Melaleuca alternifolia, ext.

Acute Toxicity: oral

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

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

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:

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

Test animals

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.

Administration / exposure

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.

Results and discussion

Effect levels

Effect levels 1

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

Effect levels 2

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.