 10 days, there were significantly fewer lesions
per square centimetre of leaf in plants treated with TTO than in controls.
Studies have been conducted with herpes simplex viruses being incubated with various concentrations
of TTO, which were then using these treated viruses to infect cell mono-layers. After 4 days, the
numbers of plaques formed by TTO-treated virus and untreated control virus were determined and
compared. The concentration of TTO inhibiting 50% of plaque formation was 0.0009% for herpes
simplex virus type 1 and 0.0008% for herpes simplex virus type 2, relative to controls. These studies
also showed that at the higher concentration of 0.003%, TTO reduced herpes simplex virus-1 titres by
98.2% and HSV-2 titres by 93.0%. In addition, by applying TTO at different stages in the virus
replicative cycle, TTO was shown to have the greatest effect on free virus (prior to infection of cells).
Another study evaluated the activities of 12 essential oils, including TTO, for activity against herpes
simplex virus -1 in Vero cells. Again, TTO was found to exert most of its antiviral activity on free virus,
with 1% oil inhibiting plaque formation completely and 0.1% TTO reducing plaque formation by
approximately 10%. Pre-treatment of the Vero cells prior to virus addition or post-treatment with 0.1%
TTO after viral absorption did not significantly alter plaque formation.
TTO has an interesting antiviral activity against influenza A⁄PR⁄8 virus subtype H1N1 in Madin–Darby
canine kidney (MDCK) cells. It has been found that TTO had an inhibitory effect on influenza virus
replication at doses below the cytotoxic dose; terpinen-4-ol, terpinolene, and α-terpineol were the
main active components (Garozzo et al. 2009).
The mechanism of action of TTO and its active components against Influenza A/PR/8 virus subtype
H1N1 was investigated in MDCK cells. The effect of TTO and its active components on different steps of
the replicative cycle of influenza virus was studied by adding the test compounds at various times after
infection. These experiments revealed that viral replication was significantly inhibited if TTO was added
within 2 h of infection, indicating an interference with an early step of the viral replicative cycle of
influenza virus and suggesting that TTO could inhibit viral uncoating by an interference with
acidification of intra-lysosomal compartment (Garozzo et al. 2011).
The antifungal activity of TTO was known anecdotally especially amongst the aboriginal people of
In 1998 Hammer et al. studied the in vitro TTO activity against C. albicans and non-albicans Candida
species. The minimum killing TTO concentration for killing isolates was 0.25% and 0.5% for C. albicans
and non-albicans Candida species, respectively.
Mondello et al. (2003 ) investigated the in vitro antifungal activity of TTO (ISO 4730-2004) against
itraconazole, as well as the in vivo activity in an experimental vaginal infection using fluconazole–
itraconazole-susceptible or -resistant strains of C. albicans. The susceptibility testing of Candida spp.,
and Cryptococcus neoformans to TTO, fluconazole and itraconazole was conducted using a microbroth
Assessment report on Melaleuca alternifolia (Maiden and Betch) Cheel, M. linariifolia Smith, M. dissitiflora F. Mueller
method according to the National Committee for Clinical Laboratory Standards (NCCLS 1997) for both
dilution antifungal susceptibility testing of yeasts (Liu X et al. 2009).
TTO was active against all tested strains, with MICs ranging from 0.03% (for C. neoformans) to 0.25%
(for some strains of C. albicans and other Candida spp.). Fluconazole- and/or itraconazole-resistant C.
albicans isolates had TTO MIC
s and MIC
s of 0.25% and 0.5%, respectively. The MIC
same fungus using a TTO mixture with relatively similar proportions of terpinen-4-ol and 1,8-cineole.
Moreover neither fungistatic nor fungicidal activities were strongly influenced by lowering the pH of the
incubation medium to pH 5, thus supporting the use of TTO for skin and mucosal infections.
The results of the in vivo investigations on the animal model (oophorectomized – ovary removal
surgery female rats of the Wistar strain) of vaginal candidiasis demonstrated that TTO administered
intravaginally using a dose volume of 0.1 ml at concentrations of 1%, 2.5% and 5% is effective in
resolving experimental C. albicans infection, with both fluconazole-susceptible and –resistant isolates.
In the case of the fluconazole-susceptible organism, treatment with TTO was comparable to a standard
treatment with fluconazole, used as positive control, whereas no effect was observed in rats treated
with TTO diluted with polisorbate 80 (Tween-80
) used as negative control. The results showed that
significant decrease in CFU counts in the first 2 weeks after the vaginal treatment, with a substantial
TTO dose dependence of fungal clearance, although the difference was not statistically significant. With
all dose regimens, the infection was cleared in 3 weeks, whereas the untreated control rats remained
infected. TTO (5%) also caused a rapid clearance of the fluconazole-resistant strain from the vagina of
experimentally infected rats. There was a statistically highly significant difference at all time-points
considered between control (or fluconazole-treated rats) and those treated with TTO. Again the
infection was resolved in 3 weeks by TTO, whereas all other animals, either untreated or fluconazole-
treated, were still infected at the end of the 3 week period.
In a follow up study, Mondello et al. (2006) confirmed the previous result with the animal experimental
model as reported on the in vivo activity of terpinen-4-ol, considered the main bioactive component of
TTO. Using the same methodology as detailed in their previous paper they concluded that terpinen-4-ol
was a likely mediator of the in vitro and in vivo activity of TTO and claimed that their results were the
first to demonstrate that terpinen-4-ol could control C. albicans vaginal infections. They concluded that
the purified compound held promise for the treatment of vaginal candidiasis, particularly the azole-
Antimycotic properties of TTO and its principal components were compared with the activity of 5-
fluorocytosine and amphotericin B. The majority of the organisms were sensitive to the essential oil,
with TTO and terpinen-4-ol being the most active oils showing antifungal activity at minimum inhibitory
concentration values lower than other drugs (Oliva et al. 2003).
The in vitro activities of TTO against Malassezia species were shown. M. furfur was the least
susceptible species. M. sympodialis, M. slooffiae, M. globosa, and M. obtusa showed similar
susceptibilities (Hammer et al. 2000).
In another study investigating in vitro antifungal activity of TTO components, the highest activity, with
minimum inhibitory concentrations and minimum fungicidal concentrations of <0-25%, was shown by
terpinen-4-ol, α-terpineol, linalool, α-pinene and β-pinene, followed by 1,8-cineole. All TTO
components, except β-myrcene, had antifungal activity. This study identified that most components of
TTO have activity against a range of fungi (Hammer et al. 2003b).
Carson et al. (2006) summarised the antifungal activity of TTO against a range of fungal species
published by a number of researchers obtained from over 15 papers: MICs were in the range between
0.03 and 0.5% and fungicidal concentrations from 0.12 to 2%. The exception to these ranges was
conducted with fungal conidia that are known to be relatively impervious to chemical agents.
Subsequent assays show that germinated conidia are significantly more susceptible to TTO than non-
germinated conidia. They also noted that TTO vapours have also been demonstrated to inhibit fungal
growth and affect sporulation.
Hammer et al. (2004) investigated the mechanism of action of TTO and its components against C.
at one or more concentrations, for up to 6 hours. During that time, alterations in permeability were
assessed by measuring the leakage of 260 nm absorbing materials and by the uptake of methylene
blue dye. Membrane fluidity was measured by 1,6-diphenyl-1,3,5-hexatriene fluorescence. The effects
of TTO on glucose-induced medium acidification were quantified by measuring the pH of cell
suspensions in the presence of both TTO and glucose. The results showed that treatment of C. albicans
with TTO and its components at concentrations of between 0.25 and 1.0% altered both permeability
and membrane fluidity. Membrane fluidity was also increased when C. albicans was cultured for 24
hours with 0.016%-0.06% TTO, as compared with control cells. For all three organisms, glucose-
induced acidification of the external medium was inhibited in a dose-dependent manner in the
presence of TTO at concentrations of 0.2%, 0.3% and 0.4%. It was concluded that the data from the
study supported the hypothesis that TTO and components exert their antifungal actions by altering
membrane properties and compromising membrane-associated functions.
Antiseptic and disinfectant activity
Effective skin antisepsis and disinfection are key factors in preventing many healthcare-acquired
infections associated with skin microorganisms, particularly Staphylococcus epidermidis. The
antimicrobial efficacy of chlorhexidine digluconate, a widely used antiseptic in clinical practice, alone
and in combination with TTO was studied. Chlorhexidine digluconate exhibited antimicrobial activity
against S. epidermidis in both suspension and biofilm (MIC 2–8 mg/l) as well as TTO (2–16 g/l), but no
synergistic effect was found for combination of chlorhexidine digluconate with TTO (Karpanen et al.
rifampicin occurred amongst the Gram-positive organisms S. aureus, S. epidermidis and Enterococcus
. Single-step mutants resistant to TTO
MIC for the remaining S. aureus strains, including a clinical MRSA isolate. Similarly, no mutants were
recovered at 2× MIC for S. epidermidis or at 1× MIC for E. faecalis. Resistance frequencies determined
in vitro for rifampicin (8× MIC) ranged from 10
for all isolates, with the exception of the S.
organisms such as Staphylococcus spp. and Enterococcus spp. have very low frequencies of resistance
to TTO (Hammer et al. 2008).
An investigation was carried out to determine the effect of Burnaid, a commercial TTO preparation,
against Enterococcus faecalis ATCC29212, S. aureus ATCC29213, E. coli ATCC25922 and Pseudomonas
aeruginosa ATCC27853.The organisms were suspended in sterile saline (density of 0.5 McFarland
Standard) and inoculated onto horse blood agar (E. faecalis and S. aureus) or Mueller-Hinton agar (E.
(Tinasolve™) were placed in duplicate in wells cut into the agar plates. Sterility and inactivation
cultures were also performed on the samples. None of the samples were found to be contaminated
with bacteria prior to testing. Only S. aureus and E. coli showed zones of growth inhibition around the
Burnaid and Tinasolve. Zones of growth inhibition (22 mm) were similar for the active product
(Burnaid) and the base (Tinasolve™). There was no bactericidal activity against E. faecalis or P.
fibroblasts and epithelial cells, it is recommended that this product should not be used on burn wounds
(Foagali et al. 1997).
Assessor’s comment: This study suggests not using TTO preparations for the care of burn wounds.
Carson et al. (2006) reported that results have been published showing that TTO has antiprotozoal
activity. TTO caused a 50% reduction in growth (compared to controls) of the protozoa Leishmania
major and Trypanosoma brucei at concentrations of 403 mg/ml and 0.5 mg/ml, respectively. TTO at
high concentration corresponding to 300 mg/ml killed all cells of Trichomonas vaginalis and there is
also anecdotal in vivo evidence that TTO may be effective in treating T. vaginalis infections.
The potential anti-tumoral activity of TTO, distilled from M. alternifolia, was analysed against human
melanoma M14 WT cells and their drug-resistant counterparts, M14 adriamicin-resistant cells. Both
sensitive and resistant cells were grown in the presence of TTO at concentrations ranging from 0.005
to 0.03%. Both TTO and its main active component terpinen-4-ol were able to induce caspase-
dependent apoptosis of melanoma cells and this effect was more evident in the resistant variant cell
population. Freeze-fracturing and scanning electron microscopy analyses suggested that the effect of
the crude oil and of the terpinen-4-ol was mediated by their interaction with plasma membrane and
subsequent reorganization of membrane lipids. In conclusion, TTO and terpinen-4-ol were able to
impair the growth of human M14 melanoma cells and appear to be more effective on their resistant
variants, which express high levels of P-glycoprotein in the plasma membrane, overcoming resistance
to caspase-dependent apoptosis exerted by P-glycoprotein-positive tumour cells (Calcabrini et al.
Human melanoma cells (M14 WT) grown in the presence of the antitumor drug adriamycin (M14 ADR)
growth of melanoma cells and of overcoming multidrug resistance. The major inhibitory effect was
found after treatment with 0.01% terpinen-4-ol. The effect of TTO on melanoma cells appears to be
mediated by its interaction with the lipid bilayer of the plasma membrane. The experiments indicate
that TTO and its main active component, terpinen-4-ol, can also interfere with the migration and
invasion processes of drug-sensitive and drug-resistant melanoma cells (Bozzuto et al. 2011).
Liu et al. (2009) reported that TTO showed strong in vitro cytotoxicity towards human lung cancer cell
line (A549), human breast cancer cell line (MCF-7) and human prostate cancer cell line (PC-3) with
IC50 values (24 hr incubation) of 0.012%, 0.031% and 0.037%, respectively.
The antioxidant activity of Australian TTO was determined using two different assays. In the 2,2-
diphenyl-1-picrylhydrazyl assay, 10 µl/ml crude TTO in methanol had approximately 80% free radical
scavenging activity, and in the hexanal/hexanoic acid assay, 200 µl/l crude TTO exhibited 60%
inhibitory activity against the oxidation of hexanal to hexanoic acid over 30 days. The results indicate
that TTO has an antioxidant activity. Inherent antioxidants, i.e., R-terpinene, R-terpinolene, and γ-
terpinene were separated from crude TTO and identified chromatographically using silica gel open
chromatography, C18-high-pressure liquid chromatography, and gas chromatography-mass
spectrometry. Their antioxidant activities decreased in the following order in both assays: α-terpinene
> α-terpinolene > γ-terpinene (Kim et al. 2004).
Overview of available pharmacokinetic data regarding the herbal
substance(s), herbal preparation(s) and relevant constituents thereof
TTO contains terpenes, sesquiterpenes, hydrocarbons, and related alcohols. Because of its lipophilic
nature, TTO is readily absorbed through the skin.
The major compound of TTO, terpinen-4-ol, is able to permeate human epidermis. The permeation
depends on the applied preparation whereas a semisolid O/W emulsion or an ointment is superior to a
cream (Reichling et al. 2006). The skin absorption rate of TTO was investigated in vitro using diffusion
cell permeation experiments with heat separated human epidermis to evaluate the capability of
terpinene-4-ol, the main component of the oil, to permeate human skin. Flux values (the absorption
rate per unit area, μl/cm
h) of three different semisolid preparations containing 5% TTO were 0.067
constants (Papp cm/s) can be calculated from flux values, taking the applied drug concentration into
account. Papp values for the cream (2.74) and pure oil (1.62) were quite comparable, whereas white
petrolatum (6.36) and the semi-solid oil/water emulsion (8.41) gave higher values indicating
penetration enhancement (Reichling et al. 2006).
It has been postulated from the high lipophilicity of its components that TTO is likely to be rapidly and
completely absorbed from the skin and mucous membranes (ESCOP 2009). On the other hand, in vitro
experiments indicated that, after application of TTO to human epidermal membranes mounted in
diffusion cells in the pure form and as a 20% solution in ethanol, only a small proportion of the applied
amount (2-4% and 1.1-1.9% respectively) penetrated into or through human epidermis (Cross et al.
Considerable research has been done on the metabolism of monoterpenes. After dermal and/or oral
absorption, liver P450 mono-oxygenases are involved in biotransformation. Subsequently, 60-80% of
absorbed monoterpenes are excreted as glucuronides (Villar et al. 1994).
Cal and Krzyaniak (2006), Cal et al. (2006) and Cal (2008) studied the penetration behaviour of TTO
and pure constituents using a flow-through diffusion cells, human skin preparations and in vivo human
studies which represented infinitive dose and occlusive application conditions. Application times of 1, 4
or 8 hours. Neat TTO, neat terpene-4-ol and 5% terpene-4-ol (grape seed oil/carbomer hydrogel and
o/w emulsion) were tested. After the exposure period, the receptor fluid and skin layers were analysed
in the in vitro studies and the skin layers in the in vivo studies. TTO or pure terpene-4-ol caused a
significant increase in the skin accumulation of terpene-4-ol in the hydrophilic skin layers (dermis and
epidermis). In contrast to the results of Cross et al. (2008) and Reichling et al. (2006) which used only
epidermis, terpene-4-ol was not detected in the receptor fluid at any stage of the study of Cal et al.
(2006) which utilised epidermal and dermal layers. TTO or pure terpene-4-ol caused a significant
increase in the skin accumulation of terpene-4-ol in the hydrophilic skin layers (dermis and epidermis).
These sets of data, accumulation in the skin layers and diffusion into the acceptor fluid, suggest that in
The acute oral LD50 in rats has been reported as 1.9-2.6 ml/kg (1.4-2.7 g/ kg of body weight)
(Hammer et al. 2006, Carson et al. 1998, Halcon & Milkus 2004). Rats receiving 1.5 g/kg or more
appeared lethargic and ataxic 72 hours post dose. By day 4 all but one animal at this dose had
regained locomotor function (Hammer et al. 2006). Postulated lethal dose for a 3-year-old child was
calculated to be 26 ml (Halcon & Milkus 2004).
The dermal LD50 in rabbits is > 5 g/kg (Council of Europe Committee of Experts on Cosmetic Products
No deaths or toxic effects were reported in a 30 days-skin irritation study in rabbits using a 25% TTO
in liquid paraffin other than slight initial irritation (Council of Europe Committee of Experts on Cosmetic
Some repeat dose toxicity data are available on some of the main TTO components. Nielsen (2005)
derived an estimated NOEL for TTO based on component data.
Terpinen-4-ol did not induce changes in the morphology or function of the kidneys of male Sprague-
Dawley rats following 28 days of repeated oral exposure to 400 mg/kg b.w. and was considered to be
non-toxic (Schilcher & Leuschner 1997). Thus the NOAEL after oral exposure may be estimated to be
400 mg/kg. As terpinen-4-ol on average constitutes 40% of TTO, this NOAEL for terpinen-4-ol could be
translated to a theoretical oral NOAEL for TTO of 1000 mg/kg.
Cineole given to B6C3F1 mice by gavage for 28 days at doses up to 1200 mg/kg/day did not result in
any changes. When given encapsulated at doses corresponding to 600 – 5607 mg/kg/day, some
hypertrophy of hepatocytes was seen, but was not considered significant (National Toxicology
Program, cited in De Vincenzi et al. 2002). Cineole (8 or 32 mg/kg/body weight) was given by gavage
to male SPF CFLP mice 6 days per week for 80 weeks. No changes were evident in mice given cineole
when compared to control mice (Roe et al. 1979). Based on the studies on hepatic and renal toxicity
evaluated by BIBRA (British Industrial Biological Research Association), a NOAEL might be estimated
as 300 mg/kg body weight, which is in agreement with the evaluation from the Norwegian Food
Control Authorities in 1999 (EFSA 2012).
Several reports of oral toxicity can be found in the literature. Data indicate that due to its systemic
toxicity, TTO should only be used as a topical agent.
General toxicology profile of TTO indicates that severe reactions would be extremely rare if TTO is not
ingested (Halcon & Milkus 2004).
TTO produced a negative result in the in-vitro Ames test (Saller et al. 1998). In December 2004 the
Scientific Committee on Consumer Products (SCCP) noted that TTO is not mutagenic in the Ames test
although they stated that there were insufficient details of the study and the study was deemed
inadequate. They further noted that, as TTO has antimicrobial properties, an Ames test would be of
limited value (SCCP 2004).
In 2005 Evandri et al. evaluated the mutagenic and antimutagenic activity of essential oils TTO and
Lavandula angustifolia (lavender oil) the bacterial reverse mutation assay in Salmonella typhimurium
TA98 and TA100 strains and in Escherichia coli WP2 uvrA strain, with and without an extrinsic
metabolic activation system. The results showed that neither essential oil had mutagenic activity on
the two tested Salmonella strains or on E. coli, with or without the metabolic activation system,
providing further evidence of the lack of mutagenic potential of TTO.
These results were also supported by a paper published by Fletcher et al. (2005) using Salmonella
strains TA102, TA100 and TA98 in the Histidine Reversion Assay Ames test: neither TTO nor terpinen-
4-ol, one of the major constituents of TTO, induced reverse mutations in any of the tester strains
examined with or without metabolic activation, confirming that they are not mutagens.
Two papers were found evaluating the mutagenic potential of TTO components:
Gomes-Carneiro et al. (2005) investigated the genotoxicity of β-myrcene, α-terpinene and (+) and (-)-
α-pinene by the Salmonella/microsome assay (TA100, TA98, TA97a and TA1535 tester strains), using
a plate incorporation procedure without and with addition of an extrinsic metabolic activation system
(rat liver S9 fraction induced by Aroclor 1254) and concluded that these common constituents of
essential oil are not mutagenic in the Ames test.
Hammer et al. (2006) in a review noted that the following components were non-mutagenic in the
Salmonella/microsome (Ames) test or the Bacillus subtilis rec- assay: terpinen-4-ol, α-terpinene, 1,8-
cineole, cymene, limonene, α -pinene, β-pinene, linalool and β -myrcene. In contrast, terpineol caused
a slight but dose related increase in the number of revertants with the TA102 tester strain both with
and without S9 mixture. However, no significant effect was seen in the other three bacterial strains,
indicating that terpineol induced a base-pair substitution affecting an A–T base pair.
In tests with mammalian cells, γ-terpinene did not increase DNA strand breakage in human
lymphocytes at 0.1 mM but did at 0.2 mM. Cineole, D-(+)-limonene, linalool, l-phellandrene and β -
pinene at concentrations ranging from 10 to 1000 μM did not increase the frequency of spontaneous
sister-chromatid exchanges in Chinese hamster ovary cells. Another study showed linalool to be non-
mutagenic using a Chinese hamster fibroblast cell line. β-myrcene did not have mutagenic activity
when tested with human lymphocytes and was not genotoxic in bone marrow cells of rats administered
β -myrcene orally.
They concluded that, overall, the available data on the mutagenicity of TTO and its individual
constituents indicate low mutagenic potential, using both bacterial and mammalian test systems.
An in vivo Mouse Micronucleus Assay (ICP Firefly Pty Ltd. 2005) was conducted according to OECD Test
Guideline No. 474, which was conducted under GLP, TTO was administered by gavage at 1000, 1350
and 1750 mg/kg b.w. TTO. There were no increases in the frequency of micronucleated cells in any of
the dose groups. There was a statistically significant depression of PCE viability and PCE+NCE ratio
(P<0.001) in the high dose group in both sexes when compared with the vehicle control groups at 48
hours. This finding is an indication that there was sufficient exposure of the bone marrow to the test
substance to elicit a response. Clinical signs in the high dose group included depressed weight gain,
wobbly gait, laboured breathing and rough coat.
No available data.
The Scientific Committee on Consumer Products in its updated “Opinion on Tea Tree Oil” in 2008 stated
This statement follows the EMEA “Public statement on the use of herbal medicinal products containing
methyleugenol” (2005) reporting a content of 0.28 to 0.9% of the natural potential carcinogen
methyleugenol in TTO. However HMPC has concluded that “the present exposure to methyleugenol
resulting from consumption of herbal medicinal products (short time use in adults at recommended
posology) does not pose a significant cancer risk.”
The Australian TTO industry reports that these levels of methyleugenol refer to Melaleuca bracteata,
whereas commercial TTO is derived solely from Melaleuca alternifolia; analytical surveys conducted by
Australian TTO industry show that Melaleuca alternifolia contains only trace levels of methyleugenol.
Southwell et al. (2011) quantified the traces of methyleugenol previously reported in TTO ranging from
less than 0.01% to 0.06% (mean 0.02%).
Reproductive and developmental toxicology
No data available on TTO.
However, exposure to α-terpinene (125 or 250 mg/kg b.w.), present at approximately 9% in TTO, for
nine consecutive days caused decreased body weight gain in pregnant Wistar rats. The offspring of
dams given 60 mg/kg b.w. from day 6 to day 15 of pregnancy had delayed ossification and skeletal
malformations. At 30 mg/kg b.w. no effects were seen on either dams or offspring. Effects at doses
higher than 60 mg/kg b.w. were accompanied by maternal toxicity. The authors suggested a NOAEL
for embryofoetotoxicity of 30 mg/kg b.w. for oral exposure of rats to α-terpinene (Araujo et al. 1996).
These limited data suggest that TTO is potentially embryofoetotoxic, although only if ingested at
relatively high levels (Araujo et al. 1996).
Hammer et al. (2006), noted that the embryofoetotoxicity of α-terpinene (normally present in TTO at
9%) has been evaluated and found that at oral doses of greater than 60 mg/kg b.w. there was delayed
ossification and skeletal malformations in the foetuses and this was accompanied by maternal toxicity.
The test material was administered to rats from day 6 to day 15 of gestation. The authors concluded
that TTO is potentially embryofoetotoxic although only if orally ingested at relatively high doses
Two studies were conducted on groups of 3 female rabbits of the New Zealand strain according to the
methodology detailed in OECD guideline 404 and were GLP compliant. In the first study TTO (100%)
was applied undiluted on 4x4 cm patches. In the second study, dilutions of 75-12.5% TTO were
applied for 4 hours with a semi-occlusive patch application followed by a 14 days observation period.
The results showed that, in the first study, TTO (100%) was found to be a mild irritant at 60 minutes
post exposure, a severe irritant at 24 and 48 hours, a moderate irritant at 72 hours and a mild irritant
7 and 14 days following a 4 hour semi-occlusive patch application on intact skin. At 21 days the skin
had returned to normal. In the second study, TTO (75%) was found to be a mild to moderate irritant,
TTO (50%) was found to be a minimal irritant. TTO (at 25% and 12.5%) was found to be a non-irritant
(SCCP December 2004).
Draize skin irritancy index was found to be 5.0, based on application of 100% TTO to intact and
abraded skin of albino rabbits, thus signifying that TTO could cause dermatitis in some users (Halcon &
The acute dermal LD
in rabbits was recorded as in excess of 5.0 g/kg since this dose resulted in 2/10
skin abnormalities in rabbits patch tested at this dose for 24 h with occlusion. Pure (100%) TTO
Assessment report on Melaleuca alternifolia (Maiden and Betch) Cheel, M. linariifolia Smith, M. dissitiflora F. Mueller
applied to the skin of albino rabbits and maintained at 2 g/kg for 24 hours resulted in no signs of
toxicity (Halcon & Milkus 2004).
A 30-day dermal irritation test in rabbits using 25% TTO in paraffin on shaved skin did not result in
visible signs of irritation. Therefore, TTO should not be used for conditions where skin irritability is
already present (e.g. dermatitis) (Halcon & Milkus 2004).
The primary eye irritation of TTO was also studied in the rabbit (female, Japanese White) under GLP
conditions. Two groups of three rabbits were given a single ocular dose (0.1 ml) of TTO (1% or 5% in
liquid paraffin). After instillation of the test substance, no abnormal signs in the clinical conditions were
observed among the rabbits. Ocular responses using Draize’s criteria demonstrated a conjunctival
discharge lasting for up to six hours following instillation of 1% TTO and conjunctival redness and
discharge for up to 24 hours following instillation of 5% TTO. In both groups, the maximal response
was observed after one hour. Based on these observations, the author concludes, that both TTO
solutions can be classified as “minimally irritating” (SCCP 2008).
TTO was found to produce ototoxicity when applied in the ears of guinea pigs at 100% concentration,
but no ototoxicity was found for 2% solutions (Halcon & Milkus 2004).
Skin sensitisation potential
In order to test the potential for TTO to cause skin sensitisation guinea pigs were pre-treated 2 times
via intradermal injections and an epidermal induction application of the oil. Two weeks after the
induction application, the animals were tested on one flank with the maximum sub-irritant
concentration of the oil. No irritant response was observed (Halcon & Milkus 2004).
A guinea pig maximization assay using the Magnusson and Kligman method (Pharmaceutical
Consulting Service 1989) and albino guinea pigs (20 per group) has been conducted with TTO. During
the induction phase, two 0.1 ml intradermal injections were given to the animals. One week later, 5%
TTO was applied to the skin at the injection site under occlusion for 48 hours. After a two week period,
a 30% TTO challenge dose was applied to the skin under occlusion for 48 hours. There was no
evidence of sensitisation in this assay. In a published report, TTO of unknown quality was tested in 10
guinea pigs using an adjuvant maximization protocol. The induction concentration was not given. At an
elicitation concentration of 30%, 3/10 guinea pigs gave positive reactions at the 48-hour reading. At
10%, no reactions were observed. The main component of TTO, terpinen-4-ol, gave no response when
cross-challenged in the reacting animals. These results may indicate that TTO may be a weak skin
sensitiser. The disagreement between the two studies cannot be explained, other that it could have
been the result of different quality and oxidation state of the TTO tested.
Three samples of TTO were tested in the Mouse Local Lymph Node Assay (LLNA) (RCC Ltd. Study
A69041, Study A78682, Study A78816 2006). Two of the samples were non-oxidised, undegraded oil,
while the third was a severely oxidised and degraded. The EC3 (calculated concentration of the test
substance which elicited a three-fold increase in the Stimulation Index) values of 24.3% and 25.5%
were obtained with the two undegraded oil samples, while the EC3 of the degraded oil was 4.4%.
There was a clear dose-response in each case. Another sample of undegraded TTO was sent to a
different laboratory (MB Research Laboratories 2007) which could perform immunophenotyping of the
lymphocytes. An EC3 value of 8.3% was calculated in this LLNA. Similarly the %B cells, %T cells, and
B:T ratio indicated a sensitising response. Overall, these results show that undegraded TTO has a weak
potential for sensitisation in this assay system. Degraded TTO had 5-times higher potency, but would
still be regarded as a moderate sensitiser.
The peroxide value and p-cymene content are particular useful indicators of the age of the oil and the
extent of degradation (Southwell 2006). The peroxide value is a measure of available oxygen, i.e. how
much one or more components of the oil have absorbed oxygen in the form of peroxide. Therefore, the
Peroxide Value is an indicator of the presence of peroxides. Generally, good quality fresh oils will have
a peroxide value below 10 µeq O
. Peroxides degrade over time and the degradation products, such as
will fall as the peroxides decompose. A very old (>10 years old), decomposed oil could have a low
peroxide value. Such an oil will have elevated levels of the decomposition products and potentially
elevated p-cymene (16% plus). p-Cymene, occurs naturally in TTO (typically 0.5 – 8%).
Southwell (2006) examined 26 TTO samples and demonstrated that the presence of 1,2,4-
trihydroxymenthane in TTO is a rare event and in the cases where this breakdown product was found,
the oils were extremely old and severely degraded to the extent where the oils would not be compliant
to the ISO Standard. Even in extremely degraded oils, 1,2,4-trihydroxymenthane concentrations were
less than 5%. Consequently, although 1,2,4-trihydroxymenthane can be detected by GC and GC/MS in
aged TTOs, when concentrations are low (<1%), the triol peak can easily be hidden by other 37 oil
peaks in the same region and therefore the presence of 1,2,4-trihydroxymenthane is not suitable to
check possible degradation of TTO. The use of other degradation products as degradation markers is
even more difficult as it has not been possible to consistently and positively identify ascaridole,
ascaridole glycol, the keto-epoxide and the di-epoxide that have been tentatively identified in degraded
TTOs as well as these products being present in even smaller concentrations than the triol.
Furthermore, Southwell also demonstrated a relationship between the levels of p-cymene and 1,2,4-
trihydroxymenthane. Thus, Southwell has proposed monitoring the degradation status of TTO by using
p-cymene as a reliable marker.
For many years, 1,8-cineole was regarded as an undesirable constituent in TTO due to its reputation as
a skin and mucous membrane irritant. However, other studies suggested that this component is not
responsible for a large proportion of sensitivity reactions (Carson et al. 1998).
Oxidation products are the likely allergens. Since oxidized TTO appears to be a more potent allergen
than fresh TTO, human adverse reactions may be minimized by reducing exposure to aged, oxidized oil
(Carson & Riley 2001).
Although some irritation was observed, undiluted TTO did not produce phototoxic effects on the skin of
hairless mice (Carson et al. 1998).
A case report documented TTO poisoning after a single dermal application of 120 ml of undiluted TTO
to 3 adult intact female purebred Angora cats, one of which died. The cats were severely infested with
fleas, so they were shaved and the oil was applied directly to the cats’ skin. The shaving produced no
nicks on the skin; however, numerous flea bites were visible. The product used to eliminate fleas was
labelled for use as a spot treatment for skin lesions, but a catalogue advertised that it would repel fleas
when diluted and used as a dip. All animals exhibited hypothermia, incoordination, dehydration and
trembling. The surviving 2 cats recovered after 1-2 days (Bischoff & Guale 1998). Neurotoxicity and
death have been observed in cats exposed to very high doses of TTO by the dermal route. However,
the possibility that these animals were also exposed by the oral route by licking of the skin and fur of
the application area cannot be ruled out.
Villar et al. (1994) reported that cases of TTO toxicosis have been reported by American veterinarians
to the National Animal Poison Control Centre when the oil was applied on derma of dogs and cats. They
noted that, in most cases, the oil was used to treat dermatologic conditions at inappropriate high
doses. The typical signs observed were depression, weakness, incoordination and muscle tremors.
Treatment of clinical signs and supportive care was sufficient to achieve recovery without sequelae
within 2-3 days.
TTO and components of TTO was tested on several human cell lines in vitro. Cytotoxicity with 100%
TTO ranged from 0.02 to 2.8 g/l, with epithelial-like cells being the most robust, and liver-derived cells
being the most susceptible. Cytotoxicity for the components of TTO was as follows: 1,8-cineole, from
0.14 to 4.2 g/l; terpinen-4-ol, from 0.06 to 2.7 g/l; α-terpineol, 0.02 to 1.1 g/l. These data suggest
that topical use of TTO is suitable, as epithelial cell seem to be the most resistant cells to its potential
cytotoxicity (Halcon & Milkus 2004).
Overall conclusions on non-clinical data
Studies on TTO demonstrate that adequate doses have broad spectrum antimicrobial activity with little
evidence for inducing tolerance and resistance. There is also some evidence of TTO possessing anti-
The cytotoxic activity towards a range of cancer cell types shown by means of in vitro studies is not
considered relevant for the purpose of this assessment.
The published pharmacokinetic data on TTO are minimal. In vitro skin permeation studies using human
skin preparations demonstrate that the extent of penetrating of TTO components is very low, with the
more polar terpenen-4-ol and α-terpineol being the only components which penetrate to any
appreciable levels. The total penetration of TTO is 2-4% and 7% of applied dose under non-occluded
and partly occluded conditions. Under infinite dose, occluded conditions terpenen-4-ol can cumulate
within the skin which may act as a reservoir for gradual elimination into the circulation. However, these
conditions are not representative of the typical use pattern of TTO. As TTO oil is a semi-volatile
substance, the majority of the applied dose rapidly evaporates from the surface of the skin before it
has the chance to absorb into the skin.
TTO has been reported to cause mild to moderate skin irritation in rabbit studies. Local lymph node
greater potential for skin sensitisation due to the presences of oxidation by-products. Proper storage
and handling of TTO and its formulated products should avoid the development of these by-products
and reduce the risk of skin irritation and sensitisation in sensitive individuals (Nielsen 2005).
There are no oral repeat dose toxicity studies available for TTO. However, there are no known
indications which require oral administration of TTO. The main route of administration is by dermal
application. Repeat dose data are available on some of the main components of TTO. Renal toxicity has
been observed in separate studies following oral administration of terperne-4-ol, cineole and cumene
(similar to p-cymene). Taking into consideration the typical levels of these components in TTO, a NOEL
of 117 mg/kg/day has been theoretically estimated for TTO (Nielsen 2005).
TTO was negative in the Ames assay using Salmonella typhimurium TA102, TA100 and TA98 examined
with or without metabolic activation and it did not induce clastogenicity in the in vivo mouse
micronucleus assay (Fletcher et al. 2005).
While TTO contains trace levels of methyleugenol, the typical use pattern in adults, being short-term
dermal use is not expected to pose a significant cancer risk (Nielsen 2005).
The mechanisms of antimicrobial action elucidated so far reflect the terpenic hydrocarbon composition
and indicate that cytoplasmic membrane integrity is compromised by treatment with TTO or some of
its major components. Alterations in eukaryotic cell membranes have also been observed with TTO and
terpinen-4-ol treatment (Longbottom et al. 2004).
Pharmacological studies in humans
Pharmacological studies conducted in humans have been discussed in ESCOP Monograph Supplement
2009. Messager et al. 2005 reported on the antimicrobial activity of TTO for hand cleansing. Koh et al.
2002 and Pearce et al. 2005 reported on the anti-allergenic and anti-inflammatory effects of TTO on
histamine and nickel-induced skin reactions.
Khalil et al. 2004 have also investigated the regulation of wheal and flare by undiluted TTO on
histamine-induced skin responses in human skin. 18 subjects had 25 μl of 100% TTO applied topically
to the histamine-induced reaction site at 10 minutes and 20 minutes after histamine injection
intradermally to the inner forearm skin. One arm of each subject was the study arm and the other arm
(randomly allocated) was the control arm with no control oil applied
to the reaction site. The TTO
after histamine injection. No adverse effects were reported.
Canyon & Speare 2007 conducted head lice (Pediculus humanus var. capitis) avoidance experiments
were applied to a test area. These test materials consisted of 100% TTO, a variety of other oils, neem
insect repellent, N,N-Diethyl-3-methylbenzamide (DEET) 69.75 g/l (positive control) and KY-Jelly, inert
lubricant gel (negative control). After 2 minutes, 15 lice were placed onto each treated area. TTO
repelled 55% of head ice from treated area, followed by peppermint oil (34%) and DEET (26%). TTO
was most effective at preventing lice from feeding (60%) followed by lavender oil (40%), peppermint
(28%) and DEET (23%).
A summary of these studies is presented in Table 3.