Melaleuca alternifolia, ext.

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: The test was carried out to determine whether Tea Tree Oil is a skin sensitiser, according to the Magnusson & Kligmann method (Magnusson & Kligmann 1969, 1970, Magnusson 1980).
- Short description of test conditions: Test animals were exposed intradermally and epidermally to the test material, along with an adjuvant to enhance the immune reaction. The guinea pigs were subsequently exposed to a lower concentration of the test material, and the incidence of skin reaction noted.
- Parameters analysed / observed: Erythemal reactions, signs of toxicity and abnormal behaviour.

GLP compliance:

no

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

This study was conducted prior to development of the LLNA method and its introduction as the default in-vivo method for REACH registration.

Specific details on test material used for the study:

Batch no.: 88/375.

Purity: 100%.

Species:

guinea pig

Strain:

other: HA strain - albino

Sex:

not specified

Details on test animals and environmental conditions:

- Source: Not specified.
- Age at study initiation: Not specified.
- Weight at study initiation: 200 - 500 g.
- Housing: Not described.
- Diet: Not specified.
- Water: Not specified.
- Acclimation period: Not described.
- Environmental conditions: Not described.

Route:

intradermal

Vehicle:

other: saline / Freund's complete adjuvant

Concentration / amount:

0.1 ml. 1:1:1 mixture of Tea Tree Oil, saline, and Freund's complete adjuvant.

Adequacy of induction:

not specified

Route:

intradermal

Vehicle:

paraffin oil

Concentration / amount:

0.1 ml. 5% Tea Tree Oil in paraffin oil B.P.

Adequacy of induction:

not specified

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

Not specified

Day(s)/duration:

48 hours

Adequacy of induction:

not specified

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Concentration / amount:

30% w/w test sample in petroleum jelly

Day(s)/duration:

24 hours

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

20 test animals and 20 control animals.

Details on study design:

- Induction procedure: The induction procedure consisted of 2 intradermal injections (0.1ml each) and an epidermal induction application. In the case of the epidermal induction application, the substance was applied epicutaneously over the injection sites one week after injection and held under occlusion for 48 hours.
- Induction test concentrations: One induction injection consisted of 5% Tea Tree Oil in paraffin oil B.P. The other was a 1:1:1 mixture of Tea Tree Oil, saline, and Freund's complete adjuvant. The undiluted test sample was used for the epidermal induction.
- Challenge exposure: Two weeks after the induction application, the animals were tested on one flank with the maximum subirritant concentration of the test compound in petroleum jelly (occlusive administration for 24 hours).
- Challenge concentrations: The subirritant dose used for challenge was 30% w/w test sample in petroleum jelly.
- Control: Control animals were treated with the vehcles alone during the induction period.

Challenge controls:

The control group was challenged with the vehicle and the test compound in subirritant concentration.

Positive control substance(s):

no

Positive control results:

No positive controls were used in this study.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

Challenge: 30% w/w test sample in petroleum jelly

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

None

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

Challenge: 30% w/w test sample in petroleum jelly

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

None

Remarks on result:

no indication of skin sensitisation

The guinea pigs appeared normal during the test period. The dermal challenge irritations did not produce irritant responses in either treated or control animals.

Interpretation of results:

not sensitising

Conclusions:

The test sample did not induce sensitization reactions after dermal challenge.

Executive summary:

Skin sensitization studies were carried out to determine whether Tea Tree Oil (Batch 88/375) was a skin sensitizing agent in the guinea pig. The test was carried out according to the Magnusson & Kligmann maximisation method.  20 albino guinea pigs were used for the test and control experiments respectively. The induction procedure consisted of two intradermal injections and an epidermal induction application. Two weeks after the induction application, the challenge dose was applied (subirritant dose, 30% w/w test sample in petroleum jelly). All animals were observed for signs of toxicity and abnormal behaviour during the experimental period. The guinea pigs appeared normal during the test period. The test sample did not induce sensitization reactions after dermal challenge.

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

The objective of this study was to determine the sensitizing potential of topically applied test substances. This study is designed to be a stand-alone alternative assay for the Buehler Guinea Pig Sensitization Assay defined in the ICCVAM report (Federal Register 63 FR 37405-6, July 10, 1998), EPA Health Effects Test Guideline, OPPTS 870.2600 final guideline effective, August 1998, and OECD Test Guideline 429, effective April 2002.

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

- Description: Clear liquid.
- Lot/batch No.: MC4061
- Purity: 100%.
- Composition of test material: Composition meets ISO Standard 4730-2004 Oil of Melaleuca, terpinen-4-ol type (Tea Tree Oil).
- Stability: Stable under storage conditions, according to Test Article Characterization.
- Storage condition of test material: Room temperature and humidity.

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

- Source: Jackson Laboratories.
- Females nulliparous and non-pregnant: Yes
- Number of animals for the main study: 25
- Age at study initiation: Approximately 11 weeks old.
- Weight at study initiation: 19.0 - 24.5 g for definitive study animals (19.3 - 24.2 g for screen animals). The weight variation of the animals at study initiation did not exceed ± 20% of the mean body weight.
- Other details on test animals: Mice were assigned to the applicable groups using a computer-based random number program.
- Identification: The animals were identified by cage notation and indelible tail marks.
- Housing: The animals were housed 1/cage in suspended wire cages. Bedding was placed beneath the cages and changed at least three times/week.
- Diet: Fresh PMI (Diet #5001), ad libitum.
- Water: Ad libitum.
- Acclimation period: At least five days.
- Temperature: The room was temperature-controlled.
- Photoperiod: 12 hour light/dark cycle.

Vehicle:

other: polyethylene glycol (PEG 400)

Concentration:

5%, 25% and 50%.

No. of animals per dose:

Five females per dose group.

Details on study design:

Dosing - Irritation Screening Study: Three groups of two CBA/J mice per group (a total of six) were treated with three concentrations (5%, 25% and 50%) of Tea Tree Oil (Melaleuca alternifolia), Lot/batch #MC4061, CAS# 85085-48-9, 68647-73-4, in PEG 400 for three consecutive days. Because the chosen vehicle, PEG 400, has not been validated, two additional groups of 2 mice each were added, one treated with the PEG 400 vehicle and one treated with 25% HCA in PEG 400. The ears were observed for edema and/or erythema and ear measurements were taken on Day 1 (pre-dose control), Day 3 (at approximately 48 hours), and Day 6 (end of in-life phase). On Day 6, the mice were sacrificed using CO2 asphyxiation. Ear thickness (ET) changes on Day 3 and Day 6 were expressed as a percent of the Day 1 pre-study values. An increase of 25% or more in ET was considered biologically significant (based on historical laboratory data) and indicative of a primary dermal irritation response. The test article concentrations used in the definitive LLNA study were chosen such that: 1) maximum concentration tested was the highest achievable solution of the test article in the vehicle, while 2) avoiding both overt systemic toxicity and excessive local irritation.

Dosing - Definitive Study: Groups of five CBA/J mice were treated by topical application of the test article concentrations, vehicle or positive control to the dorsum of each ear once daily for three consecutive days (at approximately the same time each day). The test article mixture, control or vehicle was spread over the entire dorsal surface of the ear using a micropipette at 25 ul/ear. The vehicle control, PEG 400, was chosen by the sponsor. The test article doses were requested by the sponsor and were equal to the screen doses.

Test article formulation and dosing: The test article formed a homogenous mixture in the vehicle, PEG 400. No difficulties were experienced with the application of the test articles to the ears or with the retention of test articles by the ear surface.

Ear swelling: Ear thickness measurements are performed pre-dose on Day 1, again after 48 hours on Day 3 (before the third test article application), and lastly on Day 6 before sacrifice (approximately 120 hr after first dose and 72 hr after third dose). Ear thickness changes within ± 20% are in the normal range. Changes in ear thickness of greater than 25% are considered irritating. A preliminary screening test in ear swelling measurements was performed. None of the concentrations tested was irritating (more than 25% increase in ear thickness). Concentrations of 5%, 25% and 50% of the test article were chosen by the sponsor to be used for the definitive Local Lymph Node Assay.

Positive control substance(s):

other: alpha-hexylcinnamaldehyde (HCA) 25%

Statistics:

Statistical evaluation of the calculated SI values were made by Student's t-test, using GraphPad InStat ™, c. 199-1994, v 2.05a+. However, determination of test article dermal sensitization was based on the criterion that test articles that yield a SI ≥ 3 are characterized as potential sensitizing substances.

Positive control results:

Body weight: Body weight changes were normal.
Ear swelling: The positive control, 25% HCA, had less than 25% increase in ear swelling on Day 6.
Determination of the Stimulation Index (SI): The positive control yielded a SI of 21.2 (see Table 3 in 'Any other information on results').

Parameter:

SI

Value:

1

Variability:

S.D. = +/- 0.6

Test group / Remarks:

PEG 400 (Vehicle group)

Parameter:

SI

Value:

21.2

Variability:

S.D. = +/- 7.7

Test group / Remarks:

25% HCA (Positive control group)

Parameter:

SI

Value:

2.1

Variability:

S.D. = +/- 0.7

Test group / Remarks:

5% Tea Tree Oil

Parameter:

SI

Value:

7.7

Variability:

S.D. = +/- 4.0

Test group / Remarks:

25% Tea Tree Oil

Parameter:

SI

Value:

7.9

Variability:

S.D. = +/- 3.2

Test group / Remarks:

50% Tea Tree Oil

Cellular proliferation data / Observations:

The mean number of proliferating cells in the lymph nodes of each treatment group is shown in Table 2 (see 'Any other information on results').
Mortality and systemic observations: All animals survived the in-life phase of the study and appeared normal.
Body weight: Body weight changes were normal.

Ear swelling: In the definitive study, none of the Tea Tree Oil concentrations tested resulted in dermal irritation (see Table 1 below).

Table 1. Ear Swelling.

	Mean Ear Thickness (mm)			Change (%)
Treatment	Pre-dosing (Day 1)	48 Hr (Day 3)	End In-Life (Day 6)	Day 3 - Day 1	Day 6 - Day 1
PEG 400 (Vehicle)	0.20	0.21	0.20	5.0%	0.0%
25% HCA (Positive Control)	0.20	0.22	0.23	10.0%	15.0%
5% Tea Tree Oil	0.20	0.21	0.21	5.0%	5.0%
25% Tea Tree Oil	0.20	0.21	0.23	5.0%	15.0%
50% Tea Tree Oil	0.20	0.21	0.21	5.0%	5.0%

Table 2. Mean Number of Proliferating Cells in the Lymph Nodes of each Treatment Group.

Treatment	Vehicle	Mean LNC# (x10^3)	% BrdU+	#BrdU+
PEG 400 (Vehicle)	--	1208	0.74	7254
25% HCA (Positive Control)	PEG 400	8864	1.73	153629
5% Tea Tree Oil	PEG 400	2441	0.64	15098
25% Tea Tree Oil	PEG 400	4811	1.11	55945
50% Tea Tree Oil	PEG 400	5697	1.00	57385

Table 3. Stimulation Index.

Treatment	SI	S.D.
PEG 400 (Vehicle)	1.0	(+/- 0.6)
25% HCA (Positive Control)	21.2 (a)	(+/- 7.7)
5% Tea Tree Oil	2.1	(+/- 0.7)
25% Tea Tree Oil	7.7 (a)	(+/- 4.0)
50% Tea Tree Oil	7.9 (a)	(+/- 3.2)

(a) = SI ≥ 3 indicates a sensitizing response.

EC3 Calculation: The EC3 was calculated to be 8.3%, which would classify this test article as extremely weak for sensitizing potency.

Reference: D.A. Basketter, et al., "Use of the Local Lymph Node Assay For the Estimation of Relative Contact Allergenic Potency", Contact Dermatitis (2000), 42:344-348.

Immunophenotyping of Lymph Node Cells: The %B cells, %T cells, and B:T ratio were determined by flow cytometry as described earlier. Group means and standard deviations are tabulated below (Table 4 and Table 5).

Table 4. %B220+ (B Cells), % CD3+ (T Cells).

	%B220 (B Cells)		% CD3 (T Cells)
Treatment	Mean	S.D.	Mean	S.D.
PEG 400 (Vehicle)	12.8	(+/- 0.7)	70.5	(+/- 4.0)
25% HCA (Positive Control)	30.5 (#)	(+/- 7.0)	58.4	(+/- 8.3)
5% Tea Tree Oil	14.7	(+/- 2.6)	73.1	(+/- 2.9)
25% Tea Tree Oil	19.8 (#)	(+/- 5.8)	68.3	(+/- 4.4)
50% Tea Tree Oil	22.0 (#)	(+/- 2.2)	67.7	(+/- 5.9)

Table 5: B:T Lymphocyte Cell Ratio

Treatment	Mean	S.D.
PEG 400 (Vehicle)	0.18	(+/- 0.0)
25% HCA (Positive Control)	0.55 (#)	(+/- 0.2)
5% Tea Tree Oil	0.20	(+/- 0.0)
25% Tea Tree Oil	0.29 (#)	(+/- 0.1)
50% Tea Tree Oil	0.33 (#)	(+/- 0.1)

# = greater than 1.25-fold (>25% increase) over vehicle control = positive sensitizing response

Discussion: Topical application of the test article did not significantly increase ear thickness at any of the concentrations tested, indicating it did not result in an irritating response. The Stimulation Index induced following treatment by Tea Tree Oil was 2.1 at 5% of the test article, 7.7 at 25%, and 7.9 at 50%, indicating a sensitizing response at both 25% and 50%. Immunophenotyping was also performed to examine a potential sensitizing response. In this study, the two immunophenotype criteria used to determine sensitization were (1) a 25% increase of B cells (B220+) in test article-treated group(s) compared to control, and (2) a 25% increase of B:T ratio in test article-treated group(s) compared to control. The 25% and 50% Tea Tree Oil groups met both of these criteria, again indicating a sensitizing response.

Conclusions:

Topical application of the test article Tea Tree Oil at 25% and 50% in PEG 400 resulted in a stimulation index greater than 3 (SI > 3.0), and therefore this test article is a dermal sensitizer in the Local Lymph Node Assay. The EC3 for this test article was calculated to be 8.3%, which would classify this test article as extremely weak for sensitizing potency.

Executive summary:

This GLP-compliant study followed MB Research Protocol # 5650A-06 and determined the sensitizing potential of topically applied Melaleuca alternifolia.  A preliminary screening test was conducted with three groups of healthy female CBA/J mice (2 per group) to determine the concentrations of the test article to be used in the main study.  Because the chosen vehicle, PEG 400, had not been validated, two additional groups of 2 mice each were added, one treated with the PEG 400 vehicle and one treated with 25% HCA in PEG 400.  The initial screening study showed no irritation at any of the test article concentrations tested, including the maximum soluble concentration of 50%.  No irritation was detected following treatment of the PEG 400 vehicle, whereas 25% HCA in PEG 400 elicited irritation.  Concentrations of 5%, 25% and 50% of the test article were chosen for the definitive Local Lymph Node study by the sponsor.

For the definitive study, three separate groups of five healthy female CBA/J mice were treated with increasing concentrations of Tea Tree Oil by topical application to the dorsum of each ear, once daily for three consecutive days.  A vehicle control group of five mice was treated with PEG 400 and another group of five mice were treated with the positive control, 25% HCA (in PEG 400), in exactly the same manner.

Five days following the initial dose, and five hours prior to sacrifice, the mice were given an intraperitoneal injection of the thymidine analog 5-bromo-2’deoxy-uridine (BrdU), and at sacrifice the auricular lymph nodes were isolated and single-cell suspensions of lymph node cells (LNC) were generated.  For each animal, the LNC suspension was analysed for BrdU incorporation and total number of LNC by flow cytometry.  The amount of proliferating (#BrdU+) LNC was determined as a measure of the proliferative response of the local lymph node.  The stimulation index (SI) was calculated by dividing the proliferative response (BrdU incorporation) of each test article group by the proliferative response of the vehicle control group.  Test articles that yielded a SI ≥ 3 were characterized as sensitizing substances.

All animals survived the in-life phase of the study and appeared normal.  Body weight changes were normal.  Ear swelling measurements and individual animal observations indicated that none of the treatments resulted in dermal irritation.

The SI of the positive control, 25% HCA, was 21.2, similar to 25% HCA in more common vehicles.  The SI values for the test article at 5%, 25% and 50% were 2.1, 7.7 and 7.9, respectively.  Since topical application of the test article at 25% and 50% in PEG 400 resulted in a stimulation index greater than 3, this test article is considered to be a dermal sensitizer in the Local Lymph Node Assay.  The EC3 for this test article was calculated to be 8.3%, which would classify it as extremely weak for sensitizing potency.

Reference 3

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Qualifier:

according to guideline

Guideline:

other: ICCVAM Immunotoxicology Working Group Recommended Protocol for the Murine Local Lymph Node Assay (LLNA): Testing of Chemicals for Contact Sensitizing (Allergic Contact Dermatitis [ACD]) Potential (January, 2001).

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

- Physical state: Liquid.
- Batch No.: 1215.
- Purity: 100%.
- Composition of test material: Composition meets ISO Standard 4730-2004 Oil of Melaleuca, terpinen-4-ol type (Tea Tree Oil).
- Expiration date of the batch: 2015
- Stability: Stable under storage conditions.
- Storage condition of test material: At room temperature (range of 20 ± 5°C), light protected. Cap tightly - subject to oxidation.

Species:

mouse

Strain:

other: CBA/CaHsdRcc(SPF)

Sex:

female

Details on test animals and environmental conditions:

- Source: RCC Ltd., Laboratory Animal Service, CH-4414 Füllinsdorf/Switzerland.
- Number of animals for the main study: 20
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 8 - 12 weeks (beginning of acclimatization).
- Weight at study initiation: 16g - 24g (ordered).
- Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.
- Identification: Each cage by unique cage card.
- Randomization: Randomly selected by computer algorithm at time of delivery.
- Temperature: Air-conditioned with ranges for room temperature 22 ± 3°C. Room temperature was monitored continuously.
- Humidity: Relative humidity 30 - 70%. Humidity was monitored continuously.
- Air changes: 10 - 15 per hour.
- Photoperiod: 12 hour fluorescent light/12 hour dark cycle.
- Housing: Individual in Makrolon type-2 cages with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz).
- Diet: Pelleted standard Kliba 3433, batch no. 01/06 mouse maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst) available ad libitum. There was no contamination of the diet.
- Water: Community tap water from Itingen, available ad libitum. There was no contamination of water.
Other: There were at least 8 hours of music during the light period.

Vehicle:

other: polyethylene glycol 300

Remarks:

See 'Any other information on materials and methods' for further details.

Concentration:

2%, 20% (w/v) in polyethylene glycol 300 (PEG 300) and 100% (undiluted).

No. of animals per dose:

Five female mice per dose group (3 dose groups and 1 vehicle-only negative control group).

Details on study design:

Test item preparation: The test item was placed into a volumetric flask on a tared Mettler balance and the vehicle PEG 300 was quantitatively added. The weight/volume (w/v) dilutions were prepared individually using a magnetic stirrer as homogenizer. Test item formulations were made freshly before each dosing occasion and no more than 4 hours prior to application to the ears. Homogeneity of the test item in the vehicle was maintained until start of treatment using an appropriate homogenizer. The test item in the main study was assayed at three consecutive concentrations selected by the Sponsor. Concentrations were in terms of material as supplied.

Validation/positive control study: The sensitivity and reliability of the experimental technique employed was assessed by use of a substance which is known to have skin sensitization properties in CBA/Ca mice. The validation-/positive control study was performed with alpha-hexylcinnamaldehyde in acetone/olive oil (4/1, v/v) using CBA/Ca mice (RCC Study Number A51085) from 11-JAN-2006 to 25-JAN-2006.

Treatment procedures
Topical application: Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 2%, 20% (w/v) in PEG 300 and 100% (undiluted). The application volume, 25 μl, was spread over the entire dorsal surface (diameter ~ 8mm) of each ear lobe once daily for three consecutive days.

Administration of ³HTdR: Five days after the first topical application, all mice were administered with 250 μl of 83.31μCi/ml ³HTdR (equal to 20.8 μCi ³HTdR) by intravenous injection via a tail vein.

Determination of incorporated 3HTdR: Approximately five hours after treatment with 3HTdR all mice were euthanized by inhalation of CO2 (dry ice). The draining lymph nodes were rapidly excised and pooled for each animal (2 nodes per animal). Single cell suspensions (phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 μm mesh size). After washing twice with phosphate buffered saline (approx. 10 ml) the lymph node cells were resuspended in 5% trichloroacetic acid (approx. 3 ml) and incubated at approximately +4°C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5% trichloroacetic acid (1ml) and transferred to glass scintillation vials with 10 ml of 'Irga-Safe Plus' scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured on a β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 ml-aliquots of 5% trichloroacetic acid. The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (dpm).

Positive control substance(s):

other: alpha-hexylcinnamaldehyde

Statistics:

Dunnett-test was conducted for assessment of the difference significance between the test item groups and the negative control (vehicle) group. A statistical analysis was conducted for assessment of the dose-response relationship, and the EC3 value was calculated according to the equation EC3 = (a-c) [(3-d)/(b-d)] + c, where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately below and above the S.I. value of 3 on the local lymph node assay dose response plot.

Positive control results:

The proliferative capacity of pooled lymph node cells was determined by the incorporation of ³H-methyl thymidine measured on a ß-scintillation counter (see Table 1 in 'Any other information on results').
No deaths occurred during the study period. Neither clinical / local signs nor other findings were observed in any animals of the control group. On the second application day, slight to severe ear swelling was observed at both dosing sites in all mice of Groups 2-4, persisting for a total of four days or for the remainder of the in-life phase of the study, respectively. In addition, on the second application day, slight to severe ear erythema was also noted at both dosing sites in all mice of Groups 3-4, persisting for a total of four days or for the remainder of the in-life phase of the study, respectively. The body weight of the animals, recorded prior to the first application and prior to necropsy, was within the range commonly recorded for animals of the strain and age.

Parameter:

SI

Remarks:

Mean

Value:

1.6

Variability:

SD = 0.4

Test group / Remarks:

Test Group 2 (2%)

Remarks on result:

other: See Table 2 in 'Any other information on results'.

Parameter:

SI

Remarks:

Mean

Value:

2.8

Variability:

SD = 0.7

Test group / Remarks:

Test Group 3 (20%)

Remarks on result:

other: See Table 2 in 'Any other information on results'.

Parameter:

SI

Remarks:

Mean

Value:

5.7

Variability:

SD = 1.6

Test group / Remarks:

Test Group 4 (100%, undiluted)

Remarks on result:

other: See Table 2 in 'Any other information on results'.

Parameter:

other: DPM per LN

Remarks:

Mean

Value:

249

Variability:

SD = 116

Test group / Remarks:

Control Group 1

Remarks on result:

other: See Table 2 in 'Any other information on results'.

Parameter:

other: DPM per LN

Remarks:

Mean

Value:

397

Variability:

SD = 106

Test group / Remarks:

Test Group 2 (2%)

Remarks on result:

other: See Table 2 in 'Any other information on results'.

Parameter:

other: DPM per LN

Remarks:

Mean

Value:

700

Variability:

SD = 186

Test group / Remarks:

Test Group 3 (20%)

Remarks on result:

other: See Table 2 in 'Any other information on results'.

Parameter:

other: DPM per LN

Remarks:

Mean

Value:

1 430

Variability:

SD = 389

Test group / Remarks:

Test Group 4 (100%, undiluted)

Remarks on result:

other: See Table 2 in 'Any other information on results'.

Cellular proliferation data / Observations:

Mortality: No deaths occurred during the study period.
Clinical signs: No clinical signs of local toxicity at the ears of the animals and no systemic findings were observed during the study period.
Body weights: The body weight of the animals, recorded prior to the first application and prior to necropsy, was within the range commonly recorded for animals of the strain and age.
Size of the draining lymph nodes: The size of the draining lymph nodes of Groups 3 - 4 was 2 - 3 fold larger than that of the control group.

Table 1. Positive control results.

Group	Test item concentration (w/v)	S.I.
Group 2	5	1.8
Group 3	10 *	2.9 *
Group 4	25 *	6.2 *
EC3 = 10.5 %
A clear dose-response relationship was observed. * This value was used in calculation of EC3.

The proliferative capacity of pooled lymph node cells was determined by the incorporation of ³HTdR measured on a β-scintillation counter.

Table 2. Summary of results.

 

Group	% (w/v)	dpm/LN M (SD)	S.I. M (SD)	EC3 % (w/v)	Statistical Analysis Dunnett-test (G = 4, N = 20, t = 2.59)
					t value	Conclusion
CG 1	-	249 (116)	-	-	-	-
TG 2	2	397 (106)	1.6 (0.4)	25.5	1.02	--
TG 3	20*	700 (186)	2.8* (0.7)		3.11	**
TG 4	100*^a)	1430 (389)	5.7* (1.6)		8.14	**

a)           undiluted

*            This value was used in calculation of EC3.

**          significant difference at p ≤ 0.05 (two sides)

--           not significant difference at p ≤ 0.05 (two sides)

Conclusions:

A test item is regarded as a sensitizer in the LLNA if the exposure to one more test concentrations resulted in 3-fold or greater increase in incorporation of ³HTdR compared with concurrent controls, as indicated by the S.I. In this study S.I. of 1.6, 2.8 and 5.7 were determined with the test item at concentrations of 2 %, 20 % (w/v) in PEG 300 and 100 % (undiluted), respectively. Tea Tree Oil was therefore found to be a potential skin sensitizer and an EC3 value of 25.5 % (w/v) was derived. Based on Basketter et al. (2002) and Butler et al. (2003), Tea Tree Oil has weak sensitisation potential (EC3 value > 10-30 %).

Executive summary:

A GLP-compliant study was carried out to determine the skin sensitization potential of Tea Tree Oil. The study followed the requirements of EU method B.42, OECD method 429, ICCVAM Immunotoxicology Working Group Recommended Protocol for the Murine Local Lymph Node Assay (LLNA): Testing of Chemicals for Contact Sensitizing (Allergic Contact Dermatitis [ACD]) Potential (January, 2001) and OPPTS method 870.2600 without significant deviation. Three groups of five female mice were treated with the test item at concentrations of 2%, 20% (w/v) in polyethylene glycol 300 (PEG 300) and 100% (undiluted) by topical application to the dorsum of each ear lobe (left and right) on three consecutive days. A negative control group of five mice was treated with an equivalent volume of the vehicle PEG 300 only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine, 3HTdR). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per animal. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were washed subsequently and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph nodes was determined by the incorporation of 3HTdR measured in a β-scintillation counter. All treated animals survived the scheduled study period. Neither clinical/local signs nor other findings were observed. No significant difference of dpm/LN was determined at the test item concentration of 2% in PEG 300 compared with the vehicle control group at p ≤ 0.05 (two sides). A significant difference of dpm/LN was determined at the test item concentrations of 20% (w/v) in PEG 300 and 100% (undiluted) compared with the vehicle control group at p ≤ 0.05 (two sides).

A test item is regarded as a sensitizer in the LLNA if the exposure to one more test concentrations resulted in 3-fold or greater increase in incorporation of ³HTdR compared with concurrent controls, as indicated by the S.I. In this study S.I. of 1.6, 2.8 and 5.7 were determined with the test item at concentrations of 2 %, 20 % (w/v) in PEG 300 and 100 % (undiluted), respectively. Tea Tree Oil was therefore found to be a potential skin sensitizer and an EC3 value of 25.5 % (w/v) was derived. Based on Basketter et al. (2002) and Butler et al. (2003), Tea Tree Oil has weak sensitisation potential (EC3 value > 10-30 %).

Reference 4

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Qualifier:

according to guideline

Guideline:

other: ICCVAM Immunotoxicology Working Group Recommended Protocol for the Murine Local Lymph Node Assay (LLNA): Testing of Chemicals for Contact Sensitizing (Allergic Contact Dermatitis [ACD]) Potential (January, 2001).

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

- Physical state: Liquid.
- Batch No.: # RP-05-346
- Purity: 100%.
- Composition of test material: Composition meets ISO Standard 4730-2004 Oil of Melaleuca, terpinen-4-ol type (Tea Tree Oil).
- Expiration date of the batch: 2015
- Stability: Stable under storage conditions.
- Storage condition of test material: At room temperature (range of 20 ± 5°C), light protected. Cap tightly, kept out of sunlight and direct heat. Subject to oxidation after opening.

Species:

mouse

Strain:

other: CBA/CaHsdRcc(SPF)

Sex:

female

Details on test animals and environmental conditions:

- Source: RCC Ltd., Laboratory Animal Service, CH-4414 Füllinsdorf/Switzerland
- Females nulliparous and non-pregnant: Yes
- Number of animals for the main study: 20
- Age at study initiation: 11 - 12 weeks (beginning of acclimatization).
- Weight at study initiation: 17.7g - 20.7g (at delivery).
- Identification: Each cage by unique cage card.
- Randomization: Randomly selected by computer algorithm at time of delivery.
- Housing: Individual in Makrolon type-2 cages with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz).
- Diet: Pelleted standard Kliba 3433, batch no. 001/06 mouse maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst) available ad libitum. Results of analyses for contaminants are archived at RCC. There was no contamination of the diet.
- Water: Community tap water from Itingen, available ad libitum. Results of representative bacteriological, chemical and contaminant analyses are archived at RCC. There was no contamination of water.
- Acclimatization: 7 days prior to the first topical application under test conditions after health examination. Only animals without any visible signs of illness were used for the study.
- Temperature: Air-conditioned with ranges for room temperature 22 ± 3°C. Room temperature was monitored continuously and values outside of this range occasionally occured, usually following room cleaning. These transient variations are considered not to have any influence on the study.
- Humidity: Relative humidity was 30 - 70%. Humidity was monitored continuously and values outside of this range occasionally occured, usually following room cleaning. These transient variations are considered not to have any influence on the study.
- Air changes: 10 - 15 air changes per hour.
- Photoperiod: 12 hour fluorescent light/12 hour dark cycle with at least 8 hours music during the light period.

Vehicle:

other: polyethylene glycol 300 (PEG 300)

Remarks:

See 'Any other information on materials and methods' for further details on the vehicle.

Concentration:

2%, 20% (w/v) in PEG 300 and 100% (undiluted).

No. of animals per dose:

5 females per dose group (3 test groups and 1 negative control group).

Details on study design:

- Test item preparation: The test item was placed into a volumetric flask on a tared Mettler balance and the vehicle PEG 300 was quantitatively added. The weight/volume (w/v) dilutions were prepared individually using a magnetic stirrer as homogenizer. Test item formulations were made freshly before each dosing occasion and no more than 4 hours prior to application to the ears. Homogeneity of the test item in the vehicle was maintained until start of treatment using an appropriate homogenizer. The test item in the study was assayed at three consecutive concentrations selected by the Sponsor. Concentrations were in terms of material as supplied.

- Validation/positive control study: The sensitivity and reliability of the experimental technique employed was assessed by use of a substance which is known to have skin sensitization properties in CBA/Ca mice. The validation-/positive control study was performed with alpha-hexylcinnamaldehyde in acetone/olive oil (4/1, v/v) using CBA/Ca mice from 11-JAN-2006 to 25-JAN-2006. The reliability check with alpha-hexylcinnamaldehyde indicated that the local lymph node assay as performed at RCC is an appropriate model for testing contact hypersensitivity.

Treatment Procedures
- Topical application: Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 2%, 20% in PEG 300 and 100% (undiluted). The application volume, 25 μl, was spread over the entire dorsal surface (diameter ~ 8 mm) of each ear lobe daily for three consecutive days.

- Administration of ³HTdR: Five days after the first topical application, all mice were administered with 250 μl of phosphate-buffered saline (PBS) containing 20.2 μCi/ml ³HTdR (equal to 80.62 μCi ³HTdR) by intravenous injection via a tail vein.

- Determination of incorporated ³HTdR: Approximately five hours after treatment with ³HTdR all mice were euthanized by inhalation of CO2 (dry ice). The draining lymph nodes were rapidly excised and pooled for each animal (2 nodes per animal). Single cell suspensions (phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 μm mesh size). After washing twice with phosphate buffered saline (approx. 10 ml) the lymph node cells were resuspended in 5% trichloroacetic acid (approx. 3 ml) and incubated at approximately +4°C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5% trichloroacetic acid (1 ml) and transferred to glass scintillation vials with 10 ml of 'Irga-Safe Plus' scintillation liquid and thoroughly mixed. The level of ³HTdR incorporation was then measured on a β-scintillation counter. Similarly, background ³HTdR levels were also measured in two 1ml-aliquots of 5% trichloroacetic acid. The β-scintillation counter expresses ³HTdR incorporation as the number of radioactive disintegrations per minute (dpm).

Positive control substance(s):

other: alpha-hexylcinnamaldehyde

Statistics:

The mean values and standard deviations were calculated in the body weight tables. Dunnett-test was conducted for assessment of the difference significance between the test item groups and the negative control (vehicle) group. A statistical analysis was conducted for assessment of the dose-response relationship, and the EC3 value was calculated according to the equation EC3 = (a-c) [(3-d)/(b-d)] + c, where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately below and above the S.I. value of 3 on the local lymph node assay dose response plot.

Positive control results:

Not provided.

Parameter:

SI

Remarks:

Mean

Value:

2.4

Variability:

SD = 1.4

Test group / Remarks:

Test Group 2 (2%)

Remarks on result:

other: See Table 1 in 'Any other information on results'.

Parameter:

SI

Remarks:

Mean

Value:

6.9

Variability:

SD = 2.0

Test group / Remarks:

Test Group 3 (20%)

Remarks on result:

other: See Table 1 in 'Any other information on results'.

Parameter:

SI

Remarks:

Mean

Value:

16

Variability:

SD = 6.3

Test group / Remarks:

Test Group 4 (100%, undiluted)

Remarks on result:

other: See Table 1 in 'Any other information on results'.

Parameter:

other: dpm per LN

Remarks:

Mean

Value:

228

Variability:

SD = 115

Test group / Remarks:

Control Group 1

Remarks on result:

other: See Table 1 in 'Any other information on results'.

Parameter:

other: dpm per LN

Remarks:

Mean

Value:

552

Variability:

SD = 323

Test group / Remarks:

Test Group 2 (2%)

Remarks on result:

other: See Table 1 in 'Any other information on results'.

Parameter:

other: dpm per LN

Remarks:

Mean

Value:

1 573

Variability:

SD = 467

Test group / Remarks:

Test Group 3 (20%)

Remarks on result:

other: See Table 1 in 'Any other information on results'.

Parameter:

other: dpm per LN

Remarks:

Mean

Value:

3 659

Variability:

SD = 1440

Test group / Remarks:

Test Group 4 (100%, undiluted)

Remarks on result:

other: See Table 1 in 'Any other information on results'.

Cellular proliferation data / Observations:

Viability / Mortality: No deaths occurred during the study period.
Clinical signs: Neither clinical/local signs nor other findings were observed in any animals of the control group. One day after the first or the second topical application, a slight ear erythema was observed at both dosing sites in all mice of Group 2 (2%), Group 3 (20%) and Group 4 (100%, undiluted), persisting for the remainder of the in-life phase of the study (Groups 3-4), or persisting for a total of two days (Group 2 ). In addition, two days after the third topical application (Day 5) and prior to sacrifice (Day 6), scales were found on both ears in all mice of Group 4 (100%, undiluted).
Body weights: The body weight of the animals, recorded prior to the first application and prior to sacrifice, was within the range commonly recorded for animals of the strain and age.
Size of the draining lymph nodes: The size of the draining lymph nodes of animals Nos. 13 – 15 (Group 3, 20%) and animals Nos. 16 – 20 (Group 4, 100%, undiluted) was 2-3 fold larger than that of the control group.

The proliferative capacity of pooled lymph node cells was determined by the incorporation of ³HTdR measured on a β-scintillation counter.

Table 1. Summary of results.

 

Group	% (w/v)	dpm/LN M (SD)	S.I. M (SD)	EC3 % (w/v)	Statistical Analysis Dunnett-test (G = 4, N = 20, t = 2.59)
					t value	Conclusion
CG 1	-	228 (115)	-	-	-	-
TG 2	2 *	552 (323)	2.4* (1.4)	4.4	0.66	--
TG 3	20 *	1573 (467)	6.9* (2.0)		2.74	**
TG 4	100 (undiluted)	3659 (1440)	16.0 (6.3)		6.99	**

**          significant difference at p ≤ 0.05 (two sides)

--            not significant difference at p ≤ 0.05 (two sides)

A dose-response relationship was observed.

* This value was used in calculation of EC3.

Conclusions:

A test item is regarded as a sensitizer in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in incorporation of ³HTdR compared with concurrent controls, as indicated by the S.I. In this study S.I. of 2.4, 6.9 and 16.0 were determined with the test item at concentrations of 2%, 20% (w/v) in PEG 300 and 100% (undiluted), respectively. Tea Tree Oil was therefore found to be a potential skin sensitizer and an EC3 value of 4.4% (w/v) was derived.

Executive summary:

A GLP-compliant study was carried out to determine the possible contact allergenic potential of Tea Tree Oil.  The study followed the requirements of EU method B.42, OECD method 429, ICCVAM Immunotoxicology Working Group Recommended Protocol for the Murine Local Lymph Node Assay (LLNA): Testing of Chemicals for Contact Sensitizing (Allergic Contact Dermatitis [ACD]) Potential (January, 2001) and OPPTS method 870.2600 without significant deviation.

Three groups each of five female mice were treated with the test item at concentrations of 2%, 20% (w/v) in PEG 300 and 100% (undiluted) by topical application to the dorsum of each ear lobe (left and right) on three consecutive days. A negative control group of five mice was treated with an equivalent volume of the vehicle polyethylene glycol 300 (PEG 300) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (³H-methyl thymidine, ³HTdR). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per animal. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were washed subsequently and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of ³HTdR measured in a β-scintillation counter. All treated animals survived the scheduled study period. Neither clinical/local signs nor other findings were observed in any animals of the control group.

One day after the first or the second topical application, a slight ear erythema was observed at both dosing sites in all mice of Group 2 (2%), Group 3 (20%) and Group 4 (100%, undiluted), persisting for the remainder of the in-life phase of the study (Groups 3-4), or persisting for a total of two days (Group 2).  In addition, two days after the third topical application (Day 5) and prior to sacrifice (Day 6), scales were found on both ears in all mice of Group 4 (100%, undiluted). No significant difference of dpm/LN was determined at the test item concentration of 2% (w/v) in PEG 300 compared with the vehicle control group at p ≤ 0.05 (two sides). A significant difference of dpm/LN was determined at the test item concentrations of 20% in PEG 300 and 100% (undiluted) compared with the vehicle control group at p ≤ 0.05 (two sides).

A test item is regarded as a sensitizer in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in incorporation of ³HTdR compared with concurrent controls, as indicated by the S.I.  In this study S.I. of 2.4, 6.9 and 16.0 were determined with the test item at concentrations of 2%, 20% (w/v) in PEG 300 and 100% (undiluted), respectively.  Tea Tree Oil was therefore found to be a potential skin sensitizer and an EC3 value of 4.4% (w/v) was derived.

Reference 5

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Qualifier:

according to guideline

Guideline:

other: ICCVAM Immunotoxicology Working Group Recommended Protocol for the Murine Local Lymph Node Assay (LLNA): Testing of Chemicals for Contact Sensitizing (Allergic Contact Dermatitis [ACD]) Potential (January, 2001).

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

- Physical state: Liquid.
- Batch No.: 1219
- Purity: 100%.
- Composition of test material: Composition meets ISO Standard 4730-2004 Oil of Melaleuca, terpinen-4-ol type (Tea Tree Oil).
- Expiration date of the batch: 2015
- Stability: Stable under storage conditions.
- Storage condition of test material: At room temperature (range of 20 ± 5°C), light protected. Cap tightly, kept out of sunlight and direct heat. Subject to oxidation after opening.

Species:

mouse

Strain:

other: CBA/CaHsdRcc(SPF)

Sex:

female

Details on test animals and environmental conditions:

- Source: RCC Ltd., Laboratory Animal Service, CH-4414 Füllinsdorf/Switzerland.
- Number of animals for the main study: 20
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 11 - 12 weeks (beginning of acclimatization).
- Weight at study initiation: 17.5g - 20.8g (at delivery).
- Identification: Each cage by unique cage card.
- Randomization: Randomly selected by computer algorithm at time of delivery.
- Housing: Individual in Makrolon type-2 cages with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz).
- Diet: Pelleted standard Kliba 3433, batch no. 001/06 mouse maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst) available ad libitum. Results of analyses for contaminants are archived at RCC. There was no contamination of the diet.
- Water: Community tap water from Itingen, available ad libitum. Results of representative bacteriological, chemical and contaminant analyses are archived at RCC. There was no contamination of water.
- Acclimatization: 7 days prior to the first topical application under test conditions after health examination. Only animals without any visible signs of illness were used for the study.
- Temperature: Air-conditioned with ranges for room temperature 22 ± 3°C. Room temperature was monitored continuously and values outside of this range occasionally occurred, usually following room cleaning. These transient variations are considered not to have any influence on the study and, therefore, these data are not reported but are retained at RCC.
- Humidity: Relative humidity 30 - 70%. Humidity was monitored continuously and values outside of this range occasionally occurred, usually following room cleaning. These transient variations are considered not to have any influence on the study and, therefore, these data are not reported but are retained at RCC.
- Air changes: 10 - 15 air changes per hour.
- Photoperiod: 12 hour fluorescent light/12 hour dark cycle.
- Other information on conditions: At least 8 hours music during the light period.

Vehicle:

other: polyethylene glycol 300 (PEG 300)

Remarks:

See 'Any other information on materials and methods'.

Concentration:

2 %, 20 % in PEG 300 and 100 % (undiluted).

No. of animals per dose:

5 females per dose group (3 dose groups and 1 vehicle-only negative control group).

Details on study design:

Test Item Preparation: The test item was placed into a volumetric flask on a tared Mettler balance and the vehicle PEG 300 was quantitatively added. The weight/volume (w/v) dilutions were prepared individually using a magnetic stirrer as homogenizer. Test item formulations were made freshly before each dosing occasion and no more than 4 hours prior to application to the ears. Homogeneity of the test item in the vehicle was maintained until start of treatment using an appropriate homogenizer. The test item in the study was assayed at three consecutive concentrations selected by the Sponsor. Concentrations were in terms of material as supplied.

Validation / positive control study: The sensitivity and reliability of the experimental technique employed was assessed by use of a substance which is known to have skin sensitization properties in CBA/Ca mice. The validation-/positive control study was performed with alpha-hexylcinnamaldehyde in acetone/olive oil (4/1, v/v) using CBA/Ca mice from 11-JAN-2006 to 25-JAN-2006. The reliability check with alpha-hexylcinnamaldehyde indicated that the local lymph node assay as performed at RCC is an appropriate model for testing for contact hypersensitivity.

Treatment Procedures
Topical application: Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 2%, 20% in PEG 300 and 100% (undiluted). The application volume, 25 μl, was spread over the entire dorsal surface (diameter ~ 8 mm) of each ear lobe once daily for three consecutive days.

Administration of ³HTdR: Five days after the first topical application, all mice were administered with 250 μl of phosphate-buffered saline (PBS) containing 20.2 μCi/ml ³HTdR (equal to 80.62 μCi ³HTdR) by intravenous injection via a tail vein.

Determination of incorporated ³HTdR: Approximately five hours after treatment with ³HTdR all mice were euthanized by inhalation of CO2 (dry ice). The draining lymph nodes were rapidly excised and pooled for each animal (2 nodes per animal). Single cell suspensions (phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 μm mesh size). After washing twice with phosphate buffered saline (approx. 10 ml) the lymph node cells were resuspended in 5% trichloroacetic acid (approx. 3 ml) and incubated at approximately +4°C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5% trichloroacetic acid (1 ml) and transferred to glass scintillation vials with 10 ml of 'Irga-Safe Plus' scintillation liquid and thoroughly mixed. The level of ³HTdR incorporation was then measured on a β-scintillation counter. Similarly, background ³HTdR levels were also measured in two 1ml-aliquots of 5% trichloroacetic acid. The β-scintillation counter expresses ³HTdR incorporation as the number of radioactive disintegrations per minute (dpm).

Positive control substance(s):

other: alpha-hexylcinnamaldehyde

Statistics:

The mean values and standard deviations were calculated in the body weight tables. Dunnett-test was conducted for assessment of the difference significance between the test item groups and the negative control (vehicle) group. A statistical analysis was conducted for assessment of the dose-response relationship, and the EC3 value was calculated according to the equation EC3 = (a-c) [(3-d)/(b-d)] + c, where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately below and above the S.I. value of 3 on the local lymph node assay dose response plot.

Positive control results:

For positive control results, see Table 1 in 'Any other information on results'.
No deaths occurred during the study period. Neither clinical/local signs nor other findings were observed in any animals of the control group. On the second application day, slight to severe ear swelling was observed at both dosing sites in all mice of Groups 2-4, persisting for a total of four days or for the remainder of the in-life phase of the study, respectively. In addition, on the second application day, slight to severe ear erythema was also noted at both dosing sites in all mice of Groups 3-4, persisting for a total of four days or for the remainder of the in-life phase of the study, respectively. The body weight of the animals, recorded prior to the first application and prior to necropsy, was within the range commonly recorded for animals of the strain and age.

Parameter:

SI

Remarks:

Mean

Value:

1.8

Variability:

SD = 0.4

Test group / Remarks:

Test Group 2 (2%)

Remarks on result:

other: See Table 2 in 'Any other information on results'.

Parameter:

SI

Remarks:

Mean

Value:

2.8

Variability:

SD = 1.2

Test group / Remarks:

Test Group 3 (20%)

Remarks on result:

other: See Table 2 in 'Any other information on results'.

Parameter:

SI

Remarks:

Mean

Value:

6.5

Variability:

SD = 2.3

Test group / Remarks:

Test Group 4 (100%, undiluted)

Remarks on result:

other: See Table 2 in 'Any other information on results'.

Parameter:

other: DPM per LN

Remarks:

Mean

Value:

272

Variability:

SD = 68

Test group / Remarks:

Control Group 1

Remarks on result:

other: See Table 2 in 'Any other information on results'.

Parameter:

other: DPM per LN

Remarks:

Mean

Value:

502

Variability:

SD = 98

Test group / Remarks:

Test Group 2 (2%)

Remarks on result:

other: See Table 2 in 'Any other information on results'.

Parameter:

other: DPM per LN

Remarks:

Mean

Value:

754

Variability:

SD = 314

Test group / Remarks:

Test Group 3 (20%)

Remarks on result:

other: See Table 2 in 'Any other information on results'.

Parameter:

other: DPM per LN

Remarks:

Mean

Value:

1 768

Variability:

SD = 633

Test group / Remarks:

Test Group 4 (100%, undiluted)

Remarks on result:

other: See Table 2 in 'Any other information on results'.

Cellular proliferation data / Observations:

Mortality: No deaths occurred during the study period.
Clinical signs: Neither clinical/local signs nor other findings were observed in any animals of the control group. One day after the second topical application (Day 3) and on Day 4, a slight ear erythema and hypersensitivity to touch on both ears were observed at both dosing sites in all mice of Group 2 (2%). One day after the first topical application (Day 2), a slight ear erythema was observed at both dosing sites in all mice of Group 3 (20%), persisting for the remainder of the in-life phase of the study. On Days 3-4, the ears of all mice in this group were hypersensitive to touch (both ears). On Day 6 (prior to necropsy), scales were found on both ears in all mice of Group 3 (20%). One day after the first or the second topical application, a slight to moderate ear erythema and/or slight ear swelling were observed at both dosing sites in all mice of Group 4 (100%, undiluted), persisting for the remainder of the in-life phase of the study. On Days 3-4, the ears of all mice in this group were hypersensitive to touch. On Day 4, scales were found on both ears in all mice of this group, persisting for the remainder of the in-life phase of the study.
Body weights: The body weight of the animals, recorded prior to the first application and prior to necropsy, was within the range commonly recorded for animals of the strain and age.
Size of the draining lymph nodes: The size of the draining lymph nodes of animals Nos. 16-20 (Group 4, 100%, undiluted) was 2-fold larger than that of the control group.

The proliferative capacity of pooled lymph node cells was determined by the incorporation of ³H-methyl thymidine measured on a β-scintillation counter.

Table 1. Positive control results.

Group	Test item concentration (w/v)	S.I.
Group 2	5	1.8
Group 3	10 *	2.9 *
Group 4	25 *	6.2 *
EC3 = 10.5%
A clear dose-response relationship was observed. * This value was used in calculation of EC3.

The proliferative capacity of pooled lymph node cells was determined by the incorporation of ³HTdR measured on a β-scintillation counter.

Table 2. Summary of results.

Group	% (w/v)	dpm/LN M (SD)	S.I. M (SD)	EC3 % (w/v)	Statistical Analysis Dunnett-test (G = 4, N = 20, t = 2.59)
					t value	Conclusion
CG 1	-	272 (68)	-	-	-	-
TG 2	2	502 (98)	1.8 (0.4)	24.3	1.01	--
TG 3	20 *	754 (314)	2.8 * (1.2)		2.13	--
TG 4	100 * (undiluted)	1768 (633)	6.5 * (2.3)		6.60	**

**          significant difference at p ≤ 0.05 (two sides)

--            not significant difference at p ≤ 0.05 (two sides)

A dose-response relationship was observed.

*            This value was used in calculation of EC3.

Conclusions:

A test item is regarded as a sensitizer in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in incorporation of ³HTdR compared with concurrent controls, as indicated by the S.I. In this study S.I. of 1.8, 2.8 and 6.5 were determined with the test item at concentrations of 2%, 20% (w/v) in PEG 300 and 100% (undiluted), respectively. Tea Tree Oil was therefore found to be a potential skin sensitizer and an EC3 value of 24.3% (w/v) was derived.

Executive summary:

A GLP-compliant study was carried out to determine the possible contact allergenic potential of Tea Tree Oil.  The study followed the requirements of EU method B.42, OECD method 429, ICCVAM Immunotoxicology Working Group Recommended Protocol for the Murine Local Lymph Node Assay (LLNA): Testing of Chemicals for Contact Sensitizing (Allergic Contact Dermatitis [ACD]) Potential (January, 2001) and OPPTS method 870.2600 without significant deviation.

Three groups each of five female mice were treated with the test item at concentrations of 2%, 20% (w/v) in PEG 300 and 100% (undiluted) by topical application to the dorsum of each ear lobe (left and right) on three consecutive days. A negative control group of five mice was treated with an equivalent volume of the vehicle polyethylene glycol 300 (PEG 300) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (³H-methyl thymidine, ³HTdR). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per animal. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were washed subsequently and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of ³HTdR measured in a β-scintillation counter. All treated animals survived the scheduled study period. Neither clinical/local signs nor other findings were observed in any animals of the control group.

One day after the second topical application (Day 3) and on Day 4, a slight ear erythema and hypersensitivity to touch on both ears were observed at both dosing sites in all mice of Group 2 (2%).  One day after the first topical application (Day 2), a slight ear erythema was observed at both dosing sites in all mice of Group 3 (20%), persisting for the remainder of the in-life phase of the study. On Days 3-4, the ears of all mice in this group were hypersensitive to touch (both ears). On Day 6 (prior to necropsy), scales were found on both ears in all mice of Group 3 (20%). One day after the first or the second topical application, a slight to moderate ear erythema and/or slight ear swelling were observed at both dosing sites in all mice of Group 4 (100%, undiluted), persisting for the remainder of the in-life phase of the study. On Days 3-4, the ears of all mice in this group were hypersensitive to touch. On Day 4, scales were found on both ears in all mice of this group, persisting for the remainder of the in-life phase of the study. No significant difference of dpm/LN was determined at the test item concentration of 2% or 20% (w/v) in PEG 300 compared with the vehicle control group at p ≤ 0.05 (two sides). A significant difference of dpm/LN was determined at the test item concentration of 100% (undiluted) compared with the vehicle control group at p ≤ 0.05 (two sides).

A test item is regarded as a sensitizer in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in incorporation of ³HTdR compared with concurrent controls, as indicated by the S.I.  In this study S.I. of 1.8, 2.8 and 6.5 were determined with the test item at concentrations of 2%, 20% (w/v) in PEG 300 and 100% (undiluted), respectively.  Tea Tree Oil was therefore found to be a potential skin sensitizer and an EC3 value of 24.3% (w/v) was derived.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

A total of four murine Local Lymph Node Assays (LLNA) are available for tea tree oil.  EC3 values obtained in the LLNAs ranged between 25.5% and 4.4%, suggesting that tea tree oil has weak to moderate skin sensitising potential.  However, a principal confounding factor for the LLNA test concerns the fact that tea tree oil is classified as a Cat. 2 irritant in contact with skin. (see section 5.3.4).  It is known that both sensitisers and irritants can induce lymphocyte proliferation.  Whereas true sensitisers stimulate the proliferation of antigen-specific lymphocytes, the response for irritants is nonspecific.  Measurement of lymphocyte proliferation in the LLNA using 3H-T incorporation does not allow for a differentiation of these effects.  Because of this, it is recognised that, taken in isolation, testing of non-sensitising, irritating substances using the LLNA can give rise to false positive results.

A single Guinea Pig Maximisation Test (GPMT) conducted in accordance with the Magnusson and Kligmann method is also available (Bolt, 1989).  No positive reactions were seen in any of the twenty test animals evaluated.  The guinea pig provides a better model for the human immune system than does the mouse.  Given the strengths of the GPMT method, and its ability to differentiate between specific and non-specific lymphocyte proliferations with a degree of confidence not possible in the LLNA, the results of the existing study should be taken into account when a GPMT study is already available.

In view of the very clear negative result obtained in the GPMT, it is concluded that tea tree oil does not meet the criteria for classification as a skin sensitiser.