Melaleuca alternifolia, ext.

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

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:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

GLP compliance:

yes

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:

- Description: Clear colourless liquid.
- Source: Not specified.
- Batch No. of test material: D266
- Purity: 100%
- Expiration date of the batch: 30 November 2014
- Storage condition of test material: Approximately 4°C in the dark.

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Remarks:

Hsdlf:NZW

Details on test animals or tissues and environmental conditions:

- Source: Harlan Laboratories UK Ltd., Leicestershire, UK
- Age at study initiation: Twelve to twenty weeks old
- Weight at study initiation: 2.79 kg or 2.85 kg
- Housing: The animals were individually housed in suspended cages.
- Diet: Free access to food (2930C Teklad Global Certified Rabbit diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study. The diet was considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
- Water: Free access to mains drinking water was allowed throughout the study. The drinking water was considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
- Acclimation period: At least five days.
- Identification: Each animal was given a number unique within the study which was written with a black indelible marker-pen on the inner surface of the ear and on the cage label.
- Temperature: The temperature was set to achieve limits of 17 to 23°C. Any occasional deviations from this target were considered not to have affected the purpose or integrity of the study.
- Humidity: The relative humidity was set to achieve limits of 30 to 70%. Any occasional deviations from this target were considered not to have affected the purpose or integrity of the study.
- Air changes: At least 15 changes per hour.
- Photoperiod: Lighting was controlled by a time switch to give 12 hours continuous light (06:00 to 18:00) and 12 hours darkness.
- Enrichment: The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
- Justification for chosen species: The albino rabbit has been shown to be a suitable model for this type of study and is recommended in the test method. The results of the study are believed to be of value in predicting the likely irritancy potential of Tea Tree Oil to man.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

Amount applied: 0.1 mL Tea Tree Oil

Duration of treatment / exposure:

Tea Tree Oil was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of Tea Tree Oil, and then released.

Observation period (in vivo):

Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment.

Number of animals or in vitro replicates:

Two rabbits.

Details on study design:

- Scoring system: Immediately after administration of Tea Tree Oil, an assessment of the initial pain reaction was made according to the six point scale shown in the attached Appendix 1. Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the Draize Scale for Scoring Ocular Irritation (see the attached Appendix 2).

- Tool used to assess score: Light source from a standard opthalmoscope.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

cornea opacity score

Basis:

animal #1

Remarks:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Results 2

Irritation parameter:

iris score

Basis:

animal #1

Remarks:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Results 3

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #1

Remarks:

mean

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 72 h

Results 4

Irritation parameter:

chemosis score

Basis:

animal #1

Remarks:

mean

Time point:

24/48/72 h

Score:

0.33

Max. score:

1

Reversibility:

fully reversible within: 48 h

Results 5

Irritation parameter:

cornea opacity score

Basis:

animal #2

Remarks:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Results 6

Irritation parameter:

iris score

Basis:

animal #2

Remarks:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Results 7

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #2

Remarks:

mean

Time point:

24/48/72 h

Score:

0.67

Max. score:

1

Reversibility:

fully reversible within: 72 h

Results 8

Irritation parameter:

chemosis score

Basis:

animal #2

Remarks:

mean

Time point:

24/48/72 h

Score:

0.33

Max. score:

1

Reversibility:

fully reversible within: 48 h

Irritant / corrosive response data:

Individual and group mean scores for ocular irritation are given in Table 1 and Table 2 (see 'Any other information on results'). No corneal or iridial effects were noted during the study. Moderate conjunctival irritation was noted in both treated eyes one hour after treatment with minimal conjunctival irritation noted at the 24 and 48-hour observations. Both treated eyes appeared normal at the 72-hour observation.

Other effects:

Individual bodyweights and bodyweight changes are given in Table 3 (see 'Any other information on results'). One animal showed a slight bodyweight loss while the other animal showed expected gain in bodyweight during the study.

Any other information on results incl. tables

Table 1. Individual Scores and Individual Total Scores for Ocular Irritation.

Rabbit Number and Sex	72426 Male				42463 Male
	IPR = 2				IPR = 2
Time After Treatment	1 Hour	24 Hours	48 Hours	72 Hours	1 Hour	24 Hours	48 Hours	72 Hours
CORNEA
E = Degree of Opacity	0	0	0	0	0	0	0	0
F = Area of Cornea Involved	0	0	0	0	0	0	0	0
Score (E x F) x 5	0	0	0	0	0	0	0	0
IRIS
D	0	0	0	0	0	0	0	0
Score (D x 5)	0	0	0	0	0	0	0	0
CONJUNCTIVAE
A = Redness	2	2	1	0	2	1	1	0
B = Chemosis	1	1	0	0	1	1	0	0
C = Discharge	1	0	0	0	2	0	0	0
Score (A + B + C) x 2	8	6	2	0	10	4	2	0
Total Score	8	6	2	0	10	4	2	0

IPR = initial pain reaction

Table 2. Individual Total Scores and Group Mean Scores for Ocular Irritation.

Rabbit Number and Sex	Individual Total Scores At:
	1 Hour	24 Hours	48 Hours	72 Hours
72426 Male	8	6	2	0
42463 Male	10	4	2	0
Group Total	18	10	4	0
Group Mean Score	9.0	5.0	2.0	0.0

Table 3. Individual Bodyweights and Bodyweight Changes.

Rabbit Number and Sex	Individual Bodyweight (kg)		Bodyweight Change (kg)
	Day 0	Day 3
72426 Male	2.85	2.82	-0.03
42463 Male	2.79	2.93	0.14

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Tea Tree Oil produced a maximum group mean score of 9.0 and was classified as a mild irritant (Class 4 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system. Tea Tree Oil does not meet the criteria for classification according to the Globally Harmonised System of Classification and Labelling of Chemicals or Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Dangerous Substances.

Executive summary:

A GLP-compliant study was performed to assess the irritancy potential of Tea Tree Oil to the eye of the New Zealand White rabbit following the in vitro Bovine Corneal Opacity and Permeability study (Harlan Laboratories Ltd Project number 41104699) which showed the test item not be corrosive or severely irritating to the eye. The method was designed to be compatible with OECD Testing Guideline No. 405 "Acute Eye Irritation/Corrosion" and EU Method B.5 Acute Toxicity (Eye Irritation). A single application of Tea Tree Oil to the non-irrigated eye of two rabbits produced moderate conjunctival irritation. Both treated eyes appeared normal at the 72-hour observation. Tea Tree Oil produced a maximum group mean score of 9.0 and was classified as a mild irritant (Class 4 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system. Tea Tree Oil does not meet the criteria for classification according to the Globally Harmonised System of Classification and Labelling of Chemicals or Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Dangerous Substances.