Melaleuca alternifolia, ext.

Acute Toxicity: inhalation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1300 (Acute inhalation toxicity)

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Test guideline 4

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

Test material

Test material information

Constituent 1

Reference substance name:

Melaleuca alternifolia, ext.

EC Number:

285-377-1

EC Name:

Melaleuca alternifolia, ext.

Cas Number:

85085-48-9

Molecular formula:

Not applicable (a generic molecular formula cannot be provided for this specific UVCB substance).

IUPAC Name:

Essential oil of melaleuca alternifolia

Test material form:

liquid

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.

Test animals

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.

Administration / exposure

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.

Results and discussion

Effect levels

Effect levels 1

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

Effect levels 2

Sex:

male

Dose descriptor:

LC50

Effect level:

5.23 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Effect levels 3

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.

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

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