• To evaluate the ability of tea tree oil and its major components to cause skin sensitivity.

The prick test involves an immediate allergic reaction. It is the type of test used in the diagnosis of grass and dust-mite allergy amongst other things, and we tested these allergens as well as tea tree oil in order to determine if our volunteer population was more or less allergic than average. 

The patch test detects a delayed reaction that develops on the skin surface. It is used to diagnose allergic contact dermatitis, but sometimes causes an irritant reaction. There are some substances that commonly cause allergic dermatitis. 

We tested the volunteers to some of these in addition to tea tree oil in order to determine their overall reactivity in this type of test. Any volunteer who exhibited a reaction to tea tree oil was tested again with 100% oil, 10% oil and oil components.

The tests for immediate allergy indicated that while we had an overly reactive group of people, only 1.4% of people (3 of 219) displayed any reaction to tea tree oil, and reactions were to only 1 or 2 of 10 oils. 

The volunteers also appeared to be slightly more reactive to contact allergens than might be expected. A total of 52 of the 219 volunteers displayed some reaction to tea tree oil although, in a number of these people, this consisted only of a questionable reaction to 1 or 2 of the 10 oils. Secondary testing was performed on 33 volunteers. Results were classified as allergic contact dermatitis, mild or marked irritant contact dermatitis, indistinguishable between allergy and irritancy, or no response. 

The prevalence of allergy in the whole group was 2.9% up to a possible 4.8% if indistinguishable is included. To have an allergy requires a previous exposure to the substance being tested, and the levels of allergy amongst those who had previously used tea tree oil was 4.6% up to a possible 7.6% including the indistinguishable reactions. The prevalence of marked irritancy to 100% tea tree oil ranged from 2.4-4.3% (with or without the indistinguishable reactions), with any level of irritancy ranging from 7.2-10.1%. Irritant reactions are particularly concentration dependent, which means that many of these reactions should be avoidable if a lower concentration of oil is used. Very few reactions occurred in response to tea tree oil components, with a single mild response to each of 2 components, and 4 significant reactions to another component.

• To develop a dinucleotide microsatellite DNA library for tea tree and 
• To characterise 10 microsatellite loci. The most useful microsatellite loci and primer sequences for fingerprinting breeding lines identified by this research will be documented in a Commercial-in-Confidence report to the RIRDC /ATTIA Tea Tree Breeding Project meeting of December 1999.

A DNA library enriched for dinucleotide microsatellites has been constructed. One hundred and twenty one clones were sequenced and microsatellites located in nearly all clones. Primers were made for 59 microsatellites and 50 have been tested. To date, 23 microsatellite loci are working on the ABI sequencer in a number of multiplexes. The 8 parents of the controlled cross families have been assayed for their genotypes at the 23 microsatellite loci. The genetic inheritance of the loci is currently being assessed using the controlled cross families.

• Continuing current, and establishing new selection trials. 
• Production of improved seed lines with yields up to 17% higher than current industry selections by 1997, 30% higher by 1999, and 60% higher by 2003.

The success of these sales, together with the reports of increased yields augers well for the third release expected next month. For this third release, 2.8kg of seed is available. A fully subscribed sale of this seed would return about $90,000 to the research partners, making this programme almost self-funding.

The annual sale of selected bush seed will continue until we can supply highly improved seed from the Seedling Seed Orchard (SSO).

To promote heavy flowering in the SSO, irrigation and a chemical hormone have been trailed. From the 1998 flowering, mature seed will be available in early 2000. Based on work with other species, a 30% increase in yield from using this seed should be achievable. This level of improvement will be maintained for several years then in about 2004 the first harvests giving a 60% improvement should be made.

• To determine the current nutritional status and corresponding yields of tea tree grown in Australia (North Queensland and Northern New South Wales).
• To determine some ball park optimum fertiliser recommendations for tea tree.
• To improve the knowledge base of the Australian producers on the nutritional requirements of tea tree.

Table 1: Range in leaf nutrient levels in tea tree plantations.

Nutrient   Range in Level

N %               0.9 - 2.2
P%                 0.06 - 0.40
K%                0.6 - 1.8
Ca%               0.2 - 0.8
Mg%             0.1 - 0.5
Zn mg/kg      5 - 60
Fe mg/kg       0 - 120

Soil and leaf nutrient analysis of productive plantations is allowing the optimum soil and leaf nutrient levels to be determined. There is a reasonably good relationship between leaf nutrient levels and harvested biomass nutrient levels.

With the range in biomass nutrient levels and the yield of harvested biomass, approximate levels of nutrient removal can be calculated. The nutrient removal will allow estimates of fertiliser requirements to be made: Initial results indicate the following approximation.

These results compare favourably (although generally higher) with those found in some preliminary work by Bede Clarke (N.S.W. Dept. of Ag.) who found nutrient removal levels of 105 kg/ha N, 21 kg/ha P, 126 kg/ha S, 36 kg/ha Ca and 36 kg/ha Mg.

Information is now being collected on the relationship between fertiliser inputs and soil and leaf nutrient levels and yields achieved. A good relationship is expected in North Queensland where soil types are uniform and temperature, moisture and radiation levels are not limiting plant growth. In Northern New South Wales where soil types are more variable and often plant growth is limited by temperature, moisture or radiation the relationship between yields and fertiliser inputs maybe much weaker.

Discussions with local fertiliser companies and Agronomists indicates growers are starting to check leaf and soil nutrient levels of their trees. In the samples which have been analysed Mg, Ca, Zn, and B are often low, soil P levels have been declining and many growers have been using lime.

The most commonly observed nutrient deficiency in North Queensland is following harvest when early coppice regrowth is yellow to very pale yellow. In many cases this has been traced back to low Cu, Fe or Zn levels in the plant even when soil levels appear adequate. It is believed during fast regrowth, root uptake cannot keep up with shoot demands.

Leaf oil concentration determinations have indicated that oil concentration declines in trees under nutrient stress. The decline can be as great as 30% so that not only is yield lost through smaller biomass but also lower oil content.

• Develop efficacy and safety data packages to support the registration of Tea Tree Oil for use as a medical product by providing sound epidemiological data to support its acceptance as an effective and safe antimicrobial by national and international registration authorities.

The numbers of those infected in each treatment group were similar. The most common site of MRSA isolation was skin accounting for 48% of all isolates. All infected participants received IV Vancomycin in addition to the topical decolonisation regimen. 50% of participants successfully completed the initial treatment regimen. RC participants were older (average age 74 years) and had a shorter average treatment period (5 d) than IC participants (58 years and 9.2d).

Five of the IC group were initially cleared of MRSA carriage compared to two from the RC group. No adverse events were recorded for either the tea tree oil body wash or the mupirocin nasal ointment. Adverse events for the tea tree oil nasal ointment ranged from mild swelling of the nasal mucosa to acute burning sensation upon application. One participant complained of skin tightness after using the Triclosan body wash.

Initial results appear promising, however due to small sample size, an additional clinical trials with a larger sample is strongly recommend to confirm the clinical efficacy and safety of TTO as an alternative topical therapeutic agent for MRSA carriage. Currently there are plans to commence a large clinical trial at Westmead Hospital in Sydney, NSW.

• To develop and validate methods suitable for testing the antiviral activity of tea tree oil in vitro and to use these methods to assess its potential as a topical antiviral therapeutic agent. 
• The outcomes will be the development of methods, the validation of these methods and an assessment of the suitability of tea tree as a topical antiviral agent.

Both infections occur on mucosal surfaces and therefore are potentially treatable with tea tree oil preparations. A major issue that has been resolved is that of solublising tea tree oil and avoiding toxicity in the test system. Working with bacteria, it is necessary to add an agent such as Tween 80 to enhance the solubility of the oil. However, too much Tween can compromise the activity of tea tree oil. 

Appropriate dilutions of Tween 80 have been investigated in the assay system to achieve adequate solubility without affecting cell viability. In addition, Tween 80 concentrations of 0.001% had the desired effect on solubility but did not affect virus concentration. 

At present various concentrations of tea tree oil are being tested in a plaque reduction assay to determine virus susceptibility.

• To assess the anti inflammatory activity of tea tree oil in vitro and evaluate its potential as a safe, topical anti inflammatory therapeutic agent.

In contrast, if TTO is mixed with serum-free medium in a plastic tube, the toxicity of TTO is not demonstrable in the culture medium, presumably because the toxic components are present in the hydrophobic phase adherent to the plastic wall of the tube. Addition of 10% FCS or 0.001% Tween to the diluting medium increased the stability of the oil-soluble components in the tissue culture medium and resulted in monocyte toxicity at concentrations of tea tree oil of 0.06% and above. When TTO was diluted to 0.125% concentration in plastic tubes in serum-free culture medium, potent anti-inflammatory activity was detected with dose-dependent suppression of lipopolysaccharide-induced tumour necrosis factor-alpha, interleukin-1 (IL-1), IL-10 and prostaglandin production, i.e. mediators produced by inflammatory stimuli. 

Chemical analyses by Dr IA Southwell of the Wollongbar Agricultural Institute showed that the water soluble components of TTO are terpinen-4-ol, alpha-terpineol and 1,8, cineole. When these chemicals were tested in isolation, there was no toxicity and terpinen-4-ol (42% of TTO) was shown to have strong anti-inflammatory properties. The analyses by Dr Southwell showed that the components of TTO that moved from the oil to the aqueous phases with addition of FCS to solutions in plastic tubes were p-cymene and gamma-terpinene. 

In isolation, we have not found these components sufficiently toxic for monocytes but hypothesise that toxicity may be caused by their synergistic activity.