Melaleuca alternifolia, ext.

Endpoint summary

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

acute toxicity: oral

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

Remarks:

Non-GLP study

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: The study was conducted to determine the acute oral LD50 value of tea tree oil in the albino rat, according to the method of Litchfield and Wilcoxon (A Simplified Method of Evaluating Dose-Effect Experiments, J.T. Litchfield Jr and F. Wilcoxon, J. Pharmacol. 1949).
- Short description of test conditions: The test item (diluted with peanut oil) was administered by gavage over a range of dose levels to groups of five male and female rats. Toxicity to both SPF and non-SPF animals was investigated.
- Parameters analysed / observed: Mortality and clinical signs of toxicity or abnormal behaviour.

GLP compliance:

no

Test type:

standard acute method

Limit test:

no

Specific details on test material used for the study:

- Batch no.: 88/375
- Purity: 100%

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: The source of the SPF (specific pathogen free) rats was the Animal Resources Centre, W.A. The source of the non-SPF (non specific pathogen free) rats was Animal Services, Monash University, Victoria.
- Age at study initiation: The age of the test animals was not reported.
- Weight at study initiation: Between 146 and 219 grams.
- Fasting period before study: The animals were starved 24 hours before administration of the test sample.
- Housing: Housing conditions were not reported.
- Diet and water: Rat and mouse cubes and tap water ad libitum.

Route of administration:

oral: gavage

Vehicle:

peanut oil

Details on oral exposure:

- Concentration in vehicle: The test sample was diluted with peanut oil w/w at 3 different concentrations: 1/3, 1/4 and 1/5.
- No justification was provided for the choice of vehicle (although it is considered to be appropriate).

Doses:

SPF Rats: Four groups of SPF rats were administered 3, 2.75, 2.6 and 2.5 ml/kg of test sample by gavage, using an intragastric cannula attached to a Yale glass syringe.
Non-SPF Rats: Five groups of non-SPF rats were administered 2.4, 2.25, 2.15, 2.10 and 1.70 ml/kg of test sample by gavage, using an intragastric cannula attached to a Yale glass syringe.

No. of animals per sex per dose:

5 male and 5 female per dose.

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days.
- The mortality rates and observed toxic signs were recorded for each group.

Statistics:

LD50 values were determined with 95% confidence limits.

Sex:

male/female

Dose descriptor:

LD50

Effect level:

2.6 mL/kg bw

Based on:

test mat.

95% CL:

>= 2.5 - <= 2.704

Remarks on result:

other: SPF rats

Sex:

male/female

Dose descriptor:

LD50

Effect level:

1.9 mL/kg bw

Based on:

test mat.

95% CL:

>= 1.83 - <= 1.98

Remarks on result:

other: Non-SPF rats

Mortality:

SPF Rats: The groups which received 3, 2.75, 2.6 and 2.5 ml/kg of test material yielded 70%, 70%, 90% and 30% deaths respectively (See Table 1 in 'Any other information on results').
Non-SPF Rats: The groups which received 2.4, 2.25, 2.15, 2.10 and 1.70 ml/kg of test material yielded 100%, 100%, 80%, 30% and 60% deaths respectively.

Clinical signs:

other: SPF Rats: Animals which survived generally exhibited a lack of tonus in the forelimbs, which disappeared gradually. Moribund animals had weeping eyes and bloodied noses. Non-SPF Rats: The surviving animals exhibited similar symptoms to the SPF animals.

Other findings:

The major non-lethal reaction caused by the test sample was a complete lack of muscular tone in the forelimbs.

SPF rats: The slope function of the line, determined with 95% confidence limits, was found to be 1.08 (0.99 to 1.18).

Non-SPF rats: The slope function of the line, determined with 95% confidence limits, was found to be 1.09 (1.007 to 1.174).

There was a difference in toxicity observed between the two sources of rat used in the study, however it was noted that the slopes of the dose-response relationships were found to be almost identical (see attached Fig. 1).

Table 1. Summary of mortalities and observations in SPF and non-SPF rats.

	Dose (ml/kg)	Sex (M/F)	Number of mortalities per 5 animals	Day of death	Observations
SPF rats	2.5	M	0	N/A	30% deaths in 14 days. One male and one female had impaired mobility in forelimbs (no tonus) after 24 hours, which disappeared after 48 hours. Moribund animals had weeping eyes and bloodied noses.
		F	3	1-2
	2.6	M	4	1-2	90% deaths in 14 days. The remaining rat had impaired mobility in forelimbs (no tonus) which disappeared after 4 days. Moribund animals had weeping eyes and bloodied noses.
		F	5	1-2
	2.75	M	4	1	70% deaths in 14 days. Surviving animals had their mobility restricted (no tonus) which disappeared after 72 hours. Moribund animals had weeping eyes and bloodied noses.
		F	3	1, 3
	3.0	M	4	1	70% deaths in 14 days. Surviving animals had their mobility restricted in their forelimbs (no tonus) which gradually disappeared over 4 days. Moribund animals had weeping eyes and bloodied noses.
		F	3	1, 3, 6
Non-SPF rats	1.7	M	3	1-2	60% deaths in 14 days. All survivors had a lack of tonus in front limbs, which disappeared after 3 days. Moribund animals had weeping eyes and bloodied noses.
		F	3	3-4
	2.1	M	1	3	30% deaths in 14 days. All survivors had a lack of tonus which disappeared after 4 days, except one female who did not recover throughout the experimental period. Moribund animals had weeping eyes and bloodied noses.
		F	2	1
	2.15	M	4	1-2	80% deaths in 14 days. Surviving male and female had lack of tonus in forelimbs, which disappeared after 4 days. Moribund animals had weeping eyes and bloodied noses.
		F	4	1-2
	2.25	M	5	1, 3-4	100% deaths in 14 days. Moribund animals had weeping eyes and bloodied noses.
		F	5	2-3
	2.4	M	5	1-3	100% deaths in 14 days. Moribund animals had weeping eyes and bloodied noses.
		F	5	1, 3

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

The LD50 of Tea Tree Oil was determined in two environmentally derived types of Sprague Dawley rat, and was found to be 2.6 ml/kg in SPF rats and 1.9 ml/kg in non-SPF rats (equivalent to 1691 mg/kg bw). The sample caused weeping eyes, bloodied noses and a lack of tonus in the forelimbs of those animals which survived.

Executive summary:

The acute toxicity of Tea Tree Oil by the oral route to rats was assessed according to the method of Litchfield and Wilcoxon (1949). Groups of 5 male and 5 female Sprague Dawley rats (SPF and non-SPF) were used for the study. The test sample was diluted with peanut oil w/w at 3 different concentrations: 1/3, 1/4, 1/5. Four groups of SPF rats were administered 2.5, 2.6, 2.75 and 3 ml/kg of the test sample by gavage. Five groups of non-SPF rats were administered 1.70, 2.10, 2.15, 2.25 and 2.4 ml/kg of test sample by gavage. All animals were observed for any signs of toxicity or abnormal behaviour during the experimental period of 14 days. The LD50 of Tea Tree Oil was determined to be 2.6 ml/kg bodyweight in SPF rats and 1.9 ml/kg bodyweight in non-SPF rats (equivalent to 1691 mg/kg bw). The sample caused weeping eyes, bloodied noses and a lack of tonus in the forelimbs of those animals which survived.

Reference 2

Endpoint:

acute toxicity: oral

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 423 (Acute Oral toxicity - Acute Toxic Class Method)

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

GLP compliance:

yes

Test type:

acute toxic class method

Limit test:

yes

Specific details on test material used for the study:

- Physical state: Liquid.
- Colour: Clear-pale straw.
- Purity: 100%.
- Composition of test material: Composition meets ISO Standard 4730-2004 Oil of Melaleuca, terpinen-4-ol type (Tea Tree Oil).
- Batch No.: A352
- Expiration date of the batch: 17 December 2011
- Production date: 17 December 2008
- Storage condition of test material: Room temperature (15-25°C), protected from direct sunlight, with nitrogen headspace.
- Log Pow: 3.4-5.5 (30°C)

Species:

mouse

Strain:

other: CRL:(NMRI)BR Mouse

Sex:

female

Details on test animals or test system and environmental conditions:

- Source: Charles River (Europe) Laboratories Inc. Toxi Coop Ltd. 1103 Budapest, Cserkesz u.90, Budapest, Hungary.
- Justification of species/strain: The mouse was selected in accordance with OECD TG 423, at the request of the Sponsor. CRL:(NMRI)BR mouse was selected due to previous experience with this strain, which was considered suitable for the study purposes, in agreement with the Sponsor.
- Hygienic level: SPF (Specific Pathogen Free) at arrival; standard housing conditions during the study.
- Animal health: Only healthy animals were used for the test. Health status was certified by a veterinarian.
- Age at study initiation: Young adult mice, 8 weeks old.
- Weight at study initiation: 28.7-31.2g
- Fasting period before study: On the day before treatment, the animals were fasted overnight; water was not withheld during this period. Food was returned to the animals 2h after treatment.
- Housing: Group caging (up to 3 animals/cage), with the exception of replacement female 3819, which was housed individually after its assignment to the study following the death of female 3815 (euthanised due to a dosing error (misgavage)). Cage type was Type II polypropylene/polycarbonate.
- Bedding: Certified laboratory wood bedding for laboratory animals (Lignocel Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (Rosenberg, Germany) was made available to animals during the study. A copy of the Certificate of Analysis is retained in the archive at LAB Research Ltd.
- Diet: Animals received ssniff SM R/M-Z+H "Autoclavable complete feed for rats and mice - breeding and maintenance" (ssniff Spezialdiäten GmbH Soest, Germany) ad libitum. The food is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
- Water: Tap water from the municipal supply (500 mL water bottle) was available ad libitum. Water quality control analysis was performed every three months and microbiological assessment was performed monthly by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u.36., Hungary).
- Acclimation period: At least 7 days.
- Temperature: 20.6-24.3°C. The temperature was recorded twice daily during the study and the acclimation period. No deviations from the target ranges occurred.
- Relative humidity: 33-67%. The relative humidity was recorded twice daily during the study and the acclimation period. No deviations from the target ranges occurred.
- Air changes: 15-20 air exchanges.
- Photoperiod: 12 hours daily, from 06.00 to 18.00.
- Enrichment: Group-housing allowed social interaction and the deep wood sawdust bedding allowed digging and other normal rodent activities.
- Identification: Animals were individually identified with numbers written on the tail with an indelible marker pen. The numbers were assigned on the basis of LAB Research Ltd.'s Master File, for each animal allocated to the treatment groups. The cages were identified by cards, containing information on the study code, sex, dose group, cage number and individual animal numbers.
- Other: Females were nulliparous and non-pregnant.

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

Polyethylene glycol 400 - PEG 400, Ph. Eur

Details on oral exposure:

- Dosing procedure: Animals received a single oral gavage administration of Tea Tree Oil followed by a fourteen-day observation period. Animals were weighed immediately prior to treatment. Tea Tree Oil was administered by oral gavage in the morning and a constant treatment volume of 10 mL/kg bw was utilised for each dose group.
- Concentration of Tea Tree Oil in dose formulation: 200 mg/mL
- Volume administered to study animals: 0.29-0.31 mL/animal
- Vehicle Lot No.: 14 35799
- Manufacturer of vehicle: Fluka/Sigma-Aldrich
- Storage of vehicle: Room temperature.
- Rationale for the selection of the starting dose: The initial dose level was selected based on information provided by the Sponsor. The LD50 value was expected to be approximately 2000 mg/kg body weight (bw). The stepwise procedure recommended by OECD Test Guideline 423 was followed.
- Formulation: Tea Tree Oil was freshly formulated in PEG 400 prior to each treatment, at a concentration of 200 mg/mL, by the Central Dispensary Unit of LAB Research Ltd. On each occasion, 2.00 g of Tea Tree Oil was accurately weighed into a labelled glass beaker. PEG 400 (10g, weighed accurately) was added to the beaker and the contents stirred continuously with the aid of a magnetic stirring bar to ensure homogeneity of the dose formulation. Separate dose formulations were prepared on 30 September, 08 and 09 October 2009.

Doses:

Initially, three females (Step 1) were treated at 2000 mg/kg bw. As no mortality occurred in this dose group, a confirmatory group was similarly treated with Tea Tree Oil at the same dose level (Step 2).

No. of animals per sex per dose:

3 animals per Step (2 Steps, as outlined in 'Doses' above).

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days.
- Necropsy of survivors performed: Yes.
- Clinical observations: All animals were observed individually after dosing during the first 30 minutes, then 1, 2, 3, 4 and 6 hours after the treatment and once each day for 14 consecutive days thereafter. Additional clinical observations were conducted in female 3826 one day after the treatment (Day 1) in order to provide additional information on the animal's clinical condition. Observations were performed on the skin and fur, eyes and mucous membranes and also respiratory, circulatory, autonomic and central nervous systems, somatomotor activity and behaviour pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
- Body weight: Body weight was recorded for all animals on the day prior to treatment (Day -1), immediately before treatment (Day 0) and Days 7 and 14. A single Step 1 animal (3826) was also weighed on Days 5 and 6 for health evaluation purposes, with the approval of the Study Monitor.
- Terminal procedures: All animals were sacrificed by exsanguination under pentobarbital anaesthesia (Euthasol 40%) on Day 14, with the exception of female 3815 that was euthanised on Day 0. For all animals, after examination of the external appearance, the cranial, thoracic and the abdominal cavities were opened and the organs and the tissues were examined. Any macroscopic abnormalities were recorded.
- Other: As no Tea Tree Oil-related mortality was noted in the second group, no further testing was required, in accordance with OECD TG 423.

Statistics:

The test method used was not intended to allow the calculation of a precise LD50 value. Tea Tree Oil was placed into the appropriate category of the Globally Harmonised Classification System (GHS) as described in OECD TG No. 423.

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No Tea Tree Oil-related mortality was recorded during the course of the study. Animal No. 3815 was euthanised on Day 0 as it was in poor clinical condition, suggesting a dosing error (misgavage). Prior to its death, this animal showed severely decreased activity and dyspnoea 30 min after dosing and became moribund 1h post-treatment, when it was euthanised for humane reasons.

Clinical signs:

other: Treatment with Tea Tree Oil at a dose level of 2000 mg/kg bw resulted in decreased activity (6/6) animals, hunched back position (6/6 animals), uncoordination (4/6 animals), piloerection (6/6 animals), decreased grip reflex (2/6 animals), decreased respir

Gross pathology:

There were no macroscopic findings related to treatment with Tea Tree Oil at a dose level of 2000 mg/kg bw. In animals surviving to the end of the observation period, uterus in estrus was observed in 3/6 females (3816, 3819 and 3821) and was considered unrelated to treatment. Collapsed red discoloured left lobe of the lungs, pale frothy material in the trachea and red frothy material in the nose were observed in female 3815 euthanised pre-terminally, confirming the suspicion of misdosing. Additionally, red coloured urine was also noted in this animal and was considered incidental.

Interpretation of results:

Category 5 based on GHS criteria

Remarks:

Migrated information

Conclusions:

Under the conditions of this study, the acute oral LD50 value of Tea Tree Oil was higher than 2000 mg/kg bw following administration to female CRL:(NMRI)BR mice. According to the Globally Harmonised Classification System (GHS) criteria, Tea Tree Oil should be classified as "Category 5".

Executive summary:

A GLP-compliant study was conducted in accordance with OECD Guideline 423 (acute toxic class method) to determine the acute oral toxicity of Tea Tree Oil to female CRL:(NMRI)BA mice. In this two-step study, three animals were dosed in the initial step with Tea Tree Oil (formulated in PEG 400) at a dose level of 2000 mg/kg bw, followed by an observation period of 14 days. In the absence of any mortailities, a confirmatory group of three animals was then tested at the same dose level. There were no mortalities or macroscopic findings related to treatment and no clear indications of effects on bodyweight. Clinical signs included decreased activity, hunched back position, uncoordination, piloerection, decreased grip reflex, decreased respiratory rate and/or dyspnoea, none of which persisted beyond day 8 of treatment. In conclusion, under the conditions of this study, the acute oral LD50 of Tea Tree Oil was > 2000 mg/kg bw, when administered to female mice. According to the Globally Harmonised Classification System (GHS) criteria, tea tree oil should be classified as "Category 5".

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

1 691 mg/kg bw

Quality of whole database:

Bolt (1989) has been assigned a reliability of 2. Kubaszky (2010) has been assigned a reliability of 1. Both studies meet the information requirements of REACH.

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1300 (Acute inhalation toxicity)

Qualifier:

according to guideline

Guideline:

EU Method B.2 (Acute Toxicity (Inhalation))

Qualifier:

according to guideline

Guideline:

other: MAFF Japan Testing Guidelines for Toxicity Studies 59 Nohsan Number 4200

GLP compliance:

yes

Test type:

traditional method

Limit test:

no

Specific details on test material used for the study:

- Source: Tweed Ti-Tree Produce. Address: PO BOX 1990, Kingscliff 2487, New South Wales, Australia.
- Batch No.: A352
- Purity: 100%
- Storage condition of test material: Ambient (< 30°)
- Appearance: Colourless to pale yellow liquid
- Production date of the batch: 18 December 2008
- Expiration date of the batch: 18 December 2011
- Preparation: The test item was used as supplied and no preparation was required.

Species:

rat

Strain:

Wistar

Remarks:

Wistar CRL:(WI) BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Toxi-Coop Kft, 1103 Budapest, Hungary and Charles River (Europe) Laboratories Inc.
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: Young adults (9 to 12 weeks old).
- Weight at study initiation: Weight ranged from 231-366g.
- Housing: Animals were housed in groups of five, by sex, in solid-floor cages (Type III) with stainless steel mesh lids and softwood flake bedding.
- Diet: The animals were provided with ssniff SM R/M-Z+H “Autoclavable Complete Feed for Rats and Mice – Breeding and Maintenance” (ssniff Spezialdiäten GmbH, D-59494 Soest Germany).
- Water: Tap water, as for human consumption, ad libitum.
- Acclimation period: At least 5 days.
- Temperature: The environmental controls were set to achieve target values of 22 ± 3°C. During the exposure periods, the temperature values deviated from the required range prescribed in the study plan. The temperature ranged from 19.6 to 26°C. This deviation had no effect on the purpose and integrity of the study.
- Humidity: The environmental controls were set to achieve target values of 30-70% relative humidity. During the exposure periods and on some other occasions, the humidity deviated from the required range. Humidity ranged from 5.0 to 46.0%. This deviation had no effect on the purpose and integrity of the study.
- Air changes: The animal room was ventilated at a rate of at least 15 air exchanges per hour.
- Photoperiod: The lighting was controlled to give 12 hours of continuous artificial light in each twenty-four hour period.
- Identification: Each animal was identified by a unique number marked on the tail. The animal number was assigned on the basis of the LAB Research Ltd.'s laboratory master file. Cages were identified by cage card, giving details of study code, sex, dose-group, cage number and individual animal numbers.
- Justification for species selection: Rats are the preferred species as historically they have been used for this type of study and they are specified by the appropriate regulatory authorities.
- Other: After arrival, the animals' health was certified by the resident veterinarian. The animals were randomised to groups according to body weight.

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 2.31 - <= 3.51 µm

Geometric standard deviation (GSD):

>= 2.05 - <= 2.42

Remark on MMAD/GSD:

Group 1: MMAD = 3.51 µm; GSD = 2.05
Group 2: MMAD = 2.31 µm; GSD = 2.09
Group 3: MMAD = 3.40 µm; GSD = 2.42

Details on inhalation exposure:

- Technical trials: Prior to animal exposures, test material atmospheres were generated within the exposure chamber. During these technical trials, air-flow settings and test material input rates were varied to achieve the required atmospheric characteristics.

- Exposure system and apparatus: The animals were exposed, nose-only, to an atmosphere of the test item using a TSE Rodent Exposure System (TSE Systems GmbH, Bad Homburg, Germany). This system comprises of two, concentric anodised aluminium chambers and a computer control system incorporating pressure detectors and mass flow controllers. Fresh aerosol from the generation system was constantly supplied to the inner plenum (distribution chamber) of the exposure system from where, under positive pressure, it was distributed to the individual exposure ports. A schematic diagram of the exposure system is presented in Figure 1 (see attached). Homogeneity of the test atmosphere within the test chamber and amongst the exposure ports was not specifically determined during this study. However, chambers of this design have been fully validated and shown to produce evenly distributed atmospheres in the animals' breathing zones (Pauluhn, 1994).

- Method of holding animals in test chamber: The animals were individually held in tapered polycarbonate restraint tubes located around a single tier of the exposure chamber which allowed only the animal’s nares to enter the exposure port.

- Rate of air flow: The flow of air through each port was at least, approximately, 0.7 L/min. This flow rate was considered adequate to minimise re-breathing of the test atmosphere as it is higher than the respiratory minute volume of a rat. Chamber airflow rates were monitored continuously and recorded every minute during each exposure period by the TSE-DACO monitoring system integrated into the exposure system.

- System of generating aerosols: The test item formulation was aerosolised using a stainless steel concentric jet nebuliser (TSE Systems GmbH, Bad Homburg, Germany) located at the top of the exposure chamber. The rate of formulation use was controlled by a syringe pump and compressed air was supplied by means of an oil-free compressor and passed through a suitable filter system prior to introduction to the nebuliser.

- Method of particle size determination: The particle size of the test atmosphere was determined three times during the exposure period using a 7-stage impactor of Mercer style (TSE Systems GmbH, Bad Homburg, Germany). Such devices employ an inertial separation technique to isolate particles in the discrete aerodynamic size ranges. Samples were taken from an unoccupied exposure port (representing the animal’s breathing zone). The collection substrates and the backup filter were weighed before and after sampling and the weight of test item, collected at each stage, calculated by difference. Samples were collected by pulling a suitable, known volume of test atmosphere, from an unoccupied exposure mask (representing the animals’ breathing zone), through the collection device and measuring the amount of Tea Tree Oil collected on each stage using a validated HPLC technique. In this way, the proportion (%) of aerosol less than 0.33, 0.5, 0.77, 1.21, 1.93, 3.13 and 5.09 μm was calculated. From these data, using software supplied with the impactor (TSE Systems GmbH, Bad Homburg, Germany), the Mass Median Aerodynamic Diameter (MMAD), and Geometric Standard Deviation were calculated. In addition, the proportion (%) of aerosol less than 4μm (considered to be the inhalable portion) was determined.

- Treatment of exhaust air: Spent aerosol entered the outer cylinder from where it was exhausted through a suitable filter system.

- Pressure and temperature in air chamber: Airflows and relative pressures within the system were constantly monitored and controlled by the computer system thus ensuring a uniform distribution and constant flow of fresh aerosol to each exposure port (breathing zone). Test atmosphere temperature was monitored continuously and recorded every minute during each exposure period by the TSE-DACO monitoring system integrated into the exposure system.

- Other: Following an equilibration period of at least the theoretical chamber equilibration time (T99) (Silver, 1946), each group was exposed to an atmosphere of the test material for a single period of four hours. Test atmosphere relative humidity, carbon dioxide concentration and oxygen concentration were monitored continuously and recorded every minute during each exposure period by the TSE-DACO monitoring system integrated into the exposure system.

Analytical verification of test atmosphere concentrations:

yes

Remarks:

See 'Any other information on materials and methods'.

Duration of exposure:

4 h

Concentrations:

The limit exposure of 5.0 mg/L was selected as the target concentration for the first group. Subsequent target exposures were chosen based on the results of the preceding exposure(s) in an attempt to produce a range of mortality rates.

No. of animals per sex per dose:

5 males and 5 females per dose group.

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days.
- Frequency of observations: Animals were checked for morbidity and/or mortality, five times on the day of exposure (day 0) and twice daily (early and late in the working day) during the remainder of the observation period.
- Necropsy of survivors performed: Yes - Surviving animals and the four moribund animals from Group 1 were euthanised by exsanguination under anaesthesia induced by an intra-peritoneal injection of pentobarbital solution (Euthasol® 40%; Lot No.: i) 07K05 7; ii) 10C25 7; Expiry: i) 10-2010; ii) 02-2013; Produced by AST Beheer B.V. Oudewater Netherlands (Produlab Pharma, Raamsdonksveer)), and a gross macroscopic examination was performed. All animals, including those that died during the study, were subject to a detailed examination of the abdominal and thoracic cavities. Special attention was given to the respiratory tract for macroscopic signs of irritancy or local toxicity.
- Clinical signs: Animals were observed for clinical signs at hourly intervals during exposure, as soon as practicable following removal from restraint at the end of exposure, one hour after exposure and subsequently once daily for fourteen days.
- Body weight: Individual bodyweights were recorded prior to treatment on the day of exposure and on days 7 and 14 or at death.

Statistics:

Data evaluation included the relationship, if any, between the animals’ exposure to the test item concentrations and the level of mortality, behavioural or clinical observations, bodyweight changes, macroscopic abnormalities or any other toxicological effects. The four-hour LC50 values were calculated using SPSS software and a log/probit method.

Sex:

male/female

Dose descriptor:

LC50

Effect level:

4.78 mg/L air

Based on:

test mat.

95% CL:

>= 3.94 - <= 5.32

Exp. duration:

4 h

Sex:

male

Dose descriptor:

LC50

Effect level:

5.23 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Sex:

female

Dose descriptor:

LC50

Effect level:

4.29 mg/L air

Based on:

test mat.

95% CL:

>= 3.41 - <= 6.41

Exp. duration:

4 h

Mortality:

Mortality data are presented in Table 1 in 'Any other information on results'. A single four-hour nose-only exposure of rats led to the death or pre-terminal euthanasia of seven animals dosed at 5.04 mg/L on day 1. A specific cause of death was not clearly determined for animals found dead. Necropsy of the surviving animals after a 14-day observation period did not reveal any test item-related macroscopic findings up to a concentration of 5.04 mg/L.

Clinical signs:

other: Wet fur was recorded both during and for several hours after exposure, whilst fur staining on the head was recorded on removal from restraint and persisted for several days. In addition, fur staining by the test item was detected in all groups during exp

Body weight:

The surviving males from Group 1 and all animals from Group 3, showed bodyweight loss during the first week of the observation period.

A summary of the mortality data is shown below in Table 1.

Table 1.  Mortality rates.

Group Number	Mean Achieved Atmosphere Concentration of Tea Tree Oil (mg/l)	Male Deaths	Female Deaths	Total Deaths
1	5.04	2/5	5/5	7/10
2	1.94	0/5	0/5	0/10
3	3.70	0/5	0/5	0/10

 

The test atmosphere was sampled at approximately hourly intervals during each exposure and the actual concentration of Tea Tree Oil was calculated. A summary of the mean values obtained is displayed in Table 2 below. The individual data are presented graphically in Figures 2 to 4 (see attached).

Table 2.  Test atmosphere concentration.

Group Number	Mean Achieved Atmosphere Concentration of Tea Tree Oil (mg/l)	Standard Deviation	Nominal Atmosphere Concentration of Tea Tree Oil (mg/l)
1	5.04	0.28	21.25
2	1.94	0.60	7.79
3	3.70	0.55	10.62

 

A summary of the particle size distribution of the test atmosphere is displayed in Table 3 below. The data are presented graphically in Figures 5 to 7 (see attached). Particle size distribution was satisfactory for the purposes of the study.

Table 3.  Particle Size Analysis.

Group Number	Mean Achieved Atmosphere Concentration of Tea Tree Oil (mg/l)	Mean Mass Median Aerodynamic Diameter (MMAD) (μm)	Geometric Standard Deviation	Inhalable Fraction (% < 4μm)
1	5.04	3.51	2.05	57.1
2	1.94	2.31	2.09	77.2
3	3.70	3.40	2.42	57.2

 

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

The acute inhalation median lethal concentrations (4-hr LC50) and 95% confidence limits of Tea Tree Oil in rats were calculated to be:
- Male & Female (Number of animals - 30): 4.78 (3.94 - 5.32) mg/L
- Male only (Number of animals - 15): 5.23 mg/L
- Female only (Number of animals - 15): 4.29 (3.41 - 6.41) mg/L

Executive summary:

A GLP-compliant study was carried out to determine the acute inhalation toxicity of Tea Tree Oil to rats.  The study followed the requirements of OECD guideline 403, OPPTS 870.1300, EU Method B.2 and MAFF Japan Testing Guidelines for Toxicity Studies 59 Nohsan Number 4200 without significant deviation.  Three groups of ten Wistar rats (five males and five females) were exposed to an aerosol atmosphere.  The animals were exposed for a single four-hour period using a nose-only exposure system, followed by a fourteen day observation period.  Seven mortalities occurred at the highest test concentration; a specific cause of death was not clearly determined. No mortalities occurred at the two lower test concentrations. The surviving males from Group 1 and the majority of surviving males from Group 3 showed bodyweight loss during the first week of the observation period. Necropsy of the surviving animals on completion of the fourteen day observation period did not reveal any test item-related gross findings up to a concentration of 5.04 mg/L. The acute inhalation median lethal concentrations (4-hr LC50) and 95% confidence limits of Tea Tree Oil in rats were calculated to be:

- Male & Female (Number of animals - 30): 4.78 (3.94 - 5.32) mg/L

- Male only (Number of animals - 15): 5.23 mg/L

- Female only (Number of animals - 15): 4.29 (3.41 - 6.41) mg/L

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LC50

Value:

4 780 mg/m³ air

Quality of whole database:

This study has been assigned a reliability of 1 and meets the information requirements of REACH.

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Non-GLP study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

GLP compliance:

no

Test type:

standard acute method

Limit test:

yes

Specific details on test material used for the study:

Batch No. of test material: 88/375
Purity: 100%

Species:

rabbit

Strain:

New Zealand White

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Test animals: 5 male and 5 female albino rabbits.
- Weight at study initiation: 2.44 to 3.03 kg
- Age at study initiation: Not reported.
- Source: Not specified.
- Housing conditions: Not reported.
- Environmental conditions: Not reported.

Type of coverage:

not specified

Vehicle:

unchanged (no vehicle)

Details on dermal exposure:

Area of exposure: Approximately 175 cm².

Duration of exposure:

Duration of exposure: 24 hours.

Doses:

A single dose of 2000 mg/kg.

No. of animals per sex per dose:

5 males and 5 females.

Control animals:

no

Details on study design:

The animals were observed during the 24 hour exposure period and daily for 14 days after the exposure period. Observations were made for any signs of toxicity and abnormal behaviour. The animals were weighed on days 0, 7 and 14 (see Table 1 in 'Any other information on materials and methods').

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

There were no mortalities.

Clinical signs:

other: One in ten animals exhibited slight diarrhoea on Day 3. There were no other signs of toxicity or abnormal behaviour.

Interpretation of results:

GHS criteria not met

Conclusions:

A group of 5 male and 5 female rabbits was treated with Tea Tree Oil Batch 88/375 at a rate of 2000 mg/kg dermally. Slight diarrhoea occurred in one animal. The acute dermal LD50 was determined to be > 2000 mg/kg.

Executive summary:

A study was conducted in accordance with OECD Guideline 402 to determine the acute dermal toxicity of Tea Tree Oil in the rabbit. A group of 5 male and 5 female rabbits was treated with Tea Tree Oil Batch 88/375 at a rate of 2000 mg/kg dermally.  Slight diarrhoea occurred in one animal. There were no mortalities and the acute dermal LD50 was determined to be > 2000 mg/kg. Tea Tree Oil does not meet the criteria for classification under GHS.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

This study has been assigned a reliability of 2 and meets the information requirements of REACH.

Justification for classification or non-classification

Acute Oral toxicity:

An acute oral toxicity study conducted in non-SPF male and female rats resulted in an LD50 of 1.9 mL/kg bw (equivalent to 1691 mg/kg bw) (Bolt, 1989).  Based on CLP/GHS criteria, it is concluded that tea tree oil should be assigned the classification Acute Tox. 4, H302: Harmful if swallowed.

Acute Inhalation toxicity:

An acute inhalation toxicity study conducted in male and female rats resulted in an LC50 in females of 4.29 mg/L (Nagy, 2011).  Based on CLP/GHS criteria,, it is concluded that tea tree oil should be assigned the classification Acute Tox. 4, H332: Harmful if inhaled.

Acute Dermal toxicity:

An acute oral toxicity study conducted in male and female rats resulted in an LD50 of > 2000 mg/kg bw (Bolt, 1989).  Based on CLP/GHS criteria, it is concluded that tea tree oil should not be classified on the basis of acute dermal toxicity.