p-mentha-1,3-diene

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

Examination of a -terpinene on Primary Eye Irritancy and Skin Sensitization

Author:

Park B.S.

Year:

2007

Bibliographic source:

Mol. Cell. Toxicol. Vol. 3(1), 68-75, 2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Korean Food and Drug Administration (KFDA) guidelines (98-116)

Principles of method if other than guideline:

The experiments were conducted in accordance with the Korean Food and Drug Administration (KFDA) guidelines (98-116). In this study, rabbits were divided into two groups. One group of three rabbits was washed with 20 mL of warmed distilled saline (W group). A second group of six rabbits did not have their eyes flushed with saline (NW group). The eyes or both groups were subsequently examined for indication of ocular and periocular trauma and/or inflammation at 1, 2, 3, 7, 10 and 13 days after treatment.

Eye tissues were sampled by biopsy at 14 days after treatment. Rabbits were sedated with intramuscular xylazine hydrochloride (5 mg/kg) and anesthetized with ketamine hydrochloride (35 mg/kg). Biopsies of the eyes were sampled and fixed in Bouin solution for several days. The eyes then underwent ethanol series dehydration and the tissue was embedded in paraffin. Sections were stained with he-matoxylin-eosin for observation of the cornea, iris and retina.

After treatment, I.I.O.I (the individual index of ocular irritation), M.I.O.I. (Mean index of ocular irritation) and I.A.O.I. (the index of acute ocular irritation) were calculated following a guideline of Korea Institute of Toxicology (Daejeon, Korea).

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

p-mentha-1,3-diene

EC Number:

202-795-1

EC Name:

p-mentha-1,3-diene

Cas Number:

99-86-5

Molecular formula:

C10H16

IUPAC Name:

1-isopropyl-4-methylcyclohexa-1,3-diene

Test material form:

liquid

Details on test material:

Clear colourless liquid.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL

- Source of test material: alpha terpinene purchased from Sigma (St. Louis, MO)

Repellent solution prepared using 0.5 g (2.0%) of alpha terpinene.

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Remarks:

males and females

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: New Zealand white, Sejin Laboratory Co. (Seoul, South Korea)
- Age at study initiation: 12-18 weeks of age
- Weight at study initiation: male (2.8-3.3 kg) and female (2.7-3.4 kg)
- Acclimation period: yes, for 5 days before testing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16-21.6 °C
- Humidity (%): of 40-70%
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12 h photoperiod

Test system

Vehicle:

other: Repellent solution

Controls:

yes

Amount / concentration applied:

Repellent solution (0.1 mL) of 0.5 g (2.0%) of alpha terpinene

Duration of treatment / exposure:

Eye tissues were sampled by biopsy at 14 days after treatment.

Observation period (in vivo):

1, 2, 3, 4, 7,10 and 13 days

Number of animals or in vitro replicates:

9 animals (6 animals and 3 animals used as a satellite group of animals to investigate the influence of washing).

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: yes, performed on one group (W group):
2 groups of animals:
- 3 rabbits were washed with 20 mL of warmed distilled saline (W group)
- 6 other rabbits did not have their eyes flushed with saline (NW group)
- Time after start of exposure: Washed group – 20-30 seconds of treatment following the guideline of Korea Institute of Toxicology (Daejeon, Korea).

SCORING SYSTEM: yes
I.I.O.I (the individual index of ocular irritation), M.I.O.I. (Mean index of ocular irritation) and I.A.O.I. (the index of acute ocular irritation) were calculated following a guideline of Korea Institute of Toxicology (Daejeon, Korea).

TOOL USED TO ASSESS SCORE: Sections of the eyes were stained with he-matoxylin-eosin for observation of the cornea, iris and retina.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

cornea opacity score

Basis:

animal: 6

Remarks:

non washed group

Time point:

other: days 1, 2, 3, 4, 7, 10 and 13

Score:

> 1 - < 4

Max. score:

4

Reversibility:

fully reversible within: 10 days

Remarks on result:

positive indication of irritation

Results 2

Irritation parameter:

iris score

Remarks:

redness

Basis:

animal: 6

Remarks:

non washed group

Time point:

other: days 1, 2, 3, 4, 7, 10 and 13

Score:

> 1 - < 3

Max. score:

3

Reversibility:

fully reversible within: 4 days

Remarks on result:

positive indication of irritation

Results 3

Irritation parameter:

chemosis score

Basis:

animal: 6

Remarks:

non washed group

Time point:

other: days 1, 2, 3, 4, 7, 10 and 13

Score:

> 1 - < 4

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

positive indication of irritation

Results 4

Irritation parameter:

other: Discharge

Basis:

animal: 6

Remarks:

non washed group

Time point:

other: days 1, 2, 3, 4, 7, 10 and 13

Score:

> 1 - < 3

Max. score:

3

Reversibility:

fully reversible within: 10 days

Remarks on result:

positive indication of irritation

Results 5

Irritation parameter:

cornea opacity score

Basis:

animal: mean of 6

Remarks:

non washed group

Time point:

24/48/72 h

Score:

ca. 2

Reversibility:

fully reversible

Remarks on result:

positive indication of irritation

Results 6

Irritation parameter:

iris score

Basis:

animal: mean of 6

Remarks:

non washed group

Time point:

24/48/72 h

Score:

> 1 - < 2

Reversibility:

fully reversible

Remarks on result:

positive indication of irritation

Results 7

Irritation parameter:

conjunctivae score

Basis:

animal: mean of 6

Remarks:

non washed group

Time point:

24/48/72 h

Score:

ca. 2

Reversibility:

fully reversible

Remarks on result:

positive indication of irritation

Results 8

Irritation parameter:

chemosis score

Basis:

animal: mean of 6

Remarks:

non washed group

Time point:

24/48/72 h

Score:

ca. 2

Reversibility:

fully reversible

Remarks on result:

positive indication of irritation

Other effects:

Other observations:
Ocular discharge was collected using pasteurized swabs at 1, 2, 3, 4, 7, 10 and 13 days after treatment. Ocular discharges were smeared on to a glass slide and fixed for 20-30 min using methanol. After fixing, slides were stained with Giemsa solution for 20-25 min. Stained slides were observed under a light
microscope. Cellular components of ocular discharges were determined and numbers of each component were scores.

Any other information on results incl. tables

Table 1. Grading of ocular lesions – Cornea
OECD TG 405 guideline	Scoring OECD TG 405	KFDA guideline	Scoring KFDA
Opacity: degree of density (readings should be taken from most dense area)*		Opacity - Degree of density (area which is most dense is taken for reading)
No ulceration or opacity	0	Normal	0
Scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible	1	Scattered or diffuse - details of iris clearly visible	1
Easily discernible translucent area; details of iris slightly obscured	2	Easily discernible translucent areas details of iris slightly obscured	2
Nacrous area; no details of iris visible; size of pupil barely discernible	3	Opalescent areas no details of iris visible, siae of pupilbarely discernible	3
Opaque cornea; iris not discernible through the opacity	4	Opaque. iris invisible	4
		Area of cornea involved
		Zero	0
		One quarter (or less) but not zero	1
		Greater than one-quarter and less than one -half	2
		Greater than one-half and less than three quarters	3
		Greater than three quarters up to whole area	4

* The area of corneal opacity should be noted

 

 

Table 2. Grading of ocular lesions – Iris
OECD TG 405 guideline	Scoring OECD TG 405	KFDA guideline	Scoring KFDA
Normal	0	Normal	0
Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia; or injection; iris reactive to light (a sluggish reaction is considered to be an effect	1	Folds above normal, congestion, swelling, circumcorneal injection (anyone or all of these or combination of any thereof), iris still reacting to light	1
Hemorrhage, gross destruction, or no reaction to light	2	No reaction to light, hemorrhage, gross destruction (any one or all of these)	2

 

 

Table 3. Grading of ocular lesions – Conjunctivae
OECD TG 405 guideline	Scoring OECD TG 405	KFDA guideline	Scoring KFDA
redness (refers to palpebral and bulbar conjunctivae; excluding cornea and iris)		Redness (refers to palpebral conjuctiva only)
Normal	0	Normal	0
Some blood vessels hyperaemic (injected)	1	Vessels definitely injected above normal	1
Diffuse, crimson colour; individual vessels not easily discernible	2	More diffuse deeper crimson red (individual vessels not easily discernible)	2
Diffuse beefy red	3	Diffused beefy red	3

 

 

Table 4. Grading of ocular lesions – Chemosis
OECD TG 405 guideline	Scoring OECD TG 405	KFDA guideline	Scoring KFDA
Swelling (refers to lids and/or nictating membranes)
Normal	0	Normal	0
Some swelling above normal	1	Any swelling above normal (inclused nictitating membrane)	1
Obvious swelling, with partial eversion of lids	2	Obvious swelling with partial eversion of lids	2
Swelling, with lids about half closed	3	Swelling with lids about half closed	3
Swelling, with lids more than half closed	4	Swelling with lids about half closed to completely closed	4

 

 

Table 5. Grading of ocular lesions – Discharge
OECD TG 405 guideline	Scoring OECD TG 405	KFDA guideline	Scoring KFDA
	0	Normal	0
	1	Any amount different from normal (does not include small amount observed in inner canthus of normal animals)	1
	2	Discharge with moistening of the lids and hairs just adjacent to the lids	2
	3	Discharge with moistening of the lids and considerable area around the eye	3

 

 

Table 6.Results of eye reactions – NON-washed group (NW)
Observation	Mean Animal No.	Days	Mean value of the scoring
Degree of opacity	6	1	2.6
	6	2	1.6
	6	3	1.3
	6	4	0.83
	6	7	0
	6	10	0
	6	13	0
Diffuse are of opacity	6	1	2.5
	6	2	2
	6	3	1.3
	6	4	0.3
	6	7	0
	6	10	0
	6	13	0
Iris	No change
Conjuctiva - redness	6	1	2.3
	6	2	2.16
	6	3	1.3
	6	4	0.3
	6	7	0.16
	6	10	0
	6	13	0
Chemosis	6	1	2.3
	6	2	1.5
	6	3	0.5
	6	4	0.16
	6	7	0
	6	10	0
	6	13	0
Discharge	6	1	2.5
	6	2	2.5
	6	3	1.83
	6	4	1.16
	6	7	0.16
	6	10	0
	6	13	0

 

Table 7. Results of eye reactions – washed group (W)
Observation	Mean Animal No.	Days	Mean value of the scoring
Degree of opacity	3	1	1.3
	3	2	1
	3	3	0
	3	4	0
	3	7	0
	3	10	0
	3	13	0
	3	1	1.3
	3	2	1
	3	3	0
	3	4	0
	3	7	0
	3	10	0
	3	13	0
Iris	No change
Conjuctiva - redness	3	1	1
	3	2	1
	3	3	0
	3	4	0
	3	7	0
	3	10	0
	3	13	0
Chemosis	3	1	1.3
	3	2	0.3
	3	3	0
	3	4	0
	3	7	0
	3	10	0
	3	13	0
Discharge	3	1	1.6
	3	2	1
	3	3	0
	3	4	0
	3	7	0
	3	10	0
	3	13	0

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Based on the results of this study, alpha terpinene is classified as irritant cat. 2 according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.

Executive summary:

In the key study of Park (2007), the primary eye irritation test procedure was performed according to the “Guidelines for Toxicity Studies of Drugs” provided by Korea Food and Drug Administration (KFDA). The corneal opacity, iris and conjunctival redness, edemas were recorded and classified according to the guideline of KFDA.

Repellent solution (0.1 mL) of 2.0% of alpha terpinene was dropped in the right eye of nine rabbits. Rabbits were divided into two groups. One group of three rabbits was washed with 20 mL of warmed distilled saline (W group, satellite group of animals to investigate the influence of washing). A second group of six rabbits did not have their eyes flushed with saline (NW group). The eyes or both groups were subsequently examined for indication of ocular and periocular trauma and/or inflammation at 1, 2, 3, 7, 10 and 13 days after treatment.

The application of alpha terpinene to the eye could cause the redness to the conjunctiva. However, the redness disappeared after 3 days in the W group and 7 days in the NW group, with exception of one rabbit that took 10 days. No permanent damage has been observed. Moreover, the redness could easily be prevented with saline washing shortly after any accidental exposure.

Chemosis and discharge of the conjunctiva were observed 1 day after treatment in all tested animals. Most discharge the formation on the treated eyes first one to 3 days after treatment. Similarly to the redness, chemosis and discharge of the conjunctiva eventually disappeared after exposure and could be prevented with saline washing.

Eye tissues were sampled by biopsy at 14 days after treatment. Rabbits were sedated with intramuscular xylazine hydrochloride (5 mg/kg) and anesthetized with ketamine hydrochloride (35 mg/kg). Biopsies of the eyes were sampled and fixed in Bouin for several days. The eyes then underwent ethanol series dehydration and the tissue was embedded in paraffin. Sections were stained with hematoxylin-eosin for observation of the cornea, iris and retina.

The individual index of ocular irritation (I.O.I. score) was calculated as follows:

I.O.I = (Degree of opacity X Diffuse are of opacityX5) + (IrisX5) + 2(Redness+ Chemosis+ Discharge)

Based on the rating system provided on sums of all scores obtained from ocular lesions, it was concluded that alpha terpinene was irritating.

For the purpose of this registration, the scores obtained according to the KFDA guideline were compared to the scoring system of the OECD TG 405 - grading of ocular lesions. The mean grades of ocular reaction (conjunctivae, cornea and iris) recorded after 24, 48 and 72 hours were calculated and the mean score for six animals, non-washed group was approx. 2. Furthermore, the potential eye irritation caused by a 2% solution of alpha terpinene was significantly curtailed by an immediate application of saline solution as demonstrated in the washed group.

Based on these results, alpha terpinene is classified as an eye irritant, cat. 2 according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.